JP2003522118A - Death domain containing receptor - Google Patents

Abstract

  (57) [Summary] A novel death domain containing receptor (DR3 and DR3) protein that is a member of the tumor necrosis factor (TNF) receptor family is provided. In particular, there is provided an isolated nucleic acid molecule encoding human DR3 and DR3 protein. DR3 and DR3 polypeptides are also provided, along with vectors, host cells, and recombinant methods for producing the same. Furthermore, the present invention relates to a search method for identifying an agonist and an antagonist of DR3 and DR3 activity. Antagonists or agonists of these receptors are useful for treating diseases or disorders associated with excess or deficiency of apoptosis and inflammatory diseases or disorders.

Description

Detailed Description of the Invention

TECHNICAL FIELD The present invention relates to novel members of the tumor necrosis factor family of receptors. In particular, isolated nucleic acid molecules encoding human death domain containing receptors (DR3 and DR3-V1) are provided. Death domain containing receptor (Death / Domain / C
ontaining receptor) polypeptides are also provided, along with vectors for producing them, host cells, and recombinant methods. The present invention further provides DR3
A search method for identifying active agonists and antagonists.

BACKGROUND OF THE INVENTION Many biological actions, such as the response to certain stimuli and natural biological processes, are controlled by factors such as cytokines. Many cytokines act through receptors by engaging with the receptors and causing intracellular reactions. For example, tumor necrosis factor (TNF) alpha and beta are cytokines that act via TNF receptors to regulate a myriad of biological processes including the induction of infection and shock and protection against inflammatory diseases. This TNF molecule belongs to the "TNF-ligand" superfamily and acts together with its receptor or counterligand, the "TNF receptor" superfamily. So far, 9 have been identified in the TNF ligand superfamily, and 10 in the TNF receptor superfamily have been studied.

Among the ligands are TNF-α, lymphotoxin-α (also known as LT-α, TNF-β), LT-β (present in the LT-α2-β complex heterotrimer), FasL, CD40L, CD27L, CD30L, 4-1BBL, O
X40L, and neurotrophic factor (NGF) are included. The TNF receptor superfamily includes p55TNF receptor, p75TNF receptor, TNF receptor-related protein, FAS antigen or APO-1, CD40, CD27, CD30, 4-1B.
B, OX40, low affinity p75 and NGF receptor (Meager,
A, Biologicals, 22: 291-295 (1994)).

Many of the members of the TNF ligand superfamily are expressed by activated T cells and they are required for the interaction between T cells and other cell types that underlie the ontogeny and function of the cell. (Meager, A., supra). Considerable insight into the essential function of several members of the TNF receptor family has come from the identification and creation of mutants that do not express these proteins. For example, natural variants in the FAS antigen and its ligands cause lymphoproliferative disorders, which probably reflect a failure of programmed cell death (Watanabe-Fukunag).
a.R. Nature, 356: 314 (1992)). CD40
The ligand mutations are high level immunoglobulin M and low level immunoglobulin G in plasma.
And X-link immunodeficiency, characterized by a defect in T cell-dependent B cell activation (Allen, RC et al., Science, 259: 990.
Page (1993)). Site-directed mutations of low affinity neurotrophic factor receptors cause diseases characterized by defects in peripheral structure sensor innovation (Lee, KF, et al., Cell 69: 737 (1992)).

TNF and LT-α are two types of TNF receptors (55 kd- and 75 kd-TN
F receptor). TNF and L acting through these receptors
Numerous biological effects induced by T-α include hemorrhagic necrosis of transplanted tumors, cytotoxicity,
It includes a role in endotoxic shock, inflammation, immune regulation, proliferation, and antiviral responses, as well as protection from the dangerous effects of ionizing radiation. TNF and LT-α are endotoxin shock, cerebral malaria, tumor, autoimmune disease, AIDS and graft-
It is involved in the pathogenesis of a wide range of diseases, including host rejection (Beutler, B. and Von Huffel, C, Science, 264: 667-668 (1994)). Mutations in the p55 receptor cause increased susceptibility to microbial infections. Furthermore, a domain of approximately 80 amino acids near the C-terminus of TNFR1 (p55) and Fas has been reported as a "death domain" capable of signaling for programmed cell death (Tartglia et al., Cell,
74: 845 (1993)).

Apoptosis or programmed cell death is one of the physiological processes
Essential for normal development and homeostasis of multicellular organisms (H. Steller
, Science, 267: 1445-1449 (1995)). Perturbation of apoptosis contributes to the pathogenesis of several human diseases including cancer, neurodegenerative diseases, and acquired immunodeficiency syndrome (CB Thompson, Science, 267).
Volume: 1456-1462 (1995)). Recently, cell surface death receptors (de
Ath receptor), Fas / APO-1 and TNFR-1 have received attention for their signaling and biological functions (JL Clevelan).
d, et al., Cell 81: 479-482 (1995); Frase
R., et al., Cell 85: 781-784 (1996); Nagat
a, et al., Science, 267: 1449-1456 (1995)).
Both are members of the TNF receptor family, which also includes TNFR-2, low affinity NGFR, CD40 and CD30, among others (C.A.S.
Smith et al., Science, 248: 1019-1023 (1990); Edited by Purton, Heldin, Carl, "Standard Textbooks of Molecular Biology and Cell Biology (Modular / Texts / Molecular).
・ And ・ Cell ・ Biology) 」, Chapman ・ and ・ Hall
Inc., London, 1995), M. Tewari). Members of this family are defined by the presence of cysteine-rich repeats in their extracellular domains, while Fas / APO-1 and TNFR-1 are intracellular homologs appropriately named “death domains”. Share the sex domain. This domain is closely related to the suicide gene reaper in Drosophila (P. Goldstein et al., C
Ell, 81: 185-6 (1995); Scene, such as White
nce, 264: 677-83 (1994)). The shared death domain suggests that both receptors interact with a recently identified group of related signaling molecules. Activation of Fas / APO-1 incorporates FADD / MORT1 which is a death domain containing adapter molecule (AM Chinnaiyan et al., Cell 81: 505-12 (1995); MP Boldin.
Etc. Biol. Chem. 270: 7795-8 (1995);
F. C. Kischkel et al., EMBO, 14: 5579-5588 (1
995)), which in turn binds and probably activates FLICE / MACH1, a pro-apoptotic protease of the ICE / CED-3 family.
． Muzio et al., Cell, 85: 817-827 (1996);
P. Boldin et al., Cell, 85: 803-815 (1996)).
. While the central role of Fas / APO-1 is to trigger cell death,
TNFR-1 can signal a wide array of biological activities, often due to its ability to activate NF-kB (LA Tartaglia et al., Immun.
ol. Today, 13: 151-3 (1992)). Therefore TNFR-
1 incorporates TRADD, which is a multivalent adapter molecule, which also contains a death domain as in FADD (H. Hsu et al., Cell, 81: 495-495).
504 (1995); Hsu et al., Cell, 84: 299-308
Page (1996)). TRADD can signal both apoptosis and NF-kB activation through association with a number of signaling molecules including FADD, TRAF2, and RIP (H. Hsu et al., Cell, 84: 299-308.
Page (1996); Hsu et al., Immunity, 4 volumes: 387-396.
Page (1996).

Altered effects of TNF family ligands and TNF family receptors affect a myriad of normal and abnormal functions during the biological processes of mammalian systems. Therefore, there is a clear need for identification and characterization of such receptors and ligands that affect biological activity in both normal and diseased states. In particular, new members of the TNF receptor family need to be isolated and characterized.

SUMMARY OF THE INVENTION The present invention provides an isolated nucleic acid molecule comprising a nucleic acid sequence encoding the amino acid sequences set forth in SEQ ID NO: 2 and SEQ ID NO: 4 or 199 as ATCC Deposit No. 97456.
March 1, 2006 and October 1, 1996 as ATCC Deposit No. 97757
Provided is the amino acid sequence encoded by the cDNA clone deposited in the bacterial host on day 0.

The invention also includes vectors and host cells for recombinant expression of the nucleic acid molecules described herein, as well as methods of making the vectors and host cells and DR3.
And DR3-Variant 1 (DR3-V1) (formerly known as DDCR)
Methods of using them for recombinant production of polypeptides or peptides are provided.

The invention further provides an isolated DR3 or DR3-V1 polypeptide having an amino acid sequence encoded by the polynucleotides described herein. The present invention also provides diagnostic assays, such as quantitative or diagnostic assays for detecting DR3 and DR3-V1 protein levels. So, for example,
The diagnostic assay according to the present invention for detecting overexpression of DR3 and DR3-V1 or its soluble form compared to normal control tissue samples may also be used to detect the presence of tumors.

Tumor necrosis factor (TNF) family ligands are known to be the most pleiotropic cytokines that induce numerous cellular responses including cytotoxicity, antiviral activity, immunomodulatory activity and transcriptional regulation of several genes. Responses of cells to TNF family ligands include not only normal physiological responses but also diseases associated with enhanced or inhibited apoptosis. Apoptosis-programmed cell death-is a physiological mechanism associated with the removal of peripheral T lymphocytes of the immune system, the dysregulation of which can lead to a number of different etiological processes. Diseases associated with increased cell survival or diseases associated with inhibition of apoptosis include cancer, autoimmune diseases, viral infections, inflammation,
Includes graft-versus-host disease, acute graft rejection, and chronic graft rejection. AIDS, neurodegenerative diseases, myelodysplastic syndromes,
Includes ischemic injury, toxin-induced liver disease, septic shock, cachexia and anorexia.

Therefore, the present invention further provides a method for enhancing TNF family ligand-induced apoptosis, which comprises administering an effective amount of an agonist capable of increasing DR3-mediated signal transduction to cells expressing DR3 polypeptide. To do. Preferably DR3
Treats diseases in which decreased apoptosis is seen by increasing mediated signal transduction.

In another aspect, the invention provides a method of inhibiting TNF family ligand-induced apoptosis comprising administering to a cell expressing a DR3 polypeptide an effective amount of an antagonist capable of reducing DR3-mediated signaling. Be oriented. Preferably DR3
Treats diseases in which increased apoptosis is found by reducing mediated signal transduction.

Whether any of the "agonist" or "antagonist" candidates of the present invention can enhance or inhibit apoptosis is known in the art, including those detailed below. TNF family ligands / receptors It can be determined using a somatic reaction assay. Thus, in yet another aspect, there is provided a search method for determining whether a candidate agonist or antagonist can enhance or inhibit a cellular response to a TNF family ligand. The method involves contacting cells expressing a DR3 or DR3-V1 polypeptide with a candidate compound and a TNF family ligand, assaying the cellular response, and comparing the cellular response to a standard cellular response. To do. The standard is assayed when the contact with the ligand is made in the absence of the candidate compound, and an increased cellular response over the standard indicates that the candidate compound is an agonist of this ligand / receptor signaling pathway and decreases over the standard. The cellular response shown indicates that the candidate compound is an antagonist of this ligand / receptor signaling pathway. DR3 or DR in this invention
Cells expressing the 3-V1 polypeptide can be contacted with either endogenous or exogenously administered TNF family ligand.

(Detailed Description of Preferred Embodiments) The present invention relates to DR having the amino acid sequences shown in SEQ ID NO: 2 and SEQ ID NO: 4, respectively.
Provided is an isolated nucleic acid molecule comprising (or consisting of) a nucleic acid sequence encoding a 3-V1 or DR3 polypeptide or a fragment of that polypeptide. The DR3-V1 and DR3 polypeptides of the present invention share sequence homology with human TNFRI and human Fas (Figure 4). The nucleotide sequence set forth in SEQ ID NO: 1 is the American Type Culture C of University Boulevard 10801, Manassas, Virginia 20110-2209.
obtained by sequencing the HTTNB61 clone, deposited with the collection on Mar. 1, 1996 and given accession number 97456. The deposited clone is pBluescript SK (-) plasmid (Stratagene,
La Jolla, CA). The nucleotide sequence set forth in SEQ ID NO: 3 is the American Type Culture Culture of University Boulevard 10801, Manassas, Virginia 20110-2209.
It was obtained by sequencing a clone obtained from the HUVEC library, deposited with the Collection on October 10, 1996 and given accession number 97757. The deposited clone is pBluescript SK (-) plasmid (S
Tratagene, La Jolla, CA).

Nucleic Acid Molecules Unless otherwise indicated, all nucleotide sequences determined by sequencing DNA molecules herein are by automated DNA sequencers (eg, App.
Lied Biosystems, type 373), and the amino acid sequences of the polypeptides encoded by the DNA molecules determined herein are all of the DNA sequences determined as described above. Predicted by translation.
Therefore, as is known in the art for any DNA sequence determined by this automated procedure, any nucleotide sequence determined herein may have some error. The nucleotide sequence determined by automation is typically at least about 90% or greater, more typically at least about 95% or greater and at least about 99.9% identical to the actual nucleotide sequence of the sequenced DNA molecule. There is a nature. Other techniques, including manual DNA sequencing methods well known in the art, allow the true sequence to be determined more accurately. This is also known in the art, but a single insertion or deletion in the determined nucleotide sequence causes a frameshift in the translation of the nucleotide sequence when compared to the actual sequence, starting at the point of insertion or deletion. The predicted amino acid sequence encoded by the nucleotide sequence will be quite different from the amino acid sequence actually encoded by the sequenced DNA molecule.

By “isolated” polypeptide or protein is intended a polypeptide or protein that has been displaced from its natural environment. For example, recombinantly produced polypeptides and proteins expressed in host cells are described, for example, in Smith and Johnso.
n, Gene, 67: 31-40 (1988), as well as native or recombinant polypeptides substantially purified by a suitable technique, such as the one-step purification method disclosed herein. For the purposes of, it is considered isolated.

Using the information provided herein, eg, the nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3, a nucleic acid molecule of the invention encoding a DR3-V1 or DR3 polypeptide may be derived, for example, from It can be obtained using standard cloning and screening procedures, such as cDNA cloning using mRNA as the substance. As an illustration of the invention, the nucleic acid molecule set forth in SEQ ID NO: 1 was discovered in a cDNA library derived from human testicular tumor cells. Also, as an illustration of the invention, the nucleic acid molecule set forth in SEQ ID NO: 3 was discovered in a human HUVEC cDNA library. In addition to this, the gene of the present invention has also been identified in a cDNA library of the following tissues: fetal liver, fetal brain, tonsils and leukocytes. Furthermore, DR3 transcript polymorphisms were found in Northern blots and PCR reactions, indicating that there are transcript polymorphic variants, probably due to differing message splicing.

The DR3-V1 (formerly known as DDCR) gene contains an open reading frame encoding a protein of about 428 amino acid residues, the initiation codon of which is located at positions 198 to 200 of the nucleotide sequence shown in SEQ ID NO: 1. It has a leader sequence of about 35 amino acid residues and has a deduced molecular weight of about 47 kDa. Among the known members of the TNF receptor family, the DR3-V1 polypeptides of the invention share the highest degree of homology with human TNFR1. The DR3-V1 polypeptide shown in SEQ ID NO: 2 is human T
It has about 20% identity and about 50% similarity with NFR1.

The DR3 gene contains an open reading frame encoding a protein of about 417 amino acid residues,
Here, the start codon is located at positions 1 to 3 of the nucleotide sequence shown in SEQ ID NO: 3, has a leader sequence of about 24 amino acid residues, and has a deduced molecular weight of about 43 kDa.
Within the known TNF receptor family, the DR3 polypeptides of the invention are human TN
It shares the highest degree of homology with FR1. The DR3 polypeptide set forth in SEQ ID NO: 3 has about 20% identity and about 50% similarity with human TNFR1.

As indicated, the invention also provides the mature forms of the DR3-V1 and DR3 proteins of the invention. According to the signal hypothesis, proteins secreted by mammalian cells have a signal sequence or secretory leader sequence that cleaves from the mature protein once transport of the growing protein chain across the rough endoplasmic reticulum is initiated. To be done. Most mammalian cells and even insect cells cleave secreted proteins with the same specificity. However, in some cases the cleavage of the secreted protein is not completely homogeneous, resulting in two or more mature species of the protein. It has been known for quite some time that the cleavage specificity of a secreted protein is ultimately determined by the primary structure of the complete protein, ie it is unique to the amino acid sequence of the polypeptide. Therefore, the present invention provides a nucleic acid encoding a mature DR3-V1 or DR3 polypeptide having the amino acid sequence encoded by the cDNA contained in the host identified as ATCC Deposit No. 97456 or 97757 shown in SEQ ID NO: 2 and SEQ ID NO: 4, respectively. Provide an array. ATCC Deposit No. 97456 or 97757 respectively
The mature DR3-V1 or DR3 protein having the amino acid sequence encoded by the cDNA contained in the host identified as (1) is a mammalian cell in the complete open reading frame encoded by the human DNA sequence of the clone contained in the vector in the deposited host (for example, COS
Cell, the mature form of the DR3-V1 or DR3 protein produced by expression in cells (as described below). As mentioned above, ATCC Deposit No. 97456
And the mature DR3-V1 or DR3 having the amino acid sequence encoded by the cDNA clones contained in 97757, respectively, depended on the accuracy of the cleavage site predicted based on computer analysis, and predicted "mature" SEQ ID NO: 2 (about 36 From about 42
DR3-V1 protein shown in (up to 8 amino acids) or "mature" SEQ ID NO: 4 (
DR3 protein, which is from about 24 to about 417 amino acids).

Methods are available to predict whether a protein will have a secretory leader as well as a breakpoint for that leader sequence. For example, McGeoch (Virus ·
Res. 3, Vol. 271-286 (1985)) and von Hein
je (Nucleic Acids Res., 14: 4683-4690 (1986)). For each of these methods, the predictive breakpoints of known mammalian secretory proteins are in the range of 75-80% accuracy.
von Heinje, supra. However, these two methods do not always give the same predicted breakpoint for a given protein.

In the present case, a computer program (“PSORT”) (K. Nakai), which is an expert system for predicting the intracellular position of a protein based on the predicted complete amino acid sequences of the DR3-V1 and DR3 polypeptides of the present invention based on the amino acid sequences. And M. Kanehisa, Genomics, 14: 897-911 (199
2 years))). As part of this computer location prediction,
McGeoch and von Heinje have been introduced. The cleavage site was predicted by the PSORT program to be between amino acid 35 and amino acid 36 of SEQ ID NO: 2 and between amino acid 24 and amino acid 25 of SEQ ID NO: 4. afterwards,
The complete amino acid sequence was further analyzed by visual inspection applying the simple form of the von Heinje (-1, -3) rule. von Heinje, supra. Thus D
The leader sequence of the R3-V1 protein consists of amino acid residues 1-35 of SEQ ID NO: 2, while the predicted mature DR3-V1 protein consists of residues 36-428. The leader sequence of the DR3 protein is predicted to be composed of amino acid residues 1-24 of SEQ ID NO: 4, while the predicted mature DR3 protein is residues 25-41.
It consists of 7.

As will be appreciated by those in the art, due to the possible sequencing errors described above as well as the diversity of various leader cleavage sites in previously known proteins, the actual cDNA encoded by the deposited cDNA may be encoded. The DR3-V1 polypeptide contains about 428 amino acids, but may be anywhere within the range of 410-440 amino acids, and the actual leader sequence for this protein is about 35 amino acids, but about 25 to about 4 amino acids.
It may be somewhere within the range of 5 amino acids. The actual DR3 polypeptide encoded by the deposited cDNA contains about 417 amino acids, but may be anywhere in the range of 400-430 amino acids, although the actual leader sequence of this protein is about 24 amino acids, But it may be somewhere in the range of about 14 to about 34 amino acids.

As pointed out, the nucleic acid molecules of the invention may be in the form of RNA such as mRNA, or cDNA and genomic DNA produced, for example, by cloning or synthetically.
It may also be in the form of DNA containing This DNA may be double-stranded or single-stranded.
Single-stranded DNA may be the coding strand, known as the sense strand, or the non-coding strand, known as the antisense strand.

An “isolated” nucleic acid molecule is DNA or RNA that has been removed from its natural environment.
Nucleic acid molecule is intended. For example, recombinant DNA molecules contained in a vector are considered isolated for the purposes of the present invention. Another example of an isolated DNA molecule includes
It includes recombinant DNA molecules retained in a heterologous host cell or purified (partially or substantially) DNA molecules in solution. The isolated RNA molecules include
Includes in vivo or in vitro RNA transcripts of the DNA molecules of the invention. Isolated nucleic acid molecules according to the present invention further include such molecules produced synthetically.

However, a nucleic acid (including a clone) that is a member of a library (such as a genomic or cDNA library) that has not been isolated from other members of the library (
Homogeneous solution forms containing clones and other members of the library) or chromosomes isolated or removed from cells or cell lysates (such as "chromosome spreads" in karyotype) are isolated for the purposes of the invention. Not done. As discussed further herein, the isolated nucleic acid molecules of the invention can be made naturally, recombinantly or synthetically.

The isolated nucleic acid molecule of the present invention comprises an open reading frame (ORF) as set forth in SEQ ID NO: 1.
It includes a DNA molecule of R3-V1 and further has an initiation codon at positions 198 to 200 of the nucleotide sequence shown in SEQ ID NO: 1, but still encodes the DR3-V1 polypeptide or a fragment thereof due to the degeneracy of the genetic code. DNA molecules containing sequences that are substantially different from all or part of the ORF. The isolated nucleic acid molecule of the present invention also includes a DNA molecule of DR3 containing the open reading frame (ORF) shown in SEQ ID NO: 3, and further, there are initiation codons at positions 1 to 3 of the nucleotide sequence shown in SEQ ID NO: 3. Comprises a sequence that is substantially different from all or part of the ORF encoding the DR3 polypeptide or fragment thereof due to the degeneracy of the genetic code (or
DNA molecules). Of course, the genetic code is well known in the art. Therefore, it is a routine work for those skilled in the art to make these degenerate mutants.

[0029] In one of its other aspects, the invention relates to ATCC Deposit No. 974 on March 1, 1996.
56 provides an isolated nucleic acid molecule encoding a DR3-V1 polypeptide having the amino acid sequence encoded by the cDNA clone contained in the plasmid deposited as 56. The present invention relates to a DR having an amino acid sequence encoded by the cDNA contained in the plasmid deposited under ATCC Deposit No. 97757 on October 10, 1996.
An isolated nucleic acid molecule encoding a 3 polypeptide is provided. Preferably, these nucleic acid molecules encode the mature polypeptide encoded by the deposited cDNA. The invention further provides an isolated nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3 or the nucleotide sequence of the cDNA of DR3-V1 or DR3 contained in said deposited clone, or complementary to one of said sequences. Nucleic acid molecules having different sequences are provided. This isolated DNA molecule and its fragments are used to map DR3-V1 or DR3 in human tissues (including testicular tumor tissue) for genetic mapping by in situ hybridization with chromosomes and by Northern blot analysis.
It is useful as a DNA probe for detecting gene expression.

Expression of DR3 is expressed in endothelial cells, hepatocytes, hepatocellular tumors, lymph nodes, Hodgkin lymphoma, tonsils, bone marrow, spleen, heart, thymus, pericardium, healing wound (skin), brain, pancreatic tumor, burns. Skin, U937 cells, testis, colorectal cancer (metastasis to liver), pancreas, rejection kidney, fat, ovary, olfactory endothelium, striatum, HeLa cells, LNCAP (+30 nM androgen treatment), 8-week-old embryo tissue, 9 Week-old embryo tissue, fetal brain tissue, fetal kidney tissue, fetal heart tissue, fetal thymus tissue, fetal lung tissue, fetal liver tissue, fetal spleen tissue, helper IIT cell, activated T cell (16 hours), activated T cell (24 Time), primary dendritic cells, eosinophils, monocytes, keratinocytes and HUVECs (human umbilical vein endothelial cells) have been detected in a wide range of tissues and cell types.

The invention further relates to a polynucleotide comprising (or consisting of) a fragment of the isolated nucleic acid molecule described herein. A fragment of the isolated nucleic acid molecule having one nucleotide sequence of the deposited cDNA or the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3 has a length of at least about 15 nt, more preferably at least about 20 nt, even more preferably at least Contemplated DNA fragments of about 30 nt, and more preferably at least about 40 nt, useful as diagnostic probes and primers herein. In this context, "about" includes the specifically recited values and values that are several (5, 4, 3, 2 or 1) nucleotides greater or less than that value. Of course, at least 50, 75, 100, 125, 150, 175, 200, 2 corresponding to most, if not all, of one nucleotide sequence of the deposited cDNA or of SEQ ID NO: 1 or SEQ ID NO: 3
25, 250, 275, 300, 325, 350, 375, 400, 425, 4
50, 475, 500, 525, 550, 575, 600, 625, 650, 6
75, 700, 725, 750, 775, 800, 825, 850, 875, 9
00, 925, 950, 975, 1000, 1025, 1050, 1075, 1
Large fragments containing (or consisting of) 100, 1125, 1150, 1175, 1200, 1225, 1250 or 1283 are useful in the invention. For example, a fragment of at least 20 nt in length is intended to be a fragment comprising one nucleotide sequence of the deposited cDNA or 20 or more bases from the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3.

The invention further relates to a polynucleotide comprising (or consisting of) a fragment of an isolated nucleic acid molecule encoding a subclone of DR3-V1 and DR3.
In particular, the invention relates to SEQ ID NO: 1 198-257, 208-267, 218-27.
7, 228-287, 238-297, 248-307, 258-317, 26
8-327, 278-337, 288-347, 298-357, 308-36.
7, 318-377, 328-387, 338-397, 348-407, 35.
8-417, 368-427, 378-437, 388-447, 398-45
7, 408-467, 428-487, 458-517, 478-537, 49
8-557, 518-577, 538-597, 558-617, 578-63
7, 598-657, 638-697, 658-717, 698-757, 70.
8-767, 718-777, 728-787, 738-797, 748-80
7, 758-817, 778-837, 788-847, 808-867, 82
8-887, 848-907, 868-927, 878-937, 898-95.
7, 908-967, 918-977, 928-987, 948-1007, 9
68-1047, 988-1047, 998-1067, 1018-1077,
1038-1097, 1058-1117, 1068-1127, 1088-1
147, 1098-1157, 1118-1177, 1138-1197, 11
58-1217, 1178-1237, 1198-1257, 1218-127.
7, 1238-1297, 1258-1317, 1278-1337, 1298.
-1357, 1318-1377, 1338-1397, 1358-1417,
1378-1437, 1398-1457, 1418-1477 and 1428.
A polynucleotide comprising (or consisting of) a nucleotide sequence of a member selected from the group consisting of -1481 nucleotides is provided.

The invention further relates to a polynucleotide comprising (or consisting of) an isolated nucleic acid molecule encoding the domains of DR3-V1 and DR3. In one aspect, the invention provides a polynucleotide comprising (or consisting of) a nucleic acid molecule encoding the beta-sheet region of the DR-3V1 protein set forth in Table 2. Representative of such polynucleotides are from about 24 to about 3 in SEQ ID NO: 2.
2, about 53 to about 58, about 133 to about 142, about 202 to about 234, about 28
Comprises (or consists of) 1, 2, 3, 4, 5 or more amino acid sequences selected from the group consisting of 1 to about 288, about 304 to about 312, and about 346 to about 350 amino acid residues. Nucleic acid molecules that encode the polypeptides are included. In this context, “about” means in particular the listed values and a number (5, 4, 3
2 or 1) Include values with greater or lesser amino acids. Polypeptides encoded by these polynucleotides are also included in the invention.

A preferred nucleic acid fragment of the invention is a polypeptide comprising (or consisting of) a DR3-V1 extracellular domain (amino acid residues from about 36 to about 212 in SEQ ID NO: 2); DR3-V1 membrane A polypeptide containing (or consisting of) a transmembrane domain (amino acid residues from about 213 to about 235 in SEQ ID NO: 2); a polypeptide containing (or consisting of) DR3-V1 intracellular domain (SEQ ID NO: 2) From about 236 to about 428 amino acid residues); and a polypeptide comprising (or consisting of) the DR3 death domain (from about 353 to about 419 amino acid residues in SEQ ID NO: 2) Nucleic acid molecules encoding 1, 2, 3, 4, 5 or more amino acid sequences. In this context, "about" includes those values specifically mentioned and those that are several (5, 4, 3, 2 or 1) amino acids greater or less than that value. The positions of these domains are predicted by computer graphics, and the skilled artisan will appreciate that the amino acid residues that make up these domains may vary somewhat (eg, from about 1 to 15) depending on the criteria used to define this domain. It should be recognized that residues can vary. Polypeptides encoded by these polynucleotides are also included in the invention.

The present invention also relates to nucleic acids encoding from about 1 to about 125, about 30 to about 215, about 215 to about 240, about 240 to about 428, and about 350 to about 420 amino acid residues in SEQ ID NO: 2. A polynucleotide comprising (or consisting of) a molecule is provided. In this context, “about” means in particular the listed values and a number (5,
4, 3, 2 or 1) Includes values greater or lesser amino acids. Polypeptides encoded by these polynucleotides are also included in the invention.

Preferred nucleic acid fragments of the present invention further include nucleic acid molecules that encode the epitope-bearing portion of the DR3-V1 protein. In particular, this nucleic acid fragment of the invention comprises a polypeptide comprising (or consisting of) about 1 to about 22 amino acid residues in SEQ ID NO: 2, about 33 to about 56 amino acid residues in SEQ ID NO: 2. A polypeptide comprising (or consisting of) amino acid residues in SEQ ID NO: 2 from about 59 to about 82 (or
(Comprising) a polypeptide comprising (or consisting of) about 95 to about 112 amino acid residues in SEQ ID NO: 2, about 122 amino acid residues in SEQ ID NO: 2.
To about 133, or a polypeptide comprising (or consisting of) about 133 to amino acid residues 161 to about 177 of SEQ ID NO: 2, or about 179 of amino acid residue in SEQ ID NO: 2 Contains (or consists of)
Polypeptides and nucleic acid molecules that encode polypeptides that comprise (or consist of) amino acid residues from about 196 to about 205 in SEQ ID NO: 2 are included. In this context "about" means in particular the listed values and a number (5, 4, 3, 2 or 1 above that value).
) Includes amino acid values greater or lesser. The present inventors have shown that the polypeptide fragment is D
It has been determined to be the antigenic region of the R3-V1 protein. The method for determining the other portion having the epitope in the DR3-V1 protein is described in detail below. Polypeptides encoded by these polynucleotides are also included in the invention.

Suitable nucleic acid fragments of the present invention also include nucleic acid molecules that encode the epitope-bearing portion of the DR3 protein. In particular, such nucleic acid fragments of the present invention include the DR3-V1
Nucleic acid molecules that encode regions corresponding to the epitope-bearing portion of the protein are included.
Methods for determining other epitope-bearing portions of the DR3 protein are detailed below.

In one of its other aspects, the invention provides a polynucleotide that hybridizes to a portion of the polynucleotide in a nucleic acid molecule of the invention under stringent hybridization conditions [eg, the polynucleotides described herein. And / or consisting of the complement of a nucleotide fragment or the cDNA plasmid contained in ATCC Accession No. 97456 or ATCC Accession No. 97757. “Stringent hybridization conditions” are 50% formamide, 5 × SSC (750 mM-NaCl, 75 mM-trisodium citrate), 5
0 mM-sodium phosphate (pH 7.6), 5 × Denhardt's solution, 10% dextran sulfate, and 20 μg / mL denatured cleaved salmon sperm DNA (or
Incubation) in a solution at 42 ° C. overnight, followed by washing the filter with 0.1 × SSC at 65 ° C.

A polynucleotide that hybridizes to a “portion” of a polynucleotide is at least about 15 nucleotides (nt), preferably about 2 nucleotides, of a control polynucleotide.
0 nt, more preferably at least about 30 nt, more preferably about 30-70.
Polynucleotide that hybridizes to nt (either DNA or RNA)
Intended. These are useful as the above-mentioned diagnostic probe and primer,
Details will be described below. In this context, "about" refers to the listed values and numbers above that value (
A polynucleotide portion of "at least about 20 nt in length" that includes a value greater or less than 5, 4, 3, 2, or 1) nucleotides refers to, for example, a control polynucleotide (eg, the deposited cDNA or SEQ ID NO: 1 or SEQ ID NO: 3). 20 adjacent nucleotides obtained from the nucleotide sequence of
Intended for one or more.

Of course, with a polynucleotide that hybridizes only with the poly A sequence (such as the 3′-terminal poly (A) region of the DR3-V1 cDNA shown in SEQ ID NO: 1) or a complementary strand of T (or U) residues. Polynucleotides that only hybridize are not intended to be included in the polynucleotides of the invention that should be used to hybridize to the nucleic acid moieties of the invention because such polynucleotides are poly (A) strands or their complements. Because it hybridizes to any nucleic acid molecule, including (in fact, all double-stranded cDNA clones made from oligo-dT initiated cDNA libraries).

As indicated, the nucleic acid molecule of the present invention encoding a DR3-V1 or DR3 polypeptide is not limited to the coding sequence for the mature polypeptide,
As such; such as coding sequences for mature polypeptides and such as those encoding leader or secretory sequences, such as pre- or pro-, or prepro-protein sequences or other sequences; for example, without limitation Non-coding 5'and 3'-sequences of transcribed, non-translated sequences that play a role in introns and transcription, but not introns and transcription, mRNA processing-such as splicing and polyadenylation signals-, ribosome binding and mRNA stability. It may also include a coding sequence for a mature polypeptide having the additional coding sequences described above, as well as additional non-coding sequences to include; for example, additional coding sequences that encode additional amino acids to provide additional function. Thus, for example, the polypeptide may be fused to a marker sequence, such as a peptide that facilitates purification of the fused polypeptide. In a preferred embodiment of one aspect of this invention, the marker sequence is a hexahistidine peptide, particularly the tag provided in the pQE vector (Qiagen), many of which are commercially available. is there. Gentz et al. Natl. Acad. Sci. USA, 86: 821-824 (1989), for example hexahistidine provides convenient purification of the fusion protein. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein, and is described in, for example, Wilson et al., Cell, 37: 767 (1
984).

The present invention further relates to variants of the nucleic acid molecules of the present invention, which encode fragments, analogs or derivatives of DR3-V1 or DR3 polypeptides. Variants may also occur naturally, such as allelic variants. By "allelic variant" is intended one of several alternative forms of a gene that occupy certain positions on the chromosome of an organism.
Lewin, B .; Edited by "Genes II", John Wil
ey & Sons, New York (1985). Non-naturally occurring variants may also be produced using mutagenesis techniques known in the art.

Such variants also include those produced by the substitution, deletion or addition of nucleotides, which may involve one or more nucleotides. The variant may vary in coding or non-coding regions or both. Changes in the coding region may result in substitutions, deletions or additions with conservative or non-conservative amino acids.

Another aspect of the invention comprises (a) a nucleotide sequence encoding a full-length DR3-V1 polypeptide having a predicted leader sequence and having the complete amino acid sequence set forth in SEQ ID NO: 2, and (b) a predicted leader sequence. , A nucleotide sequence encoding a full-length DR3 polypeptide having the complete amino acid sequence shown in SEQ ID NO: 4, (c) a mature DR3-V1 polypeptide having an amino acid sequence at positions from about 36 to about 428 shown in SEQ ID NO: 2 ( A full length polypeptide with the leader removed), (d) a nucleotide sequence encoding a full length DR3-V1 polypeptide having a complete amino acid sequence including the leader encoded by the cDNA contained in ATCC Deposit No. 97456, (e ) Encoded by the cDNA contained in ATCC Deposit No. 97757 A nucleotide sequence encoding a full length DR3 polypeptide having a complete amino acid sequence including a leader, (f) cdN contained in ATCC Deposit No. 97456
A nucleotide sequence encoding the mature DR3-V1 polypeptide having the amino acid sequence encoded by A, (g) a nucleotide sequence encoding the mature DR3 polypeptide having the amino acid sequence encoded by the cDNA contained in ATCC Deposit No. 97757, (h ) A nucleotide sequence encoding the DR3 extracellular domain, (i) DR
A nucleotide sequence encoding a 3 transmembrane domain, (j) a nucleotide sequence encoding a DR3 intracellular domain, and (k) a nucleotide sequence encoding a DR3 death domain, or (l) the nucleotide sequence (a), (b) , (C)
, (D), (e), (f), (g), (h), (i), (j), or at least 80% of the nucleotide sequence complementary to (k) Identical, and preferably at least 85%, 90%, 92%, 95%, 96%, 97%,
It includes isolated nucleic acid molecules that are 98% or 99% identical. "About" in this context
Includes the values specifically mentioned and several (5, 4, 3, 2 or 1) amino acids greater or less than that value.

A polynucleotide having a nucleotide sequence that is “identical”, for example, at least 95% to a control nucleotide sequence that encodes a DR3-V1 or DR3 polypeptide is a polynucleotide whose nucleotide sequence encodes a DR3-V1 or DR3 polypeptide. It is intended that the nucleotide sequence of the polypeptide is identical to the control sequence, except that each 100 nucleotides may contain up to 5 points of mutation. In other words, to obtain a polynucleotide having a nucleotide sequence that is at least 95% identical to a control nucleotide sequence, one may delete up to 5% of the nucleotides in the control sequence or replace it with another nucleotide. Yes, or up to 5% of all nucleotides in the control sequence may be inserted into the control sequence. These mutations of the control sequence are interspersed individually between the nucleotides of the control sequence, or interspersed with one or more adjacent groups within the control sequence, either at the 5'or 3'terminal position of the control nucleotide sequence or at these positions. May occur somewhere between the terminal positions of the. The reference (question) sequence is the complete DR3 encoding the nucleotide sequence shown in SEQ ID NO: 2 and SEQ ID NO: 4.
-V1 or DR3, or any DR3-V1 or DR3 fragment (DR
Polynucleotides encoding either 3-V1 or DR3 amino acid sequences and / or C-terminal deletions as described herein).

Practically, the particular nucleic acid molecule will be at least 80%, 85%, 90%, 92%, 95% of the nucleotide sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4 or the nucleotide sequence of the deposited cDNA, for example. %, 96%, 97%, 98% or 99
% For example, the Bestfit program (Wiscons
in-Sequence-Analysis-Package, 8th edition for Unix, Genetics-Computer-Group, University
・ Research / Park, Science / Drive, Madison, WI
, 37711) and can be determined using conventional methods. Bestfit is Smith, Waterman, Adva
nces ・ in ・ Applied ・ Mathematics, Volume 2: 482-4
The localized homology algorithm of page 89 (1981) is used to find the optimal segment of homology between two sequences. To determine whether a particular sequence is, for example, 95% identical to a reference sequence of the invention using Bestfit or another sequence adjustment program, the percent identity is, of course, Parameters are set to allow gaps within 5% identity of the total number of nucleotides in the control sequence, calculated for full length.

In certain embodiments, Brutlag et al. (Comp.App.Biosci., 6: 237-245.
The FASTDB computer program based on the algorithm of (1990)) is used to determine the identity (also referred to as global sequence alignment) between the reference (query) sequence (sequence of the invention) and the subject sequence. The preferred parameters used for FASTDB alignment of DNA sequences to calculate percent identity are:
Matrix = unitary, k-tuple = 4, mismatch penalty = 1,
Joining Penalty = 30, Randomized Group Length = 0, Cutoff Score = 1, Gap Penalty = 5, Gap Size Penalty = 0.0
5, window size = 500 or the length of the nucleotide sequence of interest (whichever is shorter). In this embodiment, if the subject sequence is shorter than the query sequence due to a 5'or 3'deletion rather than an internal deletion, the FASTDB program will calculate the percent identity as 5'or 3 '. Make a manual correction to the result taking into account the fact that it does not reveal truncation. In the subject sequence truncated at the 5'or 3'end, with respect to the query sequence, of the bases of the query sequence that are 5'and 3'of the subject sequence (not matched / aligned) as a percentage of the total number of bases in the query sequence. Correct percent identity by calculating the number.
Whether the nucleotides are matched / aligned is determined by the result of FASTDB sequence alignment. Then FASTD with specific parameters
Subtract this percentage from the percent identity calculated by the B program to obtain the final percent identity score. This corrected score is used for the purposes of this example. To manually adjust the percent identity score as indicated by the FASTDB alignment, only bases outside the 5'and 3'bases of the subject sequence (no match / alignment) are calculated. For example, 90
The subject sequence of bases is aligned to a 100 base query sequence to determine percent identity. If a deletion occurs at the 5'end of the subject sequence, the FASTDB alignment will be
No match / alignment of the first 10 bases at the 5'end is shown. 1
Zero unpaired bases represent 10% of the sequence (number of unmatched 5'and 3'ends / total number of bases in the query sequence), 10% percent identity calculated by the FASTDB program. Subtract from the score. If the remaining 90
If the base pairs were perfectly matched, the final percent identity would be 90%. In another example, a 90 base subject sequence is compared to a 100 base query sequence. At this time, the deletion is an internal deletion, and there is no base on the 5 ′ or 3 ′ of the subject sequence that does not match / align with the query sequence. In this case, the percent identity calculated by FASTDB is not manually corrected. Once again, only the 5'and 3'bases of the subject sequence that are not matched / aligned with the query sequence are manually corrected.
In this specific example, no other manual correction is performed.

The present application is directed to at least 80%, 85%, 90% of the nucleic acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 3 or the deposited cDNA, regardless of whether they have DR3 functional activity. , 92%, 95%, 96%, 97%, 98% or 99
Relates to nucleic acid molecules with% identity. The present application also provides that the nucleic acid sequences described herein (30-2 in SEQ ID NO: 2 regardless of whether they have DR3 functional activity).
00 amino acids, 30 to 215 amino acids, 215 to 240 amino acids, 240 to 42
(Eg, a nucleic acid sequence encoding a polypeptide having an amino acid sequence with a N-terminal and / or C-terminal deletion as described herein, such as a nucleic acid molecule encoding 8 amino acids, 350-420 amino acids or 2-428 amino acids). At least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99
Relates to nucleic acid molecules with% identity. The reason for this is that even if a particular nucleic acid molecule does not encode a polypeptide having DR3 activity, one of skill in the art will still be able to use that nucleic acid molecule, eg, a hybridization probe or the polymerase chain reaction (P
To know how to use it as a primer for CR). Uses of the nucleic acid molecules of the invention that do not encode a polypeptide having DDCR activity include, among others, (1
B) isolating the DR3-V1 or DR3 gene or an allelic variant thereof in a cDNA library; (2) DR3-V1 or DR3 in situ hybridization to metaphase chromosome expansion (eg "FISH"). The precise chromosomal location of a gene is described in Verma et al. "Human Chromosome: Basic Technology Handbook
omosomes: A ・ Manual ・ of ・ Basic ・ Technique
s) ”, Pergamon Press, New York (1988), and (3) Northern blot analysis to detect DR3-V1 or DR3 mRNA expression in specific tissues. Including.

However, at least 80%, 85%, 90%, 92% 95% of the nucleic acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 3 encoding the polypeptide having DR3 protein activity or the deposited nucleic acid sequence Nucleic acid molecules with 96%, 97%, 98%, or 99% sequence identity are preferred. A "polypeptide having DR3 activity" is a polypeptide showing a similar activity to that of the DR3 protein (full length protein or preferably mature protein) of the present invention, although it is not necessarily the same as the activity of the DR3 protein of the present invention when measured by a specific biological assay method. Intended. For example, DR3 protein activity was essentially reported in previous reports (A
． M. Chinnaiyan et al., Cell, 81: 505-12 (199).
5 years), M. P. Boldin et al. Biol. Chem. 270 Volume: 7
795-8 (1995), F.M. C. EMBO, 14 such as Kischkel
Volume: 5579-5588 (1995) A. M. Chinnaiyan et al. Biol. Chem. 271: 4961-4965 (1996).
) Can be performed as described in Example 7 below. A plasmid encoding full-length DR3 or a reporter containing the death domain candidate is co-transfected into MCF7 cells with a pLantern reporter construct encoding a green fluorescent protein. Nuclei of cells transfected with DR3 will show apoptotic morphology as assessed by DAPI staining. Similar to TNFR-1 and Fas / APO-1, (M. Mu
Zio et al., Cell, 85: 817-827 (1996), M. et al. P. B
Oldin et al., Cell, 85: 803-815 (1996), M.M. T
Ewari et al. Biol. Chem. 270: 3255-60 (1
995)), DR3-induced apoptosis is inhibited by the inhibitors of ICE-like proteases, CrmA and z-VAD-fmk. In addition to this, D
Apoptosis induced by R3 is predominantly negative version of FADD or FLICE (FADD-DN) or (FLICE-DN / MACHalC36
0S).

The functional activity of DR3 polypeptides and fragments, variants, derivatives and analogs thereof can be assayed in a variety of ways. For example, in one embodiment of assaying the ability to bind or compete for full-length polypeptide for binding to anti-DR3 antibody, a radioimmunoassay, an ELISA (enzyme linked immunosorbent assay), a "sandwich" immunoassay, an immunoassay, Radiometric assay, gel dispersion sedimentation reaction, immunodispersion assay, in situ immunoassay (using colloidal gold, enzyme or radioisotope label, etc.), Western blot, sedimentation reaction, agglutination reaction (gel agglutination assay, hemagglutination assay, etc.) Various immunoassays known in the art can be used, such as competitive or non-competitive assay systems using techniques such as complement fixation assays, immunofluorescence assays, protein A assays and immunoelectrophoretic assays. But it is not limited thereto. In one embodiment, antibody binding is detected by detecting the label on the primary antibody. In another embodiment, the primary antibody is detected by detecting binding of a secondary antibody or reagent on the primary antibody. In yet another embodiment, the secondary antibody is labeled. Many means are known in the art for binding detection in immunoassays and are within the scope of the present invention.

In another embodiment, the ligand is identified (TNF-γ-β (International Publication W
O 00/08139, which are incorporated herein by reference in their entirety))
, Or when the multiple binding ability of the polypeptide fragments, variants or derivatives of the invention is being evaluated, the binding is performed by known means such as reducing and non-reducing gel chromatography, protein affinity chromatography and affinity blotting. It can be assayed. Generally, Phizic
See Ky, E. et al., Microbiol. Rev., 59: 94-123. In another embodiment,
The physiological correlates of binding to its substrate (signal transduction) can be assayed. Other methods are known to those of skill in the art and are within the scope of the invention.

Of course, at least 80%, 85%, 90% of the nucleic acid sequence of the deposited cDNA or the nucleic acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 due to the degeneracy of the genetic code
Those skilled in the art will recognize that a large number of nucleic acid molecules having%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity encode polypeptides that have "DR3 functional activity." Will be immediately recognized. Indeed, in many instances, degenerate variants of these nucleotide sequences all encode the same polypeptide, and will be apparent to the skilled artisan without the need to carry out the comparative assay. It is also recognized in the art that for nucleic acid molecules that are not degenerate variants, a significant number will encode a polypeptide having DR3 protein activity. As will be further described below, this does not have a clear effect on the function of the protein, or an expert is fully aware of amino acid substitutions that have little effect (for example, replacement of an aliphatic amino acid with a second aliphatic amino acid). Because there is.

