JP2002544172A - Death domain containing receptor 4 - Google Patents

Abstract

  (57) [Summary] The present invention relates to a novel death domain containing receptor 4 (DR4) protein that is a member of the tumor necrosis factor (TNF) receptor family. In particular, an isolated nucleic acid molecule encoding a human DR4 protein is provided. DR4 polypeptides are also provided, as are vectors, host cells, and recombinant methods of making DR4 polypeptides. The invention further relates to screening methods for identifying agonists or antagonists of DR4 activity, and to methods using DR4 polynucleotides and polypeptides.

Description

DETAILED DESCRIPTION OF THE INVENTION

BACKGROUND OF THE INVENTION Field of the Invention The present invention relates to novel members of the tumor necrosis factor family of receptors.
More specifically, human death domain containing receptor 4
Provided is an isolated nucleic acid molecule that encodes (sometimes also referred to herein as "DR4"). A DR4 polypeptide is also provided, comprising a vector, a host cell,
The same applies to recombinant methods for producing polypeptides. Further, the present invention provides a DR
Screening methods for identifying agonists and antagonists of the 4 activity, and methods for using DR4 polynucleotides and polypeptides.

Related Art Many biological effects (eg, response to certain stimuli and natural biological processes) are controlled by factors such as cytokines. Many cytokines act through the receptor by engaging the receptor and generating an intracellular response.

[0003] For example, tumor necrosis factor (TNF) α and tumor necrosis factor β are involved in a number of biological processes, including protection against infection and the induction of shock and inflammatory diseases.
Are cytokines that act via the TNF receptor to modulate. TN
The F molecule belongs to the "TNF ligand" superfamily and works with these receptors or their counterpart ligands ("TNF receptor" superfamily). To date, nine members of the TNF ligand superfamily have been identified, and ten members of the TNF receptor superfamily have been characterized.

[0004] Ligands include TNF-α, lymphotoxin-α (also known as TNF-β, LT-α), LT-β (found in the complex heterotrimer LT-α2-β), FasL, CD40L, CD27L, CD30L, 4-1BBL, OX4
0L, and nerve growth factor (NGF). The superfamily of TNF receptors includes p55TNF receptor, p75TNF receptor, TNF receptor-related protein, FAS antigen or APO-1, CD40, CD27, C
Including D30, 4-1BB, OX40, low affinity p75, and NGF receptor (Meager, A., Biologicals, 22: 291-295 (1
994)).

[0005] Many members of the TNF ligand superfamily are expressed by activated T cells. This means that they are required for T-cell interaction with other cell types that underlie cell ontogeny and function (Meager, A., supra).

[0006] Considerable insight into the essential functions of some members of the TNF receptor family has been gained through the identification and generation of mutants that abolish expression of these proteins. For example, naturally occurring mutations in the FAS antigen and its ligands result in lymphoproliferative disorders (Watanabe-Fukunaga,
R. Et al., Nature 356: 314 (1992)), presumably reflecting failure of programmed cell death. Mutations in the CD40 ligand result in an X-linked immunodeficiency state characterized by high levels of immunoglobulin M and low levels of immunoglobulin G in plasma. This indicates incomplete T cell-dependent B cell activation (Allen, RC, et al., Science 259: 990 (199).
3)). Targeted mutations of the low-affinity nerve growth factor receptor result in a disorder characterized by incomplete sensory innervation of peripheral structures (Lee, KF et al., Cell 69: 737 (1992). )).

[0007] TNF and LT-α are two TNF receptors (55 kd and 75 kd)
TNF receptor). TN acting through those receptors
Many biological effects elicited by F and LT-α include hemorrhagic necrosis of transplanted tumors, cytotoxicity, role in endotoxin shock, inflammation, immunomodulation, proliferation and antiviral response, and the effects of ionizing radiation. Including protection against harmful effects. TNF and LT-α are involved in the pathogenesis of a wide range of diseases including endotoxin shock, cerebral malaria, tumors, autoimmune diseases, AIDS, and graft host rejection (Beutler, B. and Von Huffel, C., Science).
ce 264: 667-668 (1994)). Mutations in the p55 receptor are:
This results in increased susceptibility to microbial infection.

In addition, TNFR1 (p55) and the approximately 80 amino acid domain near the C-terminus of Fas are responsible for transmitting signals for programmed cell death.
Death domain "(Tartagl
ia et al., Cell 74: 845 (1993)).

[0009] Apoptosis, or programmed cell death, is a physiological process that is essential for the normal development and homeostasis of multicellular organisms (H. Stelle).
r, Science 267, 1445-1449 (1995)). Disruption of apoptosis contributes to the pathogenesis of several human diseases, including cancer, neurodegenerative disorders, and acquired immunodeficiency syndrome (CB Thompson, Science 2
67, 1456-1462 (1995)). Recently, much attention has been focused on the signaling and biological function of two cell surface death receptors (Fas / APO-1 and TNFR-1) (JL Cleveland et al., Cell).
81, 479-482 (1995); Fraser et al., Cell 85,7.
81-784 (1996); Nagata et al., Science 267, 1
449-56 (1995)). Both, especially TNFR-2, low affinity NG
It is a member of the TNF receptor family that also includes FR, CD40, and CD30 (CA Smith et al., Science 248, 1019-23).
(1990); Tewari et al., Modular Texts in Mo.
circular and Cell Biology M.L. Purton, H
eldin, Carl (Chapman and Hall, London,
1995). While members of the family are defined by the presence of cysteine-rich repeats in these extracellular domains, Fas / APO-1 and TNFR-1 are also responsible for the Drosophila suicide gene reaper (P. Golstein et al.
ell 81, 185-6 (1995); White et al., Science
264, 677-83 (1994)), the death domain (deat domain).
h domain), which share the region of intracellular homology, appropriately named. This shared death domain suggests that both receptors interact with a related set of signaling molecules that remained unidentified until recently. Fas / AP
Activation of O-1 reinforces the adapter molecule FADD / MORT1 containing the death domain (AM Chinnaiyan et al., Cell 81,505-12 (19).
95); P. Boldin et al. Biol Chem 270, 7795
-8 (1995); C. Kischkel et al., EMBO 14,5579-.
5588 (1995)), an adapter molecule FADD / MORT containing a death domain.
1 then binds and probably activates FLICE / MACH1, a member of the ICE / CED-3 family of pro-apoptotic proteases (
M. Muzio et al., Cell 85, 817-827 (1996); P. B
oldin et al., Cell 85, 803-815 (1996)). Fas / AP
While the central role of O-1 is to induce cell death, TNFR-1 is a signal of a variety of organized biological activities, many of which derive from the ability to activate NF-KB. (LA Tartaglia et al., Immunol T
day 13, 151-3 (1992)). Thus, TNFR-1 reinforces the multivalent adapter molecule TRADD, which, like FADD, also contains a death domain (H. Hsu et al., Cell 81, 495-504 (1995)).
H .; Hsu et al., Cell 84, 299-308 (1996)). FADD,
Through association with many signaling molecules, including TRAF2, and RIP, T
RADD can signal both apoptosis and NF-KB activation (H. Hsu et al., Cell 84, 299-308 (1996); H. Hsu et al., Immunity 4, 387-396 (1996)).

[0010] Recently, a novel apoptosis-inducing ligand has been discovered. Wiley, S.M. R.
Have described this novel molecule as a TNF-related apoptosis-inducing ligand or ("TRA
IL ") (Immunity 3, 673-682 (1995)). Pi
tti, R .; M. Et al. Call this novel molecule an Apo-2 ligand or ("Apo
-2L "). For convenience, this molecule will be referred to herein as TRAIL.

[0011] Unlike FAS ligands, whose transcripts appear to be largely restricted to stimulated T-cells, significant levels of TRAIL are detected in many tissues and some Is constitutively transcribed by cell lines. TRAIL is FAS
Have been shown to act independently of the ligand (Wiley, SR et al.,
(1995) supra). Marsters, S.M. A. A study by TRAIL
Showed rapid activation of apoptosis within a time frame similar to death signaling by Fas / Apo-1L, but much faster than TNF-induced apoptosis (Current Biology 6,750-752). (1
996)). All studies to date suggest that the receptor for TRAIL does not fit into any of the many known TNF receptors.

[0012] The effects of TNF family ligands and TNF family receptors are altered and affect numerous functions, both normal and abnormal, in the biological processes of mammalian systems. Thus, there is a clear need for the identification and characterization of receptors and ligands that affect biological activity in both normal and disease states. In particular, there is a need to isolate and characterize receptors for newly discovered TRAIL ligands.

(Summary of the Invention) The present invention relates to a nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO: 2 or the amino acid sequence encoding the cDNA clone deposited on January 21, 1997 as ATCC Accession No. 97853. Provided is an isolated nucleic acid molecule comprising or consisting of.

The present invention also provides vectors and host cells for the recombinant expression of the nucleic acid molecules described herein, as well as methods of making such vectors and host cells, and DR4 polyp by recombinant techniques. Provided are peptides or methods of using them for the production of peptides.

[0015] The invention further provides an isolated DR4 polypeptide having an amino acid sequence encoded by a polynucleotide described herein.

[0016] The invention also provides diagnostic assays (eg, quantitative assays for detecting levels of DR4 protein, and diagnostic assays). Thus, for example, to detect overexpression of DR4 or a soluble form thereof, as compared to a normal control tissue sample, a diagnostic assay according to the invention can be used to detect the presence of a tumor.

[0017] Ligands of the tumor necrosis factor (TNF) family are most versatile in inducing many cellular responses, including cytotoxicity, antiviral activity, immunomodulatory activity, and transcriptional regulation of several genes It is known to be between cytokines. TNF
Cellular responses to family ligands include not only normal physiological responses but also diseases associated with increased apoptosis or inhibition of apoptosis. Apoptosis-programmed cell death-is a physiological mechanism involved in the loss of peripheral T lymphocytes of the immune system, and its dysregulation can lead to many different pathogenic processes. Diseases associated with increased cell survival or inhibition of apoptosis include cancer, autoimmune disorders, viral infections, inflammation, graft versus host disease, acute graft rejection,
And chronic graft rejection. Diseases associated with increased apoptosis include AIDS,
Includes neurodegenerative disorders, myelodysplastic syndrome, ischemic injury, toxin-induced liver disease, septic shock, cachexia, and eating disorders.

Accordingly, the present invention further provides a method of enhancing apoptosis induced by a ligand of the TNF family, which increases DR4-mediated signaling to cells expressing a DR4 polypeptide. Administering an effective amount of an agonist that can be administered. Preferably, DR4 mediated signaling is increased for the treatment and / or prevention of diseases that show reduced apoptosis.

[0019] In a further aspect, the invention relates to a method for inhibiting apoptosis induced by a TNF family ligand, which comprises signaling DR4-mediated signaling to a cell expressing a DR4 polypeptide. Administering an effective amount of the antagonist that can be reduced. Preferably, DR4 mediated signaling is reduced for the treatment and / or prevention of diseases showing increased apoptosis.

Whether any candidate “agonist” or “antagonist” of the present invention is capable of enhancing or inhibiting apoptosis is determined by a TNF family ligand / receptor cellular response assay known in the art (as described below). (Including those described in detail). Thus, in a further aspect, a method of screening is provided for determining whether a candidate agonist or antagonist can enhance or inhibit a cellular response to a TNF family ligand. The method comprises cells expressing a DR4 polypeptide;
Contacting the candidate compound with a ligand of the TNF family, assaying the cellular response, and comparing the cellular response to a standard cellular response. Assayed when performed with the ligand in the absence of the compound), whereby an increased cellular response over the standard is indicative that the candidate compound is an agonist of the ligand / receptor signaling pathway and compared to the standard A diminished cellular response indicates that the candidate compound is an antagonist of the ligand / receptor signaling pathway. According to the present invention, cells expressing a DR4 polypeptide can be treated with endogenous or exogenously administered T cells.
It can be contacted with any of the NF family ligands.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention provides for the isolation of an amino acid sequence comprising or consisting of a nucleic acid sequence encoding a DR4 polypeptide shown in SEQ ID NO: 2, or a fragment of the polypeptide. Provided nucleic acid molecules. The DR4 polypeptide of the present invention shares sequence homology with human TNFR-1, DR3 and Fas ligand (FIG. 2).
. The nucleotide sequence shown in SEQ ID NO: 1 is HCUDS60 (America)
n Type Culture Collection, 10801 Univ
error Boulevard, Manassas, Virginia 2
Deposit No. 0110-2209 on January 21, 1997 and accession number 97
853) (given 853). The deposited clone was designated pBK plasmid (Stratagene, La).
Jolla, CA).