For example, guidance on a method of amino acid substitution that does not show phenotype is given by Bowie,
J. U. , "Message decoding in protein sequences: Tolerance for amino acid substitution (Deciphering, the, Message, in, Protein,
Sequences: Tolerance ・ to ・ Amino ・ Acid ・ Su
bitionitions) ", Science, 247, 1306-1310.
Page (1990), the authors point out that the protein is surprisingly tolerant of amino acid substitutions.

Polynucleotide Assays The present invention also relates to the use of DR3-V1 or DR3 polynucleotides to detect complementary polynucleotides, eg as a diagnostic. DR3
-Detection of mutants associated with dysfunction of V1 or DR3 results from reduced expression, overexpression or altered expression of DR3-V1 or DR3 or soluble forms thereof, such as tumors or autoimmune diseases or It would provide a diagnostic tool by which one can decide whether to add to the diagnosis of susceptibility to the disease.

Individuals with mutations in the DR3-V1 or DR3 gene can be detected at the DNA level by a variety of techniques. Nucleic acids for diagnosis include, for example, blood, urine, saliva,
Obtained from patient cells such as tissue biopsy and autopsy material. Genomic DNA can be used directly for detection, but can also be enzymatically amplified using PCR prior to analysis (S
aiki et al., Nature, 324: 163-166 (1986)).
RNA or cDNA may be used as well. As an example, DR3-V1 or DR
PCR primers complementary to the nucleic acid encoding 3 can be used to confirm and analyze DR3-V1 or DR3 expression and mutations. For example, deletions and insertions
It can be detected by comparing the sizes of the amplification product and the normal genotype. Point mutation is a radiolabeled amplified DNA of DR3-V1 or DR3 RNA, or DR3-
It can be confirmed by hybridizing with a V1 or DR3 antisense radiolabeled DNA sequence. Perfectly matched sequences and mismatched duplexes can be distinguished by RNase A digestion or by melting temperature.

Sequence differences between control genes and genes with mutations can also be elucidated by direct DNA sequencing. In addition to this, the cloned DNA segment can be employed as a probe for detecting a specific DNA segment. The sensitivity of such methods can be greatly enhanced by the appropriate use of PCR or other amplification methods. For example, sequencing primers are used with double-stranded PCR products or single-stranded template molecules generated by modified PCR. Sequencing is a radiolabeled nucleotide,
Or, it is carried out by a usual operation method by an automatic sequencing operation method using a fluorescent tag.

Genetic testing methods based on DNA sequence differences may be accomplished by detecting alterations in electrophoretic mobility of DNA fragments in gels in the presence or absence of denaturing agents. Small sequence deletions and sequence insertions can be visualized by high resolution gel electrophoresis. DNA fragments with different sequences can be distinguished on denaturing formamide gradient gels that delay the mobility of the different DNA fragments in the gel at different positions according to their particular melting or partial melting temperatures (eg Myers et al., Science, 230. Volume: 1242 pages (1985)).

Sequence changes at specific locations may be revealed by nuclease protection assays such as RNase and S1 protection or by chemical cleavage methods (eg Cott).
on, Proc. Natl. Acad. Sci. USA, Volume 85: 4397
~ 4401 (1985)).

Thus, the detection of specific DNA sequences includes hybridization, RNase protection, chemical cleavage, direct DNA sequencing or the use of restriction enzymes (eg restriction fragment length polymorphism (“RFLP”) and Southern blotting of genomic DNA. In addition to the more conventional gel electrophoresis and DNA sequencing methods, mutations can also be detected by in situ analysis.

Chromosome Assays The sequences of the present invention are also useful for chromosome identification. This sequence is specifically targeted to and can hybridize with a particular location on the human chromosome. Chromosomal DNA mapping according to the present invention is the first important step in linking these sequences with genes associated with disease.

In certain preferred aspects in this regard, the cDNAs disclosed herein are used to clone genomic DNA for the DR3-V1 or DR3 genes. This can be accomplished using a variety of well known techniques and libraries that are generally commercially available. Genomic DNA is used for in situ chromosome mapping using well known techniques for this purpose.

In addition to this, cDNA to PCR primers (preferably 15 to 25 bp)
A sequence map can be created on the chromosome by preparing Computer analysis of the 3'-untranslated region of the gene is used to rapidly select primers that do not complicate the amplification process over one or more exons in genomic DNA. These primers are then used for PCR screening of somatic cell hybrids containing each human chromosome.

Fluorescence in situ hybridization of cDNA clones with metaphase chromosome expansion (“F
ISH ") can be used to provide the exact chromosomal location in one step. This technique can be used for 50-60 cDNAs in length. For the theory of this technology, Ve
rma, etc., "Human Chromosomes: Basic Technology Handbook (Human Chromosome
s: a ・ Manual ・ of ・ Basic ・ Techniques), Per
See gamon Press, New York (1988).

Once the sequence has been mapped to the correct chromosomal location, the physical location of the sequence on the chromosome can be associated with genetic map data. Such data may be, for example, Joh
V.n., available online via the Hopkins University and Welch Medical Library. McKusick, "Mendelian inheritance in humans
Inheritance in Man) ". The relationship between diseases and genes assigned to the same chromosomal region is then confirmed by linkage analysis (co-inheritance of physically adjacent genes).

Next, it is necessary to determine the differences in the cDNA or genomic sequence between affected and unaffected individuals. If the mutation is found in some or all of the affected individuals, but not in any of the normal individuals, the mutation is likely to be the cause of the disease.

Vectors and Host Cells The present invention also relates to vectors containing the DNA molecules of the present invention, host cells genetically engineered with the vectors of the present invention, and methods of producing the polypeptides of the present invention by recombinant techniques.

Host cells can be genetically engineered to introduce nucleic acid molecules and express polypeptides of the invention. This polynucleotide may be introduced alone or together with another polynucleotide. Such another polynucleotide may be independently introduced, co-introduced, or combined with the polynucleotide of the present invention to be introduced.

According to this aspect 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. The viral vector may be replication complete or replication defective. In the latter case, viral propagation generally will occur only in complementing host cells.

Suitable among the vectors is in one aspect for the expression of the polynucleotides and polypeptides of the invention. Generally, such vectors include cis-acting control regions operably linked to the polynucleotide to be expressed and effective for expression in the host. Appropriate trans-acting factors are either supplied by the host, supplied by a complementary vector, or supplied by the vector itself when introduced into the host.

Various expression vectors can be used to express the polypeptides of the invention.
Such vectors include chromosomal, episomal and viral derived vectors such as bacterial plasmids, bacteriophages, yeast episomes,
Vectors derived from yeast chromosomal elements, for example viruses such as baculovirus, papovaviruses such as SV40, vaccinia virus, adenovirus, fowlpox virus, pseudorabies virus and retrovirus,
And vectors derived from these conjugates, such as those derived from plasmids and bacteriophage genetic elements, such as cosmids and phagemids, all of which are of the expression according to this aspect of the invention. May be used for Generally, any vector suitable to maintain, propagate or express a polynucleotide for expressing a polypeptide in a host may be used for expression in this context.

The DNA sequence in the expression vector is operably linked to a suitable expression control sequence including, for example, a promoter that directs mRNA transcription. Representative examples of such promoters include some of the well known promoters: phage lambda PL promoter, E. coli lac, trp, and tac promoters, SV40 early and late promoters, and retroviral LTR promoters. Includes. In general, expression constructs will include sites for transcription, initiation, and termination, and, within the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcript expressed by the construct contains the translation initiation AUG at the start site and suitably a stop codon (UAA, UGA or UAG) near the end of the translating polypeptide.

In addition, the constructs may contain control regions that regulate as well as engender expression. This region generally operates by controlling transcription, such as repressor binding sites and enhancers.

Vectors for propagation and expression generally will include selectable markers.
This marker may be suitable for amplification, or the vector may contain another marker for this purpose. In this regard, expression vectors preferably contain one or more selectable marker genes and provide phenotypic traits for selection of transformed host cells. Suitable markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture and the tetracycline or ampicillin resistance genes for culture of E. coli and other bacteria.

Vectors containing suitable DNA sequences, as well as suitable promoters and other suitable control sequences, as described elsewhere herein, are suitable for expression in the appropriate host in which the desired polypeptide is expressed. It can be introduced using well known techniques. Representative examples of suitable 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. Hosts for various expression constructs are well known and the present disclosure will enable one of skill in the art to readily select a host for expressing a polypeptide according to this aspect of the invention. Suitable culture media and conditions for the above host cells are known in the art.

Among the suitable vectors for use in bacteria are pQE70, pQE60 and pQE-9 available from Qiagen, pBS vector available from Stratagene, Phagescript vector, Bluescript.
t vector, pNH8A, pNH16a, pNH18A, pNH46A, and ptrc99a, pKK223-3, pK available from Pharmacia.
K233-3, pDR540, and pRIT5. Among the suitable eukaryotic vectors are pWLNEO, pSV2CAT, available from Stratagene.
There are pOG44, pXTI and pSG, and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. These vectors are listed solely for the purpose of illustration of a number of vectors that are commercially available and well known to those of skill in the art.

Selection of the appropriate vector and promoter for expression in a host cell is a well known procedure for constructing an expression vector, introducing the vector into a host and expressing in the host. The required techniques are those of ordinary skill in the art.

The present invention also relates to host cells containing the above constructs. 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 organism such as a bacterial cell. It can also be a cell.

Introduction of the construct into the host cell includes calcium phosphate gene transfer, DEAE-dextran mediated gene transfer, cationic lipid mediated gene transfer, electroporation, transduction,
It can be carried out by infection or other methods. These methods are described in a number of standard laboratory manuals, such as Davies, “Basic Methods in Molecular Biology (Basi).
c ・ Methods ・ in ・ Molecular ・ Biology), (19
1986).

In addition to including host cells containing the vector constructs described herein,
The present invention deletes or replaces primary, secondary and endogenous genetic material (such as DR3 coding sequences) and / or is operably linked to DR3-V1 or DR3 polynucleotides of the invention. Or a host cell of immortalized vertebrate origin, particularly mammalian origin, engineered to include endogenous genetic material (such as a heterologous polynucleotide sequence) that activates, alters and / or amplifies the DR3 polynucleotide. Include. For example, techniques known in the art can be used to operably link heterologous control regions (such as promoters and / or enhancers) and endogenous DR3-V1 or DR3 polynucleotide sequences via homologous recombination (US Patent 5461670, published June 24, 1997;
International publication WO94 / 12650, published August 4, 1994; Koller et al., Proc. Nat
l.Acad.Sci.USA, 86: 8932-8935 (1989); and Zijlstra et al.
Nature, 342: 435-438 (1989), which are incorporated herein by reference in their entirety. ).

The polypeptide may be expressed in a modified form, such as a fusion protein, and may contain not only secretory signals, but other heterologous functional regions. Therefore,
For example, additional amino acids, especially regions of charged amino acids, may be added to the N-terminus of the peptide to improve stability and durability inside the host cell, during purification, or during subsequent manipulation and storage. In addition, a region for promoting purification may be added to the polypeptide. Such regions may be removed prior to final preparation of the polypeptide. Addition of peptide moieties to polypeptides, especially to produce secretion or excretion, to improve stability, or to facilitate purification, is conventional and routine in the art. Preferred fusion proteins include a heterologous region from immunoglobulin that is useful for lysing the protein. For example, EP-A-O46
4533 (Canadian counterpart 2045869) discloses fusion proteins comprising various portions of the constant region of immunoglobulin molecules together with another human protein or portion thereof. In many cases, the Fc part in the fusion protein is of complete benefit for use in therapy and diagnosis, eg improving the pharmacokinetic properties (European patent A-0232).
262). On the one hand, for some purposes it may be desirable to be able to remove the Fc part after the fusion protein has been expressed, detected and purified in the advantageous manner described above. For example, when using the fusion protein as an antigen for immunization,
This is the case when the Fc part is found to interfere with its use in therapy and diagnosis. In drug discovery, an Fc portion is fused to a human protein such as hIL-5 for the purpose of conducting an efficient search in order to identify an antagonist to hIL-5. D. Journal, of, Molecular, such as Bennett
-Recognition, 8: 52-58 (1995) and K.K.
Johann, etc., The, Journal, of, Biological
-Chemistry, Volume 270 [No. 16]: 9459-9471 (1995)
Year).

DR3 and DR3-V1 polypeptides are derived from recombinant cell culture by ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyl. It can be recovered and purified by well known methods including apatite chromatography and lectin chromatography. Most preferably high performance liquid chromatography ("HPLC") is employed for purification. Well-known techniques for refolding proteins may be employed to regenerate the active conformation when the polypeptide is denatured during isolation and / or purification.

The polypeptides of the present invention may be constructed from purified natural products, products of chemically engineered manipulations, and prokaryotic or eukaryotic hosts, including, for example, bacterial, yeast, higher plant, insect and mammalian cells. Included are products produced by alternative techniques. Depending on the host employed, the recombinant production procedures may or may not be glycosylated with the polypeptides of the invention. In addition, the polypeptides of the present invention may optionally contain an initial modified methionine residue as a result of host-mediated processes.

DR3-V1 or DR3 polynucleotides and DR3-V1 or DR3
Polypeptides may be used in accordance with the present invention for a variety of applications, particularly the chemical and biological properties of DR3. These include treatment and / or prevention of tumors, resistance to parasites, bacteria and viruses, proliferation of T cells, endothelial cells and certain hematopoietic cells, thus leading to restenosis, graft-versus-host disease. DR to treat and / or prevent, to modulate antiviral response, and by agonists
There is an application to prevent certain autoimmune diseases after stimulation of 3. Additional applications relate to prognosis, diagnosis, prophylaxis and / or treatment of cellular, tissue and biological diseases. These aspects of the invention are described further below.

DR3 Polypeptides and Fragments The invention further provides an isolated DR3-V1 or DR3 polypeptide or fragment thereof having the amino acid sequences set forth in SEQ ID NO: 2 and SEQ ID NO: 4, respectively. It will be appreciated in the art that certain amino acid sequences of DR3-V1 or DR3 can be altered without any apparent effect on the structure or function of the protein. If such sequence alterations are intended, it should be borne in mind that there are determinant regions on the protein that determine activity. Such regions usually include the residues that make up the ligand binding site or death domain or those that form the three-dimensional structure affecting these domains.

Thus, the present invention further comprises DR3-V1 or DR3-regions, such as the protein fragments described below, which exhibit substantially DR3 protein activity.
Variants of V1 or DR3 proteins are included. Deletion in such mutants,
Includes insertions, inversions, repeats, and type substitutions. As described above, for indications regarding which amino acid change phenotype is not manifest, see Bowie, J. et al. U. And so on
Ce, 247: 1306-1310 (1990).

Of particular interest is the replacement of a charged amino acid with another charged amino acid and the charged amino acid with a neutral or negatively charged amino acid. The latter is DR3-V
1 or a protein with a reduced positive charge which improves the properties of the DR3 protein.
Inhibition of aggregation is highly desirable. Protein aggregation can cause problems because it not only leads to loss of activity but can also be immunogenic when producing pharmaceutical formulations (P
Inclind et al., Clin. Exp. Immunol. Volume 2: 331-3
40 (1967); Robbins et al., Diabetes, 36:83.
8 to 845 (1987); Cleland et al., Crit. Rev. The
rapeutic / Drug / Carrier / Systems, 10:30
7-377 (1993)).

Amino acid substitutions can also alter the selectivity of binding to cell surface receptors. Ostad
e, et al., Nature, 361: 266-268 (1993), wherein certain mutations bind to only one of the two known types of TNF receptors.
It has been described that the selective binding of Therefore, DR3-V1 or DR of the present invention
The 3-receptor may include one or more amino acid substitutions, deletions or additions due to natural mutations or artificial manipulations.

As mentioned above, this modification preferably has a slight difference in character, such as a conservative amino acid substitution that does not significantly affect the folding or activity of the protein (see Table 1).

[Table 1] Of course, the number of amino acid substitutions that a person skilled in the art will make depends on many factors as described above. Generally, the number of substitutions in any known DR3-V1 or DR3 polypeptide is 50, 40, 30, 25, 20, 15, 10, 5 or 3.
It is said to be the following.

Amino acids essential for function in the DR3-V1 or DR3 proteins of the invention may be, for example, site-directed mutagenesis or alanine scanning mutagenesis.
It can be confirmed by methods known in the art (Cunningham and Wells, Science, 244: 1081-1085 (1989).
Year)). The latter procedure introduces a single alanine mutation at every residue in the molecule.
The resulting mutant molecules are then tested for receptor binding or biological activity such as in vitro or in vivo proliferative activity. The site that determines ligand-receptor binding can also be determined by structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith et al., J. Mol. Biol., 224: 899-904 (1).
1992) and de Vos et al., Science, 255: 306-31.
Page 2 (1992)).

The polypeptides of the present invention are preferably provided in isolated form and are preferably substantially purified. Recombinantly produced DR3-V1 or DR3 polypeptides are described by Smith and Johnson, Gene 67: 31-40 (198).
8 years) and substantially purified by the one-step method described.

Polypeptides of the invention include those encoded by the deposited cDNA including the leader, the mature polypeptide encoded by the deposited cDNA minus the leader (ie, the mature protein), SEQ ID NO: 2 containing the leader. Or a polypeptide of SEQ ID NO: 4, a polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4 excluding a leader, an extracellular domain, a transmembrane domain, an intracellular domain, all or part of an extracellular and intracellular domain (or Comprises a soluble polypeptide which does not contain a transmembrane domain, as well as at least 80%, preferably at least 8% identical to the deposited cDNA clone, the polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4.
0% or 85% identical, more preferably at least 90%, 92%, 95%
, 96%, 97%, 98% or 99% identical polypeptides, and those with at least 30 amino acids, and more preferably parts of those polypeptides with at least 50 amino acids. Polynucleotides encoding these polypeptides are also included in the invention.

A polypeptide having at least, eg, 95% “identical” amino acid sequence to a control amino acid sequence that is a DR3-V1 or DR3 polypeptide has the amino acid sequence of that polypeptide, and the polypeptide sequence has DR3-V1 or DR3. It is intended to be identical to the control sequence, except that it may contain up to 5 amino acid changes every 100 amino acids for the control amino acids of the polypeptide. In other words, in order to obtain a polypeptide having an amino acid sequence which is at least 95% identical to the reference amino acid sequence, up to 5% of the amino acid residues in the control sequence have been deleted or replaced with another amino acid or the control Up to 5% of all amino acid residues in the sequence may be inserted in the control sequence. These changes in the control sequence may occur at the amino-terminus or the carboxy-terminus of the control amino acid sequence or at any position between these terminal positions, whether the residues are interspersed individually or one of the adjacent groups in the control sequence or It can happen more than that.

Indeed, a particular polypeptide may be at least 80%, 85%, 90%, 92% of the amino acid sequence shown, for example, in SEQ ID NO: 2 or SEQ ID NO: 4 or the amino acid sequence encoded by the deposited cDNA. , 95%, 96%, 97%, 98% or 99% identical, for example, the Bestfit program (Wisc
onsin / Sequence / Analysis / Package, Unix
Edition, Genetics Computer Computer Group, Univers
It can be routinely determined using known computer programs such as ity Research Park, 575Science Drive, Madison, WI53711). When determining whether a particular sequence is, for example, 95% identical to a reference sequence according to the invention using Bestfit or another sequence preparation program, of course, the percent identity is calculated over the entire length of the control amino acid sequence. The parameters are set to allow gaps with homology up to 5% of the total number of amino acid residues in the control sequence.

In certain embodiments, Brutlag et al. (Comp.App.Biosci., 6: 237-245.
The FASTDB computer program based on the algorithm of (1990)) is used to determine the identity (also referred to as global sequence alignment) between the reference (query) sequence (sequence of the invention) and the subject sequence. The preferred parameters used for FASTDB amino acid alignment to calculate percent identity are: Matrix = PAM, k-tuple = 4, mismatch penalty = 1, joining penalty = 20, randomized group length = 0, cutoff.・ Score =
1, Gap Penalty = 5, Gap Size Penalty = 0.05, Window Size = 500 or the length of the nucleotide sequence of interest (whichever is shorter). In this embodiment, if the subject sequence is shorter than the query sequence due to N- or C-terminal deletions rather than internal deletions, the FASTDB program will calculate N- or C-terminal truncations when calculating percent identity. Make a manual correction to the result taking into account the fact that it does not reveal. For a subject sequence truncated at the N and C termini, with respect to the query sequence, the query sequence being the N and C termini of the subject sequence (not matched / aligned) with the corresponding subject residue as a percentage of the total number of bases in the query sequence. Correct percent identity by calculating the number of residues in. Whether the nucleotides are matched / aligned is FASTD
Determined by the results of the B sequence alignment. This percentage is then subtracted from the percent identity calculated by the FASTDB program using the specified parameters to obtain the final percent identity score. This final percent identity score is used for the purposes of this example. To manually adjust the percent identity score, only the residues to the N- and C-termini of the query sequence and the subject sequence (not matched / aligned) are considered. That is, only the query residues are located furthest outside the N and C termini 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. If a deletion occurs at the N-terminus of the subject sequence, the FASTDB alignment does not show a match / alignment of the first 10 residues at the N-terminus. Ten unpaired residues represent 10% of the sequence (number of unmatched N- and C-terminal residues / total number of residues in the query sequence) with 10% identity calculated by the FASTDB program. Subtract from the percent score. If the remaining 90 residues were perfectly matched, the final percent identity would be 90%. In another example, a 90 residue subject sequence is compared to a 100 residue query sequence. At this time, the deletion is an internal deletion and there are no residues on the N- or C-termini of the subject sequence that do not match / align with the query sequence. In this case, the percent identity calculated by FASTDB is not manually corrected. Once again, as shown in the FASTDB alignment, only those residues outside the N and C termini of the subject sequence that are not matched / aligned with the query sequence are manually corrected. In this specific example, no other manual correction is performed.

The present inventors show that the DR3-V1 polypeptide exhibits three major structural domains 428.
It was found to be a residue protein. First, the ligand binding domain was identified at residues from about 36 to about 212 in SEQ ID NO: 2. Second, the transmembrane domain was identified at residues 213 to 235 in SEQ ID NO: 2. Third, the intracellular domain was identified at residues 236 to 428 in SEQ ID NO: 2. Importantly, the intracellular domain contains a death domain at residues 353 to about 419. Further preferred fragments of the polypeptide set forth in SEQ ID NO: 2 are from about 36 to about 4
It includes a mature protein from up to 28 residues, or a soluble polypeptide consisting of all or part of the extracellular and intracellular domains, but lacking the transmembrane domain. In this context, "about" refers to the listed values and numbers above that value (
5, 4, 3, 2 or 1) Includes values greater or lesser nucleotides. Polynucleotides encoded by these polypeptides are also included in the present invention.

The invention also relates to nucleic acids encoding amino acid residues of about 1 to about 125, about 30 to about 215, about 215 to about 240, about 240 to about 428, and about 350 to about 420 in SEQ ID NO: 2. A polynucleotide comprising (or consisting of) a molecule is provided. In this context, “about” means in particular the listed values and a number (5,
4, 3, 2 or 1) Includes values greater or lesser amino acids. Polynucleotides encoded by these polypeptides are also included in the present invention.

The present inventors have also discovered that the DR3 polypeptide is a 417 residue protein that exhibits three major structural domains. First, the ligand binding domain was identified at residues 25 to 201 in SEQ ID NO: 4. Second, the transmembrane domain was identified at residues 202 to 224 in SEQ ID NO: 4. Third, the intracellular domain was identified at residues 225 to 417 in SEQ ID NO: 4. Importantly, the intracellular domain contains a death domain at residues 342 to 408. Further preferred fragments of the polypeptide set forth in SEQ ID NO: 4 include the mature protein from residues from about 25 to about 417, or consist of all or part of the extracellular and intracellular domains, but with transmembrane domains. It includes a missing soluble polypeptide. In this context, "about" means the values specifically mentioned and a number (5, 4,
3, 2 or 1) Includes values greater or lesser nucleotides. Polynucleotides encoded by these polypeptides are also included in the present invention. Those skilled in the art will recognize that DR3-V1 or DR3cD
The full-length polypeptides encoded by NA differ only in the amino acid sequence of the leader peptide. The first 24 amino acids of the polypeptide shown in SEQ ID NO: 2 is SEQ ID NO: 4
Although the first 13 amino acids shown in are replaced, the rest of this amino acid sequence is the same. Thus, both the DR3-V1 cDNA and the DR3 cDNA encode the same mature protein with the same biological activity.

Therefore, the present invention further provides a leader, a mature protein, an extracellular domain,
DR3-V1 selected from transmembrane domain, intracellular domain and death domain
Alternatively, a DR3-V1 or DR3 polypeptide encoded by the deposited cDNA clone is provided, including a DR3 polypeptide fragment.

The polynucleotides of the present invention find use as sources of antibody production that bind the polypeptides of the present invention, and as molecular weight markers in SDS-PAGE gels or molecular sieve gel filtration columns using methods known to those of skill in the art. However, the present invention is not limited to these.

In another aspect, the invention provides a peptide or polypeptide that comprises (or consists of) an epitope-containing portion of a polypeptide described herein. The peptides and polypeptides of the invention having antigenic epitopes are therefore useful for raising antibodies, including monoclonal antibodies, that specifically bind to the polypeptides of the invention. For example, Wilson et al., Cell, 37: 767-77.
See page 8 (1984), page 777.

The peptides and polypeptides having an antigenic epitope of the present invention preferably contain at least 7 amino acids contained in the amino acid sequence of the polypeptide of the present invention,
More preferably at least 9, most preferably at least about 15 to about 30
Contains up to amino acids. In this context, "about" includes the specifically recited values and values that are several (5, 4, 3, 2, or 1) nucleotides greater or less than that value. Polynucleotides encoded by these polypeptides are also included in the present invention.

Non-limiting examples of antigenic polypeptides or peptides that can be used to raise DR3-specific antibodies are: a polypeptide consisting of about amino acid residues 1 to about 22 of SEQ ID NO: 2; SEQ ID NO: A polypeptide consisting of amino acid residues of about 33 to about 56 of 2; a polypeptide consisting of amino acid residues of about 59 to about 82 of SEQ ID NO: 2; a polypeptide consisting of about 95 to about 112 of amino acid residues of SEQ ID NO: 2 A peptide; a polypeptide consisting of amino acid residues 122 to 133 of SEQ ID NO: 2; a polypeptide consisting of amino acid residues 161 to 177 of SEQ ID NO: 2;
A polypeptide consisting of about amino acid residues 179 to about 190 of SEQ ID NO :; and a polypeptide consisting of about amino acid residues 196 to 205 of SEQ ID NO: 2;
Includes. In this context, "about" means in particular the listed values and a number (5,
4, 3, 2 or 1) Includes values greater or lesser nucleotides. Polynucleotides encoded by these polypeptides are also included in the present invention. In addition, antigenic polypeptides or peptides include polypeptides consisting of amino acid residues corresponding to residues of the previously disclosed DR3-V1 polypeptides. As mentioned above, the inventor has determined that the polypeptide fragment is the antigenic region of the DR3-V1 and DR3 proteins.

The epitope-bearing peptides and polypeptides of the invention may be produced by any conventional means. Houghten, R .; A. , "General method for rapid solid phase synthesis of a large number of peptides: specificity of antigen-antibody interaction at the level of each amino acid (Ge
general ・ method ・ for ・ the ・ rapid ・ solid-pha
se ・ synthesis ・ of ・ large ・ numbers ・ of ・ pep
tides: specificity, of, antigen-antibody
y, interaction, at, the, level, of, indivi
dual / amino / acids) ", Proc. Natl. Acad. Sc
i. USA 82: 5131-5135 (1985). This "Simultaneous Multiple Peptide Synthesis (SMPS)" process is further described in US Pat. No. 4,631,211 to Houghten et al.

Those skilled in the art will recognize that the DR3-of the present invention described above.
V1 or DR3 polypeptides and epitope-bearing fragments thereof can bind to portions of the constant domain of immunoglobulins (IgG), giving chimeric polypeptides.
These fusion proteins facilitate purification and show increased half-life in vivo. This has been demonstrated, for example, in chimeric proteins composed of the first two domains of human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins (European Patent 394827, Traunecker). Such as Natu
Re, 331: 84-96 (1988)). A fusion protein having a disulfide-bonded dimer structure derived from the IgG moiety is more efficient than a monomeric DR3-V1 or DR3 protein or protein fragment in binding and neutralizing other molecules. Can be (Funtoulakis et al., J. Bioche
m. 270: 3958-3964 (1995)).

Accordingly, the invention provides an epitope of a polypeptide having the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, or ATCC Deposit No. 97456 or 9775.
ATCC Accession No. 9745 under stringent hybridization or low stringency hybridization conditions as encoded by the polynucleotide sequence contained in the plasmid deposited as No.
6 or 97757, which includes a polypeptide comprising (or consisting of) an epitope of a polypeptide sequence encoded by a polynucleotide that hybridizes to the complement of SEQ ID NO: 1 or SEQ ID NO: 3 contained in the deposited plasmid. Furthermore, the present invention provides a polynucleotide sequence encoding an epitope of the polypeptide sequence of the present invention (such as the sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3), a polynucleotide sequence of a complementary strand of the polynucleotide sequence encoding the epitope of the present invention. And polynucleotide sequences that hybridize to complementary strands under stringent or low stringency hybridization conditions as described above. Polynucleotides encoding peptides with these antigenic epitopes are also included in the invention.

As used herein, “epitope” means that portion of a polypeptide that has antigenic or immunogenic activity in an animal, preferably a mammal, most preferably a human. In a preferred embodiment, the invention includes a polypeptide comprising an epitope as well as a polynucleotide encoding this polypeptide. An "immunogenic epitope" is defined as a portion of a protein that elicits an antibody response in an animal as measured by any method known in the art, eg, the antibody production methods described below (
Geysen et al., Proc. Natl. Acad. Sci. USA, Volume 81:
3998-4002 (1983)). An "antigenic epitope" is defined as the part of a protein to which an antibody can immunospecifically bind its antigen, as determined by any method known in the art, eg, the immunoassays described herein. Immunospecific binding excludes non-specific binding but does not necessarily exclude cross-reactivity with other antigens. An antigenic epitope does not necessarily have to be immunogenic.

Antigenic epitopes (ie those containing regions of protein molecules to which antibodies can bind)
With respect to the selection of peptides or polypeptides having a Has been. For example Sut
cliffe, J .; G. Shinnick, T .; M. Green, N .; And Leaner, R .; A. (1983), "Antibodies that react with a certain site on a protein (Antibodies / that / react / with / predet).
ermined ・ sites ・ on ・ proteins), Science,
219: 660-666. Peptides capable of generating protein-reactive sera are often represented in the primary sequence of the protein and are characterized by a series of simple chemical rules that result in the immunodominant region (ie, immunogenic epitope) of the intact protein. Is also not limited to the amino or carboxyl termini. Fragments that function as epitopes can be generated by any conventional method (Houghten, Proc. Natl. Acad. Sci. USA 82: 5131-5135 (
1985), and further described in U.S. Pat. No. 4,631,211).

In the present invention, the antigenic epitope is preferably 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, It comprises a sequence of at least 20, at least 25, most preferably 15-30 amino acids. Preferred polypeptides containing immunogenic or antigenic epitopes are at least 10
, 15,20,25,30,35,40,45,50,55,60,65,70,75,80,
It is 85, 90, 95 or 100 amino acid residues long. The antigenic epitope is
For example, it is useful for raising antibodies that specifically bind to an epitope, such as monoclonal antibodies. Antigenic epitopes can be used as target molecules in immunoassays (Wilson et al., Cell 37: 767-778 (1984).
); Sutcliffe et al., Science, 219: 660-666 (1983)).

Similarly, immunogenic epitopes can be used to elicit antibodies according to methods known in the art (Sutcliffe et al., Supra; Wilson et al., Supra; and Bittle.
Et al., J. Gen. Virol .., 66: 2347-2354 (1985)). Polypeptides containing one or more epitopes may be used to elicit an antibody response in an animal system (such as rabbit or mouse) with a carrier protein such as albumin if the polypeptide is sufficiently long ( At least about 25 amino acids), the polypeptide can be used without a carrier. However, immunogenic epitopes containing as few as 8-10 amino acids have been found to be sufficient to raise antibodies capable of binding at very small epitopes in denatured polypeptides (Western blotting).

Antibodies using the epitope-bearing polypeptides of the present invention, including but not limited to, methods known in the art such as in vivo immunization, in vitro immunization and phage display. Can be triggered (Sutcliff
e et al., supra; Wilson et al., supra; and Bittle et al., J. Gen. Virol .., 66: 2347.
2354 (1985)). When using in vivo immunization, animals are immunized with free peptides; however, anti-peptide antibody potency by coupling the peptides to keyhole limpets to macromolecular carriers such as macyanine (KLH) or tetanus toxoid. Value can be boosted. For example, maleimidobenzoyl-N-hydroxysuccinimide ester (MBS)
A peptide containing a cysteine residue can be coupled to a carrier using a linker such as, but other peptides can also be coupled using a more common linking agent such as glutaraldehyde. Animals such as rabbits, rats and mice are free or carrier coupled by, for example, intraperitoneal and / or intradermal injection of about 100 μg peptide or carrier protein and Freund's adjuvant or any other adjuvant that stimulates an immune response. Immunize with the peptide. For example, several booster injections may be required to provide a useful titer of anti-peptide antibody that can be detected by an ELISA assay using free peptides adsorbed to a solid surface, for example at intervals of about 2 weeks. unknown. The titer of anti-peptide antibody in serum from immunized animals can be determined according to methods known in the art such as selection of anti-peptide antibody, such as adsorption to a peptide on a solid support, and selection of said anti-peptide antibody. It can be enhanced by elution of the antibody.

It will be appreciated by those skilled in the art that the polypeptides of the invention containing immunogenic or antigenic epitopes may be fused to other polypeptide sequences. For example, a polypeptide of the invention may be fused to an immunoglobulin (IgA, IgE, IgG, IgM) constant domain or other portion (either CH1, CH2, CH3 or any combination thereof and portions thereof) to form a chimeric It can be a polypeptide. Such fusion proteins facilitate purification and prolong in vivo half-life. This has been demonstrated in a chimeric protein consisting of the first two domains of human CD4 polynucleotide and various domains of the constant region of the heavy or light chain of mammalian immunoglobulins. EP394827; Traunecker et al., Nature, 3
31: 84-86 (1988). An IgG fusion protein with a disulfide-bonded dimeric structure due to the disulfide bonds of the IgG moiety has also been found to be more effective in binding and neutralizing other molecules than monomeric polypeptides or fragments thereof alone. There is. Fountoulakis et al., J. Bioche
m., 270: 3958-3964 (1995) and the like. A nucleic acid encoding the above epitope should also be recombined with a gene of interest as an epitope tag (eg, hemagglutinin (“HA”) tag or flag tag) to assist in the detection and purification of the expressed polypeptide. You can For example, Jank
The system described by necht et al. facilitates the purification of non-denatured fusion proteins expressed in human cell lines (Janknecht et al., 1991, Proc. Acad. Sci. USA, 8
8: 8972-897). In this system, 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 6 histidine residues. The tag acts as a matrix binding domain for the fusion protein. Extracts from cells infected with recombinant vaccinia virus can be loaded onto a Ni2 ⁺ nitriloacetic acid-agarose column and histidine-tagged proteins can be selectively eluted with imidazole-containing buffer.

Yet another fusion protein of the invention may be applied to gene shuffling, motif shuffling, exon shuffling and / or codon shuffling techniques (
Collectively referred to as "DNA shuffling").
DNA shuffling may be used to modulate the activity of the polypeptides of the invention,
Such techniques can be used to produce polypeptides with altered activity and agonists and antagonists of the polypeptides. In general, U.S. Pat. Nos. 5,605,793, 5811238, 5830721, 5834252 and 5837458; and Patten et al., Curr. Opinion Biotechnol., 8: 724-.
33 (1997); Trends Biotechnol., Harayama, 16 (2): 76-82 (1).
998); Hansson et al., J. Mol. Biol., 287: 265-76 (1999); and Lorenzo and Blasco BioTechniques, 24 (2): 308-13 (1998) (
These patents and these are incorporated herein by reference in their entireties. Are incorporated herein by reference in their entirety). In one embodiment, SEQ ID NO: 1
Alternatively, the polynucleotide corresponding to SEQ ID NO: 3 can be modified and the polypeptide encoded by the polynucleotide can be obtained by DNA shuffling. DNA shuffling involves the assembly of two or more DNA segments by homologous or site-specific recombination to create variations in polypeptide sequence. In another embodiment, the polynucleotide of the invention or the polypeptide it encodes can be modified by subjecting it to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination. In other embodiments, components, motifs, of a polynucleotide encoding one or more polypeptides of the invention,
Sections, parts, domains, fragments, etc. can be recombined with one or more heterologous molecular components, motifs, sections, parts, domains, fragments, etc.

As mentioned above, even if the deletion of one or more amino acids from the N-terminus of the protein results in the alteration of loss of one or more biological functions of the protein. However, other functional activities (biological activity, ability to multimerize, ability to bind DR3-V1 ligand, etc.) are still retained. For example, the ability to elicit and / or bind to an antibody that recognizes the complete or mature form of a truncated DR3-V1 mutein polypeptide is dependent on When removed from the N-terminus,
Generally retained. Whether a particular polypeptide lacking the N-terminal residue of the complete polypeptide retains such immunological activity will depend on the routine methods described herein and others known in the art. It can be easily measured.
It is likely that the DR3-V1 mutein with multiple deletions in the N-terminal amino acid residue will retain biological or immunological activity. In fact, DR3-V1 peptides with fewer 6 amino acid residues often provoke an immune response.

Accordingly, the present invention further provides a polypeptide in which one or more residues are deleted from the amino terminus of the DR3-V1 amino acid sequence shown in SEQ ID NO: 2 to the arginine residue at position 423, and the polypeptide thereof. A polynucleotide encoding the polypeptide is provided. In particular, the invention relates to residues n1-428 of SEQ ID NO: 2 (where n1 is
There is provided a polypeptide comprising (or consisting of) an amino acid sequence of 2 to 423 corresponding to the position of the amino acid residue of SEQ ID NO: 2. Polynucleotides encoding these polypeptides are also included in the invention.

More specifically, the present invention includes an amino acid sequence that is a member selected from the group consisting of the following amino acid residues of the DR3-V1 sequence shown in SEQ ID NO: 2 (
Or a polynucleotide encoding a polypeptide).

[Equation 1]

[Equation 2]

[Equation 3]

[Equation 4] The present invention also provides a polypeptide having at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identity to a polynucleotide sequence encoding the above polypeptide. It relates to a nucleic acid molecule comprising (or consisting of) a nucleotide sequence. The present invention also includes the above polynucleotide sequences fused to a heterologous polynucleotide sequence. Polypeptides encoded by these polynucleotides are also included in the invention.

As mentioned above, even if the deletion of one or more amino acids from the C-terminus of the protein results in the alteration of loss of one or more biological functions of the protein. However, other functional activities (biological activity, ability to multimerize, ability to bind DR3-V1 ligand, etc.) are still retained. For example, the ability to elicit and / or bind to an antibody that recognizes the complete or mature form of a truncated DR3-V1 mutein polypeptide is dependent on When removed from the C-terminus,
Generally retained. Whether a particular polypeptide lacking the C-terminal residue of the complete polypeptide retains such immunological activity will depend on the routine methods described herein and others known in the art. It can be easily measured.
It is likely that the DR3-V1 mutein with multiple deletions in the C-terminal amino acid residue will retain biological or immunological activity. In fact, DR3-V1 peptides with fewer 6 amino acid residues often provoke an immune response.

Accordingly, the present invention further provides a poly-form in which one or more residues are deleted from the carboxy terminus of the amino acid sequence of the DR3-V1 polypeptide shown in SEQ ID NO: 2 to the glutamine residue at the 6th position. Peptides and polynucleotides encoding the polypeptides are provided. In particular, the invention comprises (or consists of) an amino acid sequence of 1-m1 residues of SEQ ID NO: 2, where m1 is an integer from 6 to 427 corresponding to the position of the amino acid residue of SEQ ID NO: 2. Provide a polypeptide. Polynucleotides encoding these polypeptides are also included in the invention.

More specifically, the invention includes (or consists of) an amino acid sequence that is a member selected from the group consisting of the following amino acid residue sequences of the DR3-V1 sequence set forth in SEQ ID NO: 2: A polynucleotide encoding the polypeptide is provided.

[Equation 5]

[Equation 6]

[Equation 7]

[Equation 8] The present invention also provides a polypeptide having at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identity to a polynucleotide sequence encoding the above polypeptide. It relates to a nucleic acid molecule comprising (or consisting of) a nucleotide sequence. The present invention also includes the above polynucleotide sequences fused to a heterologous polynucleotide sequence. Polypeptides encoded by these polynucleotides are also included in the invention.

The present invention also provides a polypeptide having one or more amino acid deletions from both amino and carboxy termini of a DR3-V1 polypeptide, which polypeptide is generally n1 of SEQ ID NO: 2. Described as having an -m1 residue, where n1 and m1 are the above integers. Polynucleotides encoding these polypeptides are also included in the invention.

As mentioned above, the alteration of loss of one or more biological functions of the protein even as a result of the deletion of one or more amino acids from the N-terminus of the extracellular domain of the protein. However, other functional activities (biological activity, ability to multimerize, ability to bind DR3-V1 ligand, etc.) are still retained. For example, the ability to induce and / or bind an antibody that recognizes the complete, mature or extracellular domain form of a truncated DR3-V1 extracellular domain mutein polypeptide is If residues that are not the majority of the polypeptide are removed from the N-terminus, then generally,
Retained. Whether a particular polypeptide lacking the N-terminal residue of the extracellular domain of the polypeptide retains such immunological activity is determined by routine methods described herein and others known in the art. It can be easily measured by the method. It is likely that the muteins of the DR3-V1 extracellular domain with multiple deletions in the N-terminal amino acid residue retain biological or immunological activity. In fact, 6
Peptides with the DR3-V1 extracellular domain that are low in individual amino acid residues often elicit an immune response.

Therefore, the present invention further has one or more residues deleted from the amino terminus of the amino acid sequence of the DR3-V1 extracellular domain shown in SEQ ID NO: 2 to the cysteine residue at position 206. Polypeptides and polynucleotides encoding the polypeptides are provided. In particular, the invention comprises (or consists of) an amino acid sequence of residues n2-212 of SEQ ID NO: 2, where n2 is an integer from 36 to 206 corresponding to the amino acid residue position of SEQ ID NO: 2. Provide a polypeptide. Polynucleotides encoding these polypeptides are also included in the invention.

More specifically, the present invention includes an amino acid sequence that is a member selected from the group consisting of the following amino acid residues of the DR3-V1 sequence shown in SEQ ID NO: 2 (
Or a polynucleotide encoding a polypeptide).

[Equation 9]

[Equation 10] The present invention also provides a polypeptide having at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identity to a polynucleotide sequence encoding the above polypeptide. It relates to a nucleic acid molecule comprising (or consisting of) a nucleotide sequence. The present invention also includes the above polynucleotide sequences fused to a heterologous polynucleotide sequence. Polypeptides encoded by these polynucleotides are also included in the invention.

As mentioned above, alterations that result in loss of one or more biological functions of the protein even as a result of deletion of one or more amino acids from the C-terminus of the extracellular domain of the protein. However, other functional activities (biological activity, ability to multimerize, ability to bind DR3-V1 ligand, etc.) are still retained. For example, the ability to induce and / or bind an antibody that recognizes the complete, mature or extracellular domain form of a truncated DR3-V1 extracellular domain mutein polypeptide is If residues that are not the majority of the polypeptide are removed from the C-terminus, then generally,
Retained. Whether a particular polypeptide lacking the C-terminal residue of the extracellular domain of the polypeptide retains such immunological activity is determined by routine methods described herein and others known in the art. It can be easily measured by the method. It is likely that the muteins of the DR3-V1 extracellular domain with multiple deletions in the C-terminal amino acid residue retain biological or immunological activity. In fact, 6
Peptides with the DR3-V1 extracellular domain that are low in individual amino acid residues often elicit an immune response.

Accordingly, the present invention further comprises one or more residues from the C-terminus to the proline residue at position 42 of the amino acid sequence of the extracellular domain of the DR3-V1 polypeptide set forth in SEQ ID NO: 2. Provided are deleted polypeptides and polynucleotides encoding the polypeptides. In particular, the invention relates to 36-m2 of SEQ ID NO: 2.
Residues (where m2 is 42 to 21 corresponding to the position of the amino acid residue of SEQ ID NO: 2)
A polypeptide comprising (or consisting of) an amino acid sequence of 2 is provided. Polynucleotides encoding these polypeptides are also included in the invention.

More specifically, the invention includes (or consists of) an amino acid sequence that is a member selected from the group consisting of the following amino acid residue sequences of the DR3-V1 sequence set forth in SEQ ID NO: 2: A polynucleotide encoding the polypeptide is provided.

[Equation 11]

[Equation 12]

[Equation 13] The present invention also provides a polypeptide having at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identity to a polynucleotide sequence encoding the above polypeptide. It relates to a nucleic acid molecule comprising (or consisting of) a nucleotide sequence. The present invention also includes the above polynucleotide sequences fused to a heterologous polynucleotide sequence. Polypeptides encoded by these polynucleotides are also included in the invention.

The invention also provides a polypeptide having one or more amino acid deletions from both amino and carboxy termini of a DR3-V1 polypeptide, which polypeptide is generally n2 of SEQ ID NO: 2. Described as having -m2 residues, where n2 and m2 are the above integers. Polynucleotides encoding these polypeptides are also included in the invention.

As described above, even if the deletion of one or more amino acids from the N-terminus of the protein results in the alteration of loss of one or more biological functions of the protein. However, other functional activities (biological activity, ability to multimerize, ability to bind DR3 ligand, etc.) are still retained. For example, the ability to induce and / or bind to an antibody that recognizes the complete or mature form of a truncated DR3 mutein polypeptide is determined by the fact that residues that are not the majority of the complete or mature polypeptide are N-terminal. When removed from, it is generally retained. Whether a particular polypeptide lacking the N-terminal residue of the complete polypeptide retains such immunological activity will depend on the routine methods described herein and others known in the art. It can be easily measured. DR3 muteins with multiple deletions in the N-terminal amino acid residue are likely to retain biological or immunological activity. In fact, DR3 peptides with fewer 6 amino acid residues often provoke an immune response.

Therefore, the present invention further provides a polypeptide in which one or more residues are deleted from the amino terminus of the DR3 amino acid sequence shown in SEQ ID NO: 4 to the arginine residue at position 412 and the polypeptide. A polynucleotide encoding the following is provided. In particular, the invention comprises (or consists of) an amino acid sequence of residues n3-417 of SEQ ID NO: 4, where n3 is an integer from 2 to 412 corresponding to the position of the amino acid residue of SEQ ID NO: 4. Provide a polypeptide. Polynucleotides encoding these polypeptides are also included in the invention.

More specifically, the present invention encodes a polypeptide comprising (or consisting of) an amino acid sequence which is a member selected from the group consisting of the following amino acid residues of the DR3 sequence shown in SEQ ID NO: 4: To provide a polynucleotide.