Nucleic Acid Molecules Unless otherwise indicated, all nucleotide sequences determined herein by sequencing a DNA molecule refer to an automated DNA sequencer (eg, Ap
Plied Biosystems, Inc. The entire amino acid sequence of the polypeptide encoded by the DNA molecule determined using Model 373) and determined herein was deduced by translation of the DNA sequence determined as described above. . Thus, as is known in the art for any DNA sequence determined by this automated approach, any nucleotide sequence determined herein may contain some errors. The nucleotide sequence determined by the automation is typically at least about 90% identical to the actual nucleotide sequence of the DNA molecule to be sequenced, more typically at least about 9%.
5% to at least about 99.9% identical. The actual sequence can be more accurately determined by other approaches, including manual DNA sequencing methods well known in the art. As is also known in the art, a single insertion or deletion in a determined nucleotide sequence, as compared to the actual sequence, causes a frameshift in translation of the nucleotide sequence, so that the determined nucleotide sequence The predicted amino acid sequence encoded is completely different from the amino acid sequence actually encoded by the DNA molecule to be sequenced, and begins at the point of such an insertion or deletion.

By “isolated” polypeptide or protein is intended a polypeptide or protein that has been removed from its natural environment. For example, recombinantly produced polypeptides and proteins expressed in host cells can be, for example,
natural or recombinant polypeptides that have been substantially purified by any suitable technique, such as the single-step purification method disclosed in Math and Johnson, Gene 67: 31-40 (1988). Therefore, it is considered isolated for the purpose of the present invention.

Using the information provided herein, such as the nucleic acid sequence shown in SEQ ID NO: 1, a nucleic acid molecule of the invention that encodes a DR4 polypeptide has m as a starting material
It can be obtained using standard cloning and screening procedures, such as those for cloning cDNA using RNA. Illustrative of the invention, the genes of the invention have also been identified in cDNA libraries of the following tissues:
Amniotic cells, heart, liver cancer, kidney, leukocytes, activated T cells, K562 and PM
A, W138 cells, Th2 cells, human tonsils and CD34 depleted buffy coat (umbilical cord blood).

The DR4 gene starts at positions 19-21 of the nucleotide sequence shown in SEQ ID NO: 1 with a leader sequence of about 23 amino acid residues (ie, a total protein length of 468 amino acids) and a predicted molecular weight of about 50 kDa. Codons include an open reading frame encoding a mature protein of approximately 445 amino acid residues. In this context, “about” is greater than or greater than a few (5, 4, 3, 2, 2, or 1) amino acid residues at the specifically recited range and at either or both termini. Including small ranges.

[0026] Of the known members of the TNF receptor family, the DR4 polypeptides of the present invention have the highest degree of relevance to the human TNFR1 and DR3 polypeptides shown in Figure 2 (including significant sequence homology across multiple cysteine-rich domains). Share homology.

In addition to the sequence homology shown between DR4 and receptors containing other death domains, DR4 has been shown to bind to TRAIL and, when transiently expressed, induce apoptosis. ing. MCF7 human breast cancer cells and 293 cells were transiently transfected with a DR4 expression construct as described in Example 5. As shown in FIGS. 5A and 5B, a significant percentage of the transfected cells underwent morphological changes characteristic of apoptosis. As expected, deletion of the death domain disrupted DR4's ability to participate in the death pathway. As can be seen in FIG. 5C, DR4-induced apoptosis was reduced by z-VAD-f
mk, broad spectrum irreversible caspase inhibitor and CrmA, F
It was efficiently blocked by inhibitors of the death protease, including cowpox virus, which encodes a serpin that preferentially inhibits apical caspases such as LICE / MACH-1 (caspase-8). TNFR-1, CD-95
And DR3-induced apoptosis is also attenuated by these same inhibitors, so that downstream death effector molecules are essentially the same.

To determine if DR4 is capable of binding TRAIL, DR4
The extracellular ligand binding domain of the fusion to the Fc region of human IgG (DR
4-Fc). It selectively binds to DR4-Fc, but TNFR-
TRAIL that does not bind to one of the corresponding extracellular domains nor to the corresponding extracellular domain of CD-95 is also expressed as an Fc fusion (data not shown).
In addition, DR4-Fc can bind to TNFα ligand or Fas ligand under conditions where both TNFα ligand or Fas ligand bind to these cognate receptors.
Did not bind to either of the ligands.

[0029] The ability of TRAIL to induce apoptosis in MCF7 cells is determined by DR4-F
c, but was not affected by TNFR1-Fc, CD95-Fc or Fc alone (FIG. 6A). Furthermore, as estimated, T
NFα-induced apoptosis was inhibited by TNFR-1-Fc while DR
It was not inhibited by 4-Fc, CD95-Fc or Fc alone (FIG. 6B).

Taken together, the above data indicates that DR4 is a death domain-containing receptor with the ability to induce apoptosis and a receptor for TRAIL, a known apoptosis-inducing ligand. .

As indicated, the invention also provides mature forms of the DR4 proteins of the invention. According to the signal hypothesis, proteins secreted by mammalian cells are:
Once export of a growing protein chain across the rough endoplasmic reticulum is initiated, it has a signal or secretory leader sequence that is cleaved from the mature protein. Most mammalian cells and even insect cells cleave secreted proteins with the same specificity. However, in some cases, cleavage of the secreted protein is not completely uniform, resulting in more than one mature species in the protein. In addition, the cleavage specificity of a secreted protein is ultimately determined by the primary structure of the entire protein, ie, it has long been known to be unique to the amino acid sequence of a polypeptide. Accordingly, the present invention provides a nucleotide sequence encoding a mature DR4 polypeptide encoded by a cDNA contained in a host identified as ATCC Accession No. 97853, and having an amino acid sequence as set forth in SEQ ID NO: 2. . CDNA contained in a host identified as ATCC Accession No. 97853
Mature DR4 protein having an amino acid sequence encoded by a mammalian cell (eg, a COS cell as described below) of a fully open reading frame encoded by human DNA contained in a deposited host vector Means the mature form of the DR4 protein produced by expression in As shown below, the mature DR4 having the amino acid sequence encoded by the cDNA contained in ATCC Accession No. 97853, depending on the accuracy of the cleavage site estimated based on computer analysis, is shown in SEQ ID NO: 2. Putative "mature" DR4
It may or may not be different from the protein (amino acids from about 24 to about 468 of SEQ ID NO: 2). In this context, "about" refers to the size indicated, especially at either or both termini, a few (5, 4, 3, 2, or 1) more amino acid residues than this. Or including small size.