[Equation 14]

[Equation 15]

[Equation 16]

[Equation 17] The present invention also provides a polypeptide having at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identity to a polynucleotide sequence encoding the above polypeptide. It relates to a nucleic acid molecule comprising (or consisting of) a nucleotide sequence. The present invention also includes the above polynucleotide sequences fused to a heterologous polynucleotide sequence. Polypeptides encoded by these polynucleotides are also included in the invention.

As described above, even if the deletion of one or more amino acids from the C-terminus of the protein results in the alteration of loss of one or more biological functions of the protein. However, other functional activities (biological activity, ability to multimerize, ability to bind DR3 ligand, etc.) are still retained. For example, the ability to induce and / or bind to an antibody that recognizes the complete or mature form of a truncated DR3 mutein polypeptide is determined by the fact that residues that do not represent the majority of the complete or mature polypeptide are C-terminal. When removed from, it is generally retained. Whether a particular polypeptide lacking the C-terminal residue of the complete polypeptide retains such immunological activity will depend on the routine methods described herein and others known in the art. It can be easily measured. DR3 muteins with multiple deletions in the C-terminal amino acid residue are likely to retain biological or immunological activity. In fact, DR3 peptides with fewer 6 amino acid residues often provoke an immune response.

Accordingly, the present invention further provides a polypeptide in which one or more residues are deleted from the carboxy terminus to the arginine residue at position 6 of the amino acid sequence of the DR3 polypeptide set forth in SEQ ID NO: 4 and A polynucleotide encoding the polypeptide is provided. In particular, the invention relates to residues 1-m3 of SEQ ID NO: 4 (where m3
Provides a polypeptide comprising (or consisting of) an amino acid sequence of 6 to 416 corresponding to the position of the amino acid residue of SEQ ID NO: 4. Polynucleotides encoding these polypeptides are also included in the invention.

More specifically, the present invention includes an amino acid sequence that is a member selected from the group consisting of the following amino acid residue sequences of the DR3 sequence shown in SEQ ID NO: 4 (
Or a polynucleotide encoding a polypeptide).

[Equation 18]

[Formula 19]

[Equation 20]

[Equation 21] The present invention also provides a polypeptide having at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identity to a polynucleotide sequence encoding the above polypeptide. It relates to a nucleic acid molecule comprising (or consisting of) a nucleotide sequence. The present invention also includes the above polynucleotide sequences fused to a heterologous polynucleotide sequence. Polypeptides encoded by these polynucleotides are also included in the invention.

The invention also provides a polypeptide having one or more amino acid deletions from both the amino and carboxy termini of the DR3 polypeptide, which polypeptide is generally n3-m3 of SEQ ID NO: 4. Described as having residues (where n
3 and m3 are the above integers). Polynucleotides encoding these polypeptides are also included in the invention.

As mentioned above, alterations that result in the loss of one or more biological functions of the protein even as a result of the deletion of one or more amino acids from the N-terminus of the extracellular domain of the protein. However, other functional activities (biological activity, ability to multimerize, ability to bind DR3 ligand, etc.) are still retained. For example, the ability of a truncated DR3 extracellular domain mutein polypeptide to elicit and / or bind to an antibody that recognizes a complete, mature or extracellular domain body is determined by the ability of the complete, mature or extracellular domain polypeptide to bind to the antibody. When less than the majority of residues are removed from the N-terminus, they are generally retained.
Whether a particular polypeptide lacking the N-terminal residue of the extracellular domain of the polypeptide retains such immunological activity is determined by routine methods described herein and others known in the art. It can be easily measured by the method. It is likely that muteins in the DR3 extracellular domain with multiple deletions in the N-terminal amino acid residue retain biological or immunological activity. In fact, peptides with the DR3 extracellular domain, which are low in 6 amino acid residues, often elicit an immune response.

Accordingly, the present invention further comprises one or two amino-terminal to the cysteine residue at position 195 of the amino acid sequence of the DR3 extracellular domain set forth in SEQ ID NO: 4.
Polypeptides having one or more residues deleted and polynucleotides encoding the polypeptides are provided. In particular, the invention relates to residues n4-201 of SEQ ID NO: 4 (where n4 is an integer from 25 to 195 corresponding to the position of the amino acid residue of SEQ ID NO: 4).
A polypeptide comprising (or consisting of) the amino acid sequence of Polynucleotides encoding these polypeptides are also included in the invention.

More specifically, the present invention encodes a polypeptide comprising (or consisting of) an amino acid sequence that is a member selected from the group consisting of the following amino acid residues of the DR3 sequence shown in SEQ ID NO: 4: To provide a polynucleotide.

[Equation 22]

[Equation 23] The present invention also provides a polypeptide having at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identity to a polynucleotide sequence encoding the above polypeptide. It relates to a nucleic acid molecule comprising (or consisting of) a nucleotide sequence. The present invention also includes the above polynucleotide sequences fused to a heterologous polynucleotide sequence. Polypeptides encoded by these polynucleotides are also included in the invention.

As mentioned above, alterations that result in the loss of one or more biological functions of the protein even as a result of the deletion of one or more amino acids from the C-terminus of the extracellular domain of the protein. However, other functional activities (biological activity, ability to multimerize, ability to bind DR3 ligand, etc.) are still retained. For example, the ability of a truncated DR3 extracellular domain mutein polypeptide to elicit and / or bind to an antibody that recognizes a complete, mature or extracellular domain body is determined by the ability of the complete, mature or extracellular domain polypeptide to bind to the antibody. When less than the majority of residues are removed from the C-terminus, they are generally retained.
Whether a particular polypeptide lacking the C-terminal residue of the extracellular domain of the polypeptide retains such immunological activity is determined by routine methods described herein and others known in the art. It can be easily measured by the method. It is likely that the muteins of the DR3 extracellular domain with multiple deletions in the C-terminal amino acid residue will retain biological or immunological activity. In fact, peptides with the DR3 extracellular domain, which are low in 6 amino acid residues, often elicit an immune response.

Accordingly, the present invention further comprises one or more residues deleted from the C-terminus to the proline residue at position 31 of the amino acid sequence of the extracellular domain of the DR3 polypeptide set forth in SEQ ID NO: 4. And a polynucleotide encoding the polypeptide. In particular, the invention relates to the 25-m4 residue of SEQ ID NO: 4 (
Here, m2 provides a polypeptide comprising (or consisting of) an amino acid sequence of 31 to 201 corresponding to the position of the amino acid residue of SEQ ID NO: 4. Polynucleotides encoding these polypeptides are also included in the invention.

More specifically, the present invention includes an amino acid sequence that is a member selected from the group consisting of the following amino acid residue sequences of the DR3 sequence shown in SEQ ID NO: 4 (
Or a polynucleotide encoding a polypeptide).

[Equation 24]

[Equation 25] The present invention also provides a polypeptide having at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identity to a polynucleotide sequence encoding the above polypeptide. It relates to a nucleic acid molecule comprising (or consisting of) a nucleotide sequence. The present invention also includes the above polynucleotide sequences fused to a heterologous polynucleotide sequence. Polypeptides encoded by these polynucleotides are also included in the invention.

The invention also provides a polypeptide having one or more amino acid deletions from both amino and carboxy termini of the DR3 polypeptide, which polypeptide is generally n4-m4 of SEQ ID NO: 4. Described as having residues (where n
4 and m4 are the above integers). Polynucleotides encoding these polypeptides are also included in the invention.

The present invention also includes n1-m1, n2-m2, n3-m3 and / or n4-m.
At least 80% relative to the DR3 polypeptide sequence described herein as 4.
, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical. In a preferred embodiment,
The present invention is directed to at least 80%, 85%, 90%, 92%, 95% of the polypeptides having the specific DR3 N- and C-terminal deleted amino acid sequences listed herein.
%, 96%, 97%, 98% or 99% identity of the polypeptides. Polynucleotides encoding these polypeptides are also included in the invention.

In another preferred embodiment, the DR3 protein of the invention comprises (or consists of) the above fusion protein, wherein the DR3 polypeptide is n1-m1, n.
2-m2, n3-m3 and / or n4-m4). In a preferred embodiment, the invention provides the specific DRs listed herein.
At least 80%, 85%, 90%, 92%, 95%, 96%, 97% relative to a nucleic acid sequence encoding a polypeptide having three N- and C-terminal deleted amino acid sequences
, 98% or 99% identity nucleic acid molecules. Polynucleotides encoding these polypeptides are also included in the invention.

One or more cysteine-rich regions of DR3-V1 and DR3 appear to be important for the interaction between DR3-V1 and DR3 and their respective ligands. Therefore, a particular embodiment of the present invention is 58-1 of SEQ ID NO: 2.
A polynucleotide encoding a polypeptide comprising (or consisting of) an amino acid sequence of 03, 106-136, 141-173 or 176-206 amino acid residues. Further embodiments of the invention DR comprising (or consisting of) any one, two, three or all four of the above cysteine rich regions.
A polynucleotide encoding a 3-V1 or DR3 polypeptide. Polypeptides encoded by these polynucleotides are also included in the invention.

It is preferred that the polynucleotide fragment of the present invention encodes a polypeptide that exhibits DR3 functional activity. A polypeptide exhibiting a "functional activity" of DR3-V1 or DR3 is a polypeptide capable of exhibiting a functional activity associated with one or more known full-length (complete) DR3-V1 or DR3 proteins. Means Such functional activity includes biological activity (eg, ability to induce cell self-destruction), antigenicity (ability to bind anti-DR3-V1 antibody or anti-DR3 antibody, or anti-DR3-V1 antibody or anti-DR3 antibody). Ability to compete with DR3-V1 or DR3 polypeptide for binding to), immunogenicity (DR3-V1 or DR
3 polypeptide), the ability to form multimers with the DR3-V1 or DR3 polypeptides of the invention and DR3-V1 or DR.
Receptor or ligand for 3 polypeptides (eg TNF-γ, TNF-
(eg, γ-β), but is not limited thereto.

DR3-V1 or DR3 polypeptides and fragments, variants, derivatives and analogs thereof can be assayed in a variety of ways. For example, one assayed for the ability to bind an anti-DR3-V1 antibody or an anti-DR3 antibody or to compete with a full-length DR3-V1 or DR3 polypeptide for binding to an anti-DR3-V1 antibody or an anti-DR3 antibody. In specific examples, radioimmunoassays, ELISAs (enzyme-linked immunosorbent assays), "sandwich" immunoassays, immunoradiometric assays,
Gel dispersion sedimentation reaction, immunodispersion assay, in situ immunoassay (using colloidal gold, enzyme or radioisotope labeling, etc.), Western blot, precipitation reaction, agglutination reaction (gel agglutination assay, hemagglutination assay etc.), complement fixation Various immunoassays known in the art can be used, including but not limited to competitive or non-competitive assay systems using techniques such as assays, immunofluorescent assays, protein A assays and immunoelectrophoretic assays. Absent. In one embodiment, the antibody is detected by detecting the label on the primary antibody. In other embodiments, the primary antibody is detected by detecting binding of the secondary antibody or reagent to the primary antibody. In yet another embodiment, 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.

In another embodiment to identify DR3-V1 or DR3 ligands or to assess the ability of a polypeptide fragment, variant or derivative of the invention to multimerize, for example, reducing and non-reducing gel chromatography. Binding can be assayed by known means such as, protein affinity chromatography and affinity blotting. Generally, Phizic
See Ky, E. et al., Microbiol. Rev., 59: 94-123. In another embodiment,
The physiological correlates of binding to its substrate (signal transduction) can be assayed.

In addition, the assays described herein (see Example 6 or known in the art)
Is used routinely for DR3-V1 or DR3 polypeptides and fragments thereof,
The ability of mutants, derivatives and analogs to induce DR3-V1 or DR3-related biological activity (such as inducing cell suicide in vitro or in vivo) can be measured. The ability of the polynucleotides and polypeptides of the invention to increase or decrease cell self-destruction can be routinely measured using techniques known in the art. For example, it can be routinely measured using a cell death assay performed essentially as described in the literature (AM Chinnaiyan et al. Cell 81: 505-505.
12 (1995), MP Boldin et al., J. Biol. Chem., 270: 7795-8.
1995), FC Kischkel et al., EMBO, 14: 5579-5588 (1995)
), AM Chinnaiyan et al., J. Biol. Chem., 271: 4961-4965 (1996).
Year)).

One or more cysteine-rich regions of DR3-V1 and DR3 appear to be important for the interaction between DR3-V1 and DR3 and their respective ligands. Therefore, a particular embodiment of the present invention is 58-1 of SEQ ID NO: 2.
03, 106-136, 141-173 or 176-206 amino acid sequences. Further embodiments of the invention relate to DR3-V1 or DR3 polypeptides comprising (or consisting of) 1, 2, 3, or all 4 combinations of any of the above cysteine-rich regions. Polynucleotides encoding these polypeptides are also included in the invention.

Among the particularly preferred fragments of the invention are those characterized by structural or functional properties of DR3-V1 or DR3. Such fragments include full (ie, full-length) DR3-V1 or DR3 (SEQ ID NO: 2 or SEQ ID NO: 4) alpha-helix and alpha-helix forming regions (“alpha regions”), betasheets and betasheet-forming regions. ("Beta area"), turns and turn forming areas ("turn areas"), coils and coil forming areas ("coil areas")
), Hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, surface forming regions and high antigen index regions (ie, as identified using the default parameters of the Jameson-Wolf program, 1 (Including one or more contiguous amino acids with an antigen index of 0.5 or more). Preferred regions, including but not limited to regions of the type described above, identified by analysis of the amino acid sequences set forth in SEQ ID NO: 2 or SEQ ID NO: 4 are shown in Figure 4; default parameters of the following computer programs: Such a preferred region is the Garnier-R
obson prediction alpha region, beta region, turn region and coil region; Chou-Fas
man prediction alpha region, beta region, turn region and coil region; Kyte-Dooli
ttle predicted hydrophilic and hydrophobic regions; Eisenberg alpha and beta amphipathic regions; Emini surface forming regions; and Jameson-Wolf high antigen index regions.

In yet another embodiment, the polynucleotide of the invention encodes a functional property of DR3-V1 or DR3. Preferred embodiments of the invention in this regard include alpha-helix and alpha-helix forming regions of DR3-V1 or DR3 (“alpha regions”), beta-sheets and beta-sheet forming regions (
“Beta region”), turns and turn forming regions (“turn regions”), coils and coil forming regions (“coil regions”), hydrophilic regions, hydrophobic regions, alpha amphiphilic regions, beta amphiphilic regions, It includes a fragment that includes a flexible region, a surface forming region and a high antigen index region.

Data representing the structural and functional properties of DR3-V1 or DR3 shown in FIG. 4 and / or Table 2 are DNA * ST set on default parameters.
Generated using various modules and algorithms of AR. In a preferred embodiment, the data presented in columns VIII, IX, XIII and XIV of Table 2 can be used to determine regions of DR3-V1 that show a high potential for antigenicity. The highly antigenic region is selected by selecting a value representing the region of the polypeptide that is likely to be exposed to the surface of the polypeptide in the environment where antigen recognition occurs in the process of initiation of the immune response, by selecting columns VIII, IX, XIII and / or Determined from the data presented in IV.

The preferred region for these is FIG. 4, but can also be revealed or identified by tabulating the data shown in FIG. 4, as shown in Table 2. The data of FIG. 4 was tabulated using the DNA * STAR computer algorithm used to generate FIG. 4 (set on the original default parameters) (see Table 2). The tabular data of FIG. 4 can be used to easily determine the particular boundaries of the preferred region.

The above preferred regions shown in FIG. 4 and Table 2 include, but are not limited to, regions of the aforementioned type identified by analysis of the amino acid sequence set forth in SEQ ID NO: 2. As shown in FIG. 4 and Table 2, such preferred regions include Garnier-Robson alpha region, beta region, turn region and coil region (
Columns I, III, V and VII of Table 2); Chou-Fasman alpha region, beta region, turn region and coil region (columns II, IV and VI of Table 2); Kyte
-Doolittle hydrophilic region (column VIII of Table 2); Hopp-Woods hydrophobic region (Table 2)
Column XI); Eisenberg alpha and beta amphipathic regions (columns X and XI of Table 2); Karplus-Schulz flexible regions (column XII of Table 2); Jameson-Wolf
High antigen index region (column XIII of Table 2); and Emini surface formation region (
Column XIV) of Table 2 is included.

[0154]

[Table 2]

[Table 3]

[Table 4]

[Table 5]

[Table 6]

[Table 7]

[Table 8]

[Table 9]

[Table 10]

[Table 11]

[Table 12]

[Table 13]

[Table 14]

[Table 15]

[Table 16]

[Table 17]

[Table 18]

[Table 19]

[Table 20]

[Table 21]

[Table 22]

[Table 23]

[Table 24]

In this regard, highly preferred fragments include regions of DR3-V1 and DR3, in which some structural features are combined, eg, some of the features set forth in Table 2 above, or It consists of them.

Further, the present invention provides a protein containing the polypeptide sequence encoded by the polynucleotide of the present invention.

The present invention is also directed to an isolated polypeptide comprising or consisting of fragments of DR3-V1 and DR3. In particular, the amino acid sequence of SEQ ID NO: 2
The following amino acids: 1-60, 11-70, 21-80, 31-90, 41-10.
0, 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, 231.
-290, 241-300, 251-310, 261-320, 271-330
, 281-340, 291-350, 301-360, 311-370, 321.
-380, 331-390, 341-400, 351-410, 361-420
And isolated polypeptides comprising or consisting of the amino acid sequence of a member selected from the group consisting of and 371-428, and polynucleotides encoding these polypeptides.

The invention also provides the following amino acids of amino acid SEQ ID NO: 4: 1-60, 11-7.
0, 21-80, 31-90, 41-100, 51-110, 61-120, 7
1-130, 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, 231-290, 241-300, 251-
310, 261-320, 271-330, 281-340, 291-350,
301-360, 311-370, 321-380, 331-390, 341-
Provided are isolated polypeptides comprising or consisting of the amino acid sequence of a member selected from the group consisting of 400, 351-410 and 361-417, and polynucleotides encoding these polypeptides.

The DR3-V1 or DR3 proteins of the invention may be monomeric or multimeric (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. In a particular embodiment, the polypeptides of the invention are monomers, dimers, trimers or tetramers. In a further aspect, the multimers of the invention are at least dimers, at least trimers,
Or at least a tetramer.

Multimers encompassed by the present invention are homomers or heteromers. In this specification,
The term homomer refers to a multimer containing only the 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 with the same or different polypeptide sequences.
In one specific embodiment, the homomers of the invention are multimers containing only DR3-V1 or DR3 proteins having the same polypeptide sequence. In another specific embodiment, the homomers of the invention have DR3 having different polypeptide sequences.
-Multimers containing V1 or DR3 proteins. As a specific aspect,
A multimer of the invention may be a homodimer (eg, containing a DR3-V1 or DR3 protein having the same or different polypeptide sequence) or a homotrimer (eg, a DR3-V1 having the same or different polypeptide sequence). V1 or DR3 protein). In a further aspect, the homomeric multimers of the invention are at least homodimers, at least homotrimers, or at least homotetramers.

As used herein, the term heteromer includes only the heterologous protein (ie, a polypeptide sequence that does not correspond to the polypeptide sequence encoded by the DR3 gene) in addition to the DR3-V1 or DR3 protein of the invention. Protein). In one specific embodiment, the multimers of the invention are heterodimers, heterotrimers, or heterotetramers. In a further aspect, the heteromeric multimers of the invention are at least heterodimers, at least heterotrimers, or at least heterotetramers.

The multimers of the invention may be the result of hydrophobic, hydrophilic, ionic and / or covalent attachment and / or may be attached indirectly, for example by liposome formation. Thus, in one embodiment, multimers of the invention, eg homodimers and homotrimers, are formed when the proteins of the invention come into contact with each other in solution. In another aspect, a multimer of the invention, eg, a heterodimer or heterotrimer, is characterized in that the protein of the invention is an antibody to a polypeptide of the invention ( Formed in contact with the solution). In yet another aspect, a multimer of the invention comprises a DR of the invention.
Formed by covalent attachment to and / or between the 3-V1 or DR3 proteins. Such covalent bonds include protein polypeptide sequences (eg, the polypeptide sequence set forth in SEQ ID NO: 2 or the deposited cDNA).
One or more of the amino acid residues contained in the polypeptide encoded by A) will be involved. One example of a covalent bond is a cross-link between cysteine residues within a protein's polypeptide sequence that interact in a native (ie, naturally occurring) polypeptide. Another example of covalent attachment is as a result of chemical or recombinant manipulation. Alternatively, such covalent attachment may involve one or more amino acid residues contained in the heterologous polypeptide sequence in the DR3-V1 or DR3 fusion protein. In one example, a covalent bond is formed with the heterologous sequence contained in the fusion protein of the invention (eg, US Pat. No. 5,478,925). In one embodiment, the covalent bond is formed with a heterologous sequence contained in a DR3-V1-Fc or DR3-Fc fusion protein of the invention (as described herein). In other embodiments, the covalent attachment of the fusion proteins of the invention can be carried out, for example, by osteoprotegrin.
) (See, eg, WO 98/49305, which is incorporated herein by reference in its entirety), and other TNFs capable of covalently binding to form multimers.
Formed between heterologous polypeptide sequences from family ligand / receptor members.

The multimers of the present invention can be produced by chemical methods known in the art. For example, a protein desired to be contained in the multimer of the present invention is
It can be chemically cross-reacted using linker molecules and linker molecular length optimization methods known in the art (see, eg, US Pat. No. 5,478,925, incorporated herein by reference in its entirety). . Further, the multimers of the present invention form one or more intermolecular crosslinks between cysteine residues known in the art that are located within the polypeptide sequence of the protein that it is desired to include in the multimer. Can be produced using the method (see, eg, US Pat. No. 5,478,925).
Incorporated herein by reference in its entirety). Furthermore, the protein of the present invention can be modified in the usual manner by adding cysteine or biotin to the C-terminus or N-terminus of the polypeptide sequence of the protein, and these can be modified by applying methods known in the art. Can be produced (see, eg, US Pat. No. 5,478,925, which is incorporated herein by reference in its entirety). In addition, methods known in the art can be applied to produce liposomes containing protein components that are desired to be included in the multimers of the invention (see, eg, US Pat. No. 5,478,925. Incorporated herein by reference in its entirety).

Alternatively, the multimers of the invention can be produced by genetic engineering techniques known in the art. In one aspect, the proteins contained in the multimers of the invention are described herein or otherwise known in the art (eg,
See U.S. Pat. No. 5,478,925. This is incorporated herein by reference in its entirety. Recombinantly produced using the fusion protein technology of (1). In one specific aspect,
The polynucleotide sequence encoding the polypeptide of the present invention is transferred in the reverse direction from the original C-terminus to the N-terminus (leader (Lack of sequence) ligation produces a polynucleotide encoding the homodimer of the invention (see, eg, US Pat. No. 5,478,925, incorporated herein by reference in its entirety). . In another embodiment, recombinant methods described herein or known in the art have been applied to contain the transmembrane region and be incorporated into liposomes by membrane reconstitution methods. Polypeptides can be produced (see, eg, US Pat. No. 5,478,925, incorporated herein by reference in its entirety).

Modification of Proteins Furthermore, methods known in the art (eg, Creighton, 1983, Proteins: St.
ructure and Molecular Principles, WH Freeman & Co., NY, and Hunkap
illar, M., et al., Nature 310: 105-111 (1984)). For example, a peptide corresponding to a fragment of DR3-V1 or DR3 polypeptide can be synthesized using a peptide synthesizer. Also, if desired, non-classical amino acids or chemical amino acid analogs may be added to the D
It can be introduced into the R3-V1 or DR3 polypeptide sequence by substitution or addition. As examples of non-classical amino acids, the D-isomer of the usual amino acids,
2,4-diaminobutyric acid, α-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, α-Abu, α-Ahx, 6-aminohexanoic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, Ornithine, norleucine, norvaline,
Hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, α-alanine, fluoroamino acid, α-methylamino acid, Ca-methylamino acid, Na-methylamino acid Design amino acids and amino acid analogs in general, but not limited thereto. Further, the amino acid may be D (dextrorotatory) or L (levorotatory).

Non-naturally occurring variants can be produced by variant induction methods known to those of skill in the art. Such methods include oligonucleotide-mediated mutagenesis, alanine scanning, PCR mutagenesis, site-directed mutagenesis (eg, Carter et al.
., Nucl. Acids Res. 13: 4331 (1986); and Zoller et al., Nucl. Acids Res.
. 10: 6487 (1982)), cassette mutagenesis (see, for example, Wells et al., Gen.
e 34: 315 (1985)), restriction selectin mutation.
agenesis) (e.g. Wells et al., Philos. Trans. R. Soc. London SerA 317:
415 (1986)) and the like, but are not limited thereto.

The present invention also relates to glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, antibody molecules or cellular ligands during or after translation. DR3-V differentially modified by binding etc.
1 and DR3 polypeptides. Any of a number of chemical modifications can be performed in a known manner. Such methods include cyanogen bromide, trypsin, chymotryprine, papain, V8 protease, specific chemical cleavage by NaBH ₄ , acetylation, formylation, oxidation, reduction, metabolic synthesis in the presence of tunicamycin. Examples include but are not limited to:

Additional post-translational modifications in the present invention include, for example, N-linked or O-linked carbohydrate chains, N-terminal or C-terminal processing, attachment of chemical moieties to amino acid backbones, N-linked or O-. Includes chemical modification of the linked carbohydrate and addition or deletion of the N-terminal methionine residue as a result of expression in a prokaryotic host. The polypeptide may also be modified with a detectable label such as an enzyme, fluorescent, isotope, or affinity label for detection and isolation of the protein.

The present invention also provides chemically modified derivatives of DR3-V1 or DR3 (eg, US Pat. Nos. 1 and 2) that provide additional advantages such as increased solubility, stability and circulation time of the polypeptide, or decreased immunogenicity. No. 4,179,337) is provided. The chemical moieties for derivatization can be selected from water soluble polymers such as polyethylene glycol, ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol. The polypeptide may be modified at random positions within the molecule or at predetermined positions within the molecule and may have one, two, three or more attached chemical moieties.

The molecular weight of the polymer is arbitrary and may be branched or unbranched. With respect to polyethylene glycol, the preferred molecular weights are, in terms of ease of handling and manipulation, about 1 kDa and about 100 kDa (the term "about" is used in the manufacture of polyethylene glycol, where one molecule is above the indicated molecular weight and Means less than that). The desired therapeutic profile (eg, desired duration in sustained release, effect on any biological activity, if any, ease of handling, degree of immunogenicity, and other known polyethylene glycol Other sizes may also be used, depending on their effect on the therapeutic protein or analog. For example, polyethylene glycol has an average molecular weight of about 200,5.
00, 1000, 1500, 2000, 2500, 3000, 3500, 400
0, 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.

As mentioned above, the polyethylene glycol may have a branched structure. Branched polyethylene glycols are described, for example, in the literature (US Pat. No. 5,643,575; Morpurgo et
al. Appl. Biochem. Biotechnol. 56: 59-72 (1999); Vorbjev et al., Nucleosi
des Nucleotides 18: 2745-2750 (1999); and Caliceti et al., Bioconjug. C
hem. 10: 638-646 (1999)). The disclosure of each of these documents is incorporated herein by reference.

The polyethylene glycol molecule (or other chemical moiety) should be attached to the protein in consideration of its effect on the functional or antigenic domains of the protein. There are numerous attachment methods available in the art. For example, EP 0401384 (GC for PEG
Coupling to SF) (incorporated herein by reference) and Malik et al., E
xp. Hematol. 20: 1028-1035 (1992) (report on PEGylation of GM-CSF using tresyl chloride). For example, polyethylene glycol can be covalently attached via an amino acid residue and through its reactive group (eg, a free amino or carboxyl group). A reactive group is a group to which an activated polyethylene glycol molecule can be attached. Lysine residues and N-terminal amino acid residues are included in amino acid residues having free amino groups, and aspartic acid residues, glutamic acid residues, and C-terminal amino acid residues are included in amino acid residues having free carboxyl groups. Groups are included. Sulfhydryl groups can also be used as a reactive group for attaching polyethylene glycol. Preferred for therapeutic purposes is attachment to the amino group, such as attachment to the N-terminus or lysine group.

As indicated above, polyethylene glycol can be attached to proteins via any of a number of amino acid residues. For example, polyethylene glycol can be attached to proteins via covalent bonds to lysine, histidine, aspartic acid, glutamic acid, or cysteine residues. Polyethylene glycol refers to a specific amino acid residue of a protein (eg, lysine, histidine, aspartic acid, glutamic acid, or cysteine) or one or more types of amino acid residues of a protein (eg, lysine, histidine, aspartic acid, glutamic acid, cysteine and them. One or more reaction chemistries can be utilized to couple the

Proteins that are chemically modified at the N-terminus may be particularly desirable. When using polyethylene glycol as an example of the composition of the present invention, one skilled in the art will choose among various polyethylene glycol molecules (in terms of molecular weight, branching, etc.) to select polyethylene glycol molecules and protein (or peptide) molecules in the reaction mixture. , The type of PEGylation reaction to be performed, and the method for obtaining the selected N-terminal PEGylated protein can be selected. The method of obtaining the N-terminal PEGylated preparation (ie, separating this moiety from other mono-PEGylated moieties, if desired) will be by purification of the N-terminal PEGylated material from the PEGylated protein population. Selection of proteins chemically modified at the N-terminal modification position takes advantage of the different reactivities of various types of primary amino groups (such as lysine versus N-terminus) available for derivatization of specific proteins. It can be carried out by reductive alkylation. Under appropriate reaction conditions, the protein N-
Substantially selective derivatization with terminal carbonyl group containing polymers is achieved.

As pointed out above, PEGylation of the proteins of the invention can be done by any number of means. For example, polyethylene glycol can be added to the protein directly or via an intervening linker. Addition of polyethylene glycol to proteins in the absence of linkers has been described in the literature (Delgado et al., Crit. Rev. Thera.
Drug. Carrier Sys. 9: 249-304 (1992); Francis et al., Intern. J. of Hema
tol. 68: 1-18 (1998); U.S. Pat.No. 4,002,531; U.S. Pat.No. 5,349,052;
WO95 / 06058; WO98 / 32466), the disclosure of each of which is incorporated herein by reference.

One system for adding polyethylene glycol to amino acid residues of proteins without an intervening linker is monomethoxy polyethylene glycol (MPEG).
Is used with tresyl chloride (ClSO ₂ CH ₂ CF ₃ ). When the protein is reacted with tresylated MPEG, polyethylene glycol is attached directly to the amine groups of the protein. Thus, the present invention includes a protein-polyethylene glycol complex (conjugate) produced by reacting the protein of the present invention with a polyethylene glycol molecule having a 2,2,2-trifluoroethanesulfonyl group.

Polyethylene glycol can also be attached to proteins using a number of different intervening linkers. For example, US Pat. No. 5,612,460 (incorporated herein by reference in its entirety) discloses urethane linkers for attaching polyethylene glycol to proteins. A protein polyethylene glycol complex in which polyethylene glycol is bound to a protein via a linker is a protein MPEG-succinimidyl succinate, 1,1′-carbonyldiimidazole-activated MPEG, MPEG-2,4, 5-trichloropenyl carbonate, MPEG-p-nitrophenol carbonate, and various MPEG-
It can be produced by reacting with a compound such as a succinate derivative. Many more polyethylene glycol derivatives and reaction chemistries for attaching polyethylene glycol to proteins are described in WO 98/32466, the entire disclosure of which is incorporated herein by reference. PEGylated protein products produced using the reaction chemistry detailed herein are included within the scope of the present invention.

The number of polyethylene glycol moieties attached to each protein of the invention (ie, the degree of substitution) can also vary. For example, the pegylated proteins of the invention have an average of 1, 2,
It may be associated with 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20 or more polyethylene glycol molecules. Similarly, the average substitution rate is 1-3, 2-4, 3-5, 4-6, 5-7, 6-8, 7-9, per protein molecule.
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. A method for determining the degree of substitution is described in the literature (Delgad
O et al., Crit. Rev. Thera. Drug Carrier Sys. 9: 249-304 (1992)).

Polypeptide Assays The present invention also provides quantitative and diagnostic methods for detecting levels of DR3-V1 or DR3 protein or soluble forms thereof in cells and tissues, including determination of normal and abnormal levels. It also relates to diagnostic assays such as assays. Therefore, for example, a diagnostic assay according to the invention for detecting overexpression of DR3-V1 or DR3 or a soluble form thereof compared to a normal control tissue sample may be used, for example to detect the presence of a tumor. it can. Assay techniques that can be used to determine the level of a protein, such as the DR3 protein of the 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 assays and ELISA assays.

An assay for DR3-V1 or DR3 protein levels in a biological sample is
This can be done using any method known in the art. Preferred for assaying DR3-V1 or DR3 protein levels in a biological sample are antibody-based methods. For example, expression of DR3-V1 or DR3 protein in tissues can be tested using classical immunohistological methods. M. Jalkanen et al., J. Cell. Biol. 101: 976-985 (1985); M. Jalkanen et al., J. Cell. Biol. 105:
3087-3096 (1987).

Other antibody-based methods useful for detecting DR3-V1 or DR3 protein gene expression include enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA). Suitable labels are known in the art and include enzymatic labels such as glucose oxidase, iodine ( ¹²⁵ I, ¹²¹ I), carbon ( ¹⁴ C), sulfur ( ³⁵ S), tritium ( ³ H), indium ( ¹¹² ). In), and technetium ( ^99m T
radioisotopes such as c), and fluorescent labels such as fluorescein and rhodamine, and biotin.

Antibodies The present invention further binds immunospecifically to a polypeptide of the present invention, preferably an epitope (as determined by immunoassays well known in the art for assaying specific antibody-antigen binding. Antibody) and T cell antigen receptor (TCR). Antibodies of the invention include, but are not limited to, polyclonal antibodies, monoclonal antibodies, multispecific antibodies, human antibodies, humanized or chimeric antibodies, single chain antibodies, Fab fragments, F (ab ') fragments. , Fab-produced libraries, anti-idiotype (anti-Id) antibodies (including, for example, anti-Id antibodies against the antibodies of the invention), and epitope binding fragments of any of the above. The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, ie, molecules that contain an antigen binding site that immunospecifically binds an antigen. The immunoglobulin molecule of the present invention may be of any type (eg IgG, IgE, IgM, IgD, IgA and IgY), class (eg IgG1, IgG2, IgG3, IgG4, IgA1 and I of immunoglobulin molecules.
gA2) or subclass.

Most preferably, the antibody is a human antigen-binding antibody fragment of the invention,
Ab, Fab ′ and F (ab ′) ₂ , Fd, single chain Fvs (scFv), single chain antibodies, disulfide linked Fvs (sdFv) and fragments containing either the V _L domain or the V _H domain. However, it is not limited to these. Antibody-binding antibody fragments, including single chain antibodies, include variable regions alone or with all or part of the following: hinge region, CH1 domain, CH2 domain and CH3 domain. The antibody of the present invention may be derived from any animal including birds and mammals. Preferably, the antibody is human, mouse,
It is of a donkey, ship rabbit, goat, guinea pig, camel, horse or chicken. As used herein, "human" antibody is referred to below as, for example, Ku
As described in US Pat. No. 5,939,598 to cherlapati et al., comprising an antibody having the amino acid sequence of a human immunoglobulin, transgenic for a human immunoglobulin library or one or more human immunoglobulins. And antibodies isolated from animals that do not express endogenous immunoglobulins.

Antibodies of the invention may be monospecific, bispecific, trispecific or more multispecific. A multispecific antibody may be specific for different epitopes of a polypeptide of the invention, or may be both a polypeptide of the invention and a heterologous epitope such as a heterologous polypeptide or a solid support material. It can be specific. For example, PCT publication WO93 / 17715; WO92 / 08802; WO9
1/00360; WO92 / 05793; Tutt et al., J. Immunol. 147: 60-69 (19.
91); U.S. Pat. No. 4,474,893; 4,714,681; 4,925,648.
No .; 5,573,920; 5,601,819; Kostelny et al., J. Immunol. 148:
See 1547-1553 (1992).

The antibodies of the invention can be described or specified based on the epitope, or the portion of the polypeptide of the invention that recognizes and specifically binds. Epitopes or portions of polypeptides may be identified as described herein, for example, by the N-terminal and C-terminal portions, by the size of adjacent amino acid residues, or in the tables and figures. it can. Antibodies that specifically bind to any epitope or polypeptide of the invention may also be excluded. Therefore, the invention may include and specifically exclude antibodies that specifically bind to the polypeptides of the invention.

The antibodies of the present invention can also be described or identified based on their cross-reactivity. Antibodies that bind to other analogs, orthologs or homologs of the polypeptides of the invention are included. 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%, at least 50% identity to the polypeptides of the present invention. Antibodies that bind to a polypeptide having: (calculated using methods known in the art and described herein) are also included in the invention. Less than 95%, less than 90%, less than 85%, less than 80%, 7 for the polypeptide of the invention
Does not bind to polypeptides with less than 5%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50% (using methods known in the art and described herein). Antibodies are also included in the present invention.

Further included in the invention is an antibody that binds to a polypeptide encoded by a polynucleotide that hybridizes to the polynucleotide of the invention under stringent hybridization conditions (described herein). The antibodies of the present invention can also be described or specified based on their binding affinity for the polypeptides of the present invention. The preferred binding affinity is 5 × 10 ⁻² M, 10 ⁻² M,
5 × 10 ⁻³ M, 10 ⁻³ M, 5 × 10 ⁻⁴ M, 10 ⁻⁴ M, 5 × 10 ⁻⁵ M,
10 ⁻⁵ M, 5 × 10 ⁻⁶ M, 10 ⁻⁶ M, 5 × 10 ⁻⁷ M, 10 ⁻⁷ M, 5 ×
10 ⁻⁸ M, 10 ⁻⁸ M, 5 × 10 ⁻⁹ M, 10 ⁻⁹ M, 5 × 10 ⁻¹⁰ M, 1
^{0 -10 M, 5 × 10 -11} M, 10 -11 M, 5 × 10 -12 M, 10 -12 M, 5 × 10 -13 M, 10 -13 M, 5 × 10 -14 M, 10 - ¹⁴ M, 5x
And those having a dissociation constant or Kd of less than 10 ⁻¹⁵ M, and 10 ⁻¹⁵ M.

The present invention also competitively inhibits binding of an antibody to an epitope of the invention as determined by methods known in the art for determining competitive binding, eg, immunoassays described herein. Antibodies are also provided. In preferred embodiments, 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%.

The antibody of the present invention acts as an agonist or antagonist of the polypeptide of the present invention. For example, the present invention includes antibodies that partially or completely interfere with receptor / ligand interactions with the polypeptides of the present invention. The present invention is
It is characterized by both receptor-specific and ligand-specific antibodies. The invention also features receptor-specific antibodies that do not interfere with ligand binding but interfere with receptor activation. Receptor activation (ie, signal transduction) can be determined by methods described herein or known in the art. For example, receptor activation can be determined by detecting phosphorylation of the receptor or its substrate (eg, tyrosine or serine / threonine) by immunoprecipitation followed by Western blot analysis (eg, those described herein). In certain embodiments, the activity of the ligand or receptor is at least 90%, at least 80%, at least 70% of the activity in the absence of antibody.
Antibodies that inhibit, at least 60%, or at least 50% are provided.

The present invention also recognizes receptor-specific antibodies that interfere with both ligand binding and receptor activation, as well as receptor-ligand complexes, preferably unbound receptor or unbound ligand is specific. It is also characterized by antibodies that do not recognize. Similarly, a neutralizing antibody that binds to the ligand and interferes with the binding of the ligand to the receptor, as well as a ligand that binds and thereby interferes with activation of the receptor,
Antibodies that do not interfere with the binding of the ligand to the receptor are included in the invention. These antibodies act as receptor agonists, ie potentiate or activate all or part of the biological activity of ligand-mediated receptor activation.

Antibodies are agonists, antagonists or inverse agonists of biological activity, including specific biological activities of the peptides of the invention disclosed herein.
agonists). Therefore, the invention further relates to antibodies that act as agonists or antagonists of the polypeptides of the invention. The antibody agonist can be prepared using methods known in the art. For example, PC
T Publication WO 96/40281; U.S. Patent No. 5,811,097; Deng et al., Blood.
92 (6): 1981-1988 (1998); Chen et al. Cancer Res. 58 (16): 3668-3678 (1998); H.
arrop et al., J. Immunol. 161 (4): 1786-1794 (1998); Zhu et al., Cancer Res. 58 (15).
: 3209-3214 (1998); Yoon et al., J. Immunol. 160 (7): 3170-3179 (1998); Prat et al., J. Cell. Sci. 111 (Pt2): 237-247 (1998); Pitard et al. , J. Immunol. Methods
205 (2): 177-190 (1997); Liautard et al., Cytokine 9 (4): 233-241 (1997); Carls
on et al., J. Biol. Chem. 272 (17): 11295-11301 (1997); Taryman et al., Neuron 14 (4).
): 755-762 (1995); Muller et al., Structure 6 (9): 1153-1167 (1998); Bartunek.
See Cytokine 8 (1): 14-20 (1996), all incorporated herein by reference in its entirety.

The antibodies of the invention can be used, for example, but not exclusively, to purify, detect and target the polypeptides of the invention, including both in vitro and in vivo diagnostic and therapeutic methods. Absent. For example, the antibody can be used in an immunoassay for qualitatively and quantitatively measuring the level of the polypeptide of the present invention in a biological sample. For example, Harlow et al., Antibodies:
See A Laboratory Manual (Cold Spring Harbor Laboratory Press, Second Edition, 1988), which is incorporated herein by reference in its entirety.

As described in more detail below, the antibodies of the invention can be used alone or in combination with other compositions. The antibody may further be recombinantly fused to the heterologous polypeptide at the N-terminus or the C-terminus, or may be chemically linked (including covalent and non-covalent) to the polypeptide or other composition. Good. For example, the antibodies of the invention can be recombinantly fused or conjugated to a molecule useful as a label in a detection assay or an effector molecule such as a heterologous polypeptide, drug or toxin. For example, PCT publication WO92 / 08495; WO91
/ 14438; WO 89/12624; U.S. Patent No. 5,314,995; and EP 396,387.

Antibodies of the invention include modified derivatives, that is, derivatives that have been modified by the covalent attachment of certain molecules to the antibody such that the antibody does not prevent it from producing an anti-idiotypic response. . For example, by way of example, antibody derivatives may be, for example, glycosylated, acetylated, pegylation, phosphorylated, amidated, derivatized with known protecting / blocking groups, proteolytic cleavage, cell ligands or other. Antibody modified by, for example, binding to a protein. Any of a number of chemical modifications can be made by known methods including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like. In addition, the derivative may contain one or more non-classical amino acids.

The antibodies of the present invention can be produced by any suitable method known in the art. Polyclonal antibodies against the desired antigen can be produced by various procedures well known in the art. For example, the polypeptide of the present invention is a rabbit,
It can be administered to a variety of host animals, including but not limited to mice, rats, etc., to induce the production of serum containing polyclonal antibodies specific for the antigen. Various adjuvants may be used to increase the immune response, depending on the host species. These adjuvants include Freund's (complete and incomplete) adjuvants, mineral gels such as aluminum hydroxide, surfactants,
For example, lysolecithin, pluronic polyol, polyanion, peptide, oil emulsion, keyhole limpet hemocyanin, dinitrophenol,
And potentially useful human adjuvants such as BCG (bacille Calmette-G
uerin) and Corynebacterium parvum. Such adjuvants are also well known in the art.

Monoclonal antibodies can be prepared using various methods known in the art, including the use of hybridoma, recombinant and phage display methods, or a combination thereof. For example, monoclonal antibodies are known in the art, eg, Harlow et al., Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2nd Edition, 1988); Hammerling et al.
It can be produced using hybridoma methods, including those taught in Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, New York, 1981), which is incorporated herein by reference in its entirety. The term "monoclonal antibody" as used herein is not limited to those produced by the hybridoma method. The term "monoclonal antibody" refers to an antibody that is derived from a single clone, including eukaryotic, prokaryotic, or phage clones, regardless of how it is produced. Therefore, the term "monoclonal antibody" is not limited to antibodies produced by the hybridoma method. The monoclonal antibody is a hybridoma method,
It can be prepared using a wide variety of methods, including the use of recombinant and phage display methods.

Methods for producing and screening for specific antibodies using the hybridoma method are routine and well known in the art and described in detail in Example 8. Briefly, mice can be immunized with a polypeptide of the invention or cells expressing the polypeptide. When an immune response is detected, eg, antibodies specific for the antigen are detected in mouse serum, the mouse spleen is removed and splenocytes isolated. The splenocytes are then fused by well known methods with suitable myeloma cells, for example cells from cell line SP20 available from the ATCC. Hybridomas are selected and cloned by limiting dilution. Then,
Hybridoma clones are assayed by methods known in the art for cells secreting antibodies capable of binding the polypeptides of the invention. Ascites fluid generally contains high levels of antibodies, but can be generated by immunizing mice with positive hybridoma clones.

Accordingly, the present invention provides a method for producing a monoclonal antibody and an antibody produced by the method, which method comprises culturing a hybridoma secreting the antibody of the present invention, wherein: Preferably the hybridoma is a hybridoma clone which secretes an antibody capable of binding the splenocytes isolated from a mouse immunized with the antigen of the present invention with myeloma cells, and then hybridizing the hybridoma obtained from the fusion with the polypeptide of the present invention. It was generated by screening.

Antibody fragments that recognize a particular epitope can be generated by known methods. For example, a Fab fragment F (ab ′) ₂ fragment of the invention can bind an immunoglobulin molecule to papain (producing a Fab fragment) or pepsin (F (a ′).
b ′) to produce ₂ fragments) and can be produced by proteolytic cleavage using an enzyme. F (ab ′) ₂ fragments contain the variable region, the constant region of the light chain and the CH1 domain of the heavy chain.

For example, the antibodies of the present invention can be generated using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles which contain the polynucleotide sequences encoding the domains. Among other things, such phage can be found in the repertoire (repe
rtoire) or combinatorial antibody libraries (eg, human or murine) to display expressed antigen binding domains. Phage expressing an antigen binding domain that binds to the antigen of interest can be selected or identified using an antigen, eg, a labeled antigen or an antigen bound or captured on a solid surface or beads. Phage used in these methods are typically phage gene II.
Fab, Fv recombinantly fused to either I or gene VIII protein
Or f expressed from a phage having a disulfide-stabilized Fv antibody domain
Filamentous phage containing d and M13 binding domains.