Methods are available for estimating whether a protein has a secretory leader as well as a breakpoint in the leader sequence. For example, McGeoch (Virus Res
. 3: 271-286 (1985)) and von Heinje (Nuclee).
ic Acids Res. 14: 4683-4690 (1986)). The accuracy of estimating the breakpoints of known mammalian secretory proteins for each of these methods is in the range of 75-80%. von Heinje, supra. However, the two methods do not always generate the same putative breakpoint for a given protein.

In the present case, the deduced amino acid sequence of the complete DR4 polypeptide of the present invention was analyzed by a computer program (“PSORT”) (K. Nakai).
And M. Kanehisa, Genomics 14: 897-911 (19
92)). PSORT is an expert system for estimating the cellular position of a protein based on the amino acid sequence. As part of the computer estimation of this position, the methods of McGeoch and von Heinje are incorporated. Analysis by the PSORT program showed that amino acid 23 of SEQ ID NO: 2
The cleavage site between and 24 was estimated. The complete amino acid sequence was then further analyzed by visual inspection and a simple form of the von Heinje (-1, -3) rule was applied. von Heinje, supra. Therefore, the leader sequence of the DR4 protein is predicted to consist of about 1-23 underlined amino acid residues of SEQ ID NO: 2,
On the other hand, the putative mature DR4 protein consists of residues about 24-468.

As the skilled artisan will appreciate, due to the possibility of sequencing errors and the variability of the cleavage site for the leader in different known proteins, the deposited cDN
The putative DR4 receptor polypeptide encoded by A contains about 468 amino acids, but may be anywhere in the range of 458-478 amino acids; and the putative leader sequence for this protein is about 40 amino acids. But about 30 ~
It may be anywhere in the range of about 50 amino acids. The domains described herein have been estimated by computer analysis and, thus, depending on the analytical criteria used to identify the various functional domains, for example, DR4
Extracellular domain, intracellular domain, death domain, cysteine-rich motif,
It is further understood that the exact “address” of the transmembrane domain and can vary slightly. For example, the exact location of the DR4 extracellular domain in SEQ ID NO: 2 can vary slightly (eg, from about 1 to about 20 residues), depending on the criteria used to define the domain. , More likely "shifts" by about 1 to about 5 residues). In this context, "about" refers to the size indicated, especially at either or both termini, a few (5, 4, 3, 2, or 1) more amino acid residues than this. Or including small size. In any case, as discussed further below, the present invention further provides polypeptides that have various residues deleted from the N-terminal and / or C-terminal of the complete DR4, including those of the DR4 polypeptide. Included are polypeptides that delete one or more amino acids from the N-terminus of the extracellular domain described herein that make up the soluble form of the extracellular domain.

As indicated, the nucleic acid molecules of the invention may be in the form of RNA (eg, mRNA),
Alternatively, it can be in the form of DNA, including, for example, cDNA and genomic DNA obtained by cloning or produced synthetically. DNA can be double-stranded or single-stranded. Single-stranded DNA can be the coding strand (also known as the sense strand) or the non-coding strand (also called the antisense strand).

By “isolated” nucleic acid molecule is intended a nucleic acid molecule (DNA or RNA) that has been removed from its native environment. For example, the recombinant D contained in the vector
NA molecules are considered isolated for the purposes of the present invention. Isolated DN
Further examples of A molecules include recombinant DNA molecules that are maintained in a heterologous host cell, or (partially or substantially) purified DNA molecules in solution.

However, members of a library of mixed clones (eg, genomic or cDNA libraries) that are not isolated from other clones of the library (eg, other members of the library) (In the form of a homogenous solution containing the clone), or a chromosome isolated or removed from a cell or cell lysate (eg, in the form of a homogeneous solution containing the clone)
"Chromosome spread" as in karyotype
) ") Are not" isolated "for the purposes of the present invention. RNA isolated
Molecules include in vivo or in vitro RNA transcripts of a DNA molecule of the present invention. Nucleic acid molecules isolated according to the present invention further include those molecules that are produced synthetically.

An isolated nucleic acid molecule of the present invention includes: a DR4 DNA molecule that comprises or consists of an open reading frame (ORF) set forth in SEQ ID NO: 1; Substantially different from all or part of the ORF at positions 19-21 of the nucleotide sequence set forth in No. 1, but still contains sequences encoding the DR4 polypeptide or fragment thereof due to the degeneracy of the genetic code. Or a DNA molecule consisting of it. Of course, the genetic code is well known in the art. Therefore, for those skilled in the art,
It is conventional to produce such degenerate variants.

[0039] In another aspect, the invention relates to ATCC accession number 97 on January 21, 1997.
An isolated nucleic acid molecule encoding a DR4 polypeptide having the amino acid sequence encoded by the cDNA contained in the plasmid deposited as 853. Preferably, these nucleic acid molecules encode the mature polypeptide encoded by the deposited cDNA described above. The present invention further provides an isolated nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO: 1 or the nucleotide sequence of the DR4 cDNA contained in the deposited plasmid as described above, or complementary to one of the above sequences. Nucleic acid molecule having a specific sequence. Such an isolated DNA molecule or a fragment thereof is obtained by Northern blot analysis.
Examples include, but are not limited to, use as a DNA probe for producing a genetic map by in situ hybridization of the DR4 gene in human tissues.

[0040] The present invention further relates to fragments of the isolated nucleic acid molecules described herein. A fragment of an isolated DNA molecule having the nucleotide sequence set forth in SEQ ID NO: 1, or an isolated DNA having the nucleotide sequence of a deposited cDNA (cDNA contained in a plasmid deposited as ATCC Accession No. 98853). A fragment of a molecule is at least 20 nt, and more preferably at least 30 nt long, and even more preferably at least about 4 nt.
0, 50, 100, 150, 200, 250, 300, 350, 400, 450
, 500, 550, 600, 650, 700, 750, 800, 850, 900
, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1
A 300, 1350, 1400, 1450, or 1500 nt long DNA fragment is contemplated. This is useful as a DNA probe, as discussed above. Of course, DNA fragments corresponding to most, if not all, of the nucleotide sequence set forth in SEQ ID NO: 1 are also useful as DNA probes. For example, a fragment having a length of about 20 nt means a fragment containing 20 or more bases derived from the nucleotide sequence of SEQ ID NO: 1. In this context, “about” means specifically, at the indicated size, at either or both termini, a few (5, 4, 3, 2, or 1) more or less nucleotides Including size.

Representative examples of DR4 polynucleotide fragments of the present invention include, for example, approximately nucleotides 19-87, 88-732, 88-138 of SEQ ID NO: 1.
, 139-189, 190-240, 241-291, 292-342, 343
705, 343-393, 394-444, 445-495, 496-546
, 546-597, 598-648, 649-699, 700-732, 733
810, 733 to 771, 772 to 810, 811 to 1422, 811 to 86
1, 862-912, 913-963, 964-1014, 1015-1065
, 1066 to 1116, 1117 to 1167, 1153 to 1284, 1153 to
1203, 1204 to 1254, 1255 to 1284, 1168 to 1218, 1
219-1269, 1270-1320, 1321-1371, and 1372
Or fragments complementary to or consisting of the cDNAs contained in the deposited plasmid. In this context, “about” means that a few (5, 4, 3, 2, or 1 or more) at the indicated range, either or both termini,
) Nucleotides larger or smaller.

The invention further relates to a polynucleotide comprising or consisting of an isolated nucleic acid molecule encoding a domain of DR4. In one aspect, the invention provides a polynucleotide comprising or consisting of a nucleic acid molecule encoding the β sheet region of the DR4 protein set forth in Table I. Representative examples of such polynucleotides include amino acid residues from about position 8 to about 17 in SEQ ID NO: 2.
Position, amino acid residue about 53 to about 60, amino acid residue about 87 to about 103, amino acid residue about 146 to about 155, amino acid residue about 161 to about 166, amino acid residue about 214 to about 221, amino acid residue about 240 to about 252, amino acid residue about 257 to about 264, amino acid residue about 274 to about 283, amino acid residue about 324 to about 329 About 349 to about 354, about 363 to about 369, about 371 to about 376, about 394 to about 399, and about 394 to about 399; 453th to about 458
Or a nucleic acid molecule encoding a polypeptide comprising or consisting of one, two, three, four, five, or more amino acid sequences selected from the group consisting of: In this context "about" includes the values specifically stated, values that are larger or smaller by several (5, 4, 3, 2, or 1) amino acids. The polypeptides encoded by these polynucleotides are also encompassed by the present invention.

In certain embodiments, polynucleotide fragments of the present invention encode polypeptides that exhibit DR4 functional activity. By a polypeptide that exhibits a “functional activity” of DR4 is meant a polypeptide that can exhibit one or more known functional activities associated with a complete (full-length) DR4 polypeptide or a mature DR4 polypeptide. Such functional activities include, but are not limited to: biological activity (eg, the ability to induce apoptosis in cells expressing the polypeptide (see, eg, Example 5). ); Antigenicity (ability to bind (ie, compete with a DR4 polypeptide for binding) to an anti-DR4 antibody), immunogenicity (ability to produce an antibody that binds a DR4 polypeptide), ability to form multimers, And the ability to bind a receptor or ligand for a DR4 polypeptide (eg, TRAIL; Wiley et al., Immunity 3, 673-
682 (1995)).

[0044] The functional activity of DR4 polypeptides, and fragments, variants, derivatives, and analogs thereof, can be assayed by various methods.

For example, in one embodiment, various immunoassays known in the art are assayed for the ability to bind to an anti-DR4 antibody or compete with a full-length (complete) DR4 polypeptide for binding to an anti-DR4 antibody. Can be used. Such assays include radioimmunoassays, ELISAs (enzyme-linked immunosorbent assays), "sandwich" immunoassays, immunoradiometric assays, gel diffusion sedimentation reactions, immunodiffusion assays, in situ immunoassays (
(Eg, using colloidal gold, enzyme or radioisotope labels), Western blots, sedimentation reactions, agglutination assays (eg, gel agglutination assays, hemagglutination assays), complement binding assays, immunofluorescence assays, protein A assays, and immunological Includes, but is not limited to, competitive and non-competitive assay systems using techniques such as electrophoresis assays. In one embodiment, antibody binding is detected by detecting a label on the primary antibody. In another embodiment, the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody. In a further embodiment, the secondary antibody is labeled. Many means are known in the art for detecting binding in immunoassays and are within the scope of the invention.

In another embodiment for identifying a DR4 ligand (eg, TRAIL) or assessing the ability of a polypeptide fragment, variant, or derivative of the invention to multimerize, binding is, for example, reduction and It can be assayed by means well known in the art, such as non-reducing gel chromatography, protein affinity chromatography, and affinity blotting. Generally, see Physicky, E .; Et al., Microbiol. Rev .. 59: 94-1
23 (1995). In another embodiment, the physiological correlation (signaling) of DR4 binding to its substrate may be assayed.

In addition, the assays described herein (see Examples 5 and 6)
Otherwise, assays known in the art indicate that DR4 polypeptides and fragments, variants, derivatives, and analogs thereof, are capable of inducing a DR4-related biological activity (eg, bind TRAIL in vitro or in vivo). (Eg, see Example 6), and can be routinely applied to determine the ability to induce apoptosis in cells expressing the polypeptide (eg, see Example 5). . For example, the biological activity can be determined essentially as previously described (Chinnaiyan et al., Cell 81: 505-51.
2 (1995); Boldin et al. Biol. Chem. 270: 7795
-8 (1995); Kischkel et al., EMBO 14: 5579-5588.
(1995); Chinnaiyan et al. Biol. Chem. 271: 4
961-4965 (1996)), and can be routinely measured using a cell death assay performed as set forth in Example 5 below. In one embodiment involving MCF7 cells, a plasmid encoding a candidate cell death domain comprising full length DR4 or the receptor is replaced with pL encoding green fluorescent protein.
Co-transfect with the antern receptor construct. Nuclei of cells transfected with DR4 show apoptotic morphology as assessed by DAPI staining.

Other methods are known to those skilled in the art and are within the scope of the present invention.

[0049] Preferred nucleic acid fragments of the present invention include nucleic acid molecules encoding members selected from the group consisting of: comprising the DR4 extracellular domain (amino acid residues from about 24 to about 238 of SEQ ID NO: 2). Or a polypeptide consisting thereof; cysteine-rich domain of DR4.
Polypeptide comprising or consisting of about 229); a polypeptide comprising or consisting of the DR4 transmembrane domain (amino acid residues about 239 to about 264 of SEQ ID NO: 2); a fragment of a putative mature DR4 polypeptide (herein Wherein the fragment has a DR4 functional activity (eg, antigenic or biological activity); the DR4 intracellular domain (about amino acid residues 265 to 265 of SEQ ID NO: 2).
About 468); a polypeptide comprising or consisting of the extracellular and intracellular domains of the DR4 receptor, wherein all or part of the transmembrane domain is deleted; a DR4 receptor death domain ( Or a polypeptide comprising or consisting of amino acid residues from about 379 to about 422 of SEQ ID NO: 2; one, two, three, four, or more of the DR4 receptor proteins A polypeptide comprising or consisting of an epitope-bearing portion. In a further embodiment, a polynucleotide fragment of the invention comprises 1, 2, 3, 4, 5, 6, 7, or all 8 combinations of any of the above polypeptide embodiments. Or a polypeptide consisting thereof. As described above, with the leader sequence,
Amino acid residues that make up the DR4 receptor extracellular, transmembrane, and intracellular domains were deduced by computer analysis. Thus, those skilled in the art will recognize that the amino acid residues making up these domains may vary slightly (e.g., on the order of about 1 to 15 residues), depending on the criteria used to define each domain. Understand that. The polypeptides encoded by these nucleic acid molecules are also encompassed by the present invention.