Examples of phage display methods that can be used to produce the antibodies of the present invention include Brinkman et al., J. Immunol. Methods 182: 41-50 (1995); Ames et al., J. Immunol.
Immunol. Methods 184: 177-186 (1995); Kettleborough et al., Eur. J. Immunol.
24: 952-958 (1994); Persic et al., Gene 187: 9-18 (1997); Burton et al. Advances.
in Immunology 57: 191-280 (1994); PCT application No. PCT / GP91 / 0113.
No. 4; PCT publication WO90 / 02809; WO91 / 10737; WO92 / 0
1047; WO92 / 18619; WO93 / 11236; WO95 / 1598
2; WO95 / 20401; and US Pat. No. 5,698,426; 5,22.
3,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427. ,
908; 5,516,637; 5,780,225; 5,658,727; 5
, 733,743 and 5,969,108, which are incorporated herein by reference in their entirety.

After phage selection, isolating the antibody coding region from the phage as described in the above references and using it to generate whole antibodies, including human antibodies, or other desired antigen binding fragments. , For example, as detailed below,
It can be expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria. For example, methods of recombinantly producing Fab fragments, Fab ′ fragments and F (ab ′) ₂ fragments are also described in PCT Publication WO 92/22324; Mullinax et al., BioTechniques 12 (6): 864-869 (1992);
And Sawai et al., AJRI 34: 26-34 (1995); and Better et al., Science 240: 1041-.
1043 (1988), which is incorporated herein by reference in its entirety, and can be utilized using methods known in the art.

Examples of methods that can be used to produce single chain Fvs and antibodies include US Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Me.
thods in Enzymology 203: 46-88 (1991); Shu et al., PNAS 90: 7995-7999 (1993).
And Skera et al., Science 240: 1038-1040 (1988). For some uses, including in vivo use of antibodies in humans and in vitro detection assays, it is preferable to use chimeric, humanized or human antibodies.
A chimeric antibody is a molecule in which different parts of the antibody are derived from different animal species, such as an antibody having a variable region derived from a mouse monoclonal antibody and a constant region of human immunoglobulin. Methods for producing chimeric antibodies are known in the art. For example, Morrison, Science 229: 1202 (1985); Oi et al., BioTechniques 4: 214 (
1986); Gillies et al., (1989) J. Immunol. Methods 125: 191-202; US Pat.
See 807,715; 4,816,567; and 4,816,397, which are incorporated herein by reference in their entirety.

Humanized antibodies are one or more complementarity determining regions (CDRs) from a non-human species.
And an antibody from a non-human species antibody that binds to the desired antigen having framework regions from human immunoglobulin molecules. Often, 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. These framework substitutions are performed by methods well known in the art, eg, by modeling the interaction of CDR residues with framework residues to identify those framework residues that are important for antigen binding. , Or by comparing the sequences to identify unusual framework residues at specific positions (eg, Queen et al., US Pat. No. 5,585,089; Riechmann et al., Nature 332: 323 (1988). (See herein in its entirety for reference). Humanization of antibodies can be accomplished, for example, by CDR grafting (EP 239,400; PCT Publication WO 91/09967; US Pat. No. 5,225,539; 5,530,101; and 5,585,089).
, Veneering or Resurfacing (EP592106; EP519596; Padlan, Molecular Immunology 28 (4/5): 489-498 (199).
1); Studnicka et al., Protein Engineering 7 (6): 805-814 (1994); Roguska et al., PN.
AS 91: 969-973 (1994)) and chain shuffling (US Pat. No. 5,565,332).

Fully human derived antibodies are particularly desirable for the treatment of human patients. Production of human antibodies can be carried out by various methods known in the art, including the above-described phage display method using an antibody library derived from human immunoglobulin sequences. U.S. Pat. Nos. 4,444,887 and 4,716,111; and PC
T publication WO98 / 46645, WO98 / 50433, WO98 / 2489
3, WO98 / 16654, WO96 / 34096, WO96 / 3373.
See also No. 5, and WO 91/10741, which is incorporated herein by reference in its entirety.

Human antibodies can also be produced using transgenic mice that are incapable of expressing functional endogenous immunoglobulins but are capable of expressing human immunoglobulin genes. For example, human heavy and light chain immunoglobulin gene complexes can be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively,
Human variable, constant, and diversity regions can be introduced into mouse embryonic stem cells in addition to human heavy and light chain genes. The mouse heavy and light chain immunoglobulin genes can be rendered non-functional independently or simultaneously with the introduction of the human immunoglobulin loci by homologous recombination. In particular, homozygous deletions in the JH region prevent the production of endogenous antibodies. The modified embryonic stem cells are expanded and microinjected into blastocysts to obtain chimeric mice. The chimeric mice are then grown to obtain homozygous offspring which express human antibodies. The transgenic mice are immunized in the usual manner with a selected antigen, eg, all or part of a polypeptide of the invention. A monoclonal antibody against the antigen can be obtained from the immunized transgenic mouse using a conventional hybridoma method.

The human immunoglobulin transgene carried by the transgenic mouse is B
It relocates during cell differentiation and then undergoes class switching and somatic mutations. Therefore, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies using such methods. For a review of this technique for producing human antibodies, see Longberg and Huszar (1995, Int.
ev. Immunol. 13: 65-93). For details of this technique for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, eg, PCT Publication WO 98/24893; WO 96 /
34096; WO96 / 33735; US Pat. No. 5,413,923; 5
5,625,126; 5,633,425; 5,569,825; 5,661,01
No. 6, No. 5,545,806; No. 5,814,318; and No. 5,939,59.
No. 8 (incorporated herein by reference in its entirety). In addition, Abgenix, I
Companies such as nc. (Fremont, Calif.) and GenPharm (San Jose, Calif.) can be reserved to provide human antibodies to selected antigens using techniques similar to those described above.

Antibodies of fully human origin that recognize selected epitopes are referred to as “guided selection”.
selection) ”). This approach uses selected non-human monoclonal antibodies, such as mouse antibodies, to guide the selection of fully human-derived antibodies that recognize the same epitope (Jespers et al., Bio / tec
hnology 12: 899-903 (1988)).

Further, using antibodies to the polypeptides of the invention, in turn anti-idiotypic antibodies that “mimic” the polypeptides of the invention are well known to those of skill in the art (eg, Greenspan & Bona). , FASEB J. 7 (5): 437-444 (1989) and Nis.
sinoff, J. Immunol. 147 (8): 2429-2438 (1991)). For example, an antibody that binds and competitively inhibits polypeptide multimerization and / or binding of a polypeptide of the invention to a ligand may be "mimicked" of a polypeptide multimerization and / or binding domain. , So that an anti-idiotype that binds and neutralizes the polypeptide and / or its ligand can be produced. Such neutralizing anti-idiotypes or Fab fragments of such anti-idiotypes can be used in therapeutic regimens that neutralize polypeptide ligands. For example, such anti-idiotypic antibodies can be used to bind to a polypeptide of the invention and / or to its ligand / receptor, thereby blocking its biological activity.

A. Polynucleotides Encoding Antibodies The invention further provides polynucleotides and fragments thereof that include nucleotide sequences that encode the antibodies of the invention. The invention also preferably provides stringent hybridization to a polynucleotide encoding an antibody 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. Also included are polynucleotides that hybridize under conditions or lower stringency hybridization conditions, such as those described above.

The polynucleotide can be obtained by a number of methods known in the art, and the nucleotide sequence of the polynucleotide can be determined by a number of methods known in the art. For example, if the nucleotide sequence of the antibody is known, the polynucleotide encoding the antibody can be assembled from chemically synthesized oligonucleotides (eg, Kutmeier et al., BioTechniques 17
: 242 (1994)), which, in brief, synthesizes overlapping oligonucleotides containing a portion of the sequence encoding the antibody, anneals and ligates the oligonucleotides, and then Amplifying the ligated oligonucleotide by PCR.

Alternatively, antibody-encoding polynucleotides can also be generated from nucleic acids from suitable sources. Although clones containing the nucleic acid encoding the particular antibody are not available, if the sequence of the antibody molecule is known, the nucleic acid encoding the immunoglobulin can be obtained from a suitable source (eg, an antibody cDNA library, or A cDNA library produced from a tissue or cell expressing said antibody, such as a hybridoma selected to express the antibody of the invention, or a nucleic acid isolated from said tissue or cell, preferably polyA + RNA), 3'end of said sequence And a cDNA clone from a cDNA library encoding the antibody, for example, by PCR amplification with synthetic primers capable of hybridizing to the 5'end or by cloning with an oligonucleotide probe specific for a particular gene sequence. Can be obtained. The amplified nucleic acid obtained by PCR can then be cloned into a replicative cloning vector using any method well known in the art.

Once the antibody nucleotide sequence and corresponding amino acid sequence have been determined, the antibody nucleotide sequence can be analyzed by methods well known in the art for manipulation of nucleotide sequences (eg, Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual).
, Second Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, and Ausubel et al., 1998, Current Protocols.
in Molecular Biology, John Willie & Sons, NY (both of which are incorporated herein by reference in their entirety) and have different amino acid sequences, such as amino acid substitutions, deletions and / or insertions. Antibodies can be generated.

In a particular embodiment, the amino acid sequences of the variable domains of the heavy and / or light chains are examined and complemented by methods well known in the art, eg by comparison with other heavy and light chain variable regions. The sex determining region (CDR) sequence can be identified and the hypervariable region of the sequence can be determined. As described above, using routine recombinant DNA techniques, one or more CDRs within the framework regions,
For example, a non-human antibody can be humanized by inserting within a human framework region. The framework regions may be naturally occurring or consensus framework regions, preferably human framework regions (for a list of human framework regions, see, eg, Chothia et al., J. Mol. Biol. 278: 457-
479 (1998)). The polynucleotide produced by the combination of the framework regions and CDRs preferably encodes an antibody that specifically binds to a polypeptide of the invention. As noted above, it is preferred that one or more amino acids are substituted within the framework regions, such amino acid substitutions preferably improving the binding of the antibody to its antigen. Further, such a method provides an antibody that lacks one or more intrachain disulfide bonds by making amino acid substitutions or deletions on one or more variable region cysteine residues participating in the intrachain disulfide bond. It can be used to generate molecules. Other modifications to the polynucleotide are encompassed by the present invention and within the skill of those in the art.

In addition, methods developed to produce "chimeric antibodies" by splicing genes from mouse antibody molecules of suitable antigen specificity with genes from human antibody molecules of suitable biological activity ( Morrison et al., 1984, Proc. Natl. Acad
Sic. 81: 851-855; Neuberger et al., 1984, Nature 312: 604-608; Takeda et al., 19
85, Nature 314: 452-454) can be used. As described above, a chimeric antibody has a variable region derived from a mouse monoclonal antibody and a constant region of human immunoglobulin, for example, a molecule in which different portions are derived from different animal species, such as a humanized antibody. Is.

Alternatively, the method described for the production of single chain antibodies (US Pat. No. 4,694)
4,778; Bird, 1988, Science 242: 423-42; Huston et al., 1988, Proc. Natl.
Acad. Sci. USA 85: 5879-5883; and War et al., 1989, Nature 334: 544-554).
Can be adapted to produce single chain antibodies. A single chain antibody is produced by linking a heavy chain fragment and a light chain fragment of the Fv region with an amino acid bridge to form a single chain polypeptide. Methods for assembling functional Fv fragments in E. coli can also be used (Skerra et al., 1988, Science 242: 1038-.
1041).

B. Method for Producing Antibody The antibody of the present invention can be produced by a method known in the art as a method for synthesizing an antibody, particularly by chemical synthesis, or preferably by recombinant expression method. Recombinant expression of an antibody of the invention, or a fragment, derivative or analog thereof, such as a heavy or light chain of the antibody of the invention requires the construction of an expression vector containing a polynucleotide encoding the antibody. . Once the polynucleotide of the invention encoding the antibody molecule or the heavy or light chain of the antibody or a part thereof (preferably comprising the variable domain of the heavy or light chain) is obtained, Vectors can be produced by recombinant DNA methods using methods well known in the art. Therefore, described herein is a method of making a protein by expressing a polynucleotide that comprises a nucleotide sequence that encodes an antibody.

Methods which are well known to those skilled in the art can be used to construct expression vectors containing antibody coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA methods, synthetic methods, and in vivo genetic recombination. Therefore, the invention provides a replicable vector comprising a nucleotide sequence encoding an antibody molecule of the invention or a heavy or light chain thereof or a variable domain of a heavy or light chain operably linked to a promoter. Such a vector may include a nucleotide sequence encoding the constant region of an antibody molecule (eg, PCT Publication WO86 / 05807; PCT Publication WO89 / 01).
036; and US Pat. No. 5,122,464), the variable domain of the antibody can 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 a conventional method, and then the transfected cell is cultured by a conventional method to produce the antibody of the present invention. Therefore, the invention includes a host cell containing a polynucleotide encoding an antibody of the invention, or a heavy or light chain thereof, operably linked to a heterologous promoter. In a preferred embodiment for expression of double-chain antibodies, a vector encoding both heavy and light chains is co-expressed in a host cell for expression of whole immunoglobulin molecules, as described in detail below. be able to.

A variety of host-expression systems may be used to express the antibody molecule of the present invention. Such host-expression systems are not only vehicles that produce and subsequently purify the coding sequence of interest, but also cells that express the antibody molecule of the present invention in situ when transformed or transfected with the appropriate nucleotide sequence. Also stands for.
These host-expression systems include, but are not limited to, recombinant bacteriophage DNA, plasmid DNA or cosmid D containing antibody coding sequences.
Microorganisms such as bacteria transformed with NA expression vector (eg, E. coli, B. subtilis); yeast transformed with recombinant yeast expression vector containing antibody coding sequence (
For example, Saccharomyces, Pichia); insect cell lines infected with recombinant viral expression vectors containing antibody coding sequences (eg baculovirus); recombinant viral expression vectors containing antibody coding sequences (eg cauliflower mosaic virus, CaMV; tobacco). Mosaic virus, TMV), or transformed with a recombinant plasmid expression vector containing antibody coding sequences (eg Ti plasmid), plant cell line; or derived from mammalian cell genome (eg metallothionein promoter) or A mammalian cell line (eg, COS, having a recombinant expression construct containing a promoter from a mammalian virus (eg, adenovirus late promoter; vaccinia virus 7.5K promoter)).
CHO, BHK, 293, 3T3 cells). In particular, for expression of whole recombinant antibody molecule, preferably bacterial cells such as E. coli, more preferably eukaryotic cells can be used for expression of recombinant antibody molecule. For example, mammalian cells such as Chinese hamster ovary cells (CHO) in combination with vectors such as the major intermediate early gene promoter from human cytomegalovirus are effective expression systems for antibodies. (Foecking et al., 1986, Gene 45: 101; Cockett et al., 1990, Bio / Technology 8: 2).

In bacterial systems, many expression vectors may be advantageously selected based on their use in the antibody molecule to be expressed. For example, when producing large quantities of such proteins for producing pharmaceutical compositions of antibody molecules, vectors that direct high levels of expression of fusion protein products that are easily purified are desirable. Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruther et al. 1983, EMBO J. 2: 1791); pI
N Vector (Inouye & Inouye, 1985, Nucleic Acids Res. 13: 3101-3109; Van
Heeke & Shuster, 1989, J. Biol. Chem. 24: 5503-5509).
The pGEX vector can also be used to express foreign proteins as a fusion protein with glutathione S-transferase (GST). Generally, such fusion proteins are soluble and can be readily purified from lysed cells by adsorption and binding to matrix glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vector is
It is designed to contain a thrombin or Factor Xa protease cleavage site so that the cloned target gene product can be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. This virus is Spodoptera f
Proliferate in rugiperda cells. The antibody coding sequences are individually labeled for the nonessential region of the virus (
For example, it can be cloned into the polyhedrin gene) and placed under the control of the AcNPV promoter (eg polyhedrin promoter).

In mammalian host cells, a number of virus-based expression systems may be used. When an adenovirus is used as an expression vector, the desired antibody coding sequence is labeled with an adenovirus transcription / translation regulatory complex, such as the late promoter and 3
It can be ligated to a tripartite leader sequence. This chimeric gene is then inserted in the adenovirus genome by in vitro or in vivo recombination. Nonessential regions of the viral genome (eg region E1 or E
The insertion into 3) will result in a virus that can survive and express the antibody molecule in the infecting host (eg Logan & Shenk, 1984, Proc. Natl.
Acad. Sic. USA 81: 355-359).

Specific initiation signals may also be required for efficient expression of the inserted antibody coding sequence. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the open reading frame of the desired coding sequence to ensure translation of the entire insert. These foreign translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression can be increased by including appropriate transcription enhancer sequences, transcription terminators, etc. (Bittner et al., 1987, Metho.
See ds in Enzymol. 153: 51-544).

In addition, 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. Modifications (eg glycosylation) and processing (eg cleavage) of such protein products are important to the function of the protein. Different host cells have characteristic and specific mechanisms for 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. For this purpose, eukaryotic host cells can be used that have the cellular machinery for proper processing of the primary transcript and proper glycosylation and phosphorylation of the gene product. Such mammalian host cells include, but are not limited to, CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3,
WI38, especially breast cancer cell lines such as BT483, Hs578T, HTB2
, BT20 and T47D, and normal mammary gland cell lines such as CRL7030.
And Hs578Bst.

Stable expression is preferred for long-term, high-yield production of recombinant proteins. For example, cell lines that stably express the antibody molecule can be engineered. Transformation of host cells with DNA and a selectable marker controlled by appropriate expression control sequences (eg, promoter sequences, enhancer sequences, transcription terminators, polyadenylation sites, etc.), rather than using expression vectors with viral origin of replication can do. After the introduction of the foreign DNA, the engineered cells are
Grow for 2 days then switch to selective medium. The selectable marker in the recombinant plasmid confers resistance to selection, allowing cells to stably integrate the plasmid into their chromosomes and grow to form foci, which in turn are cloned and expanded into cell lines. can do. This method can be advantageously used to engineer cell lines that express the antibody molecule. The cell lines so engineered are particularly useful in screening and evaluating compounds that interact directly or indirectly with the antibody molecule.

Many selection systems can be used, including but not limited to the herpes simplex virus thymidine kinase gene (Wigler et al., 1977, Cell 11: 223), hypoxanthine-guanine phosphoribosyl transferase gene (Szybalska).
& Szybalski, 192, Proc. Natl. Acad. Sic. USA 48: 202) and adenine phosphoribosyl transferase gene (Lowy et al., 1980, Cell 22: 817) for tk-, hgprt- or aprt- cells, respectively. be able to. Antimetabolite resistance can also be used as the basis for selection of the following genes: dhfr (conferring resistance to methotrexate) (Wigler et al., 1980, Natl.
Acad. Sci. USA 77: 357; O'Hara et al., 1981, Proc. Natl. Acad. Sci. USA 78:
1527); gpt (conferring resistance to mycophenolic acid) (Mulligan & Berg,
1981, Proc. Natl. Acad. Sci. USA 78: 2072); neo (aminoglycoside G)
Endowed resistance to -418) (Clinical Pharmacy 12: 488-505; Wu and Wu,
1991, Biotherapy 3: 87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxic
ol. 32: 573-596; Mulligan, 1993, Science 260: 926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62: 191-217; May, TIB TECH 11 (5): 1
55-215); and hygro (conferring resistance to hygromycin) (Santer
Re et al., 1984, Gene 30: 147).

Methods commonly known in the art of recombinant DNA technology that can be used in the present invention are Ausu
bel et al., 1993, Current Protocols in Molecular Biology, John Willie and Sons, New York; Krieger, 1990, Gene Transfer and Expressio.
n, A Laboratory Manual, Stockholm Press, New York; and Dracop
oli et al. (eds.), 1994, Current Protocols in Human Genetics, John Willie and Sons, New York Chapters 12 and 13; Colberre-Garapin et al., 19
81, J. Mol. Biol. 150: 1, which is incorporated herein by reference in its entirety.

Expression levels of antibody molecules can be increased by vector amplification (reviewed by Bebbington and Hentschel, The use of vectors based on gene amplificat).
ion for the expression of cloned genes in mammalian cells in DNA cloning
, Vol. 3 (Academic Press, New York, 1987)). If the marker in the vector system expressing the antibody can be amplified, increasing the level of inhibitor present in the culture medium of the host cell will increase the copy number of the marker gene. Since the amplified gene is linked to the antibody gene, antibody production will also be increased (Crouse et al., 1983, Mol. Cell. Biol. 3: 257).

Host cells can be co-transfected with two expression vectors of the invention, a first vector encoding a heavy chain derived polypeptide and a second vector encoding a light chain derived polypeptide. . These two vectors may contain the same selectable marker to allow equal expression of heavy and light chain polypeptides. Alternatively, a single vector may be used which encodes both heavy and light chain polypeptides. In such cases, the light chain should be placed before the heavy chain to avoid excess toxic free heavy chain (
Proudfoot, 1986, Nature 322: 52; Kohler, 1980, Proc. Natl. Acad. Sci. US
A 77: 2197). The heavy and light chain coding sequences include cDNA or genomic DNA.

Once the antibody molecule of the present invention has been recombinantly expressed, methods known in the art for purification of immunoglobulin molecules, such as chromatography (eg, ion exchange chromatography, affinity chromatography, especially Affinity for a specific antigen to protein A, and sizing column chromatography), centrifugation, fractional solubility, or any other standard method for purification of proteins.

C. Antibody Conjugates The invention may be recombinantly fused or chemically linked (covalently and / or covalently) to a polypeptide of the invention (or a portion thereof, preferably at least 10, 20 or 50 amino acids of said polypeptide). The antibody is a fusion protein (including both non-covalent bonds). The fusion does not necessarily have to be direct, but via a linker sequence. These antibodies may be specific for antigens other than the polypeptides of the invention (or portions thereof, preferably at least 10, 20 or 50 amino acids of said polypeptide). For example, a polypeptide of the invention can be targeted to a particular cell type either in vitro or in vivo by fusing or conjugating it to an antibody specific for a particular cell surface receptor. The antibody can be used for.

Antibodies fused or conjugated to the polypeptides of the invention can also be used in in vitro immunoassays and purification methods using methods known in the art. For example, Harbor et al., Supra, and PCT antibody WO93 / 212232; EP.
439,095; Naramura et al., Immunol. Lett. 39: 91-99 (1994); US Pat. No. 5,474,981; Gillies et al., PNAS 89: 1428-1432 (1992); Fell et al., J. Imm.
See unol. 146: 2446-2452 (1991), which is incorporated herein by reference in its entirety.

The present invention further includes compositions comprising the polypeptides of the present invention fused or conjugated to antibody domains other than the variable regions. For example, the polypeptides of the present invention can be fused or conjugated to the Fc region of an antibody, or a portion thereof. The portion of the antibody that is fused to the polypeptide of the invention includes the constant region, hinge region, CH1 domain, CH2 domain, and CH3 domain, or the entire domain or a combination thereof. The polypeptide may also be fused or conjugated with a portion of the antibody described above to form a multimer. For example, an Fc portion fused to a polypeptide of the invention can form a dimer due to disulfide bonds between the Fc portions.

[0235] Higher order multimeric forms can be produced by fusing the polypeptide to IgA and IgM moieties. Methods for fusing or conjugating the polypeptides of the present invention to antibody moieties are known in the art. For example, US Pat.
No. 6,603; No. 5,622,929; No. 5,359,046; No. 5,349,0.
No. 53; No. 5,447,851; No. 5,112,946; EP 307,434; EP 367,166; PCT publication WO 96/04388; WO 91/06
570; Ashkenazi et al., Proc. Natl. Acad. Sci. USA 88: 10535-10539 (1991).
Zheng et al., J. Immunol. 154: 5590-5600 (1995); and Vil et al., Proc. Natl. A.
cad. Sci. USA 89: 11337-11341 (1992), which is incorporated herein by reference in its entirety.

As described above, the polypeptides of the invention may be used to increase the in vivo half-life of the polypeptide, or for use in immunoassays, using methods known in the art to render the antibody moieties described above. Can be fused or conjugated to. In addition, the polypeptides of the present invention can be fused or conjugated to the antibody moieties described above to facilitate purification. One reported example is human C
It is a chimeric protein consisting of the first two domains of the D4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins (EP
394,827; Traunecker et al., Nature 331: 84-86 (1988)). Polypeptides of the invention fused or conjugated to an antibody (for IgG) having a disulfide-bonded dimer structure may also bind and neutralize other molecules than the monomer-secreted protein or protein fragment alone. Is more effective (Fountoulak
is et al., J. Biochem. 270: 3958-3964 (1995)).

In many cases, the Fc portion in the fusion protein will be of therapeutic and diagnostic benefit and thus may result in improved pharmacokinetic properties, for example (EP A232,
262). Alternatively, after the fusion protein has been expressed, detected and purified, the F
It is desirable to delete the c portion. For example, the Fc portion interferes with therapy and diagnosis when the fusion protein is used as an antigen for immunization. In the discovery of drugs, human proteins such as the hIL-5 receptor have been fused to the Fc portion for the purpose of high throughput screening assays to identify antagonists of hIL-5 (D. Bennett et al., J. Molecular Recognition. 8: 52-58 (1995);
See K. Johanson et al., J. Biol. Chem. 270: 9459-9471 (1995)).

In addition, the antibodies of the invention or fragments thereof can be fused to a marker sequence such as a peptide to facilitate purification. In a preferred embodiment,
The marker amino acid sequence is a hexahistidine peptide such as the tag provided in the pQE vector (QIAGEN, Inc., 9259 Eaton Avenue, Chatworth, CA, 91311), many of which are commercially available. For example,
Hexahistidine provides convenient purification of the fusion protein, as described in Gentz et al., Proc. Natl. Acad. Sci. USA 86: 821-824 (1989). Other peptide tags useful for purification include, but are not limited to, "HA" tags (corresponding to epitopes derived from influenza hemagglutinin protein (
Wilson et al., Cell 37: 767 (1984)), and the "flag" tag.

The invention further includes an antibody or fragment thereof conjugated to a diagnostic or therapeutic agent. Antibodies can be used for diagnostic purposes, eg, as part of a clinical trial procedure to determine the efficacy of a given treatment and / or prophylaxis regimen, eg, to monitor tumor development or progression. Detection can be facilitated by coupling the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent substances, luminescent substances, bioluminescent substances, radioactive substances, positron emitting metals using various positron emission tomography, and non-radioactive paramagnetic metal ions. Is mentioned. For metal ions that can be conjugated to antibodies for use as diagnostics according to the present invention,
See, for example, U.S. Pat. No. 4,741,900.

Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase,
Examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin, examples of suitable fluorophores include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein. , Dansyl chloride or phycoerythrin, examples of luminescent substances include luminol, examples of bioluminescent substances include luciferase, luciferin, and aequorin, examples of suitable radioactive substances include ¹²⁵ I , ¹³¹ I, ¹¹¹ In or ⁹⁹ Tc
Is mentioned.

In addition, the antibody or fragment thereof can be conjugated to a therapeutic moiety such as a cytotoxin, eg, cytostatic or cytocidal agent, therapeutic agent or radioactive metal ion. Cytotoxic or cytotoxic agents include any agent that is harmful to cells. Examples include paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine.
), Mitomycin, etoposide, tenoposide,
Vincristine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracindione, mitoxantrone, mithramycin, actinomycin D, l-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs thereof. Or a homolog can be mentioned.

Therapeutic agents include, but are not limited to, antimetabolites (eg,
Methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5
-Fluorouracil decarbazine), alkylating agents (eg mechlorethamine), thioepachlorambucil, melphalan, carmustine (BSNU) and lomustine (lomust).
ine) (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin),
Anthracyclines (eg daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (eg dactinomycin)
(Formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC), and cytostatics (eg, vincristine and vinblastine).

The conjugates of the invention can be used to modify a given biological response and the therapeutic agent or pharmaceutical moiety is not intended to be limited to classical chemotherapeutic agents. For example, the pharmaceutical moiety can be a protein or polypeptide that has the desired biological activity. Examples of such proteins include toxins such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; tumor necrosis factor, α-interferon, β-interferon, nerve growth factor, platelet-derived growth factor, tissue plasminogen. Activator, thrombotic factor (thrombot
ic agent) or an anti-angiogenic agent, eg, a protein such as angiostatin or endostatin; or, eg, lymphokine, interleukin-1 (
"IL-1"), interleukin-2 ("IL-2"), interleukin-
6 (“IL-6”), granulocyte-macrophage colony stimulating factor (“GM-CSF”)
)), Granulocyte colony stimulating factor (“G-CSF”) or other growth factors such as biological response modifiers.

Antibodies can also be attached to a solid support, which is particularly useful in immunoassays or purification of target antigens. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.

Methods for conjugating such therapeutic moieties to antibodies are well known, eg Arnon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In.
Cancer Therapy ", Monoclonal Antibodies And Cancer Therapy, Reisfeld et al.
Ed.), Pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies F
or Drug Delivery ", Controlled Drug Delivery (2nd edition), Robinson et al. (ed.)
Naka, pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of
Cytotoxic Agents In Cancer Therapy: A Review ", Monoclonal Antibodies' 8
4: Biological And Clinical Applications, Pinchera et al. (Ed.), Pp. 475-506
(1985); "Analysis, Results, And Future Prospective Of The Therapeutic U
se Of Radiolabeled Antibody In Cancer Therapy ", Monoclonal Antibodies Fo
r Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., "The Preparation And Cytotoxic Propertie.
s Of Antibody-Toxin Conjugates ", Immunol. Rev. 62: 119-58 (1982).

Alternatively, an antibody can be conjugated to a second antibody as described by Segal in US Pat. No. 4,676,980, which is incorporated herein by reference in its entirety. Antibody to form a heteroconjugate. Administering an antibody, conjugated or unconjugated with a therapeutic moiety, alone or with cytotoxic factors and / or cytokines,
It can be used as a therapeutic agent. D. Antibody Binding Assays The antibodies of the present invention can be assayed for immunospecific binding by any method known in the art. The immunoassay that can be used is the method (
For example, Western blot, radioimmunoassay, E, to name a few.
LISA (enzyme linked immunosorbent assay), "sandwich" type immunoassay, immunoprecipitation assay, precipitation enzyme reaction, gel dispersion precipitation enzyme reaction, immunodiffusion assay, agglutination assay, complement fixation assay, immunoradiation assay, fluorescent immunoassay, protein A immunoassay), but is not limited to competitive and non-competitive assay systems. These assays are routine and well known in the art (eg, Ausubel et al., Eds., 1994, Current P).
See rotocolos in Molecular Biology 1, John Wiley & Sons, Inc., New York, which are incorporated herein by reference). A typical immunoassay is briefly described below (this is not intended to be limiting).
.

Immunoprecipitation protocols typically involve lysis buffer (eg, RIPA buffer (1 % NP-40 or Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15M NaCl, 0.01M sodium phosphate, pH 7.2, 1% trasylol)). , Add the antibody of interest to the cell lysate and incubate at 4 ° C for a certain period of time (for example, 1 to 4 hours), add protein A and / or protein G sepharose bed to the cell lysate, and allow for about 1 hour Alternatively, incubate further at 4 ° C. and wash the bed in solution buffer And it includes resuspending the bed in SDS / sample buffer. The ability of the antibody of interest to immunoprecipitate a particular antigen can be assessed by, eg, Western blot analysis. One of ordinary skill in the art would be familiar with parameters that can be modified to increase antibody-antigen binding and reduce background (eg, prewash cell lysate with sepharose bed). Further description of immunoprecipitation protocols can be found in, eg, Ausubel et al., Eds., 199.
4, Current Protocolos in Molecular Biology 1st edition, John Wiley & Sons, Inc.
, New York, 10.16.1.

Western blot analysis usually involves preparing a protein sample and electrophoresing the protein sample in a polyacrylamide gel (eg 8% -20% SDS-PAGE depending on the molecular weight of the antigen). Transfer the protein sample from the polyacrylamide gel to a membrane (eg nitrocellulose, PVDF or nylon),
The membrane was blocked in a blocking solution (eg, PBS with 3% BSA or nonfat milk) and washed with wash buffer (eg, PBS-Tween 20) to remove the primary antibody (eg, of interest). Antibody), wash the membrane in wash buffer and bind to enzyme substrate (eg horseradish peroxidase or alkaline phosphatase) or radioactive material (eg ³² P or ¹²⁵ I) diluted in blocking buffer. Blocking the membrane with a secondary antibody (which recognizes a primary antibody (eg, an anti-human antibody)), washing the membrane in wash buffer, and detecting in the presence of antigen. . One skilled in the art will be familiar with parameters that can be modified to increase the signal detected and reduce background noise. Further description of Western blot protocols can be found, for example, in
Edited by Ausubel et al., 1994, Current Protocolos in Molecular Biology 1, J,
See ohn Wiley & Sons, Inc., New York, 10.8.1.

ELISA prepares the antigen, coats the wells of a 96-well microtiter plate with the antigen, binds the antibody of interest conjugated to a detectable compound such as an enzyme substrate (eg, horseradish peroxidase or alkaline phosphatase). Addition to the wells, incubation for a period of time and detection in the presence of antigen. In the case of ELISA, it is not necessary to bind the antibody of interest to the detectable compound, instead a secondary antibody conjugated to the detectable compound (which recognizes the antibody of interest) is added to the wells. May be. Further, instead of coating the well with the antigen, antibody may be coated to the well. In this case, the antigen of interest may be added to the coated wells followed by the addition of a secondary antibody conjugated to a detectable compound. One of ordinary skill in the art would be familiar with parameters that can be modified to increase the signal detected, as well as other variants of the ELISA known in the art. For further explanation of ELISA, see, eg, Ausubel et al., 1994, Current Protocolos in Molecular Biology 1, John Wiley.
& Sons, Inc., New York, 11.2.1.

Antibody-antigen binding affinity and off-rate of antibody-antigen interaction (
off-rate) can be measured by a competitive binding assay. One example of a competitive binding assay is a radioimmunoassay, which involves incubating a labeled antigen (eg, ³ H or ¹²⁵ I) with an antibody of interest while increasing the amount of unlabeled antigen, and labeling Detecting the antibody bound to the antigen. The affinity and binding off rate of the antibody of interest for an antigen can be determined from the data by Scatchard plot analysis. Competition with secondary antibodies can also be measured using radioimmunoassay. In this case, the antigen is incubated with the antibody of interest conjugated to a labeled compound (eg, ³ H or ¹²⁵ I) with increasing amounts of unlabeled secondary antibody.

E. Antibody-Based Therapy The present invention further relates to antibody-based therapy, wherein the therapy is for treating and / or preventing one or more of the disorders or conditions described herein. Administering the antibody to an animal (preferably a mammal, most preferably a human) patient. The therapeutic compounds of the present invention include, for example, the antibodies of the present invention (including fragments, analogs and derivatives thereof described herein), and nucleic acids encoding the antibodies of the invention (described herein). Including, but not limited to, fragments, analogs and derivatives thereof.

Without wishing to be bound by theory, it is believed that the DR3 receptor induces programmed cell death by association / cross-linking of the death domain between different receptor molecules. Therefore, drugs that prevent DR3 death domain association / crosslinking (
Antibody) will prevent DR3-mediated programmed cell death,
Drugs (eg, antibodies) that induce DR3 death domain association / crosslinking will induce DR3-mediated programmed cell death. In addition, DR3 ligands that trigger DR3-mediated programmed cell death (eg, TNF-γ-β) are thought to function by causing DR3 death domain association / crosslinking.

As alluded to above, the DR3 receptor has been found to bind TNF-γ-β (see PCT Publication WO / 08139, which forms a part of this specification).
The DR3 receptor is also known to be present in many tissues and on the surface of many cell types. These tissues and cell types include endothelial cells, hepatocytes, hepatocellular tumors, lymph nodes, Hodgkin lymphoma, tonsils, bone marrow, spleen, heart, thymus, pericardium, healing wounds (skin), brain, pancreatic tumors, Burned skin, U937 cells, testis, colorectal cancer (metasticized to the liver), pancreas, rejection kidney, fat, ovary, olfactory epithelium, striatum depression, HeLa cells, LNCAP
(When treated with +30 nM androgen), HUVEC (human umbilical vein endothelial cells), 8-week embryonic tissue, 9-week embryonic tissue, fetal brain tissue, fetal kidney tissue, fetal heart tissue, fetal tissue Thymus tissue, fetal lung tissue, fetal liver tissue, fetal spleen tissue, helper II T-cells, activated T-cells (16 hours), activated T-cells (2
4 hours), including primary dendritic cells, eosinophils, monocytes and keratinocytes. Furthermore,
TNF-γ-β has been shown to induce apoptosis, have anti-angiogenic activity, and suppress tumor cell growth in vivo. In addition, TNF-γ-β
The activity is believed to be altered at least somewhat by interaction with the DR3 receptor.

Antibodies that act as both agonists and antagonists of receptor function are known in the art. For example, Deng et al. (Blood 92: 1981-1988 (1988)) described human c-Mp.
Monoclonal antibodies that bind to 1 receptor and stimulate megakaryocyte formation are described. Therefore, the monoclonal antibody described by Deng et al. Is a c-Mp1 receptor agonist.

Antibodies that bind to the DR3 receptor will have altered effects on these receptors. These effects differ based on the unique portion of the DR3 receptor to which the antibody binds and the three-dimensional configuration of the antibody molecule itself. Thus, an antibody that binds to the extracellular domain of the DR3 receptor can stimulate or inhibit DR3 activity (eg, induction of apoptosis). Stimulates DR3 receptor activity (eg, DR
The antibody is a DR3 agonist that inhibits DR3 receptor activity (eg, blocks binding to TNF-γ-β, and / or DR3) by promoting association with the 3 receptor death domain. Antibodies (by inhibiting the association with the receptor death domain) are DR3 antagonists.

Antibodies of the invention that function as DR3 receptor agonists and antagonists include antibody fragments that bind antigen (eg, Fab and F (ab ′) ₂ fragments), Fd, single chain Fvs ( scFv), Fvs (sd of disulfide bond)
Fv) and fragments containing either V _L or V _H domains, as well as polyclonal, monoclonal and humanized antibodies. Each of the antibody fragments and antibodies that bind these antigens are described in more detail elsewhere herein.

In view of the above, the antibodies of the invention, as well as the other agonists, are useful for treating DR3 in endothelial cells.
It is useful in stimulating death domain activity and, consequently, in causing anti-angiogenic activity. This type of antibody is useful for rheumatoid arthritis and solid tissue cancers (eg, skin cancer, head and neck tumors, breast cancer, endothelial tumors, osteoblasts, giant cell types of bone and Kaposi's sarcoma).
It is useful for preventing and / or treating diseases and conditions associated with hypervascularization and neovascularization, such as, and diseases and conditions associated with chronic inflammation.

Diseases and conditions associated with chronic inflammation (eg, ulcerative colitis and Crohn's disease) often have histological changes associated with ingrowth of new blood vessels in inflamed tissue. Agonists of the invention that stimulate the activity of the DR3 death domain will induce apoptosis in endothelial cells. As a result, the agonists of the present invention are able to inhibit the formation of blood vessels and lymph vessels and thus are used to prevent and / or treat diseases and conditions associated with hypervascularization and neovascularization. be able to.

Other angiogenic diseases and conditions that can be prevented and / or treated with the agonists of the present invention include hypertrophic and keloid scars, proliferative diabetic retinas. Disease, congenital arteriovenous disorders, atherosclerotic plaques, hemophilic joints, pseudoarticular fractures, Osler-Weber syndrome, psoriasis, purulent granulomas, scleroderma, trachoma, menorrhagia and vascular adhesions.

As mentioned above, the DR3 receptor is also found on T-cells. Accordingly, agonists of the present invention (eg, anti-DR3 receptor antibodies) prevent diseases and illnesses that inhibit T-cell mediated immune responses and are associated with increased T-cell proliferation and Useful for treating. Diseases and conditions associated with T-cell mediated immune responses and increased T-cell proliferation include graft-versus-host responses and diseases, inflammation, autoimmune disease and T-cell leukemia.

Further, drugs that inhibit DR3 death domain activity (eg, DR3 antagonists) include:
It is also useful for preventing and / or treating a number of diseases and conditions associated with decreased angiogenesis, decreased T-cell proliferation and decreased T-cell populations. As indicated above, examples of antagonists of DR3 receptor activity include anti-DR3 receptor antibodies. These antibodies block binding to ligands (eg, TNF-γ-β) that stimulate DR3 death domain activity by binding to, for example, the DR3 receptor, or the conformation of the DR3 receptor involved in membrane signaling. It can function by inhibiting changes in configuration.

Examples of diseases associated with reduced angiogenesis that can be treated with the antagonists of the present invention include slow wound healing. In particular, older people often heal at a slower rate than younger individuals. Accordingly, the antagonists of the present invention can prevent and / or inhibit apoptosis from occurring in endothelial cells at the site of a wound, thus healing at a "normal" rate when healing a wounded individual. Wound healing can be promoted as in the case of healing of a living individual. Accordingly, the antagonists of the invention can be used to promote and / or accelerate wound healing. The antagonists of the present invention also treat diseases and conditions including restenosis, myocardial infarction, peripheral vascular disease, dangerous limb ischemia, angina, atherosclerosis, edema, cirrhosis, osteoarthritis and pulmonary fibrosis. , And / or be useful in preventing.

Antagonists of the invention (eg, anti-DR3 receptor antibodies) prevent diseases and conditions associated with increased T-cell mediated immune responses and with decreased T-cell proliferation. And / or is also useful for treating. Antibodies of the invention that block the binding of DR3 receptor ligands to the DR3 receptor or suppress the conformational changes of the DR3 receptor involved in membrane signaling inhibit DR3-mediated T-cell apoptosis. can do. Inhibition of DR3-mediated apoptosis causes, for example, an increased rate of expansion of T-cell populations in vivo, or a reduction in the size of those populations. Accordingly, the antagonists of the present invention can be used to prevent and / or treat diseases or illnesses that are associated with increased or decreased T-cell populations. Examples of such diseases and conditions are acquired immunodeficiency syndrome (AIDS) and its associated distress (eg, AIDS-related syndromes), T-cell immunodeficiency, radiation sickness, and radiation and / or chemotherapy. Includes T-cell depletion.

Furthermore, when an antagonist of the present invention is administered to an individual for the treatment and / or prophylaxis of a disease or condition associated with a reduction in T-cell populations, the antagonist activates lymphocyte proliferation. It can be administered with a drug that induces and / or induces (eg, a cytokine). Combination therapies of this nature, as well as other combination therapies, are described in more detail below.

Accordingly, anti-DR3 antibodies are useful for treating and / or preventing malignancies, disorders, diseases and / or conditions associated with tissues and cell types expressing the DR3 receptor. Furthermore, a malignant disease, which can be treated and / or prevented by inducing programmed cell death of cells expressing the DR3 receptor,
Abnormalities, diseases and / or conditions can be treated and / or prevented with the DR3 receptor agonists of the present invention. Similarly, malignancies, disorders, diseases and / or conditions that can be treated and / or prevented by inhibiting programmed cell death of cells expressing the DR3 receptor are included in the D of the present invention.
It can be treated and / or prevented with an R3 receptor antagonist.

A number of other malignancies, disorders, diseases and / or conditions that can be treated with the agonists and antagonists of the invention are described elsewhere herein (eg, in “Treatment below”). "Section").

The antibodies of the invention may be conjugated in a number of ways, either by binding the polynucleotides or polypeptides of the invention locally or systemically in the body, or by direct cytotoxicity of the antibody. (Eg complement (CDC) or effector cells (
(Mediated by ADCC))).

The antibodies of the invention may be other monoclonal or chimeric antibodies, or lymphokines, tumor necrosis factors (eg TNF-γ-β) or hematopoietic growth factors (eg IL-2, IL-3 and IL-7). ) And can be used advantageously. For example, if one intends to induce DR3-mediated cell death in cells expressing the DR3 receptor of the present invention, administer an agonistic anti-DR3 antibody in combination with TNF-γ-β. You can Combination therapies of this nature, as well as other combination therapies, are described in more detail below.

The antibodies of the invention can be administered alone or in combination with other types of treatments (eg, radiation therapy, chemotherapy, hormone therapy, immunotherapy and anti-tumor agents). In general, it is preferable to administer a product that is of the same species as the patient's species, which is of species origin or species reactivity (in the case of antibodies). Thus, in a preferred embodiment, human antibodies, fragment derivatives, analogs or nucleic acids are administered to human patients for treatment or prevention.

Polynucleotides or polypeptides of the invention, including fragments thereof
Inhibiting and / or neutralizing in vivo the polypeptides or polynucleotides, fragments or regions thereof of the invention with high affinity and / or potency in the immunoassays and therapies for disorders associated therewith. It is preferable to use an antibody that The antibodies, fragments or regions thereof preferably have an affinity for polynucleotides or polypeptides, including fragments thereof. The preferred binding affinity has an association constant or Kd of 5 × 10 ⁻⁶ M, 10 ⁻⁶ M, 5 × 10 ⁻⁷ M, 10 ⁻⁷ M, 5 ×.
10 ⁻⁸ M, 10 ⁻⁸ M, 5 × 10 ⁻⁹ M, 10 ⁻⁹ M, 5 × 10 ⁻¹⁰ M, 1
^{0 -10 M, 5 × 10 -11} M, 10 -11 M, 5 × 10 -12 M, 10 -12 M, 5 × 10 -13 M, 10 -13 M, 5 × 10 -14 M, 10 - ¹⁴ M, 5x
Including those smaller than 10 ⁻¹⁵ M and 10 ⁻¹⁵ M.

Transgenic Non-Human Animals The proteins of the invention can also be expressed in transgenic animals. Animals of any species (mouse, rat, rabbit, hamster, guinea pig,
Pigs, micropigs, goats, sheep, cows and non-human primates (eg, but not limited to baboons, monkeys and chimpanzees) can be used to obtain transgenic animals. In certain embodiments, the techniques described herein, or otherwise known in the art, can be used to express a polypeptide of the invention in humans as part of a gene therapy protocol.

Any technique known in the art can be used to introduce a transgene (eg, a nucleic acid of the invention) into an animal to establish the founder line of the transgenic animal.
) Can be used to obtain These techniques include, for example, pronuclear microinjection (Paterson et al., App. Microbiol. Biotechnol, 40: 69).
1-698 (1994); Caver et al. Biotechnology (NY): 11 1263-1270 (19903); Wri
Biotechnology (NY) 9: 830-834 by ght et al. and US Pat. No. 4,873,191 by Hoppe et al .; Germline (Proc. Natl. Avad. Sci., by Van der Putten et al.
USA, 82; 6148-6152 (1985)), retrovirus-mediated gene transfer to pulmonary blastocysts or embryos; gene targets in lung stem cells (Thompson et al. Cell 56: 3).
13-321 (1989)); Electroporation of cells or lungs (Mol. Cell by LO.
Biol. 3: 1803-1814 (1983)); introduction of the polynucleotide of the present invention using a gene gun (eg, Ulmer et al., Science 259: 1745 (1993)); introduction of nucleic acid construct into lung multifunctional stem cells. And repopulating the blastocyst with the stem cell; and sperm-mediated gene transfer (see Lavitrano et al., Cell 57: 717-723 (1989)). See Gordon for a review of those technologies.
Transgenic Animals, "Intl. Rev. Cytol. 115; 171-229 (1989), which forms a part of this specification. Further, each document cited in this paragraph forms part of this specification. US Pat. No. 5,631,764 (Positive-Ne by Capecchi et al.
gative Selection Methods and Vectors); U.S. Pat.No. 5,464,764 (Cells and Non-Human Organisms Containing Predertemined Genomic Mod by Capecchi et al.
ifications and Positive-Negative-Selection Methoudas and Vectors for Mak
U.S. Pat. No. 4,736,191 (Wagner et al., Genetic Transformation)
of Zygotes), each of which constitutes a part of this specification).