It is believed that one or both of the extracellular cysteine-rich motifs of the DR4 polypeptide disclosed in SEQ ID NO: 2 are important for the interaction between DR4 and its ligand (eg, TRAIL). Accordingly, certain embodiments of the present invention relate to amino acid residues 131-183 and / or amino acid residues 184-2 of SEQ ID NO: 2.
29. A polynucleotide encoding a polypeptide comprising or consisting of one or both amino acid sequences. In certain embodiments, a polynucleotide encoding a DR4 polypeptide of the invention comprises or consists of both of the extracellular cysteine-rich motifs disclosed in SEQ ID NO: 2. The polypeptides encoded by these polynucleotides are also encompassed by the present invention.

In a further embodiment, a polynucleotide of the invention encodes a DR4 functional property. In this regard, preferred embodiments of the present invention are 1, 2, 3,
Includes fragments comprising or consisting of 4, or more of the following functional domains: the α-helix and α-helix forming regions of DR4 (“α-region”), β-sheet and β- Sheet-forming region (“β-region”), turn and turn-forming region (“turn-region”), coil and coil-forming region (“coil-region”), hydrophilic region, hydrophobic region, α-amphiphilic region , Β amphipathic region, flexible region, surface forming region and high antigenic index region.

Certain preferred regions for these are shown in FIG. 3, which may be presented or identified by using a spreadsheet representation of the data shown in FIG. 3, as shown in Table I. . Using the DNA ^* STAR computer algorithm (set to the initial default parameters) used to generate FIG.
The data of FIG. 3 is shown in a spreadsheet format (see Table I). Using the spreadsheet format of the data in FIG. 3, the specific boundaries of the preferred region can be easily determined.

The above preferred regions shown in FIG. 3 and Table I include, but are not limited to, regions of the above type identified by analysis of the amino acid sequence shown in SEQ ID NO: 2. As shown in FIG. 3 and Table I, such preferred regions include G
arrier-Robson α-region, β-region, turn-region, and coil-region (columns I, III, V, and VII in Table I), Chou-Fas
man α-region, β-region, and turn-region (columns II, IV in Table I)
, And VI), Kyte-Doolittle hydrophilic region (column VIII of Table I)
), Hopp-Woods hydrophobic region (column IX of Table I), Eisenberg
α-amphiphilic and Eisenberg β-amphiphilic regions (columns X and XI in Table I), Karplus-Schulz flexible region (column XII in Table I)
), The Jameson-Wolf region of high antigenicity index (column XIII in Table I), and the Emini surface-forming region (column XIV in Table I). In this regard,
Particularly highly preferred polynucleotides have several structural features (eg,
It encodes a polypeptide that comprises or consists of a region of DR4 that combines several (eg, one, two, three, or four) features of the same or different regions described above. .

[0054] As described above, the data representing the structural properties or functional properties of the DR4 shown in FIG. 3 and / or Table I, set to the default parameters the DNA ^* ST
Created using various modules and algorithms of AR. In a preferred embodiment, the data shown in Table I, columns VIII, XII, and XIII can be used to determine regions of DR4 that exhibit a high degree of potential for antigenicity.
Regions of high antigenicity are selected by selecting a value that indicates the region of the polypeptide that is likely to be exposed on the surface of the polypeptide, in an environment where antigen recognition can occur in the process of initiating an immune response, column VIII, Determined from the data shown in XII and / or XIII.

[0055]

[Table 1]

A preferred nucleic acid fragment of the invention encodes a full-length DR4 polypeptide lacking the nucleotides encoding the amino-terminal methionine (nucleotides 19-21 of SEQ ID NO: 1). Because methionine is cleaved spontaneously, it is known that such sequences may be useful for genetic engineering of DR4 expression vectors. Polypeptides encoded by such polynucleotides are also included by the present invention.

Among the highly preferred fragments in this regard are fragments that include or consist of a region of DR4 that combines several structural features (eg, some of the features described above). Preferred nucleic acid fragments of the present invention also encode polypeptides comprising or consisting of one, two, three, four, five, or more epitope-bearing portions of the DR4 protein. Nucleic acid molecules, but are not limited thereto. In particular, such nucleic acid fragments of the invention include nucleic acid molecules encoding the following polypeptides: comprising from about 35 to about 92 amino acid residues of SEQ ID NO: 2;
Or a polypeptide consisting thereof; approximately 114 amino acid residues of SEQ ID NO: 2
A polypeptide comprising or consisting of about 160 to about 160; a polypeptide comprising or consisting of about 169 to about 240 amino acid residues of SEQ ID NO: 2; about 267 to about amino acid residue of SEQ ID NO: 2 298 or
Or a polypeptide consisting thereof; approximately 330 amino acid residues of SEQ ID NO: 2
A polypeptide comprising or consisting of about 364 to about 364; a polypeptide comprising or consisting of about 391 to about 404 of amino acid residues of SEQ ID NO: 2; and about 418 to about 418 to about amino acid residues of SEQ ID NO: 2 A polypeptide comprising or consisting of about 9465. In this context, "approximately (
By "about" is meant a larger or smaller size at either or both termini, especially at some (5, 4, 3, 2, or 1) amino acid residues specifically listed. The present inventors have determined that the polypeptide fragment is an antigenic region of DR4 protein. Methods for determining such epitope-bearing portions of the DR4 protein are described in detail below. The polypeptides encoded by these nucleic acids are also included in the present invention.