Any technique known in the art can be used to obtain transgenic clones containing a polynucleotide of the invention, such as, for example, rest-induced culture. Nuclear transfer from embryos, fetuses or adult cells to the enucleated oocyte (Campell et al., Nature, 380; 64-66 (1
996) and Wilmut et al., Nature 385: 810-813 (1997), each of which is incorporated herein by reference).

The present invention provides transgenic animals carrying the transgene of all of these cells, as well as animals carrying the transgene to some, but not all, of the cells (
That is, a mosaic animal or a chimeric animal) is provided. The transgene can be incorporated as a single transgene or as multiple copies such as conatamers (eg, head-to-head and head-to-tail tandems). The transgene is described, for example, by the teaching of Lasko et al.
Sture, Proc. Natl. Acad. Sci. USA 89: 6232-6236 (1992)), and can be selectively introduced into and activated in a certain cell type. The control sequences required for activation specific to those cell types will depend on the cell type of interest and will be apparent to those of skill in the art. When it is desired to introduce the polynucleotide transgene into the chromosomal site of the endogenous gene, target gene introduction is preferred. Briefly, when using these techniques, a vector containing several nucleotide sequences homologous to the endogenous gene is integrated into the nucleotide sequence of the endogenous gene by homologous recombination with the chromosomal sequence, and Design so as not to destroy the function of the nucleotide sequence. The transgene is selectively introduced into a cell type, for example according to the teaching of Gu et al. (Gu et al., Science 265: 103-106 (1994)), thus inactivating an endogenous gene in only one such cell type. You can also do it. The control sequences required for inactivation specific to those cell types depend on the cell type of interest, which will be apparent to those of skill in the art. All references cited in this paragraph form part of the present specification.

After obtaining transgenic animals, recombinant gene expression can be assayed using standard methods. Initial screening can be accomplished using Southern blot analysis or PCR methods and animal tissue is analyzed to demonstrate that transgene integration has occurred. The mRNA expression level of the transgene in the tissues of transgenic animals was determined by methods such as Northern blot analysis of tissue samples obtained from the animals, in situ hybridization and reverse transcriptase-PCR (rt-PCR). (Including but not limited to) can also be used. Tissue samples expressing transgenic genes can also be evaluated immunocytochemically or immunohistochemically using antibodies specific for the transgene product.

After generating the founder animals, they can be bred, inbred, outbred, or hybridized to obtain colonies of the animals. Examples of these breeding methods include outcrossing founder animals with one or more integration sites to establish separate lines; transgenes at high levels due to the effect of separate expression of each transgene. Breeding a deparate line to obtain a expressing compound transgene; heterozygous transgenic to increase expression and eliminate the need to screen animals by DNA analysis. Hybridizing animals to obtain animals that are heterozygous for a certain integration site; and hybridizing separate heterozygous lines to obtain complex, heterozygous or homozygous lines; and breeding Including but not limited to placing the transgene on a different background suitable for the experimental model of interest. No.

The transgenic, “knockout” animals of the invention have increased biological function of the DR3-V1 or DR3 polypeptide and are associated with aberrant DR3-V1 or D3.
Uses including but not limited to animal model systems useful for studying diseases and / or illnesses associated with R3 expression and for screening compounds effective in alleviating those diseases and / or disorders Have.

A further embodiment of the invention is a cell that has been genetically engineered to express a protein of the invention or genetically engineered not to express a protein of the invention (eg, a knockout). Is administered to a patient in vivo. The cells can be obtained from a patient (ie, an animal, including a human) or an MHC-compatible donor, and as such are fibroblasts, bone marrow cells, blood cells (eg lymphocytes), adipocytes, muscle cells, endothelium. Including but not limited to cells. The cells may be genetically engineered in vitro using recombinant DNA methods to introduce the coding sequences of the polypeptides of the invention into the cells, or for example, transduction methods (using viral vectors, but not cell genomes). Vectors incorporating a transgene therein are preferred) or transfection methods (plasmids, cosmids, YACs, naked DNs).
A, including, but not limited to, the use of electroporation, liposomes, etc.) disrupts the coding sequence and / or the endogenous regulatory sequences associated with the polypeptides of the invention. The coding sequences of the polypeptides of the invention may be placed under strong constitutive or inducible promoter or promoter / enhancer control conditions to obtain expression (preferably secretion) of the polypeptides of the invention. Express (preferably secreted) a polypeptide of the invention
It is preferred to introduce the engineered cells systemically (eg, circulatory or peritoneally) into the patient. Alternatively, the cells can be introduced into a matrix or implanted in the body, for example genetically engineered fibroblasts can be implanted as part of a skin graft or genetically engineered. Endothelial cells can be implanted as part of a lymphatic or vascular graft (
See, eg, Anderson et al., US Pat. No. 5,399,349, and Mulligan & Wilson, US Pat. No. 5,460,959, each of which is incorporated herein by reference).

If the cells to be administered are non-autologous or non-MHC compatible cells, they may be administered using well known methods that prevent the development of a host immune response against transduced cells. it can. For example, the cells can be introduced in capsule form. However, although the components may be exchanged with the extraneous environment, the introduced cells may not be recognized by the host immune system.

Therapeutic Agents The tumor necrosis factor (TNF) family of ligands are the most multifunctional inducing multiple cellular responses including cytotoxicity, antiviral activity, immunomodulatory activity and transcriptional regulation of several genes. It is known to be among various cytokines (D.V.G.
oddel, et al., “Tumor Necrosis Factor: Gene Structure and Biological Activity (Tumo
r ・ Necrosis ・ Factors: Gene ・ Structure ・ an
d. Biological.Activities) ", Symp. Quant
． Biol. 51: 597-609 (1986), Cold Spri.
ng Harbor; B. Beutler and A. Cerami, Annu. Re
v. Biochem. 57: 505-518 (1988); J. O
ld, Sci. Am. 258: 59-75 (1988); Fier
s, FEBS Lett. 285: 199-224 (1991)). This TNF family ligand induces a variety of cellular responses by binding to TNF family receptors (receptors) including DR3-V1 or DR3 of the present invention.

Expresses a DR3-V1 or DR3 polypeptide and binds to a DR3-V1 or DR3
Cells that are thought to have a strong cellular response to the ligand include lymphocytes, fibroblasts, macrophages, joint cells, activated T cells, lymphoblasts and endothelial cells. "Cellular response to a TNF family ligand" means any genotypic, phenotypic and / or morphological response to a TNF family ligand-induced cell, cell line, tissue, tissue culture or patient. Intentional change. As pointed out, this cellular response includes not only the normal physiological response to TNF family ligands, but also diseases associated with increased apoptosis or inhibition of apoptosis. Apoptosis-programmed cell death-is a peripheral T of the immune system.
A physiologic mechanism involved in lymphocyte depletion, whose dysregulation can lead to a wide variety of etiological processes (JC Ameisen, AIDS, 8: 11971-1.
213 (1994); H. Krammer et al., Curr. Opin.
Immunol. 6: 279-289 (1994)).

DR3-V1 or DR3 polynucleotides, polypeptides, agonists or antagonists of the invention are for treatment and diagnosis / prognosis of any disorder mediated by a defective or inadequate amount of DR3 (directly or indirectly). It can be used for assay development. The DR3-V1 or DR3 polypeptide, agonist or antagonist can be administered to a patient (eg, a mammal, preferably a human) suffering from such a disorder. Alternatively, gene therapy approaches can be applied to the treatment and / or prevention of such disorders. The disclosure of the DR3-V1 or DR3 nucleotide sequences herein allows the detection of defective DR3 genes and their replacement with genes encoding normal DR3. Defective genes are detected in in vitro diagnostic assays and by comparing the DR3-V1 or DR3 nucleotide sequences disclosed herein with sequences of the DR3 gene from patients suspected of having a deficiency in this gene. be able to.

Diseases associated with increased cell survival or inhibition of apoptosis include cancers (eg, follicular lymphoma, carcinomas with mutations of p53, and hormone-dependent such as breast cancer, prostate cancer, Kaposi's sarcoma, and ovarian cancer). Such as tumors), autoimmune diseases (eg multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, cirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemic lupus erythematosus, immune-related glomerulonephritis and Such as rheumatoid arthritis) and viral infections (such as herpes virus, poxvirus and adenovirus), information graft-versus-host disease, acute graft rejection, and chronic graft rejection. Diseases associated with increased apoptosis include AIDS, neurodegenerative diseases (such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, retinitis pigmentosa, cerebellar degeneration), myelodysplastic syndromes (dysplastic). Idiopathic anemia), ischemic injury (such as that due to myocardial infarction, stroke and reperfusion injury), toxin-induced liver disease (such as due to alcohol), septic shock, aggravation Includes fluid quality and anorexia.

Further diseases or conditions associated with increased cell survival include malignant progression and / or metastasis and related diseases such as leukemia (acute leukemia (eg acute lymphocytic leukemia, acute myelocytic leukemia (myeloblast). , Promyelocytic, myelomonocytic, monocytic and erythroleukemia)) and chronic leukemia (eg chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia)), true Polycythemia, lymphoma (eg Hodgkin and non-Hodgkin's disease) multiple myeloma, Waldenstrom macroglobulinemia, heavy chain disease, and sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenesis Sarcoma, chordoma, angiosarcoma, endotheliosarcoma
), Lymphangiosarcoma, lymphatic endothelium sarcoma (lymphangioendot)
heliosarcoma), synovial tumor (synovioma), mesothelioma, Ewing's tumor
), Leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous adenocarcinoma, papillary carcinoma, papillary adenocarcinoma (papillary ad
enocarcinomas), cystadenocarcinoma, medullary carcinoma, bronchogenic cancer (b
ronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm tumor, cervical cancer, testicular tumor
tumor), lung cancer, small cell lung carcinoma, bladder cancer, epithelial cancer,
Glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, inner ear neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma And solid tumors including, but not limited to, retinoblastoma.

Diseases associated with increased apoptosis include AIDS; neurodegenerative disorders (eg Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, retinitis pigmentosa, cerebellar degeneration and brain tumors or previous related disorders); self Immune disorders (eg, multiple sclerosis, Sjogren's syndrome, Graves' disease, Hashimoto's thyroiditis, autoimmune diabetes, cirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemic lupus erythematosus, immune-related glomerulonephritis, Autoimmune gastritis, thrombocytopenic purpura and rheumatoid arthritis), myelodysplastic syndrome (eg aplastic anemia), graft-versus-host disease (acute and / or chronic), ischemic injury (eg myocardial infarction, stroke and (Caused by reperfusion injury)
, Liver damage or disease (eg liver damage associated with hepatitis, cirrhosis, ischemic / reperfusion injury, cholestosis (bile duct damage) and liver cancer); toxin-induced liver disease (eg due to alcohol) , Septic shock, ulcerative colitis, cachexia and eating disorders. In a preferred embodiment, DR3 polynucleotides, polypeptides, agonists and / or antagonists are used to treat, prevent, diagnose, and / or prognose the diseases and disorders listed above.

Thus, in one aspect, the invention provides a DR3-V1 or DR3 ligand, an analogue thereof, which is capable of enhancing DR3-V1 or DR3-mediated signaling in cells expressing a DR3-V1 or DR3 polypeptide. Alternatively, the present invention relates to a method for enhancing apoptosis induced by a TNF family ligand, which comprises administering an effective amount of an agonist (agonist). Preferably, DR3-V1 or DR3-mediated signaling is enhanced to treat and / or prevent diseases in which decreased apoptosis or decreased cytokines and adhesion molecule expression are observed. DR3 for agonist
-Soluble forms of V1 or DR3 and monoclonal antibodies against DR3-V1 or DR3 polypeptides may be included.

In yet another aspect, the invention administers to a cell expressing a DR3-V1 or DR3 polypeptide an effective amount of an antagonist capable of reducing DR3-V1 or DR3-mediated signaling. Comprising inhibiting TNF family ligand-induced apoptosis. Preferably DR3
-Reduce Vl or DR3-mediated signaling and treat and / or prevent diseases that exhibit enhanced apoptosis or expression of NF-kB. DR for antagonist
Soluble forms of 3-V1 or DR3 and monoclonal antibodies to DR3-V1 or DR3 polypeptides can be included.

In another aspect, the DR3-V1-Fc and DR3-Fc proteins and D
Soluble proteins in the extracellular domain of R3-V1 and DR3 proteins are useful for stimulating neovascularization and angiogenesis. Thus, these polypeptides are, for example, diseases and conditions associated with hypoangiogenesis (eg Turner syndrome, cardiovascular aging).
, Bronchial stenosis, depression) and / or prophylaxis.

Specifically included within the scope of the invention are DR3-V1-Fc and DR3.
-Fc protein receptors / Fc fusion proteins, and nucleic acid molecules encoding these proteins. These fusion proteins include those having the amino acid sequence of the extracellular domain of the DR3 protein of the present invention. Examples of portions of the DR3 extracellular domain that are useful in making DR3 receptor / Fc fusion proteins include amino acids 1-199 of SEQ ID NO: 4 and amino acids 1-210 of SEQ ID NO: 2,
37-210, 50-210 and 100-210 are included.

In addition, distress that may be treated and / or prevented by DR3-V1 and DR3-mediated stimulation of angiogenesis includes soft tissue trauma (eg cuts and contusions), ulcers (eg peptic, cutaneous and venous). And scleroderma.

“Agonist” means naturally occurring and synthetic compounds capable of enhancing or enhancing apoptosis. “Antagonist” means naturally occurring and synthetic compounds capable of inhibiting apoptosis. Whether any "agonist" or "antagonist" candidate of the present invention can enhance or inhibit apoptosis is known in the art, including those described in further detail below.
It can be determined by using the NF family ligand / receptor cell response assay.

One such screening procedure involves the use of melanophores that have been transfected to express the receptors of the present invention. This screening technique is described in PCT WO92 / 01810, published February 6,1992. Such assays include, for example, contacting a receptor-encoding melanophore cell with both a TNF family ligand and a candidate antagonist (or agonist) to form a receptor polypeptide of the invention. It can be employed to screen for compounds that inhibit (or enhance) activation. 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, for example, Science, 246: 181-.
296 (October 1989), including the use of receptor-expressing cells (eg, transgenic CHO cells) in a system to measure extracellular pH changes caused by receptor activation. For example, a compound is contacted with a cell expressing a receptor polypeptide of the invention and a second messenger response, such as signal transduction or pH change, is measured and the potential compound activates or inhibits its receptor. You may decide whether to do it.

Another such screening technique involves introducing receptor-encoding RNA into Xenopus orcites to transiently express the receptor. This receptor aussite is then contacted with a receptor ligand and the compound to be searched for, followed by inhibition or activation of calcium signals in the case of screening for compounds that appear to inhibit activation of that receptor. To detect.

[0295] Another screening technique involves expressing in cells a construct in which the receptor is linked to phospholipase C or D. Such cells include endothelial cells, smooth muscle cells, embryonic kidney cells and the like. This screen can be accomplished by detecting activation of the receptor or inhibition of receptor activation from the phospholipase signal, as described herein above.

Another method is for compounds which inhibit activation of the receptor polypeptide of the antagonists of the invention by measuring the inhibition of binding of labeled ligand to cells bearing the receptor on the surface. Including screening methods. Such methods include transfecting a eukaryotic cell with DNA encoding the receptor, allowing the cell to express the receptor on its surface, and compounding the cell in the presence of a labeled form of a known ligand. Including contact with. The ligand can be labeled, for example, by radioactivity. The amount of labeled ligand bound to the receptor is measured, for example, by measuring the radioactivity of the receptor. If the compound binds to the receptor, binding of the labeled ligand to the receptor is inhibited, as measured by the decrease in labeled ligand that binds to the receptor.

Yet another screening assay for agonists and antagonists of the invention is described in L. et al. A.
Tartaglia and D.M. V. Goeddel, J .; Biol. Chem. Two
67 [7]: 4304-4307 (1992).

Thus, in yet another aspect, there is provided a screening method to determine whether an agonist or antagonist candidate can enhance or prevent a cellular response to a TNF family ligand. The method comprises contacting cells expressing a 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 is assayed by contacting it with a ligand in the absence of the candidate compound, whereby an increase in cellular response relative to the standard indicates that the candidate compound is an agonist of the ligand / receptor signaling pathway, Candidate compound is ligand /
It is shown to be an antagonist of the receptor signaling pathway. "Assaying a cellular response" refers to qualitatively or by measuring a cellular response to a candidate compound and / or TNF family ligand (eg, measuring or assessing an increase or decrease in T cell proliferation or tritylated thymidine label). It means to measure quantitatively. According to the present invention, cells expressing a DR3-V1 or DR3 polypeptide can be contacted with an endogenous or exogenously administered TNF family ligand.

Agonists according to the invention include, for example, peptide fragments of TNF family ligands, transforming growth factor β, neurotransmitters (such as glutamate, dopamine, N-methyl-D-aspartate), tumor suppressors ( p53), cytolytic T cells and naturally occurring and synthetic compounds such as antimetabolites. Suitable agonists include chemotherapeutic agents such as cisplatin, doxorubicin, bleomycin, cytosine arabinoside, nitrogen mustard, methotrexate and vincristine. Others include ethanol and β-
Amyloid peptides are included (Science, 267: 1457-145).
Page 8 (1995)). Further suitable agonists include polyclonal and monoclonal antibodies raised against DR3-V1 or DR3 polypeptides, or fragments thereof. Such agonist antibodies raised against the TNF family of receptors are described in L. et al.
A. Tartaglia et al., Proc. Natl. Acad. Sci. USA
88: 9292-9296 (1991) and L.S. A. Tartagl
ia and D.I. V. Goeddel, supra. In addition, PCT application W
See also O94 / 09137.

Antagonists according to the invention include, for example, CD40 ligand, neutral amino acids, zinc, estrogens, androgens, viral genes (eg adenovirus ElB,
Baculovirus p35 and IAP, cowpox virus crmA, Epstein-
Burr virus BHRF1, LMP-1, African swine fever virus LMW5-H
L and herpes virus yl34.5), calpain inhibitors, cysteine protease inhibitors, and tumor promoters (such as PMA, phenobarbital and α-hexachlorocyclohexane) and naturally occurring and synthetic compounds.

Other potential antagonists include antisense molecules. Thus, in a particular embodiment, an antagonist according to the invention is deposited with the nucleic acid corresponding to the sequence contained in DR3-V1 or DR3 or its complementary strand, and / or ATCC Deposit Nos. 97456 and 97757. It is a nucleic acid corresponding to the nucleotide sequence contained in the cDNA. In one aspect, the antisense sequence is
Internally produced by the organism, in another embodiment, the antisense sequence is separately administered (eg, O'Connor, J., Neurochem. 56: 560 (1991) and Oligodeoxynuc).
leotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Rat
on, FL (1988)). Antisense technology can be used to regulate gene expression by antisense DNA or RNA or through triple helix formation. Antisense technology is described, for example, in Okano, J., Neurochem. 56: 560 (1
991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression,
CRC Press, Boca Raton, FL (1988). Triple helix formation is described in, for example, Lee et al., Nucleic Acids Reaearch 6: 3073 (1979); Cooney.
et al., Science 241: 456 (1988); and Dervan et al., Science., Science.
251: 1300 (1991). This method uses complementary DNA or R
It is based on the binding of the polynucleotide to NA.

For example, the 5'coding portion of the polynucleotide encoding the mature polypeptide of the invention can be used to design an antisense RNA oligonucleotide of about 10-40 base pairs in length. DNA oligonucleotides are designed to be complementary to regions of the gene involved in transcription, thereby interfering with transcription and receptor production. Antisense RNA oligonucleotides have in vivo mR
It hybridizes to NA and blocks the translation of mRNA molecules into receptor polypeptides.

In one aspect, the DR3-V1 or DR3 antisense nucleic acids of the invention are produced intracellularly by transcription from an exogenous sequence. For example, the vector or part thereof is transcribed to produce the antisense nucleic acid (RNA) of the invention. Such a vector would include sequences encoding DR3-V1 or DR3 antisense nucleic acids. Such a vector can be transcribed to produce the desired antisense RN.
As long as it can produce A, it can remain episomal or become chromosomally integrated. Such a vector can be constructed by a recombinant DNA technology state of the art. Vectors may be plasmids, viral, or others known in the art used for replication and expression in vertebrate cells. Expression of the DR3-V1 or DR3 coding sequence, or a fragment thereof, can be by any promoter known in the art to operate in vertebrate, preferably human cells. Such promoters may be inducible or constitutive. Such promoters include the SV40 early promoter region (Bernoist and Chambon, Nature 29: 304-310 (1981)), the promoter contained in the 3'long terminal repeat of Rous sarcoma virus (Yamamoto et al., Ce.
ll 22: 787-797 (1980)), herpes thymidine promoter (Wagner et al., Proc
Natl. Acad. Sci. USA 78: 1441-1445 (1981)), metallothionein regulatory sequences (Brinster et al., Natere 296: 39-42 (1982)), and the like, but are not limited thereto.

The antisense nucleic acids of the invention comprise or consist of sequences complementary to at least a portion of the RNA transcript of the DR3 gene. However, perfect complementarity is preferred but not required. The sequence “complementary to at least part of RNA” described herein means a sequence having sufficient complementarity to hybridize with this RNA and forming a stable duplex; Chain DR3-V1 or D
In the case of R3 antisense nucleic acids, single strands of double DNA can be tested or triplex formation can be assayed. The ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid. Generally, the longer the hybridizing nucleic acid, the more base mismatches with the DR3-V1 or DR3 RNA may be contained and still be able to form stable duplexes (and optionally triplexes). One of ordinary skill in the art can determine the tolerance for mismatches by measuring the melting point of the hybridized complex using standard techniques.

Oligonucleotides complementary to the 5'end of the message, eg, from the 5'untranslated sequence to the AUG initiation codon, inclusive, should work most effectively in inhibiting translation. However, it was shown that a sequence complementary to the 3'untranslated sequence of mRNA was similarly effective in inhibiting translation of mRNA. Generally, Wagner, R.,
See 1994, Nature 372: 333-335. Therefore, SEQ ID NO: 2 and SEQ ID NO: 4
Oligonucleotides complementary to either the 5'- or 3'-untranslated, non-coding regions of DR3-V1 or DR3 shown in Figure 3 are used in the antisense approach to direct translation of endogenous DR3-V1 or DR3 mRNA Can be inhibited. The oligonucleotide complementary to the 5'untranslated region of this mRNA is
It should include the complement of the AUG start codon. Antisense oligonucleotides complementary to the mRNA coding region are less effective translation inhibitors,
It can be used according to the invention. When designed to hybridize to the 5'-, 3'- or coding region of DR3-V1 or DR3 mRNA, the antisense nucleic acid comprises an oligonucleotide ranging in length from at least 6 nucleotides, preferably from 6 to about 50 nucleotides. Should be In specific terms,
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 regard, "about" means in particular the stated value and a number (5, 4, 3,
2 or 1) Includes values greater or lesser nucleotides.

The polynucleotides of the present invention may be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof (single-stranded or double-stranded). The oligonucleotide can be modified at the base moiety, sugar moiety or phosphate backbone to improve eg molecular stability, hybridization etc. This oligonucleotide includes other additional groups such as peptides (eg for targeting host cell receptors in vivo), or cell membranes (eg Letsinger et al.
al., Proc Natl. Acad. Sci. USA 86: 6553-6556 (1989); Lemaitre et al.,
Proc. Natl Acad. Sci. 84: 648-652 (1987); PC published on December 15, 1988.
T Publication No. WO88 / 09810) or the blood-brain barrier (eg, April 2, 1988).
Substances that facilitate transport through PCT Publication No. WO 89/10134 published on the 5th, degrading substances due to hybridization (eg, Krol et al., Bio
Techniques 6: 958-976 (1988)) or intercalating substances (eg Zon, Pha
rm. Res. 5: 539-549 (1988)). To this end, the oligonucleotide can be conjugated to another molecule, such as a peptide, a cross-linking agent due to hybridization and a transport agent, a degradation agent due to hybridization, and the like.

Antisense oligonucleotides include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyhydroxylmethyl) uracil, 5
-Carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosyl qu eosin, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosin, 2,2 -Dimethylguanine, 2-methyladenine, 2-methylguanine,
3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D-mannosylqueosin, 5-methoxycarboxymethyluracil 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine,
Uracil-5-oxyacetic acid (v), wybutoxosine (wybutoxosine), pseudouracil, que eosin (queosine), 2-thiocytosine, 5-methyl-2-
Thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 5
-Methyl-2-thiouracil, 3- (3-amino-3-N-2-carboxypropyl) uracil, (acp3) W and at least selected from the group including but not limited to 2,6-diaminopurine One modified base moiety may be included.

Antisense oligonucleotides may also include at least one modified sugar moiety selected from the group including, but not limited to, arabinose, 2-fluoroarabinose, xylulose and hexose.

In yet another embodiment, the antisense oligonucleotides are phosphorothioates, phosphorodithioates, phosphoramidothioates, phosphoramidates, phosphordiamidates, methylphosphonates, alkylphosphotriesters and formacetals. Or at least one modified phosphate backbone selected from the group including, but not limited to, analogs thereof.

In yet another aspect, the antisense oligonucleotide is an α-anomeric oligonucleotide. The α-anomeric oligonucleotides form specific double-stranded hybrids with complementary RNA, where contrary to the normal β-unit,
The chains run parallel to each other (Gautier et al., Nucl. Acids Res. 15: 6625-664.
1 (1987)). This oligonucleotide is a 2-0-methyl ribonucleotide (Inoue
et al., Nucl. Acids Res. 15: 6131-6148 (1987)) or chimeric RNA-DNA
It is an analog (Inoue et al., FEBS Lett. 215: 327-330 (1987)).

Polynucleotides of the invention may be prepared using standard methods known in the art, eg automated DNA.
It can be synthesized by using a synthesizer (for example, those commercially available from Biosearch, Applied Biosystems). As an example, phosphorothioate oligonucleotides are described by Stein et al., (Nucl. Acids Res. 16: 3209 (1988).
Can be synthesized according to the method described in US Pat.
Sci. USA 85: 7448-7451 (1988)).

Although antisense nucleotides complementary to the DR3-V1 or DR3 coding region sequences can be used, those complementary to the transcribed untranslated region are most preferred.

Potential antagonists of the invention also include catalytic RNA, or ribozymes (eg, PCT International Publication WO 90/11364, published October 4, 1990; Sarv.
er et al., Science 247: 1222-1225 (1990)). Using a ribozyme that decomposes mRNA at a specific recognition sequence site, DR3-V1 or DR3m
Although RNA can be destroyed, the use of hammerhead ribozymes is preferred. Hammerhead ribozymes cleave mRNAs at positions defined by flanking regions that form complementary base pairs with the target mRNA. The only requirement is the target mRNA
Has the following two base sequence: 5'-UG-3 '. The construction and production of hammerhead ribozymes is well known in the art and can be found in Haseloff and Gerlach, Nature
334: 585-591 (1988). DR3-V1 (SEQ ID NO: 2
) Or DR3 (SEQ ID NO: 4) within the nucleotide sequence, there are a number of potential hammerhead ribozyme cleavage sites. Preferably, the ribozyme is engineered so that its degradation recognition site is located near the 5'end of the DR3-V1 or DR3 mRNA; Minimize accumulation.

As with the antisense approach, the ribozymes of the invention can be composed of modified oligonucleotides (eg, for improved stability, targeting, etc.) that are DR3-V1 or in vivo. Delivered to cells expressing DR3. The DNA construct encoding the ribozyme is introduced into cells in the same manner as above, and the antisense-encoding DNA is introduced. A preferred method of delivery uses a DNA construct that "encodes" a ribozyme under the control of a strong constitutive promoter, such as the pol III or pol II promoter,
It is included so that the transfected cells disrupt the endogenous DR3-V1 or DR3 message and produce sufficient amounts of ribozymes to inhibit translation. Unlike antisense molecules, ribozymes are catalytic and require lower intracellular concentrations for efficacy.

Endogenous gene expression has also been demonstrated using targeted homologous recombination, DR3 gene and / or
Alternatively, it can be reduced by inactivating or “knocking out” its promoter (eg Smithies et al., Nature 317: 230-23).
4 (1985); Thomas & Capecchi, Cell 51: 503-512 (1987); Thompson et al., Cell
5: 313-321 (1989); each of which is incorporated herein by reference in its entirety). For example, a mutant, non-functional polynucleotide (or completely unrelated DNA sequence) of the invention flanked by DNA homologous to an endogenous polynucleotide sequence (coding or regulatory region of the gene). ),
Cells with or without a selectable marker and / or a negative selectable marker can be used to transfect cells expressing a polypeptide of the invention in vivo. In another embodiment, techniques known in the art are used to create knockouts in cells that contain but do not express the gene of interest. Insertion of the DNA construct via targeted homologous recombination inactivates the targeted gene. Such an approach is particularly suitable in the field of research and agriculture, where modifications to embryonic stem cells can be used to generate offspring of animals with inactivated targeted genes (eg Thomas & Capecchi 1987 and Thompson. 1989, see above). However, this approach is used in humans if the recombinant DNA construct is administered directly to the required site in vivo using a suitable viral vector, which is apparent to those skilled in the art, or is targeted. Can be mechanically adapted to. The contents of each of the documents cited in this paragraph are incorporated herein by reference in their entirety.

In another aspect, antagonists of the invention include soluble forms of DR3-V1 or DR3 (eg, DR3-V1 set forth in SEQ ID NO: 2 and fragments of DR3 set forth in SEQ ID NO: 4) ( These include the ligand binding domain from the extracellular region of the full length receptor). Such soluble forms, either naturally occurring or synthetic, of DR3-V1 or DR3 antagonize cell surface bound receptors for binding to TNF family ligands, and DR3
Antagonize V1- or DR3-mediated signaling. The antagonists of the invention also include antibodies specific for TNF family ligands and DR3
Both -V1-Fc and DR3-Fc fusion proteins are included.

“TNF family (family) ligands” refers to naturally occurring ligands, recombinant ligands and recombinant ligands that have the ability to bind to members of the TNF receptor family and induce and / or block ligand / receptor signaling pathways. It means a synthetic ligand. Members of the TNF ligand family include TNF-α
, Lymphotoxin-α (known as LT-α, or TNF-β), L
T-β (found in the complex heterotrimer LT-α2-β), FasL, T
NF-γ (International Publication WO96 / 14328), TNF-γ-α, TNF-γ-β
(International Publication WO00 / 08139), AIM-I (International Publication WO97 / 3389)
9), AIM-II (International Publication WO97 / 34911), APRIL (J. Exp.
Med. 188 (6): 1185-1190), endokine-α (international publication WO9
8/07880), Neurokine-α (International Publication WO98 / 18921), C
D40L, CD27L, CD30L, 4-1BBL, OX40L and nerve growth factor (NGF) are included, but are not limited to.

The antibodies of the invention can be produced by any of a variety of standard methods using the DR3-V1 or DR3 receptor immunogens of the invention. DR like this
3-V1 or DR3 receptor immunogens include DR3-V shown in SEQ ID NO: 2.
1 protein and the DR3 protein shown in SEQ ID NO: 4 (each of which may or may not contain a leader sequence) and a polypeptide fragment of the receptor, which is a ligand of DR3-V1 or DR3 Those that include or consist of binding domains, extracellular domains, transmembrane domains, intracellular domains, or any combination thereof.

Polyclonal and monoclonal antibody agonists or antagonists of the invention may be prepared according to the methods disclosed herein and / or known in the art, eg, Tartaglia and Goeddel, J. Biol. Chem. 267 (7). : 4304-4307 (1992); T
artaglia et al., Cell 73: 213-216 (1993) and PCT application WO94 / 091.
37 (the contents of each of these three applications are hereby incorporated by reference in their entirety), which are of the invention having the amino acid sequence of SEQ ID NO: 2 or 4. It is preferably specific for the polypeptide.

Further included in the antagonists of the invention are soluble forms of DR3-V1 or DR3.
, Ie, DR3-V1 or DR3 fragments, which contain the ligand binding domain from the extracellular region of the full length receptor. Such soluble forms of the receptor, which may be naturally-occurring or synthetic, include T
Cell surface DR3-V1 or DR3 for binding to NF-family ligands
Antagonizes DR3-V1 or DR3-mediated signaling by antagonizing Therefore, the soluble form of the receptor containing this ligand binding domain is a novel cytokine with the ability to inhibit apoptosis induced by TNF-family ligands. These are preferably expressed as dimers or trimers. This is because they have been shown to be superior as antagonists over monomeric forms of soluble receptors such as IgG-Fc-TNF receptor family fusions. Other such cytokines are known in the art and include Fas B, a soluble form of the murine Fas receptor, which acts physiologically and limits Fas ligand-induced apoptosis. Hughes and IN Crispe, J. Exp. Med. 182: 1395
-1401 (1995)).

The experiments described in Examples 6 and 7 show that the death domain in which DR3 can trigger both apoptosis and NF-kB activation, two pathways that are predominant in the control of the immune system. ) Indicates a contained molecule. This experiment also demonstrates the internal signaling mechanism of this novel death receptor. The experiments described below further demonstrate that DR3-induced apoptosis is associated with ICE-like protease, CrmA.
And blocked by inhibitors of z-VAD-fmk. Importantly, DR3-induced apoptosis was also shown to predominantly negative versions of FADD (FADD-DN) or FLICE previously shown to inhibit death signaling by Fas / APO-1 and TNFR-1. (F
LICE-DN / MACHa1C360S). Therefore, inhibitors of ICE-like proteases, FADD-DN and FLICE-DN /
MACHa1C360S can also be used as an antagonist of DR3 activity.

As used herein, the term “antibody” (Ab) or “monoclonal antibody” (mAb) refers to an intact molecule and fragments thereof (eg, Fab and F (ab ′) ₂ fragments) that are capable of binding antigen. ) Is meant. Fab and F
The (ab ′) ₂ fragment lacks the Fc fragment found in intact antibodies and disappears rapidly from the circulation and appears to have less nonspecific tissue binding than intact antibodies (Wahl et al., J. Nucl. Med., 24: 316-325 (1983)).

Antibodies of the invention may be produced by any of a variety of methods using the DR3-V1 or DR3 immunogens of the invention. As mentioned above, this DR3-V1 or DR3
Immunogens include full-length DR3-V1 or DR3 polypeptides (with or without a leader sequence) and DR3-V1 or DR3, eg, ligand binding domains, transmembrane domains, intracellular domains and death domains. Polypeptide fragments are included.

Proteins and other compounds that bind to the DR3-V1 or DR3 domain are also candidate agonists and antagonists of the invention. Such binding compounds are
It can be "captured" using the yeast two-hybrid system (Fields and Song, Nature, 340: 245-246 (1989)). A modified two-hybrid system of yeast has been described by Roger Brent and co-workers (J. Gyuris et al., Cell 75: 791-803 (
1993); S. Zervos et al., Cell, Volume 72: 223-232.
Page (1993)). Preferably, the yeast two-hybrid system is used according to the invention to capture compounds that bind to the DR3-V1 or DR3 ligand binding domain or the DR3-V1 or DR3 intracellular domain. Such compounds are good candidates as agonists and antagonists of the invention.

A “TNF family ligand” binds a member of the TNF receptor family,
By naturally occurring, recombinant, and synthetic ligands capable of inducing ligand / receptor signaling pathways. Members of the TNF ligand family include, but are not limited to, DR3-V1 or DR3 ligands, TNF-α, lymphotoxin-α (also known as LT-α, TNF-β), (international Public WO9
6/14328), TNF-γ-α (PCT Publication WO00 / 08139), TN
F-γ-β (PCT Publication WO00 / 08139), LT-β (found in the complex heterotrimer LT-α2-β), FasL, CD40, CD27, CD3.
0,4-1BB, OX-40 and neurotrophic factor (NGF).

Representative therapeutic applications of the present invention are discussed in further detail below. The immunodeficient determinants of AIDS are dependent on a decrease in the number and function of CD4 ⁺ T lymphocytes.
Recent reports speculate that the number of CD4 ⁺ T cell depletion is between 3.5 × 10 ⁷ and 2 × 10 ⁹ daily (X. Wei et al., Nature, 373: 117-12).
2 (1995)). One of the reasons for CD4 ⁺ T cell depletion in the setting of HIV infection is believed to be HIV-induced apoptosis. In fact, HI
Cell death by V-induced apoptosis has been demonstrated not only in vitro but, more importantly, in infected individuals (JC Ameisen, AIDS, 8).
Volume: 1197-1213 (1994); H. Finkel and N.N. K. B
anda, Curr. Opin. Immunol. , Volume 6: 605-615 (
1995); C.I. A. Muro-Cacho et al. Immunol. 1
54: 5555-5566 (1995)). Furthermore, apoptosis and CD4 ⁺ T-lymphopenia are strongly correlated in various animal models of AIDS (T. Bruner et al., Nature, 373: 441-444 (1).
995); L. Gougeon et al., AIDS Res. Hum. Ret
Roviruses, 9: 553-563 (1993)), and apoptosis has not been observed in animal models in which viral replication does not lead to AIDS, Id. Further data point out that uninfected but sensitized or activated T lymphocytes obtained from HIV infected individuals undergo apoptosis after encountering the TNF family ligand FasL. It has been demonstrated that HIV infection of U937 cells leads to de novo expression of FasL, which mediates HIV-induced apoptosis, using a monocyte cell line that leads to death after HIV infection (AD. Badle
y., J. Virol. 70: 199-206 (1996)). Furthermore, a TNF family ligand can be detected in uninfected macrophages and its expression is elevated after HIV infection to selectively kill uninfected CD4 T lymphocytes, Id. Thus, the present invention provides a method of treating an HIV ⁺ individual that comprises administering an antagonist of the present invention to reduce the selective killing of CD4 T lymphocytes. The administration form and dose will be described in detail below.

In allograft rejection, in most cases the immune system is primed with environmental antigens, but the recipient animal's immune system is not primed to respond. . Tissues from another individual of the same species are not presented as much as, for example, viruses and bacteria are presented. In the case of allograft rejection, immunosuppressive treatments are designed to prevent the immune system from reaching the effector stage. However,
The immune profile of xenograft rejection is more similar to disease recurrence than allograft rejection. In case of disease recurrence, the immune system is already activated as evidenced by the destruction of the original islet cells. Therefore, in disease recurrence, the immune system is already in the effector stage. Lymphocytes are activated and differentiate into effector cells, DR3-V1
Alternatively, the agonists of the invention may suppress immune responses against both allografts and xenografts, as they would be sensitive to compounds that express the DR3 polypeptide and thus enhance apoptosis. . Therefore, the present invention further provides a method for producing an immune privileged tissue. The antagonists of the present invention can further be used for the treatment and / or prevention of inflammatory bowel disease.

DR3 activates the NF-kB transcription factor as well as TNFR1. This transcription factor is very closely related to cytokine (eg IL-8) and adhesion molecule (eg ELAM) transcriptional stimulation. DR3 and DR3-V1 here like TNF
Ligands (or agonists) are sometimes pro-inflammatory and antagonists may be useful agents to block this response. Therefore, DR3 and DR3-V1
May be useful in the treatment, prevention, diagnosis and / or prognosis of inflammatory diseases such as rheumatoid arthritis, osteoarthritis, psoriasis, sepsis, and inflammatory bowel disease.

In addition, because of DR3 lymphoblast expression, soluble DR3, agonist or antagonist mABs may be used to diagnose, prognose, treat and / or prevent this type of cancer. In addition, 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, sepsis, and inflammatory bowel disease.

DR3 polynucleotides, polypeptides, agonists or antagonists of the invention are used to diagnose, prognose, treat and / or prevent cardiovascular disorders including peripheral arterial disease such as limb ischemia. be able to.

Cardiovascular disease includes cardiovascular abnormalities such as arterio-arterial fissures.
tula), arteriovenous fistula, cerebral arteriovenous malformation, congenital heart failure, pulmonary atresia and Scimita
Includes r syndrome. Congenital heart failure includes aortic coarctation and cortriatriatam (c
ortriatriatum), coronary blood vessel abnormality, crisscross heart, right thoracic heart, patent ductus arteriosus, Ebstein abnormality, Eisenmenger complex, aplastic left heart syndrome,
Left chest, tetralogy of Fallot, large blood vessel dislocation, double outlet right ventricle (double out
let right ventricle), tricuspid regurgitation, persistent truncus
arteriosus) and septal defects, such as aortopulmonary septal defect, endopericardial cushion deficiency, Lutembacher syndrome, trilogy of Fallot, ventricular septal defect.

Cardiovascular disorders also include heart diseases 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, cardiac hypertrophy, congestive cardiomyopathy, left ventricular hypertrophy, right ventricular hypertrophy, post-infarction cardiac rupture,
Rupture of ventricular septum, heart valve disease, myocardial disease, myocardial ischemia, pericardial effusion, pericarditis (including contractile and tuberculous), pericarditis, postpericardiotomy syndrome, pulmonary heart disease , Rheumatic heart disease, ventricular failure, hyperemia, cardiovascular pregnan
cy complications), Scimitar syndrome, cardiovascular syphilis and cardiovascular tuberculosis.

Arrhythmias include sinus arrhythmia, atrial fibrillation, atrial flutter, bradycardia, premature systole, Adams-Stroke.
s syndrome, leg block, sinoatrial block, long QT syndrome, parasyst
ole), Lown-Ganong-Levine syndrome, Mahaim-type pre-excitation syndrome, Wolff-Parkinson-White syndrome, sick sinus syndorome, tachycardia and ventricular fibrillation. Tachycardia includes paroxysmal tachycardia, supraventricular tachycardia, accelerated ventricular rhythm, atrioventricular nodal reentrant tachycardia, ectopic atrial tachycardia, ectopic junctional tachycardia, sinoatrial nodal reentry. Includes tachycardia, sinus tachycardia, torsades de pointes and ventricular tachycardia.

Heart valve disease includes aortic valve insufficiency, aortic valve stenosis, heart murmurs, aortic valve prolapse, mitral valve prolapse, tricuspid valve prolapse, mitral valve insufficiency, mitral valve stenosis, pulmonary atresia, pulmonary valve Insufficiency, pulmonary artery stenosis, tricuspid valve closure, tricuspid valve failure and tricuspid stenosis.

Cardiomyopathy includes alcoholic cardiomyopathy, congestive cardiomyopathy, hypertrophic cardiomyopathy, subaortic stenosis, pulmonary subvalvular stenosis, restrictive cardiomyopathy, Chagas cardiomyopathy, endocardial elastic fibrosis, myocardium Includes intimal fibrosis, Kearns syndrome, myocardial reperfusion injury and myocarditis.

Myocardial ischemia includes coronary artery diseases such as angina, coronary aneurysms, coronary atherosclerosis, coronary thrombosis, coronary vasospasm, myocardial infarction and myocardial collapse.

Cardiovascular diseases also include vascular diseases such as aneurysms, angiogenesis, hemangiomatosis, bacillary hemangiomatosis, Hippel-Lindau disease, Klippel-Trenaunay-Weber syndrome, Sturge-W.
eber syndrome, angioedema, aortic disease, Takayasu arteritis, aortitis, Leic
he syndrome, arterial occlusion disease, arteritis, enarteritis, polyarteritis nodosa, cerebrovascular disease, diabetic vasculopathy, diabetic retinopathy, embolism, thrombosis, erythromelalgia, Hemorrhoids, hepatic vein occlusion disease, hypertension, hypotension, ischemia, peripheral vascular disease, phlebitis, pulmonary vein occlusion disease, Raynaud's disease, CREST syndrome, retinal vein occlusion,
Scimitar syndrome, superior vena cana syndrome, telangiectasia, atacia telangiectasia, hereditary hemorrhagic telangiectasia, varicocele, nodular vein, nodular ulcer, vasculitis and Includes venous insufficiency.

Aneurysms include dissecting aneurysms, pseudoaneurysms, inflammatory aneurysms, and ruptur aneurysms.
ed aneurysms), aortic aneurysm, cerebral aneurysm, coronary aneurysm, cardiac aneurysm and iliac aneurysm.

Arterial occlusive disease includes arteriosclerosis, intermittent claudication, carotid stenosis, fibromuscular dysplasia, mesenteric duct obstruction, moyamoya disease, renal artery obstruction, renal artery occlusion and occlusive thrombosis. Includes vasculitis.

Cerebrovascular disorders include carotid artery disease, cerebral amyloid angiopathy, cerebral aneurysm, cerebral anoxia, cerebral arteriosclerosis, cerebral arteriovenous malformation, cerebral artery disease, cerebral embolism and thrombosis, carotid artery thrombosis, Sinus thrombosis, Wallenberg syndrome, cerebral hemorrhage, epidural hematoma, subdural hematoma, subaraxhnoid hemorrhage, cerebral infarction, cerebral ischemia (including transient), subclavian steel (steal) syndrome, chamber Periventricular leuko
malacia), vascular headache, cluster headache, migraine and vertebrobasilar insufficiency.

Embolism includes air embolism, amniotic fluid embolism, cholesterol embolism, blue toes (
blue toe) syndrome, fat embolism, pulmonary embolism and thromboembolism. Thromboses include coronary thrombosis, hepatic vein thrombosis, retinal vein occlusion, carotid thrombosis, sinus thrombosis, Wallenberg syndrome and thrombophlebitis.

Ischemia includes cerebral ischemia, ischemic colitis, compartment syndrome, anterior hiatus syndrome, myocardial ischemia, reperfusion injury and peripheral limb ischemia.
Vasculitis includes aortitis, arteritis, Behcet syndrome, Churg-Strauss syndrome, mucocutaneous lymph node syndrome, thromboangiitis obliterans, hypersensitivity vasculitis, Schoenlein-Henoch purpura, allergic cutaneous vasculitis and Wegener granulation. Includes neoplasia.

In one aspect, the DR3 polynucleotides, polypeptides, agonists or antagonists of the invention are used to diagnose, prognose, treat and / or thrombotic microangiopathy.
Or prevent. One such disease is thrombotic thrombocytopenic purpura (TTP)
(Kwaan, HC, Semin. Hematol. 24:71 (1987); Thompson et al., Blood.
80: 1890 (1992)). An increase in TTP-related mortality was reported by the US Centers for Disease Control (Torok et al., Am. J. Hematol. 50:84 (1995)). TT
Plasma of patients suffering from P (including HIV + and HIV− patients) induces apoptosis of human endothelial cells of cutaneous microvascular origin but not macrovascular origin (Laurence et al., Blood 87: 3245. (1996)). Therefore, plasma of TTP patients is thought to contain one or more factors that directly or indirectly induce apoptosis. Another thrombotic microangiopathy is hemolytic uremic syndrome (HUS)
(Moake, JL, Lancet, 343: 393, 1994; Melnyk et al., Arch. Intern.
Med., 155: 2077, 1995; Thompson et al., Supra). Thus, in one aspect,
The present invention relates to the use of DR3 to diagnose, prognose, treat and / or prevent the condition often referred to as "adult HUS" (although it can occur in children as well). The disorder known as pediatric / diarrhea-related HUS is caused by adult H
Different from US. In another aspect, the condition characterized by coagulation of small blood vessels is DR3
Can be used to diagnose, prognose, treat and / or prevent. 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 thought to involve small vessel coagulation. Disruption of the microvasculature in the heart has been reported in patients with multiple sclerosis. As a further example, treatment of systemic lupus erythematosus (SLE),
Prevention, diagnosis and / or prognosis are considered.

The DR3 polynucleotides, polypeptides, agonists or antagonists of the invention can be used in combination with other agents useful in treating, preventing, diagnosing and / or prognosing particular disorders. For example, Laurence et al. (Blood 87:32
45 (1996)) reported that TTP plasma-mediated apoptosis of microvascular endothelial cells was somewhat reduced by using normal plasma depleted with anti-Fas blocking antibodies, aurintricarboxylic acid or cold precipitates. . Thus, a patient may combine a polynucleotide and / or a polynucleotide of the invention in combination with a substance that inhibits Fas ligand-mediated apoptosis of endothelial cells, such as those listed above.
Alternatively, it can be treated with a polypeptide. In one aspect, both the DR3 polynucleotide, polypeptide, agonist or antagonist, and anti-FAS blocking antibody are administered to a patient suffering from a disorder characterized by thrombotic microangiopathy, such as TTP or HUS. Examples of blocking monoclonal antibodies against Fas antigen (CD95) are described in International Patent Application Publication No. WO 95/10540, which is incorporated herein by reference.

The naturally occurring balance between endogenous stimulants and inhibitors of angiogenesis is such that the inhibitory effect predominates (Rastinejad et al., Cell 56: 345-355 (1989)). In the rare instances where neovascularization occurs under normal physiological conditions, such as wound healing, organ regeneration, embryonic development and female reproductive processes, angiogenesis is stringently regulated and spatially and temporally delimited. Under the conditions of pathological angiogenesis, which is characterized by solid tumor growth, these regulatory controls are defective.

Uncontrolled angiogenesis becomes pathological and sustains the progression of numerous neoplastic and non-neoplastic diseases. A number of serious diseases are dominated by abnormal neovascularization, including solid tumor growth and metastasis, arthritis, some types of ocular disorders and psoriasis. For example, review Moses et al., Biotech. 9: 630-634 (1991); Folkma.
n et al., N. Engl. J. Med., 333: 1757-1763 (1995); Auerbach et al., J. Mi
crovasc. Res. 29: 401-411 (1985); Folkman, Advances in Cancer Research, e.
ds. Klein and Weinhouse, Academic Press, New York, pp. 175-203 (1985); P
atz, Am. J. Opthalmol. 94: 715-743 (1982); and Folkman et al., Science.
221: 719-725 (1983). In many pathological conditions, the progression of angiogenesis contributes to the disease state. For example, significant data has been accumulated showing that solid tumor growth depends on angiogenesis. Folkman and Klagsbrun, Science 235: 442-447 (19
87).

The present invention provides the DR3 polynucleotides and / or polypeptides (D
(Including R3 agonists and / or antagonists),
Provide treatment, prevention, diagnosis and / or prognosis of diseases or disorders associated with neovascularization. The malignant and metastatic conditions that can be diagnosed, prognosed, treated and / or prevented with the polynucleotides and polypeptides of the invention are described herein or otherwise known in the art (such as: For a summary of disabilities, Fish
man et al., Medicine, 2d Ed., JB Lippincott Co., Philadelphia (1985)
), Malignant, solid tumors and cancer.

In addition, neovascularization-related ocular disorders that 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: Vascular glaucoma, diabetic retinopathy, retinoblastoma, posterior lens fibroplasia, uveitis, retinopathy of premature macular degeneration, corneal graft neovascularization, and other ocular inflammatory diseases, eye tumors, and Diseases associated with neovascularization of the choroid plexus or iris are included, but are not limited to. See, eg, review Waltman et al., Am. J. Ophthal. 85: 704-710 (1978) and Gartner et al., Surv. Ophthal. 22: 291-312 (1978).

Furthermore, diseases that 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 hemangiomas, arthritis, psoriasis, vascular fibers. These include tumors, atherosclerotic plaques, delayed wound healing, granulation, hemorrhagic joints, hypertrophic scars, pseudoarticular fractures, Osler-Weber syndrome, pyogenic granulomas, scleroderma, trachoma and vascular adhesions. Not limited to.

The 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. Such diseases and conditions include cancer (eg, cancer associated with immune cells, breast cancer, prostate cancer, ovarian cancer, follicular lymphoma, glioblastoma, cancer associated with mutations or changes in p53, brain tumor, bladder). Cancer, cervical cancer, colorectal cancer, colorectal cancer, non-small cell lung cancer, small cell lung cancer, gastric cancer, etc., lymphoproliferative disorders (eg lymphadenopathy and lymphoma (eg EBV-induced lymphoproliferative) And Hodgkin's disease)), microbial (eg, viral, bacterial, etc.) infection (eg, HIV-1 infection, HIV-2 infection, herpes virus infection (HSV-1, HSV-2, CMV, VZV, HHV-6). , HHV
-7, including but not limited to EBV), adenovirus infection, poxvirus infection, human papillomavirus infection, hepatitis virus infection (eg HAV).
, HBV, HCV, etc.), Helicobacter pylori infection, invasive Staphylococcia
, Etc.), parasitic infections, nephritis, bone diseases (eg osteoporosis), atherosclerosis, pain, cardiovascular disorders (eg neovascularization, hypovascularization or hypocirculation (eg ischemic diseases (eg myocardial infarction)). , Seizures)), AIDS, allergies, inflammation, neurodegenerative diseases (eg Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, pigmentary retinitis, cerebellar degeneration, etc.), transplant rejection (eg
Acute or chronic), graft-versus-host disease, diseases caused by myelodysplasia (eg aplastic anemia), joint tissue destruction in rheumatism, liver disease (eg acute and chronic hepatitis, liver injury and cirrhosis), autoimmunity Diseases (eg, multiple sclerosis, myasthenia gravis, rheumatoid arthritis, systemic lupus erythematosus, immune complex glomerulonephritis, autoimmune diabetes, autoimmune thrombocytopenic purpura, Graves' disease, Hashimoto's thyroiditis, inflammation Autoimmune disease), cardiomyopathy (eg dilated cardiomyopathy), diabetes, diabetic complications (eg diabetic nephropathy, diabetic neuropathy, diabetic retinopathy), influenza, asthma, psoriasis, glomerulonephritis, Includes but is not limited to septic shock and ulcerative colitis.

Polynucleotides and / or polypeptides of the present invention, and / or agonists and / or antagonists thereof, may induce angiogenesis, promote wound healing (eg, injury, burns and fractures), and control bone formation, and osteoporosis. Useful for treatment and / or prevention.

The polynucleotides and / or polypeptides of the invention, and / or agonists and / or antagonists thereof, are also useful as adjuvants to enhance immunoreactivity and / or antiviral immune response to a particular antigen.

More generally, the polynucleotides and / or polypeptides of the invention, and / or their agonists and / or antagonists, regulate immune responses (
That is, it is useful for strengthening or reducing). For example, the polynucleotides and / or polypeptides of the present invention may be useful in the preparation or recovery of surgery, trauma, radiation therapy, chemotherapy and transplantation, or immune response and / or in elderly and immunocompromised individuals. Or it can be used to boost recovery. Alternatively, the polynucleotides and / or polypeptides of the invention, and / or agonists and / or antagonists thereof, are useful as immunosuppressive agents, eg, in the treatment and / or prevention of autoimmune diseases, or in the prevention of transplant rejection. is there. In a particular embodiment, the polynucleotides and / or polypeptides of the invention are used to chronically inflammatory, allergic or autoimmune, such as those described herein or known in the art. Diagnose, prognose, treat and / or prevent symptoms.

Gene Therapy In certain embodiments, antibodies or functions thereof for treating, suppressing and / or preventing diseases or disorders associated with aberrant expression and / or activity of the polypeptides of the invention by gene therapy methods. A nucleic acid containing a sequence encoding a sex derivative is administered. Gene therapy refers to treatment performed by administering an expressed or expressible nucleic acid to a subject. In this aspect of the invention, the nucleic acid produces its encoded protein that mediates a therapeutic effect.

Any method for gene therapy available in the art may be used in accordance with the present invention. A typical method is described below.

For a general review of gene therapy methods, Goldspiel et al., 1993, Clin.
ical Pharmacy 12: 488-505; Wu and Wu, 1991, Biotherapy 3: 87-95; Tols
toshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32: 573-596; Mulligan, 1993,
Science 260: 926-932; and Morgan and Anderson, 1993, Ann. Rev. Bio.
chem. 62: 191-217; May, 1993, TIBTECH 11 (5): 155-215). Methods generally known in the field of recombinant DNA technology that can be used are described by Ausubel et al. (Eds.
), 1993, Current Protocols in Molecular Biology, John Wiley & Sons, NY;
And Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual,
Listed in Stockton Press, NY.

In a preferred embodiment, the compound comprises a nucleic acid sequence that encodes an antibody, which nucleic acid sequence is of an expression vector that expresses the antibody or fragment or chimeric protein, or their heavy or light chains in a suitable host. It is a part. In particular, such nucleic acid sequences have a promoter operably linked to the antibody-encoding region, the promoter being inducible or constitutive, and optionally tissue-specific. In another specific aspect, the antibody coding sequence and other desired sequences are flanked by sequences that promote homologous recombination at desired sites in the genome such that the antibody nucleic acid is expressed chromosomally. Using a nucleic acid molecule (Kol
ler and Smithies, 1989, Proc. Natl. Acad. Sci. USA 86: 8932-8935; Zij
lstra et al., 1989, Nature 342: 435-438). In a particular embodiment, the expressed antibody molecule is a single chain antibody; alternatively, the nucleic acid sequence comprises sequences encoding both the heavy and light chains of the antibody, or fragments thereof.

Delivery of the nucleic acid to the patient is either directly, in this case, directly exposing the patient to the nucleic acid or a vector carrying the nucleic acid, or indirectly, here first in vitro.
Either the cells may be transformed with the nucleic acid and then transplanted into the patient.
Each of these two approaches is known as in vivo or ex vivo gene therapy.

In a particular embodiment, the nucleic acid sequences are directly administered in vivo, which is expressed to produce the encoded product. This can be accomplished by a number of methods known in the art, for example by constructing a nucleic acid sequence as part of a suitable nucleic acid expression vector, administering it and placing it in vivo, eg a defective or attenuated retrovirus or other. Virus vector (US Pat. No. 4,980,286), or by direct injection of naked DNA, or by microparticle bombardment (eg, gene gun; Biolistic, Dupont), or lipids. Coating with or using cell surface receptors or transfectants, encapsulation into liposomes, microparticles or microcapsules, or administering nucleic acid sequences linked to peptides known to enter the nucleus Targets ligands for receptor-mediated endocytosis Administering the linked nucleic acid sequence (see, for example, Wu and Wu, 1987, J. Biol. Chem. 262: 4429-4432), which specifically refers to the target cell type expressing the receptor. (Can be used)
It can be implemented by In another embodiment, a nucleic acid-ligand complex is formed with a ligand containing a fusogenic viral peptide that disrupts endosomes,
Degradation of nucleic acids by lysosomes can be avoided. In yet another embodiment, nucleic acids may be targeted for cell-specific uptake and expression by targeting specific receptors in vivo (eg PCT Publication WO 92/06180 dated April 1992).
16 days (Wu et al.); WO 92/22635 dated December 23, 1992 (Wilson et al.); WO 92/203
16 date November 26, 1992 (Findeis et al.), WO 93/14188 date July 22, 1993 (Clark
e et al.); WO 93/20221 dated 14 October 1993 (Young)). Alternatively, the nucleic acid may be introduced intracellularly and expressed by homologous recombination into host cell D.
NA (Koller and Smithies, 1989, Proc. Natl. Acad. Sci.
USA 86: 8932-8935; Zijlstra et al., 1989, Nature 342: 435-438).

In a particular embodiment, a will vector containing a nucleic acid sequence encoding an antibody of the invention is used. For example, retroviral vectors may be used (Miller et al.
, 1993, Meth. Enzymol. 217: 581-599). This retroviral vector lacks retroviral sequences that are not required for viral genome packing and integration into host cell DNA. Nucleic acid sequences encoding the antibodies used in gene therapy are cloned into one or more vectors to facilitate gene delivery to the patient. For more details on retrovirus vectors,
Boesen et al., 1994, Biotherapy 6: 291-302, which uses a retroviral vector that carries the mdr1 gene to hematopoietic stem cells to make stem cells more resistant to chemotherapy. Is listed. Other references exemplifying the use of retroviral vectors in gene therapy are:
Clowes et al., 1994, J. Clin. Invest. 93: 644-651; Kiem et al., 1994, Bl
ood 83: 1467-1473; Salmons and Gunzberg, 1993, Human Gene Therapy 4:
129-141; and Grossman and Wilson, 1993, Curr. Opin. In Genetics and
Devel. 3: 110-114.

Adenovirus is another viral vector that can be used for gene therapy. Adenovirus is a particularly attractive vehicle for delivering genes to the respiratory epithelium.
In nature, adenovirus infects the respiratory epithelium and causes mild illness. Other targets for adenovirus-based delivery systems include the liver, central nervous system, endothelial cells and muscle. Adenovirus is convenient because it has the ability to infect non-dividing cells.
Review of adenovirus-based gene therapy includes Kozarsky and Wilson,
1993, Current Opinion in Genetics and Development 3: 499-503. Bout
In et al., 1994, Human Gene Therapy 5: 3-10, the use of an adenovirus vector for transferring a gene to the respiratory epithelium of Rhesus monkeys has been demonstrated. Another example of the use of adenoviruses in gene therapy is Rosenfeld et al.,
1991, Science 252: 431-434; Rosenfeld et al., 1992, Cell 68: 143-155; Ma
strangeli et al., 1993, J. Clin. Invest. 91: 225-234; PCT publication WO 94/1
2649; and Wang et al., 1995, Gene Therapy 2: 775-783. In a preferred embodiment, adenovirus vectors are used.

Adeno-associated virus (AAV) can also be used for gene therapy (Walsh et al.,
1993, Proc. Soc. Exp. Biol. Med. 204: 289-300; U.S. Pat.No. 5,436,146.
).

Other gene therapy approaches include transferring genes to cells in tissue culture by such methods as electroporation, lipofection, calcium phosphate mediated transfection or viral infection. Usually, the transfer method involves transfer of a selectable marker to the cells. Thereafter, the cells are placed under selection, the transferred gene is taken up, and the expressing cell is isolated. The cells are then delivered to the patient.

In this aspect, the nucleic acid is introduced into cells and the resulting recombinant cells are administered in vivo. Such introduction is known in the art of transfection, electroporation, microinjection, infection with viral or bacteriophage vectors containing nucleic acid sequences, cell fusion, chromosomal mediated gene transfer, microcell mediated gene transfer. , Spheroplast fusion, and the like, but not limited thereto. Many methods are known in the art for the introduction of foreign genes into cells (eg Loeffler and Behr, 1993, Meth. Enzymol. 217: 599-6.
18; Cohen et al., 1993, Meth. Enzymol. 217: 618-644; Cline, 1985, Pharma.
c. Ther. 29: 69-92), as long as it does not disrupt essential development and physiological function in the recipient cell, it can be used according to the present invention. This technique provides for the stable transfer of nucleic acid to a cell, whereby the nucleic acid is expressed by, and preferably inherited by, the cell's progeny.

The resulting recombinant cells can be delivered to a patient by various methods known in the art. Recombinant blood cells (eg, hematopoietic stem cells or progenitor cells) are preferably administered by intravenous injection. The amount of cells envisioned for use can be determined by one of ordinary skill in the art depending on the desired effect, patient condition, etc.

Cells into which the nucleic acid can be introduced for purposes of gene therapy include any desired and available cell type, including epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; , T-lymphocytes, B-lymphocytes, mononuclear cells, macrophages,
Blood cells such as neutrophils, eosinophils, megakaryocytes, granulocytes; including various stem or progenitor cells, especially hematopoietic stem or progenitor cells, such as those obtained from bone marrow, cord blood, peripheral blood, fetal liver, etc. However, it is not limited to these.

For the preferred embodiment, the cells used for gene therapy are from a patient.

Regarding an embodiment in which a recombinant cell is used in gene therapy, a nucleic acid sequence encoding an antibody is introduced into the cell to enable expression in the cell or its progeny, and then the recombinant cell is treated with a therapeutic effect. In vivo administration for the purpose. In particular embodiments, stem cells or progenitor cells are used. Many of the stem and / or progenitor cells that can be isolated and maintained in vitro can potentially be used according to this aspect of the invention (eg, PCT Publication WO 94/08598, date April 28, 1994; Stem
ple and Anderson, 1992, Cell 71: 973-985; Rheinwald, 1980, Meth. Cell.
Bio. 21A: 229; and Pittelkow and Scott, 1986, Mayo Clinic Proc. 61.
: 771).

In a particular embodiment, the nucleic acid introduced for purposes of gene therapy contains an inducible promoter operably linked to the coding region and is controlled by the control of the presence or absence of a suitable transcription inducer. The expression can be controlled.

Mechanism of Administration The present invention provides methods of treatment, inhibition and prevention by administering to a subject an effective amount of a compound or pharmaceutical composition of the invention. In a preferred embodiment,
The compound is substantially purified (eg, substantially free of substances that limit its effect or have unwanted side effects). The subject is preferably an animal, including but not limited to, for example, cows, pigs, horses, chickens, cats, dogs, etc., preferably mammals, most preferably humans.

Formulations and methods of administration that may be practiced when the compound comprises nucleic acids or immunoglobulins are described above; in addition, suitable formulations and routes of administration are selected from among those described herein below. You can

The agonists or antagonists described herein can be administered to cells expressing the receptor of the invention in vitro, in vitro or in vivo. Administration of an "effective amount" of an agonist or antagonist means an amount of the compound, including the polypeptide, sufficient to increase or inhibit the cellular response to TNF family ligands. In particular, administration of an "effective amount" of agonist or antagonist is
By an amount effective to increase or inhibit V1- or DR3-mediated apoptosis. Of course, if apoptosis is to be increased, the agonist of the present invention
It can be administered at the same time as the NF family ligand. Those skilled in the art will recognize that the effective amount of an agonist or antagonist can be determined empirically and can be used in pure form or in the form of a pharmaceutically acceptable salt, ester or prodrug. The agonist or antagonist can be administered as a composition in combination with one or more pharmaceutically acceptable additives.

It will be appreciated that, when administered to a human patient, the total daily dose of the compounds and compositions of the invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for a particular patient will depend on factors well known in the medical arts.

As a general rule, although the total pharmaceutically effective amount of DR3 polypeptide administered parenterally per administration is about 1 μg / kg / day to 10 mg / kg / day of patient body weight. As mentioned above, this is subject to therapeutic discretion. More preferably,
This dose is at least 0.01 mg / kg / day, and for hormones most preferably for humans between about 0.01 mg / kg / day and 1 mg / kg / day.
For continuous administration, the DR3 agonist or antagonist is typically administered at a rate of about 1 μg / kg / hour to about 50 μg / kg / hour, by infusion 1 to 4 times per day, or using, for example, a minipump. And administer by continuous subcutaneous infusion. An intravenous bag solution may be used.

The dosage regimen may also be adjusted on a patient-by-patient basis to provide a predetermined concentration of the agonist or antagonist in the blood as measured by the RIA method. That is, administration to a patient can be adjusted to achieve constant, continuous blood levels on the order of about 50-1000 ng / mL, preferably 150-500 ng / mL, as measured by RIA.

For example, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing compounds, receptor-mediated endocytosis (eg, Wu and Wu, 1987, J. Biol. Chem. 262: 4429-4432), and various delivery systems are known, such as the construction of nucleic acids as part of retroviruses or other vectors, and are used to administer the compounds of the invention. You can Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural and oral routes. The compound or composition can be administered by any conventional route, such as by infusion or bolus injection, by absorption through the inner surface of the epithelium or skin mucosa (eg, oral mucosa, rectum and small intestine, etc.) It can be administered with a biologically active agent. Administration can be systemic or local. Furthermore, it may be desirable to introduce the pharmaceutical compounds or compositions of the invention to the central nervous system by any suitable route, including intracerebroventricular and subarachnoid injection. Intracerebroventricular injection can be easily performed, for example, by an intraventricular catheter attached to a reservoir such as the Ommaya storage organ. Pulmonary administration can also be employed, eg, by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.

The pharmaceutical composition is provided to include an agonist or antagonist and a pharmaceutically acceptable carrier or additive, and is orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically ( Powder, ointment, drops or transdermal patch), buccal, or as an oral or nasal spray. Importantly, co-administration of an agonist and a TNF family ligand can reduce clinical side effects by using low doses of both the ligand and agonist. It will be appreciated that, depending on the urgency of the particular therapeutic application, the agonist can be “co-administered” before, after or simultaneously with the TNF Fumilly ligand. “Pharmaceutically acceptable carrier” means a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. As used herein, "parenteral" refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrathoracic, subcutaneous, and intraarticular injection or infusion.

Pharmaceutical compositions of the invention for parenteral injection may include sterile pharmaceutically acceptable aqueous or non-aqueous solutions, dispersions, suspensions or emulsions and sterile injectable solutions or Sterile powders can be included for reconstitution of the dispersant immediately before use.

In addition to a soluble DR3-V1 or DR3 polypeptide, a DR3 containing a transmembrane region may be included when suitably solubilized by the inclusion of a detergent such as CHAPS or NP-40 with a buffer. -V1 or DR3 polypeptides can also be used.

In specific embodiments, it may be desirable to administer the pharmaceutical compounds or compositions of the invention locally to the area in need of treatment. It can be injected, catheterized, enema, or porous, non-porous or sialasti.
c) with a gelatinous or fibrous implant comprising a membrane, such as a membrane, which can be achieved, for example, by topical injection during surgery, eg topical application in combination with post-surgical wound dressing. It is not limited to this. Preferably, when administering proteins, including antibodies, of the present invention, care must be taken to use materials that do not absorb the protein.

In another embodiment, the compound or composition can be delivered in vesicles, particularly liposomes (Langer, 1990, Science 249: 1527-1533, Treat et. Al., In Lipo.
somes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein a
nd Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, i
bid, pp. 317-327, generally ibid).

In yet another aspect, the compound or composition can be delivered in a controlled release system. In one aspect, a pump can be used (Langer, supr
a; Sefton, 1987, CRC Crit. Ref. BIomed. Eng. 14: 201; Buchwald et. al.,
1980, Surgery 88: 507; Saudek et. Al., 1989, N. Engl. J. Med. 321: 574). In another embodiment, polymeric materials can be used (Medical Applicat
ion of Controlled Release, Langer and Wise (eds.) CRC Pres., Boca Raton,
Florida (1974); Controlled Drug Bioavailability, Drug Product Design an
d Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger a
See nd Peppas, J., 1983, Macromol. Sci. Rev. Macromol. Chem. 23:61; Le.
vy et. al., 1985, Science 228: 190, During et. al., 1989, Ann. Neurol. 25
: 351; Howard et. Al., 1989, J. Neurosurg. 71: 105). In yet another embodiment, a controlled release system can be placed in the vicinity of the therapeutic target, the brain, requiring only a small fraction of the systemic dose (eg Goodson, in Medica.
l Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)
See).

Other controlled release systems are discussed in a review by Langer (19
90, Science 249: 1527-1533).

In a particular embodiment where the compound of the invention is a nucleic acid encoding a protein, for example, the use of retroviral vectors (see US Pat. No. 4,980,286).
By or by direct injection, or by microparticle bombar
dment) (eg, gene gun; Biolistic, DuPont), or the use of coatings or transfectants with lipids or cell surface receptors, or homeobox-like peptides known to enter the nucleus (eg, Joliot et al. . al.,
1991, Proc. Natl. Acad. Sci. USA 88: 1864-1868), the nucleic acid is constructed as a part of an appropriate nucleic acid expression vector, and administered intracellularly. By such administration, the nucleic acid can be administered in vivo to promote the expression of the encoded protein. Alternatively, the nucleic acid can be introduced intracellularly and incorporated by homologous recombination into host cell DNA for expression.

The present invention further provides pharmaceutical compositions. Such a composition comprises a therapeutically effective amount of the compound and a pharmaceutically acceptable carrier. In a specific embodiment, the term "pharmaceutically acceptable" is for use under federal or state government regulation or in the United States Pharmacopeia, or in animals, and more specifically in humans. Means that it is on the other generally recognized Pharmacopoeia of.
The term "carrier" means a vehicle with which a diluent, adjuvant, additive, or therapeutic agent thereof is administered. Such pharmaceutical carriers are sterile liquids, such as water and oils 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 dextran and glycerol solutions can also be used as liquid carriers, especially for injectable solutions. Suitable pharmaceutical additives include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, Includes 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, emulsions, tablets, pills, capsules, powders, sustained-release preparations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. Examples of suitable pharmaceutical carriers are EW Martin
It is described in his book "Remington's Pharmaceutical Sciences". 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 will suit the mode of administration.

The compounds of the invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include salts formed with anions derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine. , A salt formed with a cation derived from 2-ethylaminoethanol, histidine, procaine, and the like.

The amount of a compound of the invention that is effective in treating, suppressing and / or preventing a disease or disorder associated with aberrant expression and / or activity of a polypeptide of the invention is determined by standard clinical techniques. can do. In addition, in vitro analysis may optionally be employed to help determine optimal dosage ranges. Precise doses for use in the formulations will also depend on the route of administration, and the severity of the disease or disorder, and should be decided according to the judgment of the practitioner and the circumstances of the individual patient. The effective dose is
It may be extrapolated from dose-response curves obtained in vitro or from animal model test systems.

For antibodies, the dose administered to a patient is typically from 0.1 mg / kg to 100 mg / kg.
mg / kg (patient weight). Preferably, the dose administered to the patient is 0.1 mg
/ Kg to 20 mg / kg (patient weight), more preferably 1 mg / kg to 10 m
It is between g / kg (patient weight). In general, human antibodies have a longer half-life within the human body than antibodies from other species because of the immune response to the foreign polypeptides. Therefore, it is often possible to administer human antibodies at lower doses and less frequently. In addition, for example, lipidation
By increasing the uptake and permeability of tissues (for example, into the brain), the dosage and frequency of administration of the antibody of the present invention can be reduced by such modifications.

In a preferred embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition suitable for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. If desired, the composition can also include a solubilizer and a local anesthetic such as lignocaine to relieve pain at the injection site. Generally, the ingredients are either dry or in a mixed form together in a single dose formulation, for example, a dry lyophilized powder or anhydrous concentrate in a sealed container such as an ampoule or sachet indicating the quantity of active ingredient. Provided as. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule 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
Before use in humans, it is tested in vivo for the desired therapeutic or prophylactic activity. For example, in vitro assays to determine the therapeutic or prophylactic utility of a compound or pharmaceutical composition include the effect of the compound on a cell line or tissue sample of a patient. The effect of a compound or composition on cell lines and / or tissue samples can be determined using techniques known to those of skill in the art including, but not limited to, rosette formation assays and cytolytic assays. In vitro analysis, which can be used in accordance with the present invention to determine if administration of a particular compound is desirable, includes culturing a tissue sample of a patient in culture, exposing to the compound or otherwise. In vitro cell analysis involves administering compounds and observing the effects of such compounds on the tissue sample.

The compositions of the present invention can be administered alone or in combination with other therapeutic agents. Therapeutic agents that can be administered in combination with the compositions of the invention include TNF.
Other members of the family include, but are not limited to, chemotherapeutic agents, antibiotics, steroidal and non-steroidal anti-inflammatory drugs, conventional immunotherapeutic agents, cytokines and / or growth factors. The combination, together (eg as an additive)
, May be administered separately (simultaneously or in parallel or sequentially). This includes a presentation in which the combined agents are administered together as a therapeutic mixture, and the combined agents separately but simultaneously (eg, to the same person through separate intravenous lines).
Also included are procedures that are administered. Administration "in combination" further includes the administration of one compound or agent first, followed by a second compound or agent administered,
Separate administration is included.

In one aspect, the compositions of the invention are administered in combination with other members of the TNF family. TNF, TNF-related or TNF-like molecules that can be administered with the compositions of the invention include TNF-α, lymphotoxin-α (LT-α, also known as TNF-β), LT-β (heterotrimer complex). LT-α2-β), OPGL, FasL, CD27L, CD30L, CD40L, 4-1.
BBL, DcR3, OX40L, TNF-γ (International Publication No. WO96 / 1432
8), TNF-γ-α (International Publication No. WO00 / 08139), TNF-γ-β.
(International publication number WO00 / 08139), AIM-I (International publication number WO97 /
33899), AIM-II (international publication number WO97 / 34911), endokine-α (international publication number WO98 / 07880), TR6 (international publication number WO9).
8/30694), OPG, and Neutrokine-α (International Publication No. WO9
8/18921), OX40, and soluble forms of nerve growth factor (NGF),
And Fas, CD30, CD27, CD40 and 4-IBB, TR2 (International Publication Number WO96 / 34095), DR4 (International Publication Number WO98 / 32856).
), TR5 (International Publication Number WO98 / 30693), TR6 (International Publication Number WO)
98/30694), TR7 (International Publication No. WO98 / 41629), TRAN
K, TR9 (International Publication Number WO98 / 56892), TR10 (International Publication Number W)
O98 / 55422), 312C2 (International Publication No. WO98 / 06842) and soluble forms of TR12, and soluble forms of CD154, CD70 and CD153.

In another aspect, the composition of the invention comprises a CD40 ligand (CD40L), CD4.
0L soluble forms (eg AVREND ™), biologically active fragments of CD40L, variants or derivatives, anti-CD40L antibodies (eg agonist or antagonist antibodies), and / or anti-CD40 antibodies (eg It is administered in combination with an agonist antibody or an antagonist antibody).

In yet another embodiment, the composition of the present invention is prepared by the following composition: Tacrolimus (Fuji
sawa), thalidomide (eg Celgene), anti-Tac (Fv) -PE40 (eg Protein Design Lab), inolimomab (Biotest), MAK-195F (Kno).
ll), ASM-981 (Novartis), interleukin-1 receptor (eg Im
munex), interleukin-4 receptor (eg, Immunex), ICM3 (ICOS)
, BMS-188667 (Bristol-Myers Squibb), anti-TNF Ab (eg T
herapeutic antibodies), CG-1088 (Celgene), anti-B7 Mab (eg, Innogetics), MEDI-507 (BioTransplant), ABX-CBL (Abg
Administered in combination with 1, 2, 3, 4, 5 or more of enix).

In certain embodiments, inventive compositions are administered in combination with an antiretroviral agent, a nucleoside reverse transcriptase inhibitor, a non-nucleoside reverse transcriptase inhibitor, and / or a protease inhibitor. Nucleoside reverse transcriptase inhibitors that can be administered in combination with the compositions of the present invention include RETROVIR ™ (zidovudine / AZT), VIDEOX ™ (didanocin / ddI), HIDVI.
D ™ (Zalcitabine / ddC), ZERIT ™ (Stavudine / d4)
T), EPIVIR ™ (lamivudine / 3TC) and COMBIVIR (
Trademark) (zidovudine / lamivudine). Non-nucleoside reverse transcriptase inhibitors that can be administered in combination with the compositions of the present invention include VIRAMUNE ™ (Nevirapine), RESCRIPTOR ™.
(Delavirdine) and SUSTIVA ™ (efavirenz), but are not limited thereto. Protease inhibitors that can be administered in combination with the compositions of the present invention include CRIXIVAN ™ (Indinavir), NORV
Includes but is not limited to IR ™ (ritonavir), INVIRASE ™ (saquinavir) and VIRACCEPT ™ (nerfinavir). In a specific embodiment, antiretroviral agents, nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors and / or protease inhibitors are used in combination with the compositions of the invention to treat, prevent, treat AIDS, And / or diagnose, and / or HIV infection can be treated, prevented, and / or diagnosed.

In other embodiments, the compositions of the invention can be administered in combination with anti-opportunistic infections. Anti-opportunistic infections that can be used in combination with the compositions of the present invention include TRIMETHOPRIM-SULFAMETHOXAZOLE (TM).
, DAPSONE ™, PENTAMIDINE ™, ATOVAQU
ONE ™, ISONIAZ1D ™, RIFAMPIN ™, P
YRAZINAMIDE ™, ETHAMUTOL ™, RIFABU
TIN ™, CLARITHROMYCIN ™, AZITHROMY
CIN (TM), GANCICLOVIR (TM), FOSCARNET (TM), CIDOFOVIR (TM), FLUCONAZOLE (TM), ITRA
CONAZOLE ™, KETOCONAZOLE ™, ACYCLO
VIR ™, FAMCICOLVIR ™, PYRIMETHAMIN
Includes, but is not limited to, E ™, LEUCOVORIN ™, NEUPOGEN ™ (filgrastin / G-CSF), and LEUKINE ™ (sarglamostin / GM-CSF). In a particular embodiment, the composition of the present invention is treated with TRIMETHOPRIM-SULFAMETHOXAZOLE.
(TM), DAPSONE (TM), PENTAMIDINE (TM), and / or ATOVAQUONE (TM) in any combination for opportunistic Pneu
Prophylactically treat, prevent, and / or diagnose mocystis carinii pneumonia infections. In another specific embodiment, the composition of the invention is treated with ISONIAZID ™, RIFA.
MPIN ™, PYRAZINA MIDE ™ and / or ETHA
Opportunistic Mycobacterium avium for use in any combination with MBATOL ™
Prophylactically treat, prevent and / or diagnose complex infections. In another specific embodiment, the composition of the invention is treated with RIFABUTIN ™, CLARITHROMYCI.
Used in any combination with N ™ and / or AZITHROMYCIN ™ to prophylactically treat, prevent and / or diagnose opportunistic Mycobacterium tuberculosis infections. In another specific embodiment, the composition of the invention is treated with GANCICLO.
VIR ™, FOSCARNET ™ and / or CIDOFOVI
Used in any combination with R ™ to prophylactically treat, prevent and / or diagnose opportunistic cytomegalovirus infections. In another specific embodiment, the compositions of the invention are used in any combination with FLUCONAZOLE ™, ITRACONAZOLE ™ and / or KETOCONAZOLE ™ to prophylactically treat, prevent and prevent opportunistic fungal infections. / Or diagnose. In another specific embodiment, the compositions of the present invention are treated with ACYCLOVIR ™ and / or FAMCIC.
It is used in any combination with OLVIR ™ to prophylactically treat, prevent and / or diagnose opportunistic herpes simplex virus type 1 and / or 2. In another specific embodiment, the compositions of the invention are used in any combination with PYRIMETHAMINE ™ and / or LEUCOVORIN ™ to opportunistic To
Prophylactically treat, prevent and / or diagnose xoplasma gondii infection. In another specific embodiment, the compositions of the invention are used in any combination with LEUCOVORIN ™ and / or NEUPOGEN ™ to prophylactically treat, prevent and / or diagnose opportunistic bacterial infections.

In a further aspect, the compositions of the invention are administered in combination with an antiviral agent.
Antiviral agents that can be administered in combination with the compositions of the present invention include, but are not limited to, acyclovir, ribavirin, amantadine and remantidine.

In a further aspect, the compositions of the invention are administered in combination with an antibiotic. Antibiotics that can be used in combination with the compositions of the present invention include amoxicillin, aminoglycosides, β-lactams (glycopeptides), β-lactamase, cindamycin, chloramphenicol, cephalosporins, ciprofloxacin. , Erythromycin, fluoroquinolone, macrolide, metronidazole, penicillin, quinolone, rifampicin, streptomycin, sulfonamide, tetracycline, trimethopurine, trimethopurine-sulfamtoxazole,
And vancomycin, but is not limited thereto.

Conventional non-specific immunosuppressive agents that can be used in combination with the compositions of the present invention include steroids, cyclosporine, cyclosporine analogs, cyclophosphamide, methylprednisone, prednisone, azathioprine, FK-506.
, 15-deoxyspergualin and other immunosuppressive agents that act by suppressing the function of responsive T cells.

In a specific embodiment, the compositions of this invention are administered in combination with an immunosuppressive agent. Immunosuppressant preparations that can be administered with the compositions of the invention include ORTHOC.
LONE (trademark) (OKT3), SANDIMMUNE (trademark) / NEORAL
™ / SANGDYA ™ (cyclosporine), PROGRAF ™ (tacrolimus), CELLCEPT ™ (mycophenolate), azathioprine, glucoruticosteroids, RAPAMUNE ™ (sirolimus), which includes Not limited. In a specific embodiment, immunosuppressive agents can be used to prevent rejection of organ or bone marrow transplants.

In one aspect, the compositions of the invention are administered in combination with steroid therapy. Steroids that can be administered in combination with the compositions of the invention include, but are not limited to, oral corticosteroids, prednisone, and methylprednisone (eg, IV methylprednisone). In a specific embodiment, the compositions of the invention are administered in combination with prednisone. In a more specific aspect, the compositions of the invention are administered in combination with prednisone and an immunosuppressant. Immunosuppressive agents that can be administered with the compositions of the present invention and prednisone are those described herein, including but not limited to azathioprine, cyclophosphamide, and cyclophosphamide IV. Not done. In another specific embodiment, the compositions of the invention are administered in combination with methylprednisone. In a more specific aspect, the compositions of the invention are administered in combination with methylprednisone and an immunosuppressant. Immunosuppressive agents that can be administered with the compositions of the invention and methylprednisone are those described herein, including azathioprine, cyclophosphamide, and cyclophosphamide IV. Not limited.

In another aspect, the compositions of the invention are administered in combination with an antimalarial drug. Antimalarial drugs that can be administered with the compositions of the present invention include, but are not limited to, hydroxychloroquine, chloroquine and / or quinacrine.

[0403]   In one aspect, the compositions of the invention are administered in combination with an NSAID.

In another aspect, the composition of the present invention is prepared by combining the following drugs: NRD-101 (Hoechst Ma
rion Roussel), diclofenac (Dimethaid), potassium oxaprozin (Mon
santo), mechasermin (Chiron), T-614 (Toyama), pemetrexed disodium (Eli Lilly), atreluton (Abbott), valdecoxib (Mon)
santo), Byten Gulden, Campus, AGM-1470 (Takeda)
, CDP-571 (Celltech Chiroscience), CM-101 (CarboMed), M
L-3000 (Merckle), CB-2431 (KS Biomedix), CBF-BS2 (
KS Biomedix), IL-1Ra gene therapy (Valentis), JTE-522 (Japan
Tobacco), Paclitaxel (Angiotech), DW-166HC (Dong Wha),
Dalbuferone mesylate (Warner-Lambert), soluble TNF receptor 1 (synerg
en; Amgen), IPR-6001 (Institute for Pharmaceutical Research),
Trocade (Hoffman-La Roche), EF-5 (Scotia Pharmaceuticals), BI
IL-284 (Boehringer Ingelheim), BIIF-1149 (Boehringer Ing
elheim), Leuko Vax (Inflammatics), MK-663 (Merck), ST
-1482 (Sigma-Tau), Butyxocortopropionate (Warner-Lambert)
1, 2, 3, 4, 5, 10 or more of the above.

In yet another aspect, the composition of the present invention is provided with the following drugs: methotrexate, sulfasalazine, sodium aurothiomaleate, auranofin, cyclosporine, penicillamine, azathioprine, antimalarial drugs (eg, herein. Listed)), cyclophosphamide, chlorambucil, gold, EMBREL ™
(Etanercept), anti-TNF antibody, and prednisolone 1, 2, 3, 4,
Administer in combination with 5 or more. In a more preferred embodiment, the compositions of the invention are administered in combination with an antimalarial drug, methotrexate, anti-TNF antibody, ENBREL ™ and / or sufrasalazine. In one aspect, the compositions of the invention are administered in combination with methotrexate. In another aspect, the compositions of the invention are administered in combination with an anti-TNF antibody. In another aspect, the compositions of the invention are administered in combination with methotrexate and an anti-TNF antibody. In another aspect, the compositions of the invention are administered in combination with sufrasalazine. In another specific aspect, the compositions of the invention are administered in combination with methotrexate, an anti-TNF antibody, and sufrasalazine. In another aspect, the composition of the invention is treated with ENBREL (
Trademark). In another aspect, the composition of the present invention is provided with ENBREL.
(TM) and methotrexate in combination. In another aspect, the compositions of the invention are administered in combination with ENBREL ™, methotrexate and sufrasalazine. In another aspect, the composition of the invention is ENBREL ™.
, Methotrexate and sofrasalazine in combination. In other embodiments, one or more antimalarial drugs are combined with one of the combinations cited above. In a specific embodiment, the compositions of the invention are administered in combination with an antimalarial drug (eg, hydroxychloroquine), ENBREL ™, methotrexate and sufrasalazine. In another specific embodiment, the compositions of the invention are administered in combination with an antimalarial drug (eg hydroxychloroquine), suflasalazine, an anti-TNF antibody and methotrexate.

In a further aspect, the compositions of the invention are administered in combination with an antibiotic. Antibiotics that can be administered in combination with the compositions of the present invention include tetracycline, metronidazole, amoxicillin, β-lactamase, aminoglycosides, macrolides, quinolones, fluoroquinolones, cephalosporins, erythromycin, ciprofloxacin, and Includes but is not limited to streptomycin.

In a further aspect, the compositions of the invention may be administered alone or in combination with anti-inflammatory agents. Anti-inflammatory agents that can be administered with the compositions of the present invention include glucocorticoids, and non-steroidal anti-inflammatory drugs, aminoarylcarboxylic acid derivatives, arylacetic acid derivatives, arylbutyric acid derivatives, arylcarboxylic acids, arylpropionic acid derivatives. , Pyrazole, pyrazolone, salicylic acid derivative, thiazinecarboxamide, e-acetamidocaproic acid, S-adenosylmethionine, 3-amino-4-hydroxybutyric acid, amixetrin, bendazac, benzidamine, bucolome, difenpyramide, ditazole, emorfazone, Guaiazulene, nabumetone, nimesulide, orgoteine, oxaceprole, paraniline, perisoxal, pifoxime, proquazone, proxazole, and tenidap. There.

In a further aspect, the compositions of the invention are administered in combination with a chemotherapeutic agent. Chemotherapeutic agents that can be administered with the compositions of the present invention include antibiotic derivatives (
For example doxorubicin, bleomycin, daunorubicin, and dactinomycin), antiestrogens (eg tamoxifen); antimetabolites (eg fluorouracil, 5-FU, methotrexate, furoxyuridine, interferon α-2b, glutamic acid, plicamycin, mercaptopurine, And 6-thioguanine); cytotoxic agents (eg, carmustine, BCNU, lomustine, CCNU, cytosine arabinoside, cyclophosphamide, estramustine, hydroxyurea, procarbazine, mitomycin, busulfan, cisplatin, and vincristine sulfate); Hormones (eg, medroxyprogesterone, estramustine sodium phosphate, ethinyl estradiol,
Estradiol, megesterol acetate, methyltestosterone, diethylstilbestol diphosphate, chlorotrianisene, and test lactone);
Nitrogen mustard derivatives (eg mephalen, chlorambucil, mechlorethamine (nitrogen mustard) and thiotepa); steroids and combinations (eg betamethasone sodium phosphate); and others (eg dicarbazine, asparaginase, mitotan, vincristine sulfate, vinblastine sulfate) And etoposide).

In a further aspect, the compositions of the invention are administered in combination with a cytokine.
Cytokines that can be administered in combination with the compositions of the invention include IL-
2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-
12, IL-13, IL-15, anti-CD40, CD40L, IFN-γ and T
NF-α is included but is not limited thereto.

In a further aspect, the compositions of the invention are administered in combination with an angiogenic protein. Angiogenic proteins that can be administered with the compositions of the present invention include:
Glioma-derived growth factor (GDG) disclosed in European Patent No. EP-399816
F), Platelet-derived growth factor-A (disclosed in European Patent No. EP-682110).
PDGF-A), platelet-derived growth factor-B (PDGF-B) disclosed in European Patent No. EP-283217, placental growth factor (PlGF) disclosed in International Publication No. WO92 / 06194; Hauser et.al., Growth Factors, 4: 259-268 (1993), placental growth factor-2 (PlGF-2); International Publication No. WO 90/136.
49. Vascular Endothelial Growth Factor (VEGF); European Patent No. EP-5064.
77. Vascular Endothelial Growth Factor-A (VEGF-A); International Publication No. WO9.
Vascular Endothelial Growth Factor-2 (VEGF-2) disclosed in 6/39515; Vascular Endothelial Growth Factor B-186 (VEGF disclosed in International Publication No. WO96 / 26736).
-B186); vascular endothelial growth factor-D (VEGF-D) disclosed in International Publication No. WO98 / 02543; vascular endothelial growth factor-D (VEGF-D) disclosed in International Publication No. WO98 / 07832; German Patent Included, but not limited to, the vascular endothelial growth factor-E (VEGF-E) disclosed in the number DE19639601. The above references form part of the present description.

In a further aspect, the compositions of the invention are administered in combination with fibroblast growth factor. Fibroblast growth factors that can be administered with the compositions of the invention include FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6.
, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF
-12, FGF-13, FGF-14 and FGF-15 are included but are not limited thereto.

In a further aspect, the compositions of the invention are administered in combination with other therapeutic or prophylactic regimes, such as radiation therapy.

The present invention further includes compositions comprising a polypeptide of the invention (eg, a DR3 polypeptide or anti-DR3 antibody associated with a heterologous polypeptide, a heterologous nucleic acid, a toxin, or a prodrug. Composition), DR3
The present invention provides a method for delivering a membrane-bound form of a target cell that expresses the DR3 receptor on its surface or expresses the DR3 receptor on its surface. DR3 polypeptides or anti-DR3 antibodies of the invention may be associated with heterologous polypeptides, heterologous nucleic acids, toxins or prodrugs by hydrophobic, hydrophilic, ionic and / or covalent interactions. You can

In one aspect, the invention provides that the composition of the invention is administered to cells by administering a polypeptide of the invention that associates with a heterologous polypeptide or nucleic acid (eg, a DR3 or anti-DR3 antibody). A specific delivery method is provided. In one example, the invention provides methods for delivering therapeutic proteins into target cells. In another example, the invention provides a single-stranded nucleic acid (eg, antisense or ribozyme) or 2
Provided are methods for delivering nucleic acids of the present acids (eg, DNA that can integrate into the cell's genome or that is episomally replicable and translatable) into target cells.

In another aspect, the invention provides for the specific destruction of cells by administering a polypeptide of the invention (eg, a DR3 polypeptide or an anti-DR3 antibody, for example) in combination with a toxin or a cytotoxic prodrug. Methods are provided for (eg, destruction of tumor cells).

In a specific aspect, the invention provides that cells that express a membrane bound form of DR3 on their surface (eg, spleen, by administration of an anti-DR3 antibody in combination with a toxin or a cytotoxic prodrug). Methods for specifically destroying bone marrow, kidney and PBL) are provided.