In certain embodiments, the polynucleotide of the invention is less than 100,000 kb, less than 50,000 kb, less than 10,000 kb, less than 1000 kb, 5
Less than 00 kb length, less than 400 kb length, less than 350 kb length, less than 300 kb length, 2
Less than 50 kb length, less than 200 kb length, less than 175 kb length, less than 150 kb length, 1
Less than 25 kb, less than 100 kb, less than 75 kb, less than 50 kb, 40 k
Less than b length, less than 30 kb length, less than 25 kb length, less than 20 kb length, less than 15 kb length, less than 10 kb length, less than 7.5 kb length, or less than 5 kb length.

In a further embodiment, the polynucleotide of the present invention comprises at least 15, at least 30, at least 50, at least 100 of the DR4 coding sequence.
, Or at least 250, at least 500, or at least 100
It contains or consists of 0 consecutive nucleotides, but does not exceed 1000 kb, 500 kb or less, 250 kb or less of the genomic DNA adjacent to the 5 ′ coding nucleotide or 3 ′ coding nucleotide shown in SEQ ID NO: 1.
kb or less, 150 kb or less, 100 kb or less, 75 kb or less, 50 kb or less, 3
It consists of 0 kb or less, 25 kb or less, 20 kb or less, 15 kb or less, 10 kb or less, or 5 kb or less. In further embodiments, the polynucleotides of the present invention comprise at least 15, at least 30, at least 50 of the DR4 coding sequences.
At least 100, or at least 250, at least 500, or at least 1000 contiguous nucleotides, but does not include all or part of any DR4 intron. In another embodiment, the nucleic acid comprising or consisting of the DR4 coding sequence is a genomic flanking gene (ie, 5 ′ or 3 ′ to the DR4 gene in the genome).
'Side) does not include the code sequence. In another embodiment, the polynucleotide of the invention has more than 1000, more than 500, more than 250, more than 100, more than 50, more than 25, more than 20, 15 Not more than 10, more than 5, more than 4, more than 3, more than 2, or not more than one genomic flanking gene coding sequence.

Further, the present invention provides a nucleic acid molecule having the nucleotide sequence for the extension of SEQ ID NO: 1 as follows: HTOIY07R (SEQ ID NO: 6) and HTX
EY80R (SEQ ID NO: 7) (both shown in FIG. 4).

Further, the present invention provides that at least about 3 of residues 365-1,422 of SEQ ID NO: 1
A polynucleotide comprising or consisting of any portion of 0 nucleotides, preferably at least about 50 nucleotides. In this context, “approximately” means that only a few (5, 4, 3, 2, or 1) of the amino acid residues listed above have a larger or smaller size at either or both termini. means.

In another embodiment, the invention hybridizes under stringent hybridization conditions to a portion of the polynucleotide in a nucleic acid molecule of the invention described above, as described herein. An isolated nucleic acid molecule comprising or consisting of a polynucleotide, eg, complementary to a coding and / or non-coding sequence shown in SEQ ID NO: 1 (ie, a transcribed sequence, a non-transcribed sequence). CDN contained in ATCC accession number 97853
A and a sequence or polynucleotide fragment encoding the DR4 domain are provided. "Stringent hybridization conditions" are 50%
Formamide, 5 × SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 × Denhardt solution,
Incubate overnight at 42 ° C. in a solution containing or consisting of 10% dextran sulfate and 20 μg / ml denatured sheared salmon sperm DNA, followed by washing the filter with 0.1 × SSC at about 65 ° C. It is intended to The polypeptides encoded by these nucleic acids are also included in the present invention.

A polynucleotide that hybridizes to a “portion” of a polynucleotide is at least about 15 nucleotides (nt) of the reference polynucleotide, and more preferably at least about 20 nt, and even more preferably at least about 30 nt.
nt, and even more preferably, polynucleotides (either DNA or RNA) that hybridize to about 30-70 nt. In this context, “approximately” refers to a size that is specifically larger or smaller by some (5, 4, 3, 2, or 1) nucleotides at either or both termini. )including. A portion of a polynucleotide "at least 20 nt long" is intended to mean, for example, 20 or more consecutive nucleotides derived from the nucleotide sequence of the reference polynucleotide (eg, the deposited cDNA or the nucleotide sequence shown in SEQ ID NO: 1). . These include, but are not limited to, use as diagnostic probes and primers as discussed in detail above and below.

Of course, the poly A sequence (eg, 3 ′ of the DR4 cDNA shown in SEQ ID NO: 1)
Polynucleotides that hybridize only to terminal poly (A) tracts) or complementary stretches of T (or U) residues are those of the invention that are used to hybridize to a portion of the nucleic acids of the invention. Not included in polynucleotide. This is because such a polynucleotide will hybridize to any nucleic acid molecule comprising a poly (A) stretch or its complement (eg, in particular, any double-stranded cDNA).

As indicated, nucleic acid molecules of the invention that encode a DR4 polypeptide include, but are not limited to: a sequence that encodes a mature polypeptide alone; A coding sequence (eg, a sequence encoding a leader or secretory sequence (eg, a preprotein sequence, or a proprotein or preproprotein sequence)); additional non-coding sequences (eg, transcription, mRNA processing (splicing, and polyadenylation signals). (Eg, ribosome binding and mRN)
Including, but not limited to, introns and non-coding 5 'and 3' sequences, such as transcribed non-translated sequences that fulfill (stability of A). Coding sequence for the mature polypeptide; additional coding sequence encoding additional amino acids (eg, amino acids that provide additional functionality). Thus, for example, a polypeptide can be fused to a marker sequence, such as a peptide that facilitates purification of the fusion polypeptide. In certain preferred embodiments of this aspect of the invention, the marker sequence is a hexahistidine peptide (eg, a tag provided in a pQE vector (Qiagen, Inc.)), many of which are commercially available. . Gentz et al., Proc
. Natl. Acad. Sci. USA 86: 821-824 (1989), for example, hexa-histidine provides for convenient purification of the fusion protein. The “HA” tag is another peptide useful for purification, corresponding to an epitope derived from the influenza hemagglutinin protein, which comprises:
For example, as described by Wilson et al., Cell 37: 767-778 (1984). As discussed below, other such fusion proteins include the DR4 receptor fused to Fc at the N-terminus or C-terminus.

The present invention further relates to variants of the nucleic acid molecules of the present invention, wherein the variants encode a fragment, analog or derivative of a DR4 polypeptide. Variants can occur naturally, for example, as natural allelic variants. "Allelic variant"
By this is intended one of several alternative forms of the gene occupying a given locus on the chromosome of the organism. Genes II, Lewin, B .; Hen, Joh
n Wiley & Sons, New York (1985). Non-naturally occurring variants can be made using mutagenesis techniques known in the art.