A “toxin” is a compound that binds to and activates an exogenous cytotoxic effector system, a radioisotope, a holotoxin, a modified toxin, a catalytic subunit of a toxin, a cytotoxin (cytotoxic agent), or a defined toxin. It means any molecule or enzyme that normally causes cell death under the conditions described above, but is not present inside the cell or on the cell surface. Toxins that can be used in accordance with the methods of the present invention include radioisotopes known in the art, such as antibodies that bind to native or induced exogenous cytotoxic effector systems (or their complement binding). (Containing portion), thymidine kinase, endonuclease, RNAse, alpha toxin, ricin, abrin, pseudomonas exotoxin A, diphtheria toxin, saporin, momordin, gelonin, pokeweed antiviral protein, α-sarcin and cholera toxin. Not limited to “Toxins” further include cytostatic or cytocidal substances, therapeutic substances or radioactive metal ions, eg α-emitters such as ²¹³ B, or ¹⁰³ Pd, ¹³³ Xe, ¹³¹ I, ⁶⁸ Ge, ⁵⁷ Co, ^65. Zn, ⁸⁵ Sr, ³² P, ³⁵ S, ⁹⁰ Y, ¹⁵³ Sm, ¹⁵³ Gd, ¹⁶⁹ Yb, ⁵¹ C
^{r, 54 Mn, 75 Se,} 113 Sn, 90 ^yttrium, 117 ^{Tin, 18 6 rhenium,} other radioisotopes such as ¹⁶⁶ Holmium, and ¹⁸⁸ rhenium; luminescent labels, such as luminol, fluorescent labels such as fluorescein and rhodamine, and biotin Is included.

Techniques known in the art can be applied to label the proteins (including antibodies) of the present invention. Such techniques include bifunctional binders (eg, US Pat. Nos. 5,756,065; 5,714,631; No. 5,696,239; No. 5,652,361; No. 5,505,931; No. 5,489,425; No. 5,435,990; No. 5,428,139. No. 5,342,604; No. 5,274,119; No. 4,994,560 and No. 5,808,003), but are not limited thereto. Cytotoxins or cytotoxic agents include any agent that is harmful to cells. Examples include paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide,
Includes vincristine, vinblastine, cortisine, doxorubicin, daunorubicin, dihydroxyanthracindione, mitozantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol and puromycin and analogs or homologues thereof. Be done. Therapeutic substances include antimetabolites (eg methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,
5-fluorouracil decarbazine), an alkylating agent (eg, mechlorethamine, thioepachlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclotosfamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and Cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (eg,
Daunorubicin (formally daumomycin) and doxorubicin), antibiotics (eg, dactinomycin (formally actinomycin), bleomycin,
Mitramycin, and anthramycin (AMC)) and cytostatics such as vincristine and vinblastine, but are not limited thereto.

“Cytotoxic prodrug” means a non-toxic compound that is converted into a cytotoxic compound by the enzymes normally present in cells. Cytotoxic prodrugs that can be used in accordance with the methods of the invention include glutamyl derivatives of mustard alkyl benzoates, phosphate derivatives of etoposide or mitomycin C, cytosine arabinoside, daunorubicin, and phenoxyacetamide derivatives of doxorubicin. Include, but are not limited to.

As described in more detail below, the invention further provides a pharmaceutical package or kit comprising one or more containers filled with one or more components of the pharmaceutical compositions of the invention. I will provide a. In some cases, such containers may be accompanied by a written notice in the form prescribed by a governmental agency that regulates the manufacture, use, or sale of pharmaceuticals or biologics, which notice is to humans. It reflects the approval by that agency for the manufacture, use or sale for administration of.

Diagnostic and Imaging Labeled antibodies that specifically bind the polypeptide of interest, and derivatives and analogs thereof, are used for diagnostic purposes and are associated with aberrant expression and / or activity of the polypeptides of the invention. Diseases and / or disorders can be detected, diagnosed or monitored. The invention comprises (a) analyzing the expression of a polypeptide of interest in a human cell or body fluid using one or more antibodies specific for the polypeptide of interest, and (b ) Detecting the aberrant expression of the polypeptide of interest, comprising comparing the level of gene expression to a standard gene expression level, whereby the level of gene expression of the analyzed polypeptide An increase or decrease as compared to the expression level is indicative of aberrant expression, providing detection.

The present invention comprises (a) analyzing the expression of a polypeptide of interest in a human cell or body fluid using one or more antibodies specific for the polypeptide of interest, And (b) a diagnostic assay for diagnosing a disorder comprising comparing the level of gene expression to a standard gene expression level, whereby the standard of gene expression levels of the analyzed polypeptide Provides a diagnostic assay in which an increase or decrease as compared to the expression level of is indicative of a particular disorder. For cancer, the presence of relatively high abundance of transcripts in biopsied tissue taken from a person may indicate a predisposition to the disease, or a means of detecting the disease before actual clinical signs appear. May be provided. This type of more definitive diagnosis allows health professionals to take prophylactic measures or earlier aggressive measures to prevent the development or further progression of cancer.

The antibodies of the invention can be used to analyze protein levels in biological samples using traditional immunohistological methods known to those of skill in the art (
For example, Jalkanen, M. et. Ai, J. Cell. Biol. 101: 976-985 (1985); Jalkan
En, M. et. al., Cell. Biol. 105: 3087-3096 (1987)). Other antibody-based methods useful for detecting gene expression of proteins include enzyme-linked immunosorbent assay (ELISA) and immunoassays such as radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and include enzyme labels such as glucose oxidase, iodine ( ¹²⁵ I, ¹²¹ I), carbon ( ¹⁴ C).
, Sulfur ( ³⁵ S), tritium ( ³ H), indium ( ¹¹² In) and techinium ( ⁹⁹ Tc) and other radioisotopes, luminescent labels such as luminol, and fluorescent labels such as fluorescein and rhodanine and biotin. .

One aspect of the invention is the detection and diagnosis of diseases or disorders associated with aberrant expression of the polypeptide of interest in animals, preferably mammals, and most preferably humans. In one aspect, diagnosis is a) administering to a subject an effective amount of a labeled molecule that specifically binds to a polypeptide of interest (eg, parenterally, subcutaneously or intraperitoneally); b) after administration. , The time interval (where the labeled molecule can be preferentially concentrated at the site in the analyte where the polypeptide is expressed (
And a time interval during which unbound labeled molecules are removed to background levels) waiting; c) measuring background levels; and d)
Comprising measuring labeled molecules in the analyte, such that detection of labeled molecules above background levels was associated with abnormal expression of the polypeptide of interest in the analyte. Indicates having a particular disease or disorder. Background levels can be measured by a variety of methods, including comparing the amount of labeled molecule detected to standard values previously measured for a particular system.

It will be appreciated in the art that the amount of contrast moiety needed to produce a diagnostic image will depend on the size of the subject and the contrast system used. In the case of a radioisotope moiety, for a human subject, the amount of radioactivity injected is 9
It is usually in the range of about 5 to 20 millicuries at 9 mTc. The labeled antibody or antibody fragment will preferentially accumulate at the site of cells containing the particular protein. In vivo tumor imaging is described by SW Burchiel et. Al., "Immunopharmacokinetics of
Radiolabeled Antibodies and Their Fragments. "(Tumor imaging Chapter 13: The Radiochemical Detection of Cancer, SW Burchiel and BA Rhodes
, eds., Masson Publishing Inc. (1982)).

Depending on a number of changes, including the type of label used and the mode of administration, the labeled molecule can be preferentially concentrated at a site in its analyte and is labeled unbound. The time interval after administration until the molecules are cleared to background levels is 6-48 hours or 6-24 hours or 6-12 hours. In another aspect, the time interval after administration is 5 to 20 days or 5 to 10 days.

In some embodiments, the method of diagnosing a disease or disorder is repeated, for example, by repeating the method 1 month after the first diagnosis, 6 months after the first diagnosis, 1 year after the first diagnosis, and the like. Or monitor the failure.

The presence of labeled molecule can be detected in a patient using methods known in the art for in vivo scanning. These methods depend on the type of label used. One of ordinary skill in the art will be able to determine the appropriate method for detecting a particular label. Methods and devices that can be used in the diagnostic methods of the invention include computer tomography (CT), whole body scans such as positron emission tomography (PET), magnetic resonance imaging (MRI) and ultrasonography. Include, but are not limited to.

In a specific embodiment, the molecule is labeled with a radioisotope and measured in a patient using a radio-responsive surgical instrument (Thurston et. Al., US Pat. No. 5,441,050). In another embodiment, the molecule is labeled with a fluorescent compound and detected in a patient using a fluorescence responsive scanning device. In another embodiment, the molecule is labeled with a positron emitting metal and detected in the patient using positron emission tomography. In yet another aspect, the molecule is labeled with a paramagnetic label and detected in the patient using magnetic resonance imaging (MRI).

Kits The present invention provides kits that can be used in the above methods. In one aspect, the kit comprises an antibody of the invention, preferably a purified antibody, in one or more containers. In a specific embodiment, the kit of the present invention comprises a substantially purified polypeptide comprising an epitope having specific immunoreactivity for the antibody contained in the kit. Preferably, the kit of the present invention further comprises a control antibody that does not react with the polypeptide of interest. In another specific embodiment, the kit of the invention comprises means for detecting the binding of the antibody to the polypeptide of interest (eg, the antibody is a fluorescent compound, a substrate for an enzyme, a radioactive compound or a luminescent compound, etc. Or a secondary antibody that recognizes the primary antibody may bind to the detectable substrate).

In another specific aspect of the invention, the kit is a diagnostic kit for use in screening sera containing antibodies specific for proliferative and / or cancerous polynucleotides and polypeptides. Is. Such a kit may include a control antibody that does not react with the polypeptide of interest. Such a kit
It may comprise a substantially isolated polypeptide antigen comprising an epitope having specific immunoreactivity to at least one anti-polypeptide antigen antibody. Further, such kits may include means for detecting the binding of the antibody to the antigen (eg, the antibody may be conjugated with a fluorescent compound such as fluorescein or rhodamine that can be detected by flow cytometry). Contains.
In a specific embodiment, the kit may include recombinantly produced or chemically synthesized polypeptide antigens. The polypeptide antigens of the kit may also be attached to a solid support.

In a more specific aspect, the detection means of the above kit comprises a solid support to which the polypeptide antigen is bound. Such a kit may include an anti-human antibody labeled with an unbound reporter. In this aspect, binding of the antibody to the polypeptide antigen can be detected by binding of the antibody labeled with the reporter.

In a further aspect, the invention includes a diagnostic kit for use in screening sera containing an antigen of a polypeptide of the invention. The diagnostic kit comprises a substantially isolated antibody having specific immunoreactivity for a polypeptide or polynucleotide antigen, and means for detecting the polynucleotide or polypeptide antigen for that antibody. In one aspect, the antibody is attached to a solid support. In a specific aspect, the antibody is a monoclonal antibody. The detection means of the kit may include a second labeled monoclonal antibody. Alternatively, or additionally, the detection means may include a labeled competing antigen.

In one of the diagnostic forms, the test serum is reacted with a solid phase reagent having the surface bound antigen obtained by the method of the invention. After binding to the antigen-antibody specific to the reagent, unbound serum components were removed by washing, and the reagent was reacted with a reporter-labeled anti-human antibody to bind to the solid support. A reporter is bound to the substance depending on the amount of anti-antigen antibody. The reagent is washed again to remove unbound labeled antibody and the amount of reporter associated with the reagent is measured. Typically,
The reporter may be any suitable fluorescent, luminescent or chromogenic substrate (Sigma, St. Louis.
, MO) is an enzyme detected by incubation in the presence of.

Solid surface reagents in the above analysis were polymer beads, dipstick, 9
Produced by known techniques for attaching protein materials to solid support materials such as 6-well plates or filter media. These coupling methods generally include
Non-specific adsorption of proteins to a support, chemically reactive groups on the solid support of the protein, typically via free amine groups, such as activated carboxyl, hydroxyl or aldehyde groups. Covalent bond of. Alternatively, streptavidin-coated plates can be used in combination with biotinylated antigen.

Thus, the present invention provides an analytical system or kit for carrying out this diagnostic method. The kit generally comprises a support having surface-bound recombinant antigens, and a reporter-labeled anti-human antibody for detecting surface-bound anti-antigen antibody.

Example 1 Expression and Purification in Escherichia coli A DNA sequence encoding the mature DR3-V1 protein in the cDNA contained in ATCC 97456 was prepared using a PCR oligonucleotide primer specific to the amino-terminal sequence of the DR3-V1 protein and its gene. Amplify using primers specific to the 3'vector sequence of Additional nucleotides, including restriction sites, are added to the 5'and 3'sequences, respectively, to facilitate cloning.

The following primers were used for expression of the DR3 extracellular domain in E. coli:
5'-primer: 5'-GCGCCATGGGGGGCCCGGCGGCAG-
The 3 '(SEQ ID NO: 7) includes an NcoI site and 15 nucleotides starting from 290 nucleotides of SEQ ID NO: 1 to 304. 3'-primer: 5'-G
CGAAGCTTTCTAGGACCCAGAACATCTGCC-3 '(SEQ ID NO: 8) is a HindIII site, a stop codon and 822 to 840 of SEQ ID NO: 1.
18 nucleotides complementary to the nucleotides up to. This protein is untagged.

The restriction site is a bacterial expression vector p used in this example for bacterial expression.
QE60 (Qiagen, 9259 Eton Avenue, Chatswo
rth, CA91311) as a restriction enzyme site. pQE6
0 codes for ampicillin antibiotic resistance (“Amp ^r ”), which is a bacterial origin of replication (“
ori "), an IPTG inducible promoter, a ribosome binding site (" RBS ").

Both the amplified DR3-V1 DNA and the vector pQE60 are digested with NcoI and HindIII, then the digested DNAs are ligated together. Limit pQE
60 Insertion of the DDCR protein DNA into the vector operably linked the region encoding the DR3-V1 protein downstream of the IPTG inducible promoter of the vector to form an initiation AUG in the proper configuration for translation of the DR3-V1 protein. Place in frame.

The ligation mixture is transformed into competent E. coli cells using standard procedures. Such an operation method is described in “Molecular Clon” by Sambrook et al.
ing: a Laboratory Manual ", 2nd Edition, Cold Spr
ing Harbor Laboratory Press, Cold Sp
ring Harbor, N.M. Y. (1989). E. coli strain M15 / rep4 carries multiple copies of the plasmid pREP4, which expresses the lac repressor and confers kanamycin resistance ("Kan ^r "), which is used in performing the exemplary examples described herein. This strain is only one of many suitable for expression of the DR3-V1 protein, but is commercially available from Qiagen.

Transformants are confirmed 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 of the cloned DNA is confirmed by restriction analysis.

Clones containing the desired construct were allowed to grow overnight (“O / N”) in ampicillin (100 μm).
g / mL) and kanamycin (25 μg / mL) are grown in liquid cultures of LB medium.

Large scale cultures are inoculated with O / N cultures at a dilution of about 1: 100 to 1: 250. The cells are cultured to an optical density at 600 nm (“OD600”) of between 0.4 and 0.6. Next, isopropyl-BD-thiogalactopyranoside ("IPTG") is added to a final concentration of 1 mM to inactivate the lacI repressor and induce transcription from the lac repressor sensitive promoter. The cells are subsequently incubated for a further 3 to 4 hours. Then using the standard method,
Cells are harvested by centrifugation and disrupted. Inclusion bodies are purified from disrupted cells using conventional recovery techniques to solubilize the protein from inclusion bodies in 8M-urea. The 8M-urea solution containing the solubilized protein is passed through a PD-10 column in 2x phosphate buffered saline ("PBS") to remove urea, replace the buffer and refold the protein. The protein is purified in a further chromatographic step to remove endotoxin. Then sterile filter. Sterile filtered protein preparation in 2 × PBS 95 μ / m
Store at L concentration.

Example 2 Expression in Mammalian Cells Most of the vectors used to transiently express a given gene sequence in mammalian cells have an SV40 origin of replication. This allows the vector to replicate to high copy numbers in cells that express the T antigen required for initiation of viral DNA synthesis (eg, COS cells). Any other mammalian cell line may be utilized for this purpose.

A typical mammalian expression vector contains promoter elements that mediate the initiation of transcription of the mRNA, protein coding sequences, and signals necessary for termination of transcription and polyadenylation of the transcript. Other elements are enhancers, Koza
It contains an intervening sequence to which the donor and acceptor sites for splicing the k sequence and RNA are attached. Very efficient transcription can be achieved with early and late promoters from SV40, such as the long terminal repeat (LTR) from retroviruses such as RSV, HTL VI, HIVI and the early promoter of cytomegalovirus (CMV). However, cellular signals (eg human actin, promoter) can also be used. Expression vectors suitable for use in practicing the present invention include, for example, pSVL and pMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC37152), pSV2dhfr.
(ATCC37146), and vectors such as pBC12MI (ATCC67109). Mammalian host cells that can be used include human HeLa, 283,
H9 and Jurkat cells, mouse NIH3T3 and C127 cells, Co
s1, Cos7 and CV1, African green monkey cells, quail QC1-3 cells, mouse L cells and Chinese hamster ovary (CHO) cells.

Alternatively, the gene of interest can be expressed in stable cell lines containing the gene integrated into a chromosome. For example, co-transfection with selectable markers such as dhft, gpt, neomycin, hygromycin can identify and separate transfected cells.

The transfected gene can also be amplified to express large amounts of the encoded protein. DHFR (dihydrofolate reductase) is a useful marker for developing cell lines that carry hundreds to thousands of copies of the gene of interest. This marker is used to culture mammalian cells with increasing amounts of methotrexate for selection and to select the cells with the highest degree of resistance. These cell lines contain an amplified gene integrated into the chromosome. Chinese hamster ovary (CHO) cells are frequently used for protein production.

The expression vectors pC1 and pC4 represent the Rous sarcoma virus strong promoter (LTR) (Cullen et al., Molecular and Cellular).
r Biology, 438: 44701 (March 1985)) and CM
Fragment of V-enhancer (Boshart et al., Cell, 41: 521-5)
30 pages (1985)). For example, restriction enzyme cleavage sites BamHI, Xba
Multiple cloning sites with I and Asp718 etc. facilitate cloning of the gene of interest. The vector further contains the 3'-intron, polyadenylation and termination signals of the rat preproinsulin gene.

Example 2A Expression of extracellular soluble domains of DR3-V1 and DR3 in COS cells Expression plasmid pDR3-V1 HA is a cDNA encoding DR3-V1.
(ATCC97456) as an expression vector pcDNAI / Amp (Invitro
(available from Gen). The expression plasmid pDR3 HA was prepared by cloning the cDNA encoding the DR3 (ATCC97757) into the expression vector pcDNAI / Amp.

The expression vector pcDNAI / amp comprises (1) an E. coli origin of replication effective for growth in E. coli and other prokaryotic cells, (2) an ampicillin resistance gene for selection of plasmid-containing prokaryotic cells, (3) SV40 origin of replication for growth in eukaryotic cells, (4) cDNA is conveniently placed under expression control of the CMV promoter utilizing a restriction site in the polylinker, the SV40 intron and polyadenyl A CMV promoter, a polylinker, an SV40 intron, and a polyadenylation signal arranged to be operably linked to the activation signal.

A DNA fragment encoding the entire DR3-V1 or Dr3 precursor and the HA tag fused in frame to its 3'-end was cloned into the polylinker region of the vector, and recombinant protein expression was directed by the CMV promoter. To do so. HA tags are described in Wilson et al., Cell, 37: 767 (1984).
Corresponding to the epitope derived from the influenza hemagglutinin protein described in 1. Fusion of the HA tag to the target protein allows easy detection of the recombinant protein by an antibody that recognizes the HA epitope.

The plasmid construction plan is as follows: The deposited cDNA DR3-V1 or DR3 cDNA was transformed into E. coli and S. Amplify using primers containing convenient restriction sites as described above for construction of expression vectors for expression of DR3-V1 or DR3 in Fuguiperda.

To facilitate detection, purification and characterization of the expressed DR3-V1 or DR3, one of the primers contains a hemagglutinin tag (“HA tag”) as described above.

Suitable primers for DR3-V1 used in this example include: 5′-primer: 5′-CGC GGATCC GCCATCATGGAGGGAG.
AGCCAGCAG-3 '(SEQ ID NO: 9) contains an underlined BamHI site, an ATG start codon followed by 5 codons.

Suitable primers for DR3 used in this example include: 5'-Primer: 5'-CGCGGATCCGCGCATCATGGAGGCAGCGGGCC.
GCGG-3 '(SEQ ID NO: 10) contains an underlined BamHI site, an ATG start codon followed by 5 codons.

The 3'-primers for both DR3 and DR3-V1 are underlined with XbaI.
It contains a site, a stop codon, a hemagglutinin tag and the last 14 nucleotides of the 3'-coding sequence (3'-end) and has the following sequence: 5'-GCG TCTAGA TCAAAGCGTAGTCTGGGACGTCGT.
ATGGGTACGGGCCGCGCTGCA-3 '(SEQ ID NO: 11).

The PCR amplified DNA fragment and the vector pcDNAI / Amp were added to BamHI.
And digest with XbaI and ligate. The ligation mixture was transformed into E. coli SURE strain (Stra
tagene · Cloning · Systems, 11099 · North ·
Available from Torrey Pines Road, La Jolla, CA 92037)
And the transformed culture is plated on a plate of ampicillin medium, which is then incubated to grow ampicillin resistant colonies. Isolation of plasmid DNA from resistant colonies and DR by restriction analysis and gel sizing
Check for the presence of fragments encoding 3-V1 or DR3.

For expression of recombinant DR3-V1 or DR3, expression vectors were used in COS cells as described above using DEAE-DEXTRAN, eg Sambrook.
k, etc., Molecular Cloning: a Laboratory M
annual, Cold Spring Harbor Laboratory
Press, Cold Spring Spring, N.M. Y. (1989) for transfection.

Cells are incubated under conditions where this vector expresses DR3-V1 or DR3.

Expression of the DR3-V1HA fusion protein or the DR3HA fusion protein can be achieved by
Antibodies: a Laboratory Manu, such as arlow
al, 2nd edition, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.M. Y. Detection by radiolabeling and immunoprecipitation using the method described in (1989). Here, 2 days after transfection, the cells are labeled by incubation for 8 hours in medium containing ³⁵ S-cysteine. Take cells and medium and place cells in Wilson et al., RIPA buffer containing detergent as described above: 1
50 mM-NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.
Wash with 5% DOC, 50 mM-TRIS pH 7.5 and dissolve. Proteins are precipitated from cell lysates and culture media using HA-specific monoclonal antibodies. The precipitated protein is then analyzed by SDS-PAGE gel and autoradiography. Expression products of the expected size are found in cell lysates,
This is not seen in the negative control.

Example 2B Expression and Purification of Human DR3-V1 and DR3 Using CHO Expression System DR3-V1 or DR3 (ATCC97456 or ATCC97 respectively)
757) Use vector pC1 for protein expression. Plasmid pC1 is a derivative of plasmid pSV2-dhfr [ATCC Accession No. 37146]. Both plasmids contain the mouse DHFR gene under the control of the SV40 early promoter. Chinese hamster ovary cells deficient in dihydrofolate activity or other cells transfected with these plasmids were transfected with selective medium (alpha minus MEM, Life Te;
chnologies). There is good literature on amplification of the DHFR gene in cells that are resistant to methotrexate (MTX) (eg, FW Alt et al., J. Biol. Chem., 253: 1.
Pp. 357-1370 (1978), J. Am. L. Hamlin and C.I. Ma, B
iochem. et Biophys Acta, 1097 vol: 107-143
Page (1990), M.G. J. Page and M.M. A. Sydenham, Bio
technology, 9: 64-68 (1991)). When cultured with increasing MTX concentration, the cells show the target enzyme DHF as a result of DHFR gene amplification.
Overproduction of R develops resistance to this drug. If the second gene is linked to the DHFR gene, it too will usually be co-amplified and overexpressed.
Developing cell lines with more than 1000 copies of the gene belongs to the current state of the art. Upon subsequent removal of methotrexate, the cell line contains the amplified gene integrated into the chromosome.

Plasmid pC1 is a strong promoter of the Rous sarcoma virus long terminal repeat (LTR) for expression of the gene of interest (Cullen et al., Molecular.
and Cellular Biology, pp. 438-447 (March 1985)
Mon)), and fragments isolated from enhancers of the human cytomegalovirus (CMV) immediate-early gene (Boshart et al., Cell 41: 521-53).
Page 0 (1985)). Downstream of this promoter there is a single restriction enzyme cleavage site capable of gene integration: BamHI, followed by the 3'-intron and the polyadenylation site of the rat preproinsulin gene. Other efficient promoters can also be used for expression, such as the human β-actin promoter, the SV40 early or late promoter, or the long terminal repeats from other retroviruses such as HIV and HTLVI. Other signals can be used as well for polyadenylation of the mRNA, such as from the human growth hormone or globin genes.

Stable cell lines carrying the gene of interest integrated into the chromosome are also found, for example in gpt,
It can be selected upon cotransfection with a selectable marker such as G418 or hygromycin. It is advantageous to initially use more than one selectable marker, eg G418 and methotrexate.

Plasmid pC1 is digested with the restriction enzyme BamHI and then dephosphorylated using engineered methods known in the art using calf intestinal phosphatase. The vector is then separated from a 1% agarose gel.

DNA encoding DR3-V1 or DR3 in the deposited cDNA clone
The sequence is amplified using a PCR oligonucleotide primer specific for the amino acid carboxyl terminal sequence of the DR3-V1 or DR3 protein and a PCR oligonucleotide primer specific for the 3'-vector sequence of 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 is BamH underlined.
Sequence containing the I restriction site: 5'-CGC GGATCC GCCATCATGGAGG
AGACGCAGCAG-3 '(SEQ ID NO: 12), which encodes an 18-nucleotide sequence of the DR3-V1 coding sequence set forth in SEQ ID NO: 1 beginning with the initiation AUG followed by the Kozak sequence and the first base of the ATG codon. .

The 5'-oligonucleotide primer for DR3 is the sequence containing the underlined BamHI restriction site: 5'-CGC GGATCC GCCATCATGGAGGCAGC.
GGCCGCGG-3 ′ (SEQ ID NO: 13), which encodes the starting AUG followed by the Kozak sequence and the 18 nucleotide sequence of the DR3 coding sequence set forth in SEQ ID NO: 3 starting at the first base of the ATG codon.

The 3'-primers for DR3 and DR3-V1 are complementary to the underlined BamHI restriction site followed by the last 14 nucleotides of the coding sequence for DR3-V1 or DR3 shown in SEQ ID NO: 1 or SEQ ID NO: 3, respectively. 17 nucleotides, and a sequence containing a stop codon: 5'-CGC GGATCC TCACGGGGCC
It has GCGCTGCA-3 '(SEQ ID NO: 14).

[0470]   This restriction site favors a restriction enzyme site in the CHO expression vector pC1.

Amplified DR3 or DR3-V1 DNA and vector pC1 were both Ba
Digest with mHI and then ligate the digested DNAs together. DR3-V1 or D
Insertion of R3 DNA into BamHI restriction vector results in DR3-V1 or DR3
The coding region is located downstream and operably of the promoter of this vector. The sequence of the inserted gene is confirmed by DNA sequencing.

Transfection of CHO-DHFR-cells Chinese hamster ovary cells lacking active DHFR enzyme are used for transfection. Using the lipofection method (Felgner et al., Supra), 5 μg of the expression plasmid C1 was added to 0.5 μg of the plasmid pSVneo.
And co-transfect. Plasmid pSV-neo contains a dominant selectable marker, the gene neo from Tn5 which encodes an enzyme that confers resistance to a group of antibiotics including G418. The cells are inoculated into Alpha Minus MEM supplemented with 1 mg / mL G418. Two days later, the cells were trypsinized and inoculated on a hybridoma cloning plate (Greiner, Germany) to give 10 cells.
Culture for ~ 14 days. After this period, single clones were trypsinized and then methotrexate was added at various concentrations (25 nM, 50 nM, 100 nM, 200 nM,
Inoculate 6-well Petri dishes added at 400 nM). The clones growing at the highest concentration of methotrexate were then transferred to the higher concentrations of methotrexate (500 nM, 1
μM, 2 μM, 5 μM) in a new 6-well plate. The same procedure is repeated until the clone grows at a concentration of 100 μM.

Expression of the desired gene product is analyzed by Western blot analysis and SDS-PAGE.

Example 3 Cloning and Expression of Soluble Extracellular Domains of DR3-V1 and DR3 in Baculovirus Expression System Deposited clones (ATCC97456 and ATCC97757, respectively)
Encoding the soluble extracellular domain of the DR3-V1 or DR3 protein in the
The NA sequence is amplified using PCR oligonucleotide primers corresponding to the 5'- and 3'-sequences of the gene.

The DR3-V1 5′-primer was underlined with a BamHI restriction enzyme site followed by K
Sequence containing many bases of ozak sequence and sequence of DR3-V1 of SEQ ID NO: 5
'-CGC GGATCC GCCATCATGGAGGAGACGCAGCAG-
It has 3 '(SEQ ID NO: 15). The 5'-end of the amplified fragment encoding DR3-V1 is inserted into an expression vector as described below to give an efficient signal peptide. M. Kozak, J .; Mol. Biol. 196: 947-950 (1
987) and an efficient signal for initiation of translation in eukaryotic cells, as described in (1987), is appropriately placed in the vector portion of the construct.

The DR3 5'-primer was underlined with a BamHI restriction site followed by Koza.
Sequence containing a large number of bases of the k sequence and the sequence of DR3 of SEQ ID NO: 3: 5′-CGCG
It has GATCCGCCATCATGGAGGCAGCGGCGCCG-3 '(SEQ ID NO: 16). The 5'-end of the amplified fragment encoding DR3 is inserted into an expression vector as described below to give an efficient signal peptide. M. Kozak,
J. Mol. Biol. 196: 947-950 (1987), an efficient signal is optionally placed in the vector portion of the construct for translation initiation in eukaryotic cells.

The 3'-primers for both DR3 and DR3-V1 are underlined with XbaI.
Restriction site followed by DR3-shown in SEQ ID NO: 1 or SEQ ID NO: 3, respectively
A sequence containing a nucleotide complementary to the V1 or DR3 nucleotide sequence, followed by a stop codon: 5′-GCG AGATCT AGTCTGGACCCAGAACA
It has TCTGCCTCC-3 '(SEQ ID NO: 17).

The amplified fragment was used in a commercially available kit ("Geneclean", BIO
101 company, La Jolla, Calif.) Is used to separate from a 1% agarose gel. The fragment is then digested with BamHI and Asp718 and again purified on a 1% agarose gel. This fragment is designated herein as F2.

Using the vector pA2, for example, Summers, A Manual, etc.
of Methods for Baculovirus Vectors
and Insect Cell Culture Procedures, T
exas Agricultural Experimental Stati
on Bulletin, 1555 (1987) using standard methods to express the DR3-V1 or DR3 protein in a baculovirus expression system. This expression vector is an Autograph California nuclear polyhedrosis virus (Autograph californica nuclear).
A powerful polyhedron of polyhedrosis virus (ACMNPV) (
polyhedron) promoter, followed by convenient restriction sites. For easy selection of recombinant viruses, the β-galactosidase gene from E. coli is inserted in the same orientation as the polyhedral promoter, followed by the polyadenylation signal of the polyhedral gene. This polyhedron sequence is flanked by viral sequences for cell-mediated homologous recombination with wild-type viral DNA to produce viable virus expressing the cloned polynucleotide at both ends.

If desired, the construct provides appropriately positioned signals for transcription, translation, trafficking, etc., such as in-frame AUG and signal peptides,
As those skilled in the art will recognize, instead of pA2, for example pAc373, pVL9
Numerous other baculovirus vectors such as 41 and pAcIM1 can be used. These vectors have been described by Virology, such as Luckow, among others.
170: 31-39 (1989).

This plasmid is digested with the restriction enzymes BamHI and XbaI using routine procedures known in the art and then dephosphorylated with bovine small intestine phosphatase. This DNA is then used in a commercially available kit ("Geneclean", B
Separation from 1% agarose gel using IO101, La Jolla, CA). This vector DNA is designated herein as "V2".

Fragment F2 and dephosphorylated plasmid V2 are ligated together with T4 DNA ligase. E. coli HB101 cells are transformed with the ligation mixture and spread on culture plates. Bacteria containing this plasmid are confirmed by the human DDCR gene by digesting the DNA from each colony with BamHI and XbaI and then analyzing the digestion products by gel electrophoresis. The sequence of the cloned fragment is confirmed by DNA sequencing. This plasmid is designated herein as pBacDR3-V1 or pBacDR3.

Felgner et al., Proc. Natl. Acad. Sci. USA, 84
Volume: 7413-7417 (1987) using the lipofection method to obtain 5 μg of pBacDR3-V1 or pBacDR3 at 1.0 μg of commercially available linear baculovirus DNA (“BaculoGold ^™ baculovirus DNA”). , Pharmingen, San Diego, CA). BaculoGold ^™ viral DNA (1 μg)
And plasmid pBacDR3-V1 (5 μg) in 50 μL of serum-free Grac
e medium (Life Technologies, Gaithersburg,
MD) in a sterile well of a microtiter plate. afterwards,
Lipofectin (10 μL) and Grace's medium (90 μL) are added, mixed and incubated for 15 minutes at room temperature. The transfection mixture is then added drop-wise to Sf9 insect cells (ATCC CRL1711) and inoculated into 35 mm tissue culture plates with 1 mL of serum-free Grace's medium. Rock the plate back and forth to mix with the freshly added solution. The plate is then incubated at 27 ° C for 5 hours. After 5 hours, the transfection solution is removed from the plate and 1 mL of Grace insect medium supplemented with 10% fetal bovine serum is added. The plate is returned to the incubator and the culture is continued at 27 ° C for 4 days.

After 4 days, supernatants are collected and plaque assayed as described by Summers and Smith, supra. "Blue Gal" (Life Technology)
The agarose gel supplemented with Ogies, Gaithersburg) is used to easily identify and isolate gal-expressing clones that produce blue-stained plaques (for a detailed description of this type of "plaque assay", see Life Technology).
can be found on pages 9-10 of the User's Guide for Insect Cell Culture and Baculovirology, distributed by Geies, Gaithersburg).

Four days after serial dilution, virus is added to the cells. After a suitable incubation, the blue stained plaques are picked up with an Eppendorf pipette tip. The agar containing the recombinant virus is then resuspended in an Eppendorf tube containing 200 μL Grace's medium. Briefly centrifuge to remove agar and use supernatant containing recombinant baculovirus to infect Sf9 cells seeded in 35 mm dishes. After 4 days, the supernatants of these culture dishes are collected and then stored at 4 ° C. D inserted correctly
Clones containing R3-V1 or DR3 are D containing restriction mapping and sequencing
Confirm by NA analysis. This is referred to as V-DR3-V1 or V-D in this specification.
Name it R3.

Sf9 cells are grown in Grace medium supplemented with 10% heat-inactivated FBS. Cells are infected with recombinant baculovirus V-DR3-V1 at a multiplicity of infection ("MOI") of about 2 (about 1 to about 3). After 6 hours, the medium was removed, and SF900II medium containing no methionine and cysteine (Life Technologies, G.
(available from aisersburg). After 42 hours, ³⁵ S-methionine (5 μCi) and ³⁵ S-cysteine (5 μCi) (Amersha).
(available from Company m). The cells are incubated for a further 16 hours, then harvested by centrifugation, lysed and the labeled protein visualized by SDS-PAGE and autoradiography.

Example 4 A. Tissue distribution of DR3-V1 gene expression. Northern blot analysis was performed and among others the DR3-V1 gene (ATCC97 in human tissues using the method described by Sambrook et al., Supra).
456) Examine expression. The cDNA probe containing the entire nucleotide sequence of the DR3-V1 protein (SEQ ID NO: 1), using the rediprime ^{T M} DNA labeling system according to the manufacturer's instructions (Amersham Life Science, Inc.) labeled with ³² P. After labeling, the probe is purified using a CHROMA SPIN-100 ™ column (Clontech Laboratories) according to the manufacturer's protocol PT1200-1. The purified labeled probe is then used to test various human tissues for DR3-V1 mRNA.

Multi-tissue Northern (MTN) blots containing various human tissues (H) or human immune system tissues (IM) were obtained from Clontech and manufactured according to the manufacturer's protocol PT.
Probing with labeled probe using ExpressHyb ^™ Hybridization Solution (Clontech) according to 1190-1. Following hybridization and washing, the blot is mounted and the film is exposed overnight at -70 ° C and the film is developed according to standard procedures. DR3-V1 expression was detected in lymphocyte-rich tissues including peripheral blood leukocytes (PBL), thymus, spleen, large intestine, and small intestine. Expression of DR3-V1 appears to be restricted to the lymphocyte fraction,
It can be foreseen that 3-V1 plays a role in lymphocyte homeostasis.

B. Tissue distribution of DR3 gene expression Northern blot analysis was performed, among which the DR3 gene in human tissues (ATCC 97757) using the method described by Sambrook et al., Supra.
Examine expression. CD containing the entire nucleotide sequence of DR3 protein (SEQ ID NO: 1)
The NA probe is labeled with ³² P using the rediprime ^™ DNA labeling system (Amersham Life Science) according to the manufacturer's instructions. After labeling, CHROMA SPIN-100 ^™ column (Clonte
ch. Laboratories) is a manufacturer's protocol PT1200-1
Purify the probe using The purified labeled probe is then used to test various human tissues for DR3 mRNA.

Multi-tissue Northern (MTN) blots containing various human tissues (H) or human immune system tissues (IM) were obtained from Clontech and manufactured according to the manufacturer's protocol PT.
Probing with labeled probe using ExpressHyb ^™ Hybridization Solution (Clontech) according to 1190-1. Following hybridization and washing, the blot is mounted and the film is exposed overnight at -70 ° C and the film is developed according to standard procedures.

DR3 expression was detected in lymphocyte-rich tissues including peripheral blood leukocytes (PBL), thymus, spleen, large intestine, and small intestine. In contrast, TNFR-1 is expressed here and there and Fas / APO-1 is expressed in lymphocytes, liver, heart, lung, kidney, and ovary (Watanabe-Fukunaga et al., J. Immunol.
． 148: 1274-1-9 (1992)).

Expression of DR3 appears to be restricted to lymphocyte compartments and one could foresee that DR3 plays a role in lymphocyte homeostasis.

C. Northern Blot Analysis of DR3 in Various Cell Lines Method Cells Unless stated otherwise, cell lines were obtained from the American Type Culture Collection (Manussass, VA). Bone marrow studied (Koeffler et al. (1980), Koeffler (1983), Harris and Ra.
lph (1985) as well as Tucker et al. (1987)) and B cell lines (Jonak et al. 1922) are representative cell types at various stages of the differentiation pathway. KG1a and PLB985 cells (Tucker et al. (1987
H). P. Obtained from Koeffler (UCLA School of Medicine). BJA
-B is Z. This is from Jonak (Smith Kline Beecham). The stromal cell line TF274, which exhibits osteoblastic properties, was prepared from bone marrow of healthy male donors (Z. Jonak and KB Tan, unpublished). Primary carotid endothelial cells were purchased from Clonetics (San Diego, Calif.) And monocytes were prepared by differential centrifugation of peripheral blood mononuclear cells and adherence to tissue culture dishes. CD19 +, C
D4 + and CD8 + cells (> 90% pure) are cell-type specific immunomagnetic beads (D
Rynal, Lake Sacees, NY).

RNA Analysis Total RNA from adult tissues was purchased from Clonetech (Palo Alto, CA). Total RNA for cell lines (exponential growth phase) and primary cells for TriReagen
t (Molecular Research Center, Cincinnati,
OH). Known method with slight modification of 5 μg to 7.5 μg of total RNA (
Wide, Mini-Sub, Cell gel tray (Sambrook, etc.)
Fractionation on a 1% agarose gel with formaldehyde cast in Bio-Rad, Hercules, CA). Reduce the formaldehyde concentration to 0.5M and add 100 μg / mL ethidium bromide to the loading buffer.
NA was stained before electrophoresis. Electrophoresis with continuous buffer recirculation (60 Volts / 9
After 0 minutes), the gel was photographed, and RNA was quantitatively transferred to Zeta-probe nylon membrane (Biorad, Hercules, CA) by vacuum blotting with 25 mM-NaOH for 90 minutes. After neutralization with 1 M Tris-HCl containing 3 M NaCl, pH 7.5 for 5-10 minutes, the blots are stained with 50% formamide, 8%.
Dextran sulfate, 6 × SSPE, 0.1% SDS and cleaved modified salmon sperm DN
Prehybridized with 100 μg / mL A at 42 ° C. for at least 30 minutes. The cDNA insert labeled with ³² P-dCTP (Stratagene, La Jolla, CA) by random priming was denatured with 0.25 M NaOH (3
(10 minutes at 7 ° C) and added to the prehybridization solution. 2 at 42 ° C
After 4-65 hours, blots were washed under high stringency conditions (Sambr).
, etc.) and exposed to X-ray film.

Results DR3 was expressed in cell lines: TF274 (bone marrow stroma); MG63, TE85 (osteosarcoma); K562 (erythrocytes); KG1a, KG1, PLB985, HL60, U9.
37, TNHP-1 (bone marrow); REH, BJAB, Raji, IM-9 (B cells); Sup-T1, Jurkat, H9, Molt-3 (T cells); RL95-
2 (endometrial cancer); MCF-7 (breast cancer); BE, HT29 (colon cancer); IMR3
2 (neuroblastoma) was evaluated by Northern blot and could only be detected with KG1a. Expression of DR3 was detected in several lymphoblastoid cell lines. DR3 was weakly expressed in CD19 + cells in the purified human hematopoietic cell population and more strongly in monocytes. However, when stimulated with PMA and PHA, T cells (C
Highest levels were observed in D4 + or CD8 +), probably indicating that DR3 plays a role in regulating T cell activation.

Example 5 Intracellular Signaling Molecules Used by DR3 Proteins In vitro and in vivo binding studies were performed to study the DR3 signaling pathway. Since DR3 contains a death domain, we have shown that by recruiting DR3 like TNFR-1 and Fas / APO-1, death domain containing adapter molecules (DAM) such as FADD, TRADD and RIP. I hypothesized that it might transmit a signal.

Experimental Design In vitro binding experiments were previously reported (AM Chinnayan et al., Ce.
11, 81: 505-12 (1995), M.A. P. Boldin, J
． Biol. Chem. 270: 7795-8 (1995), F.I. C.
It was carried out as described in Kischkel et al., EMBO 14: 5579-5588 (1995)). Briefly, the DR3 cytoplasmic domain (amino acid residues 215 to 393 (SEQ ID NO: 4)) and the death domain mutant ΔDR3 (amino acid residues 215 to 321 (SEQ ID NO: 4)) were transformed into a suitable template and pGSTAG. Amplified by PCR using primers. pGSTag and pGSTa
g-TNFR-1 has been previously reported (AM Chinnaiyan et al., Cell, 81: 505-12 (1995), MP Boldin et al., J. Biol. Chem., 270). : 7795-8 pages (1995), F
． C. Kischkel et al., EMBO, 14: 5579-5588 (19.
1995)). GST and GST fusion proteins are E. coli strain BL21 (DE3) pL
Prepared from ysS using standard published procedures, recombinant proteins were immobilized on glutathione-agarose beads. ³⁵ S-labeled FADD, RIP and T
RADD is pcDNA3AU1-FADD (AM Chinnaiyan et al., Cell, 81: 505-12 (1995), MP Boldin et al., J. Biol. Chem., 270: 7795-8 (1995). Year), F
． C. Kischkel et al., EMBO, 14: 5579-5588 (19.
1995)), pRKmyc-TRADD (H.Hsu et al., Cell, 81:
495-504 (1995)), or pRKmyc-RIP (H. Hsu).
Immunity, 4: Vol .: 387-396 (1996)) as template, prepared by in vitro transcription-translation using Promega's TNT or T7 or SP6-linked reticulocyte lysate system according to the manufacturer's instructions. did. After translation, an equivalent amount of total ³⁵ S-labeled reticulocyte lysate was added to GST binding buffer (50 mM-Tris, pH 7.6, 120 mM NaCl, 1% -NP-4).
0) Dilute to 150 μL and mix with various GST fusion proteins bound to beads at 4 ° C
Incubation for 2 hours at 37 ° C., followed by pelleting the beads by plus centrifugation, washing 3 times with GST buffer, boiling in SDS-sample buffer, 12.5
Separated on% SDS-PAGE. Bound proteins were visualized by subsequent autoradiography at -80 ° C. In vitro translated ³⁵ S-labeled RI
P, TRADD and FADD are GST alone or Fas, TNFR-1, DR
3 (215-393), or incubated with glutathione beads containing GST fusions of the plasma domain of DDR3 (215-321). After washing the beads, the retained proteins were analyzed by SDS-PAGE and autoradiography. The gel was Coomassie stained to monitor loading uniformity.

To demonstrate DR3 and TRADD association in vivo, Flag-T
Constructs encoding NFR-1 and Flag-ΔTNFR-1 were used. F
The constructs of lag-TNFR-1 and Flag-ΔTNFR-1 have been previously described (
A. M. Chinnaiyan et al. Biol. Chem. , Volume 271: 4
961-4965 (1996)). Flag-TNFR-1 and F
A construct encoding lag-ΔTNFR-1 was also previously described (AM Chinn.
aiyan et al. Biol. Chem. , 271: 4961-4965 (1996)). DR3 and ΔDR3 (1-321) in IBI using signal peptide provided by vector to facilitate tag binding of epitope
It was cloned into the Kodak FLAG plasmid (pCMV1FLAG). 293 cells (2 × ¹⁰ 6 / 100mm plate) penicillin G, streptomycin, and grown in glutamine, and non-essential amino acids added 10% heat-inactivated fetal bovine serum-containing DMEM medium. PcDNA3-CrmA (M. Tewari) for preventing cell death and maintaining protein expression using a construct encoding the designated protein.
Etc. Biol. Chem. 270: 3255-60 (1995)
Cells) using the calcium phosphate precipitation method. Lysis buffer (50 mM-HEPES, 150 mM-NaCl, 1 mM-EDTA, 1%
Cells were lysed in 1 mL of NP-40 and a cocktail of protease inhibitors). Lysates were previously described with control monoclonal or anti-Flag antibodies (
A. M. Chinnaiyan et al. Biol. Chem. , Volume 271: 4
961-4965 (1996)) at 4 ° C. for at least 4 hours. The beads were washed with lysis buffer 3X, but in case of TRADD binding NaCl
The concentration was adjusted to 1M. The precipitate was fractionated by 12.5% SDS-PAGE and transferred to nitrocellulose. Subsequent report (H. Hsu et al., Cell, 84: 299)
~ 308 (1996) A. M. Chinnaiyan et al. Biol
． Chem. , 271: 4961-4965 (1996)). After 24 to 32 hours, extracts are prepared and control monoclonal antibody or anti-Flag-monoclonal antibody (IBI Kodak)
Was immunoprecipitated. Western analysis showed that the expression levels of myc-TRADD and death receptor were similar in all samples. Co-precipitated myc-TRAD
D was detected by immunoblotting using an anti-myc-HRP conjugated antibody (Boehringer Mannheim).

Results As an initial screen, in vitro translated radiolabeled DAM was precipitated with various glutathione S-transferase (GST) fusion proteins immobilized on glutathione-sepharose beads. Previous report (AM Chinanay
An et al., Cell 81: 505-12 (1995), M.G. P. Bol
Din et al. Biol. Chem. 270: 7795-8 (1995
Year), F.I. C. Kischkel et al., EMBO, Volume 14: 5579-5588.
Page (1995), H .; Hsu et al., Cell, 81: 495-504 (1
995)), FADD was associated with the cytoplasmic domain of GST-Fas, while TRADD was associated with the cytoplasmic domain of GST-TNFR-1. In addition to this, there was a direct but weak interaction between RIP and GST-TNFR-1. Interestingly, GST-DDCR is TRA
It was specifically associated with DD but not FADD or RIP. Furthermore, a truncated death domain mutant of DR3 (GST-DDR3) did not interact with TRADD. To demonstrate the association of DR3 with TRADD in vivo, myc-epitope-tagged TRADD (myc-TRADD) and Fl.
ag-epitope-tagged DR3 (Flag-DR3), Flag-TNFR-
Plasmids directing the synthesis of 1 or mutants were transiently transfected into 293 cells. Consistent with in vitro binding studies, TRADD is associated with DR3 and TNF
Specific co-precipitation with R-1, but death domain mutants DDR3 and D
It did not precipitate with TNFR-1. Thus, DR3, like TNFR-1, appears to activate downstream signaling processes by its ability to recruit the adapter molecule TRADD.

Overexpression of TRADD induces two of the most important TNFR-1 signaling activities, apoptosis and NF-kB activation (H. Hsu.
Etc.). Oligomerization of TNFR-1 by trimeric TNF recruits TRADD to the receptor signaling complex (H. Hsu et al., Cell, 8
Volume 4: 299-308 (1996)). TRADD then recruits the following signaling molecules: (1) TRAF2, TNFR2, which mediates NF-kB activation.
And CD40-related molecules (M. Rothe et al., Cell, 78: 681-9).
P. 2 (1994), M .; Rothe et al., Science, 269 Volume: 142
4-1427 (1995); (2) RIPs that mediate NF-kB activation and apoptosis (H. Hsu et al., Immunity, 4: 387-396 (1996)), initially Fas / A protein that interacts with APO-1 was confirmed by 2-hybrid analysis (B. Z. Stanger et al., C.
ell, 81: 513-23 (1995)); and (3) FADD (AM Shinn, a Fas / APO-1 related molecule that mediates apoptosis).
Aiyan et al., Cell 81: 505-12 (1995), M.A. P.
Boldin et al. Biol. Chem. 270: 7795-8 (1
995), F.I. C. Kischkel et al., EMBO, Volume 14: 5579-5.
588 (1995)). Therefore, the inventor has proposed that RIP, TRAF2 and FA
We demonstrate that DD can co-immunoprecipitate with DR3. Only weak associations between the two molecules could be detected in 293 cells expressing DR3 and RIP. However, in the presence of TRADD, the association between RIP and DR3 was clearly enhanced. Similarly, TRAF2 and DR3 rarely coprecipitate directly in 293 cells. However, when DR3 and TRAF2 were expressed in the presence of TRADD and RIP, both of which could bind TRAF2, enhanced binding of TRAF2 to DR3 could be detected. A similar association between FADD and DR3 was also observed.
In the presence of TRADD, FADD efficiently co-precipitates with DR3.

In previous studies, FADD was an ICE / CED-3-like protease, FLIC.
It was demonstrated that E can be recruited to the Fas / APO-1 death-induced signaling complex (M. Muzio et al., Cell 85: 817-827 (1996).
, M .; P. Boldin et al., Cell, 85: 803-815 (1996).
Year)). Co-precipitation experiments in 293 cells were performed to demonstrate that FLICE can associate with TNFR-1 and DR3. Interestingly, FLICE was found to complex with TNFR-1 and DR3. FLICE-TNFR-1 / DR3 for co-transfection of TRADD and / or FADD
Since the interaction could not be strengthened, it suggests that endogenous amounts of these adapter molecules are sufficient to maintain this association.

Example 6 DR3-induced Apoptosis and NF-kB Activation Overexpression of Fas / APO-1 and TNFR-1 is similar to receptor activation in mammalian cells (M. Muzio et al., Cell, Volume 85: 817-8
P. 27 (1996), M.A. P. Boldin et al., Cell, Volume 85: 803
Pp. 815 (1996)). Therefore, the functional role of DDCR was studied using this system. Ectopic expression of DR3 in MCF7 breast cancer cells and 293 human embryonic kidney cells induced rapid apoptosis.

Experimental Design The cell death assay was essentially described in previous reports (AM Chinnaiyan et al.,
Cell, 81: 505-12 (1995), M.G. P. Boldin et al. Biol. Chem. 270: 7795-8 (1995), F.I.
C. Kischkel et al., EMBO, 14: 5579-5588 (199).
5 years), A. M. Chinnaiyan et al. Biol. Chem. , 27
Volume 1: 4961-4965 (1996)). Briefly, vector only, CrmA expression constructs (M. Tewari et al., J. Biol. C.
hem. 270: 3255-60 (1995)), or FADD-D.
N expression constructs (AM Chinnayan et al., J. Biol. Chem.
, 271: 4961-4965 (1996)) and lipofectamine (PCF3) in the presence of a 10-fold excess of pcDNA3 expression construct encoding the designated protein and an MCF-7 human breast cancer clonal cell line. GIBCO
-BRL) was used to transiently transfect pCMV-β-galactosidase. Similarly, 293 cells were transfected using the CaPO ₄ method.
ICE family inhibitor z-VAD-fmk (Enzyme / Systems /
Products, Dublin, CA) at a concentration of 10 μM was added to cells 5 hours after transfection. Cells were fixed 32 h after transfection and reported in a previous report (AM Chinnaiyan et al., Cell, 81: 5.
05-12 (1995), M.S. P. Boldin et al. Biol. Ch
em. 270: 7795-8 (1995), F.I. C. Kischkel
Etc., EMBO, 14: 5579-5588 (1995)), and stained with X-Gal. The data obtained (mean ± SD) are the percentage of circular blue cells in the total number of blue cells counted. Data were obtained from at least 3 independent experiments.

The NF-kB luciferase assay has been previously reported (H. Hsu et al., Immunit
y, 4: 387-396 (1996), M.A. D. Nats such as Adams
ure, 377: 3-174 (1995), G.A. S. Feng et al.
Biol. Chem. 271: 12129-32 (1996), M .; R
Othe et al., Cell, 78: 681-92 (1994), M.A. Rot
He et al., Science, 269: 1424-1427 (1995),
A. M. Chinnaiyan et al. Biol. Chem. , Volume 271: 4
961-4965 (1996)). Briefly, pCMV-β-galactosidase, E-selectin-by the calcium phosphate precipitation method.
Luciferase reporter gene (M. Rothe et al., Cell, 78:
681-92 (1994), M.M. Science, 269, such as Rothe
Vol .: 1424-1427 (1995)), 293 cells were co-transfected with the designated death receptor and the designated dominant negative inhibitor. In addition to this, DR3 or DDR3 was co-transfected with the pLantern expression construct (GIBCO-BRL) encoding a green fluorescent protein (luminescent insect). Cells were visualized by fluorescence microscopy using a FITC range barrier filter cube. Nuclei of transfected cells were visualized by DAPI staining and image overlay. (The cell death assay was basically a previous report (
Chinnaiyan et al., Cell, 81: 505-12 (1995).
, M .; P. Boldin et al. Biol. Chem. Volume 270: 7795
~ P. 8 (1995), Kischkel et al., EMBO, Volume 14: 5579 ~.
5588 (1995), Chinanaiyan et al., J. Am. Biol. Che
m. , 271: 4961-4965 (1996)). The dominant negative inhibitor used 4 times more than the death receptor. The amount of total DNA was kept constant.

RIP-DN (Stanger et al., Cell, 81: 513-23 (1
995)) and TRAF2-DN (Hsu et al., Cell, 81: 495.
Pp. 504 (1995), Rothe et al., Cell, 78: 681-92.
P. (1994), Rothe et al., Science, 269: 1424-1.
427 (1995)) to demonstrate that DR3 induces NF-kB activation that is dictated by the designated molecule and NF-kB luciferase reporter plasmid (Rothe et al., Cell, 78: 681-92). (1994
, 1974, Rothe et al., Science, 269: 1424-1427 (1
1993)) was co-transfected into 293 cells and luciferase activity was measured. The NF-kB luciferase assay is reported separately (Hsu et al., Immun
vol. 4: 387-396 (1996), Adams et al., Nature.
e, 377: 3-174 (1995), Feng et al., J. Am. Biol. C
hem. 271: 12129-32 (1996), Rothe et al., C.
Ell, 78: 681-92 (1994), Rothe et al., Science.
Ce, 269: 1424-1427 (1995), Chinanaiyan.
Etc. Biol. Chem. , 271: 4961-4965 (1996
Year))). Briefly, pCMV-β-galactosidase and E-selectin-luciferase reporter genes (Rothe et al., Cell, 78: 681-921 (1994)) in 293 cells by the calcium phosphate precipitation method.
, Rothe et al., Science, 269: 1424-1427 (199
5 years)), designated death receptors and designated dominant negative inhibitors were co-transfected. Dominant negative inhibitors were used in 4-fold higher amounts than death receptors. All D
The amount of NA was kept constant. A representative experiment was performed in duplicate at 3 independent time points (mean ± SD).

Results These cells displayed a typical morphological change of cells undergoing apoptosis, turning into circles, shrinking and floating from the dishes. In MCF7 cells, full length DR3 or DDR3
Was encoded with the pLantern reporter construct encoding the green fluorescent protein. The nuclei of cells transfected with DR3 but not DDR3 showed apoptotic morphology as assessed by DAPI staining.
TNFR-1 and Fas / APO-1 (M. Muzio et al., Cell, 85
Volume: 817-827 (1996), M.A. P. Boldin, Cell,
85: 803-815 (1996), M.S. Tewari et al. Bio
l. Chem. 270: 3255-60 (1955)).
3-induced apoptosis is an inhibitor of ICE-like proteases, CrmA and z-VA
It was inhibited by D-fmk. Importantly, DR3-induced apoptosis has been previously shown to inhibit Fas / APO-1 and TNFR-1 signaling, and a dominant negative version of FADD or FLICE (FADD-DN) or ( FLICE-DN / MACHalC360
S) was also inhibited (M. Muzio et al., Cell, 85: 817-.
827 (1996), M.G. P. Boldin et al., Cell, Volume 85: 80
Pages 3-815 (1996), H .; Hsu et al., Cell, Volume 84: 299-3
98 (1996), A. M. Chinnaiyan et al. Biol. C
hem. 271: 4961-4965 (1996)). Thus, FA
The DD and ICE-like protease FLICE appear to be essential components for DR3-induced apoptosis.

Since DR3 activation recruits three molecules that are associated with TNF-induced NF-kB activation, we tested whether DR3 could activate NF-kB. Transfection of control vector or expression of Fas / APO-1 did not induce NF-kB activation. In contrast, NF-kB was activated by ectopic expression of DR3 or TNFR-1, but not by the inactive signaling mutants DDR3 or DTNFR-1. Importantly, DR3-induced NF-
kB activation is a dominant negative derivative of RIP and TRAF2 and was previously TN
RIP-DN and T proven to eliminate F-induced NF-kB activation
Inhibited by RAF2-DN (H. Hsu et al., Cell, 84:29.
9-308 (1996), H. Hsu et al., Immunity, 4:38
7-396 (1996)). As expected, FADD-DN did not interfere with DR3-mediated NF-kB activation (H. Hsu et al., Cell 84: 2).
99-398 (1996) A. M. Chinnaiyan et al. Bi
ol. Chem. 271: 4961-4965 (1996)).

Thus, the experiments shown in Examples 6 and 7 indicate that DR3 is a death domain-containing molecule capable of triggering both apoptosis and NF-kB activation, two of the major pathways of immune system regulation. Prove that. These experiments also demonstrate the internal signaling mechanism of this novel cell death receptor. This DR3 signaling complex assembles the recruitment of the multivalent adapter molecule TRADD in a hierarchical fashion, which results in two distinct signaling processes, (1) TRAF2 and RIP-mediated N.
F-kB activation and (2) FADD, FLICE, and RIP-mediated cell death occur.

Example 7 Gene Therapy Using the Heterologous DR3 Gene Another gene therapy method according to the present invention is described in, for example, US Pat. No. 5,641,670.
Issue (Published June 24, 1997); International Publication Number WO96 / 29411 (199)
Published September 26, 2006); International publication number WO94 / 12650 (August 4, 1994)
Published); Koller et al., Proc. Natl. Acad. Sci. USA
86: 8932-8935 (1989); and Zijlstra et al., Nat.
ure 342: 435-438 (1989) and to operably attaching a heterologous DR3 sequence to a promoter via homologous recombination.
This method involves activation of a gene that is present in the target cell but is not expressed intracellularly or is expressed at a lower level than desired. The polynucleotide construct is constructed to include a promoter and a target sequence flanking the promoter, which is homologous to the 5'non-coding sequence of the heterologous DR3.
The target sequence is sufficiently close to the 5'end of DR3 so that the promoter will be operably linked to the heterologous sequence upon homologous recombination. The promoter and target sequence can be amplified using PCR. Preferably, the amplified promoter contains different restriction enzyme sites at the 5'and 3'ends. Preferably, the 3'end of the first target sequence contains the same restriction enzyme site as the 5'end of the amplified promoter, and the 5'end of the second target sequence is the 3'end of the amplified promoter. Contains the same restriction sites.

The amplified promoter and amplified target sequence are digested with the appropriate restriction enzymes, followed by treatment with bovine small intestinal phosphatase. The digested promoter and digested target sequence are added together in the presence of T4 DNA ligase. The resulting mixture is maintained under conditions suitable for ligation of the two fragments. The construct is size-fractionated on an agarose gel and then purified by phenol extraction and ethanol precipitation.

In this example, the polynucleotide construct was administered as a naked polynucleotide by electroporation. However, the polynucleotide construct is also used as a transfection-facility agent.
(e.g. liposomes, viral sequences, viral particles, precipitants, etc.). Such delivery methods are known in the art.

Once the cells are transfected, homologous recombination occurs resulting in a promoter operably linked to the heterologous DR3 sequence. This allows DR3-V in cells
Expression of 1 or DR3 occurs. Expression may be detected by immunostaining or any other method known in the art.

Fibroblasts are obtained from a subject by skin biopsy. The obtained tissue is DMEM +
Place in 10% fetal bovine serum. Exponentially growing or early quiescent fibroblasts are trypsinized and rinsed from a plastic surface containing nutrient medium. An aliquot of cell suspension is removed for counting and the remaining cells are subjected to centrifugation. The supernatant is aspirated off and the pellet is removed from the electrophoresis buffer (5 ml, 20 mM).
HEPES, pH 7.3, 137 mM NaCl, 5 mM KCl, 0.7 m
M Na ₂ HPO ₄ , 6 mM dextrose). The cells are centrifuged again, the supernatant is aspirated off, and the cells are resuspended in electrophoresis buffer containing 1 mg / ml acetylated bovine serum albumin. The final cell suspension contains approximately 3 × 10 ⁶ cells / ml. Electroporation should be performed immediately after resuspension.

Plasmid DNA is prepared according to standard techniques. For example, to construct a plasmid that targets the DR3 locus, plasmid pUC18 (M
BI Fermentas, Amherst, NY) is digested with HindIII. The CMV promoter was constructed with an XhaI site at the 5'end and BamH at the 3'end.
Amplify by PCR using the I site. Two DR3 non-coding sequences are amplified by PCR: one DR3 non-coding sequence (DR3 fragment 1) was added to the 5'end of Hin.
Amplified using a dIII site and a 3'end XbaI site;
Amplify using the dIII site. The CMV promoter and DR3 fragment are loaded with the appropriate enzymes (CMV promoter-XhaI and BamHI; DR3 fragment 1-Xha.
I; DR3 fragment 2-BamHI) and ligated together. The resulting ligation product was digested with HindIII and HindIII digested pU
Ligate with C18 plasmid.

Sterile cuvette (Bio Bio) with 0.4 cm electrode gap for plasmid DNA.
-Rad). The final DNA concentration is generally at least 120 μg
/ Ml. The cell suspension (0.5 ml, containing approximately 1.5 × 10 ⁶ cells) is then added to the cuvette and the cell suspension and DNA solution are gently mixed. Electroporation is performed using a Gene-Pulser device (Bio-Rad). The capacitance and voltage are 960 μF and 250-300, respectively.
Set to V. Increased voltage reduces cell survival, but dramatically increases the proportion of viable cells that have stably integrated the DNA introduced into the genome. Given these parameters, a pulse time of approximately 14-20 mSec would be obtained.

The electroporated cells are maintained at room temperature for about 5 minutes, then the contents of the cuvette are gently removed using a sterile transfer pipette. Cell 10c
Add directly to pre-warmed nutrient medium (10 ml, DMEM + 15% bovine serum) in m-dish and incubate at 37 ° C. The next day, aspirate the medium, replace with fresh medium (10 ml) and incubate for an additional 16-24 hours.

The engineered fibroblasts were then either used alone or in cytodex.
Dex) Proliferate to confluence on 3 microcarrier beads and then inject into host. Here, the fibroblasts produce the protein product. Then
Fibroblasts can be introduced into a patient as described above.

Example 8 Production of Antibodies A. Hybridoma Technology The antibody of the present invention can be produced by various methods (Ausubel et al., Ed., 1998, Current Protocols in Molecular).
Biology, John Wiley & Sons, NY, Chapter 2). As one example of such a method, cells expressing DR3-V1 or DR3 are administered to an animal to induce the production of serum containing polyclonal antibodies. In a preferred method, a DR3-V1 or DR3 protein preparation is prepared and purified to be substantially free of native contaminants. Such preparations are then introduced into animals to produce more specific polyclonal antisera.

Monoclonal antibodies specific for the proteins DR3-V1 or DR3 are produced using hybridoma technology (Kohler et al. Nature 256:
495 (1975); Kohler et al., Eur. J. Immunol. 6:51
1 (1976); Kohler et al., Eur. J. Immunol. 6: 292 (
1976); Hammerling et al., Monoclonal Antibod.
ies and T-Cell Hybridomas, Elsevier, N
． Y. , Pp. 563-681 (1981)). Generally, the animal (preferably a mouse) is treated with a DR3-V1 or DR3 polypeptide, more preferably a secreted DR3-V.
Immunize with cells expressing 1 or DR3 polypeptide. Such polypeptide-expressing cells are treated with any suitable tissue culture medium, preferably 10% fetal bovine serum (inactivated at about 56 ° C.), and non-essential amino acids (about 10 g / l), penicillin (about 1).
, 000 U / ml) and streptomycin (about 100 μg / ml) in Earle's modified Eagle's medium.

The splenocytes of such mice are extracted and fused with the appropriate myeloma cell line.
Any suitable myeloma cell line may be utilized in accordance with the present invention; however, A
It is preferred to use the parental myeloma cell line (SP2O) available from TCC. After fusion, the resulting hybridoma cells were selectively maintained in HAT medium and then Wands et al. (Gastroenterology, 80: 225-232).
(1981) as described in limiting diluti.
on). The hybridoma cells obtained by such selection are then assayed to identify clones secreting antibodies capable of binding the DR3-V1 or DR3 polypeptide.

Alternatively, additional antibodies capable of binding DR3-V1 or DR3 polypeptides can be produced in a two-step procedure using anti-idiotype antibodies. Such a method makes use of the fact that the antibody is itself an antigen, and therefore an antibody is available that binds to the secondary antibody. According to this method, protein-specific antibodies are used to immunize animals, preferably mice. The splenocytes of this animal are then used to make hybridoma cells and the hybridoma cells are screened to produce antibodies whose ability to bind to DR3-V1 or DR3 protein-specific antibodies can be blocked by DR3-V1 or DR3. Identify the clones that do. Such antibodies include anti-idiotypic antibodies directed against DR3-V1 or DR3 protein-specific antibodies to immunize animals for additional DR.
Used to induce the formation of 3-V1 or DR3 protein specific antibodies.

For in vivo use of the antibody in humans, the antibody is "humanized." Such antibodies can be produced using genetic constructs derived from hybridoma cells which produce the monoclonal antibodies described above. Methods for producing chimeric and humanized antibodies are known in the art and are described below (for a review, see Morriso
n, Science 229: 1202 (1985); Oi et al., BioTech.
questions 4: 214 (1986); Cabilly et al., U.S. Pat. No. 4,8.
16,567; Taniguchi et al., EP 171496; Morriso.
n et al., EP 173494; Neuberger et al., WO 8601533; R.
obinson et al., WO 8702671; Boulianne et al., Nature.
e312: 643 (1984); Neuberger et al., Nature 314.
: 268 (1985)).

B. Isolation of DR3-V1 and DR3-Directed Antibody Fragments from the scFv Library A large library of antibody fragments with the naturally occurring V-gene isolated from human PBL and reactive with the polypeptides of the invention. (The donor may or may not have been exposed to the protein of the invention. See, eg, US Pat. No. 5,885,793, which is incorporated by reference in its entirety. Incorporated into the present specification).

Library Rescue A rescue scFv library is constructed from human PBL RNA as described in WO92 / 01047. In order to shake off the phage display antibody fragment, about 10 ⁹ Escherichia coli carrying phagemid was used to 2 × TY ((50 ml) 2 × TY-A containing glucose (1%) and ampicillin (100 μg / ml).
MP-GLU) and incubate while shaking. D. Grow to 0.8. 5 ml of this culture was used to inoculate 2xTY-AMP-GLU (50 ml),
Delta gene 3 helper phage (2 × 10 ⁸ TU, M13 delta gene III
, WO92 / 01047), and the cultures at 37 ° C. for 45 minutes,
Incubate without shaking and then at 37 ° C. for 45 minutes with shaking. The culture was placed at 4000 r. p. m. Centrifuge at 10 min and pellet 2 × containing ampicillin (100 μg / ml) and kanamycin (50 μg / ml).
Resuspend in TY (2 liters) and grow overnight. Phage to WO92 / 01
Prepare as described in 047.

The M13 delta gene III is produced as follows: M13 delta gene II
Since the I helper phage does not encode the gene III protein, the phage displaying the antibody fragment (mid) has a higher binding activity to the antigen. The infectious M13 delta gene III particle is associated with wild type gene III during phage morphogenesis.
It is made by growing helper phage in cells which carry the protein-providing pUC19 derivative. The culture is incubated for 1 hour at 37 ° C. without shaking and then for another hour at 37 ° C. with shaking. Pellet cells (10 min, IEC-Centra 8,4000 revs / min), ampicillin (100 μg / ml) and kanamycin (25 μg / ml).
Resuspended in 2 × TY broth (2 × TY-AMP-KAN, 300 ml) containing
Grow overnight at 37 ° C with shaking. The phage particles were purified from the culture medium and 2
Concentrated to double PEG-precipitate (Sambrook et al., 1990), PBS (2
ml) and 0.45 μm filter (Minisart NML; Sa
to a final concentration of about 10 ¹³ transducing units / ml (ampicillin resistant clones).

Library Panning Immunotubes (Nunc) are coated overnight with PBS (4 ml) containing either 100 mg / ml or 10 mg / ml of a polypeptide of the invention. Tubes are blocked with 2% Marvel-PBS for 2 hours at 37 ° C and then washed 3 times with PBS. Phage (approximately 10 ¹³ TU) was added to the tube and incubated for 30 minutes at room temperature with tumbling on a turntable, then 1.
Let stand for 5 hours. The tubes were washed 10 times with PBS, 0.1% Tween-20 and 10 times with PBS. Elute the phage by adding 100 mM triethylamine (1 ml) and spinning up and down for 15 minutes on a turntable, then immediately neutralize the solution with 1.0 M Tris-HCI, pH 7.4 (0.5 ml). . The phages are then used to infect mid-log E. coli TG1 (10 ml) by incubating the eluted phage with bacteria for 30 minutes at 37 ° C. E. coli is then plated on TYE plates containing glucose (1%) and ampicillin (100 μg / ml). The resulting bacterial library is then shaken off with Delta Gene 3 helper phage as described above to prepare phage for subsequent rounds of selection. The process is then repeated for a total of 4 affinity purifications, but for the 3 rd and 4 th tube the washing of the tube is increased to 20 times with PBS, 0.1% Tween-20 and up to 20 times with PBS.

Binder Characterization E. coli HB2151 using phage eluted from the third and fourth round of selection.
To produce soluble scFv from a single colony for assay (
Marks et al. Mol. Biol 222: 581-597 (1991)).
. ELISA was performed using the polypeptide of the present invention (50 mM bicarbonate, pH 9.6).
10 pg / ml) coated microtiter plate.
Colonies positive in the ELISA are further characterized by PCR fingerprinting (see, eg, WO92 / 01047) followed by sequencing.

Example 9 Method for Determining Changes in the DR3 Gene RNA is isolated from whole-family or individual patients presenting with the phenotype of interest (eg, 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 utilizing primers in the peripheral region of interest in SEQ ID NO: 1. Proposed PCR conditions are 95 ° C for 30 seconds, 52-58 ° C for 60-120 seconds and 70 ° C for 60-1.
It consisted of 35 cycles of 20 seconds, which was described by Sidransky, D .; Et al, Sci
The buffer solution described in ence 252: 706 (1991) is used.

The PCR product was then labeled with SequiTherm polymerase (Epicen) using primers labeled at the 5'end with T4 polynucleotide kinase.
Sequencing is performed using Tre Technologies). Also, the intron-exon boundaries of the selected exons of DR3 are determined and the genomic PCR products are analyzed to confirm the results. The PCR product with the putative mutation in DR3 is then cloned and sequenced to confirm the direct sequencing results.

The DR3 PCR product was cloned from Holton, T .; A. And Graham, M .; W.
, Nucleic Acids Research, 19: 1156 (1991).
) And cloned into a T-tail vector and sequenced using T7 polymerase (United States Biochemical). Affected individuals are identified by mutations in DR3 that are not present in unaffected individuals.

Genomic rearrangements are also observed as a way to determine changes in the DR3 gene. Genomic clones isolated using techniques known in the art were transformed into digoxigenin deoxyuridine 5'-triphosphate (Boehringer Manh.
nick translation using Johnson, C. et al. Et al, Methods C
ell Biol. FISH is performed as described in 35: 73-99 (1991). Hybridization with a labeled probe was performed with a large excess of human cot-1 D due to specific hybridization to the DR3 genomic locus.
Performed using NA.

Chromosomes were cloned into 4,6-diamino-2-phenylidol (phenyidole).
) And propidium iodide to produce a combination of C- and R-bands. The images aligned for accurate mapping were combined with triple band filter sets (Chroma Technology).
, Brattleboro, VT) a cooled charge-coupled device camera (Photo
(Metrics, Tucson, AZ) and tunable excitation wavelength filters (Johnson, C., et al., Genet Anal. Tech).
． Appl. 8:75 (1991)). Image collection, analysis, and chromosomal fragment length measurement were performed using the ISee Graphical Program System (In.
vision corporation, Durham, NC). Chromosomal changes in the genomic region of DR3 (hybridized by the probe) are identified as insertions, deletions and transpositions. These changes in DR3 are used as diagnostic markers for related diseases.

Example 10 Method for Determining Abnormal Levels of DR3 in Biological Samples DR3 polypeptides can be detected in biological samples and elevated or decreased levels of DR3 are detected. If so, the polypeptide is a marker for a particular phenotype. It will be appreciated by those skilled in the art that there are numerous detection methods and therefore the following assays may be modified to suit a particular need.

For example, an antibody sandwich ELISA is used to detect DR3 in a sample, preferably a biological sample. DR3 wells of microtiter plate
With a specific antibody for 0.2 to 10 μg / ml final concentration. Antibodies are either monoclonal or polyclonal and are made using techniques known in the art. The wells are blocked to reduce non-specific binding of DR3 to the wells.

[0535] The coated wells are then> 2 with the sample containing DR3 at room temperature.
Incubate for hours. Preferably, serial dilutions of the sample should be used to confirm the results. The plate is then washed 3 times with deionized or distilled water to remove unbound DR3.

Next, specific antibody-alkaline phosphatase conjugate (50 μl, concentration 25-4).
00 ng) is added and incubated at room temperature for 2 hours. Repeat the plate 3 times,
Unbound conjugate is removed by washing with deionized water or distilled water.

4-Methylumbelliferyl phosphate (MUP) or p-nitrophenyl phosphate (NPP) substrate solution (75 μl) was then added to each well,
Incubate at room temperature for 1 hour to cleave substrate and fluorescence. Fluorescence is measured with a microtiter plate reader. A calibration curve [sample concentration is plotted on the X-axis (log scale) and fluorescence or absorbance is plotted on the Y-axis (linear scale)] using experimental results from serial dilutions of control samples. Then D in the sample
The R3 polypeptide concentration is interpolated using a calibration curve based on the measured fluorescence of the sample.

Example 11 Method of Treating Elevated Levels of DR3 The present invention relates to a method of treating an individual in need of reduced biological activity of DR3 levels in the body, which is therapeutically effective in such individuals. Administering a composition comprising an effective amount of a DR3 antagonist. A preferred antagonist for use in the present invention is a DR3-specific antibody.

Further, it will be appreciated that pathologies caused by reduced normal or normal DR3 expression levels in an individual can be treated by administering DR3, preferably in soluble and / or secreted form. Therefore, the invention also provides a method of treating an individual in need of elevated levels of a DR3 polypeptide,
The method involves administering to such an individual a pharmaceutical composition comprising an amount of DR3 to increase the biological activity level of DR3 in such individual.

For example, a patient with decreased levels of DR3 polypeptide may have a daily dose of 0.1
100 μg / kg of polypeptide is taken continuously for 6 days. Preferably the polypeptide is in soluble and / or secreted form.

Example 12 Reduction of DR3 Levels Method of Treatment The present invention also relates to a method of treating an individual in need of elevated levels of DR3 biological activity in the body, the method being therapeutic to such an individual. Administering a composition comprising an effective amount of DR3 or an agonist thereof.

Inhibition of DR3 production using antisense technology. This technique preferably results in a soluble and / or secreted form of DR3 resulting from various etiologies such as cancer.
It is one example of a method of reducing the level of a polypeptide.

For example, in patients diagnosed with abnormally elevated levels of DR3, antisense polynucleotides were added at 0.5, 1.0, 1.5, 2.0 and 3.0 mg / kg 21 days. Intravenous administration daily. After a 7 day rest, this procedure is repeated if it is determined to be very well tolerated.

Example 13 Method of Treatment with Gene Therapy—Ex Vivo In one method of gene therapy, fibroblasts capable of expressing soluble and / or mature DR3 polypeptides are transplanted into a patient. Fibroblasts are generally obtained from a subject by skin biopsy. The resulting tissue is placed in tissue culture medium and separated into small pieces. Place small tissue mass in wet surface tissue culture flasks (about 10 pieces in each flask). The flask is capped tightly, rotated up and down and left at room temperature overnight. After 24 hours at room temperature, the flask is inverted and the tissue mass remains solid at the bottom of the flask and fresh medium (eg Ham's F12 medium, 10% FBS, penicillin and streptomycin added) is added. The flask is then incubated at 37 ° C for approximately 1 week.

At this point fresh medium is added, followed by replacement every few days. After culturing for another 2 weeks, a monolayer of fibroblasts appears. Trypsinize the monolayer and scale up to a larger flask.

PMV-7 (Kirsc) flanked by the long terminal repeats of Moloney murine sarcoma virus.
hmeier, P.M. T. Et al., DNA, 7: 219-25 (1988)).
It is digested with RI and HindIII, followed by treatment with calf intestinal phosphatase. The linear vector is fractionated on an agarose gel and purified using glass beads.

The cDNA encoding DR3 can be amplified using PCR primers corresponding to the 5'and 3'end coding sequences, respectively. Preferably, the 5'primer contains an EcoRI site and the 3'primer contains a HindIII site.
Equivalent amount of Moloney murine sarcoma virus linear skeleton and amplified EcoRI and H
The indIII fragment is added together in the presence of T4 DNA ligase. The resulting mixture is maintained under conditions suitable for ligation of the two fragments. The ligation mixture was then used to transform E. coli HB101 and then the vector with the correct insert D
This is plated on kanamycin containing agar to ensure that it contains R3.

Amphoteric pA317 or GP + am12 packaging cells were placed in tissue culture at 10% bovine serum (confluent) until they reached confluent density.
CS), penicillin and streptomycin containing Dulbecco's modified Eagle
s medium (DMEM). The MSV vector containing the DR3 gene is then added to the medium and the packaging cells are transduced with the vector. Here, the packaging cells produce infectious viral particles containing the DR3 gene (hereinafter the packaging cells are referred to as producer cells). Fresh medium is added to the transduced producer cells, and then the medium is harvested from confluent producer cells (10 cm plate). Old (s) containing infectious virus particles
Pent) medium is filtered through a Millipore filter to remove detached producer cells and then used to infect fibroblasts from this medium. Remove medium from subconfluent plates of fibroblasts and quickly replace with medium from producer cells. Remove this medium and replace with new medium. At high virus titers, virtually all fibroblasts are infected and no selection is necessary. If the titer is very low then it is necessary to use a retroviral vector with a selectable marker (eg neo or his). Once the fibroblasts are efficiently infected, they are analyzed to determine if DR3 protein is being produced.

The engineered fibroblasts are then transplanted into a host, either alone or after growing to confluence on Cytodex 3 microcarrier beads.

Example 14 Method of Treatment Using Gene Therapy-In Vivo Another aspect of the invention is to treat injuries, diseases and conditions using in vivo gene therapy methods. Gene therapy methods relate to introducing naked nucleic acid (DNA, RNA and antisense DNA or RNA) DR3 sequences into an animal to increase or decrease expression of a DR3 polypeptide. The DR3 polynucleotide is
It can be operably linked to a promoter or any other genetic element essential for expression of the DR3 polypeptide by the target tissue. Such gene therapy and delivery techniques and methods are known in the art [eg, WO90 / 11
092, WO98 / 11779; US Patent Nos. 5,693,622 and 5,7.
05, 151, 5,580, 859; Tabata H .; Et Card
iovasc. Res. 35: 470-479 (1997); Chao J. et al. Et al., Pharmacol. Res. 35: 517-522 (1997); Wolf.
fJ. A. Neuromuscul. Disord. 7: 314-318 (1
997); Schwartz B. Et al., Gene Ther. 3: 405-41
1 (1996); Tsurumi Y .; Et al., Circulation 94: 3.
281-2390 (1996), which are incorporated herein by reference].

DR3 polynucleotide constructs are delivered by any method that delivers injectables to cells of an animal [eg, injection into the interstitial space of tissues (heart, muscle, skin, lungs, liver, small intestine, etc.)]. Can be done. The DR3 polynucleotide construct can be delivered in a pharmaceutically acceptable liquid or aqueous carrier.

The term “naked” polynucleotide, DNA or RNA refers to any delivery vehicle that acts to assist, facilitate or facilitate entry into a cell (including viral sequences, viral particles, Sequence without a liposomal formulation, lipofectin or precipitating agent). However, DR3 polynucleotides can also be delivered in liposomal formulations (eg, Felgner, P. et al., Ann.
NY Acad. Sci. 772: 126-139 (1995) and Abda.
Ilah, B .; Et al., Biol. Cell 85: 1-7 (1995)), which may be prepared by methods well known to those skilled in the art.

DR3 polynucleotide vector constructs used in gene therapy methods are preferably those that are not integrated into the host genome or that contain sequences that allow replication. Any strong promoter known to those of skill in the art can be used to direct the expression of DNA. Unlike other gene therapy techniques, one major advantage of introducing naked nucleic acid sequences into target cells is the transient nature of polynucleotide synthesis in the cell. Figure 6 shows studies that can provide for production of a desired polypeptide for up to 6 months when a non-replicating DNA sequence is introduced into the cell.

DR3 polynucleotide constructs may be used in muscle, skin, brain, lung, liver, spleen, bone marrow,
In the interstitial space of animal tissues including thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, bladder, stomach, intestine, testis, ovary, uterus, pancreas, rectum, nervous system, eye, gland and connective tissue Can be delivered. The interstitial space of tissues is the intercellular fluid, the mucopolysaccharide matrix between the reticulated fibers of organ tissue, the elastic fibers of the walls of vessels or chambers, the collagen fibers of fibrous tissue, or the connective tissue covering muscle cells. Alternatively, it contains the same matrix in the bone cavities. So is the space occupied by circulating plasma and lymphatic fluid of the lymphatic channels. Delivery to the interstitial space of muscle tissue is preferred for the reasons described below. Conventionally, they can be delivered by injection into the tissue containing these cells.
Delivery and expression may be due to undifferentiated or not fully differentiated cells (eg,
It can be achieved in blood or skin fibroblast stem cells), but is preferably delivered and expressed into differentiated persistent non-dividing cells. In vivo muscle cells
It is particularly competent in its ability to take up and express polynucleotides.

Effective doses of DNA or RN for naked DR3 polynucleotide injection
A ranges from about 0.05 μg / kg body weight to about 50 mg / kg body weight. Preferably, the dosage is about 0.005 mg / kg to about 20 mg / kg, more preferably about 0.05 mg / kg to about 5 mg / kg. Of course, one of skill in the art will appreciate that this dosage will vary depending on the site of the injected tissue. Appropriate and effective doses of nucleic acid sequences can be readily determined by those of skill in the art, and these may depend on the condition being treated and the route of administration. The preferred route of administration is by parenteral administration by injection into the tissue interstitial space. However, other parenteral routes may also be used, such as inhalation of airgel formulations, especially for delivery to lung or bronchial tissues, throat or nasal mucosal tissues. In addition, the naked DR3 polynucleotide construct can be delivered to the artery during angioplasty by the catheter used during the procedure.

The dose response effect of DR3 polynucleotides injected intramuscularly in vivo is determined as follows. Suitable DR3 template DNA for production of mRNA encoding DR3 polypeptide is prepared according to standard recombinant DNA methods. Mold D
NA, which can be either circular or linear, is used either as naked DNA or complexed with liposomes. Mice are then injected into the quadriceps with various amounts of template DNA.

Female and dominant Balb / C mice, 5-6 weeks old, were treated with 2.5% avertin (0
． 3 ml) and anesthetized by intraperitoneal injection. A 1.5 cm incision is made in the forefoot and the quadriceps is visualized directly. DR3 template DNA was added to 27 ml in 0.1 ml of carrier.
Inject through a gauge needle with a 1 cc syringe for 1 minute to a depth of about 0.5 cm and about 0.2 cm from the site of muscle distal attachment to the knee. The suture is placed at the injection site for previous localization and the skin is closed with a stainless steel clip.

After an appropriate incubation time (eg, 7 days), muscle extracts are prepared by excising the entire quadriceps muscle. Every five 15 μm sections of individual quadriceps are histochemically stained for DR3 protein expression. The time course for DR3 protein expression can be performed in a similar fashion, except that quadriceps from different mice were harvested at different time points. DR3 in muscle after injection after preparation of total cellular DNA and HIRT supernatant from injected and control mice
DNA persistence can be determined by Southern blot analysis. The results of the experiments in mice described above can be used to estimate accurate dosages and other treatment parameters in humans and other animals using DR3 naked DNA.

It will be appreciated that the present invention may be practiced differently than that described in detail in the above description and examples.

The myriad modifications and variations of the present invention are possible in light of the above teachings, and therefore
It is within the scope of the following claims.

With reference to the entire disclosure of the patents, patent applications, and publications cited herein,
Incorporated herein.

[Sequence list]

[Brief description of drawings]

1A-1C (SEQ ID NOS: 1 and 2) show the nucleotide sequence of DR3 and the deduced amino acid sequence therefrom. Amino acids 1 to 35 form a signal peptide, amino acids 36 to 212 form an extracellular domain, amino acids 213 to 235 form a transmembrane domain, amino acids 236 to 428 form an intracellular domain, and amino acids 353 to 353. 419 is expected to form the death domain.

2A-2B (SEQ ID NOS: 3 and 4) show the nucleotide sequence of DR3 and the amino acid sequence deduced therefrom. Amino acids 1 to 24 make up a signal peptide, amino acids 25 to 201 make up an extracellular domain, amino acids 202 to 224 make up a transmembrane domain, amino acids 225 to 417 make up an intracellular domain, and amino acids 342 to 342. 408 is expected to form the death domain.

3A-3D show DR3, human tumor necrosis factor receptor 1
, And the regions of similarity between the amino acid sequences of the Fas receptors (SEQ ID NO: 5 and SEQ ID NO: 6).

FIG. 4 shows analysis of the DR3 amino acid sequence. Alpha, beta,
Turn and coil regions, hydrophilic and hydrophobic regions, amphipathic regions, flexible regions, antigenic index and surface probabilities are shown. Amino acid residues 1-22, 33-56, 59 of SEQ ID NO: 2 in the "antigenicity index-Jameson-Wolf" graph
~ 82, 95-112, 122-133, 161-177, 179-190, 1
96-205 correspond to the highly antigenic regions of the DR3 protein shown.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61K 31/675 A61K 31/675 4H045 38/00 39/395 D 38/21 N 39/395 45/00 A61P 35/00 45/00 37/02 A61P 35/00 37/06 37/02 A61K 37/02 37/06 37/66 G // C12N 15/09 ZNA C12N 15/00 ZNAA (81) Designated country EP (AT , BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM) , GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, K , MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK , DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ , TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (71) Applicant The Regents of the University of Michigan THE REGENTS OF THE UNIVERSITY OF MICHI GAN Rica United States 48108 3003 South State Street, Ann Arbor, Michigan, Wolverine Tower, Room 2071, Technology Management Office (72) Inventor Yu Guo Liang, 242 Gravat Drive, Berkeley, CA 94705, USA (72) Inventor Ni-Jan United States 20853 Manorfield Road 5502, Rockville, MD (72) Inventor Rainer El Genz 20850 Mount Prospect Drive, Rockville, MD 13608 (72) Inventor Patrick J. Dillon United States 92009 Carlsbad, CA Snipe Court 1055 (72) Inventor Bishba M. Dixit United States 94022 Los Altos Hills, CA Shady O. Cus Court 26750 No. F Term (Reference) 4B024 AA01 BA63 CA04 DA02 EA02 HA17 4C084 AA02 AA03 BA44 DA11 DA12 DA14 DA15 DA16 DA17 DA18 DA24 DA39 MA02 ZB071 ZB081 ZB082 ZB112 ZB212 ZB261 ZB352 ZC022 4C085 ABB13 A02 CB22 DA10 DA35 MA02 MA04 MA07 ZB07 ZB08 ZB26 4C206 AA01 AA02 HA31 MA02 MA04 MA11 ZB07 ZB08 ZB26 4H045 AA10 AA20 AA30 BA10 BA57 CA40 DA51 EA28 FA74

Claims (23)

[Claims] 1. (a) 1-428 amino acids of SEQ ID NO: 2 or 1 of SEQ ID NO: 4
A second therapeutic agent selected from the group consisting of the following components: (i) a tumor necrosis factor blocker; (ii) immunosuppression. An agent; (iii) an antibiotic; (iv) an anti-inflammatory agent; (v) a chemotherapeutic agent; , A method of treating immunodeficiency or autoimmune disease. 2. The method according to claim 1, wherein the first therapeutic agent comprises an antibody that binds to a polypeptide consisting of amino acids 36 to 212 of SEQ ID NO: 2. 3. The method according to claim 1, wherein the antibody is a monoclonal antibody. 4. The method according to claim 1, wherein the antibody is a polyclonal antibody. 5. The method according to claim 1, wherein the antibody is a chimeric antibody. 6. The method according to claim 1, wherein the antibody is a humanized antibody. 7. The method according to claim 1, wherein the antibody is a single chain Fv antibody. 8. The method according to claim 1, wherein the antibody is a Fab antibody fragment. 9. The method of claim 1, wherein the first and second therapeutic agents are administered simultaneously to the individual. 10. The method of claim 1, wherein the first and second therapeutic agents are administered to the individual at different times. 11. A tumor necrosis factor blocker is (a) TNF-α; (b) TNF-β.
And (c) an antibody that binds to a protein selected from the group consisting of TNF-γ-β. 12. An immunosuppressant is (a) cyclosporine; (b) cyclophosphamide; (c) methylprednisone; (d) prednisone; (e) azathioprine; (f).
FK-506; and (g) 15-deoxyspergualine. 13. The cytokine is (a) IL-2; (b) IL-3; (c) IL.
-4; (d) IL-5; (e) IL-6; (f) IL-7; (g) IL-10; (h) IL
The method according to claim 1, which is selected from the group consisting of -12; (i) IL-13; (j) IL-15; and (k) INF-γ. 14. A first therapeutic agent comprising an antibody that binds to a polypeptide which comprises (a) a polypeptide consisting of 1 to 428 amino acids of SEQ ID NO: 2 or 1 to 417 amino acids of SEQ ID NO: 4; and (b) a group consisting of the following components: A second therapeutic agent selected from: (i) tumor necrosis factor blocker; (ii) immunosuppressive agent; (iii) antibiotic; (iv) anti-inflammatory agent; (v) chemotherapeutic agent; and (vi) cytokine A composition comprising. 15. A method for treating graft-versus-host disease, cancer, immunodeficiency or autoimmune disease, which comprises administering to the individual a therapeutically effective amount of the composition of claim 14. 16. The composition of claim 14, further comprising a pharmaceutically acceptable carrier or excipient. 17. At least 9 for amino acids 36-212 of SEQ ID NO: 2.
An isolated polypeptide comprising an amino acid sequence having 0% identity, which comprises:
3000, 4000, 5000, 6000, 7000, 8000, 9000,
A polypeptide covalently bound to polyethylene glycol having an average molecular weight selected from the group consisting of 10,000, 15,000 and 20,000. 18. At least 9 for amino acids 36-212 of SEQ ID NO: 2.
18. The isolated polypeptide of claim 17, which comprises an amino acid sequence having 5% identity. 19. The isolated polypeptide of claim 18, wherein the amino acid sequence comprises amino acids 36-212 of SEQ ID NO: 2. 20. The polypeptide is 1-3, 2-4, 3-5, 4-6, 5-
18. The isolated polypeptide according to claim 17, having an average degree of substitution with polyethylene glycol in a range selected from the group consisting of 7, 6-8, 7-9, 8-10, 9-11 and 10-12. 21. The isolated polypeptide of claim 17, produced by a recombinant host cell. 22. The isolated polypeptide of claim 17, which comprises a heterologous polypeptide. 23. A composition comprising the isolated polypeptide of claim 17 and a pharmaceutically acceptable carrier.