[0067] Such variants include those produced by nucleotide substitutions, deletions or additions, which may involve one or more nucleotides. Substitutions, deletions or additions may involve one or more nucleotides. These variants can be altered in the coding region, non-coding region, or both. Changes in the coding region can result in conservative or non-conservative amino acid substitutions, deletions or additions. Especially preferred among these are silent substitutions, additions and deletions, which are known as D
It does not alter the properties or functional activity of the R4 receptor or portions thereof. Particularly preferred in this regard are conservative substitutions.

[0068] Further embodiments of the invention include the following: (a) a nucleotide sequence encoding a full-length DR4 polypeptide (including the putative leader sequence) having the complete amino acid sequence of SEQ ID NO: 2; A nucleotide sequence encoding a full-length DR4 polypeptide having an amino acid sequence (including a putative leader sequence but lacking an amino-terminal methionine); (c) a mature DR4 polypeptide having an amino acid sequence from about position 24 to about position 468 of SEQ ID NO: 2 (Full length polypeptide with leader removed)
(D) full length D having the complete amino acid sequence including the leader encoded by the cDNA contained in ATCC Accession No. 97853.
Nucleotide sequence encoding R4 polypeptide; (e) ATCC accession number 97
Nucleotide sequence encoding a full length DR4 polypeptide having the complete amino acid sequence comprising the leader but lacking the amino terminal methionine, encoded by the cDNA contained in 853; (f) the amino acid encoded by the cDNA contained in ATCC Accession No. 97853 A nucleotide sequence encoding a mature DR4 polypeptide having the sequence: (g) an amino acid sequence at about positions 24-238 of SEQ ID NO: 2;
Or DR encoded by the cDNA contained in ATCC accession number 97853
A nucleotide sequence encoding a DR4 extracellular domain having four extracellular domains; (h) the amino acid sequence from about position 239 to about position 264 of SEQ ID NO: 2, or A
A nucleotide sequence encoding a DR4 transmembrane domain having a DR4 transmembrane domain encoded by the cDNA contained in TCC Accession No. 97853; (i)
A nucleotide sequence encoding the amino acid sequence at about positions 265 to about 468 of SEQ ID NO: 2, or the DR4 intracellular domain having the DR4 intracellular domain encoded by the cDNA contained in ATCC Accession No. 97853; (j) SEQ ID NO: 2 Amino acid sequence from about position 379 to about position 422, or ATCC accession number 9
D having a DR4 death domain encoded by the cDNA contained in 7853
Nucleotide sequence encoding the R4 death domain; (k) approximately 1 of SEQ ID NO: 2
A D4 having a DR4 cysteine-rich domain encoded by the amino acid sequence from position 31 to about position 229, or the cDNA contained in ATCC Accession No. 97853;
A nucleotide sequence encoding an R4 cysteine-rich domain; (l) a nucleotide sequence encoding a DR4 extracellular domain and an intracellular domain in which all or part of the transmembrane domain has been deleted; (m) a DR4 functional activity (eg, antigen A nucleotide sequence encoding a fragment of the polypeptide of (c) having sexual or biological activity); or (n) a (b), (c), (d), (d)
e), (f), (g), (h), (i), (j), (k), (l) or (m)
At least 80%, and more preferably at least 85%, 90%, 92%, 9% of the nucleotide sequence complementary to any of the nucleotide sequences in
Isolated nucleic acid molecules that are 5%, 96%, 97%, 98% or 99% identical. In this context, “approximately” refers to a size that is specifically larger or smaller by some (5, 4, 3, 2, or 1) nucleotides at either or both termini. )including. The polypeptides encoded by these nucleic acids are also included by the present invention.

A polynucleotide having a nucleotide sequence that is, for example, at least 95% “identical” to a reference nucleotide sequence that encodes a DR4 polypeptide is a polynucleotide that has a nucleotide sequence that is at least 95% “identical” to each of the reference nucleotide sequences that encode the DR4 polypeptide. It is intended that the nucleotide sequence of the polynucleotide be identical to the reference sequence, except that it can contain up to 5 mismatches per 100 nucleotides. In other words, up to 5% of the nucleotides of the reference sequence may be deleted or replaced with another nucleotide to obtain a polynucleotide having a nucleotide sequence at least 95% identical to the reference nucleotide sequence. ,
Alternatively, up to 5% of the total nucleotides in the reference sequence are
It can be inserted into the reference sequence. These mismatches of the reference sequence may occur at the 5 'or 3' terminal positions of the reference nucleotide sequence, or at any position between these terminal positions, and may occur alone or between nucleotides in the reference sequence, or Interspersed in any one or more of the following contiguous groups: As described herein, the reference (query) sequence is SEQ ID NO: 1 or any fragment (eg, an N-terminal and / or C-terminal deletion of DR4 as described herein). (A polynucleotide encoding the amino acid sequence of SEQ ID NO: 1).

As a practical matter, any particular nucleic acid molecule is at least 80%, 85%, 90% identical, for example, to the nucleotide sequence shown in SEQ ID NO: 1 or the nucleotide sequence of the deposited cDNA. , 92% identical, 95% identical, 96%
Whether it is identical, 97% identical, 98% identical or 99% identical is determined by Bestf
it program (Wisconsin Sequence Analysis)
Version 8, for Package, Unix (registered trademark), Genetics C
output Group, University Research Pa
rk, 575 Science Drive, Madison, WI 5371.
It can be determined conventionally using known computer programs such as 1). Bestfit uses Smith and Waterman (Advances in Appl.) To find the segment of highest homology between the two sequences.
ied Mathematics 2: 482-489 (1981)). The specific sequence is relative to a reference sequence according to the invention,
For example, when using Bestfit or any other sequence alignment program to determine if it is 95% identical, it is understood that the percent identity is calculated over the entire length of the reference nucleotide sequence, and The parameters are set such that gaps in homology of up to 5% of the total number of nucleotides in the reference sequence are tolerated.

In certain embodiments, the identity (also referred to as an overall sequence alignment) between a reference (query) sequence (sequence of the invention) and a subject sequence is also determined by Brutlag et al.
. App. Biosci. 6: 237-245 (1990)). Preferred parameters used in FASTDB alignment of DNA sequences to calculate percent identity are: Matrix = Unitary, k-tuple = 4,
Mismatch Penalty = 1, Joining Penalty = 3
0, Randomization Group Length = 0, Cutof
f Score = 1, Gap Penalty = 5, Gap Size Pen
alty 0.05, Window Size = 500 or the length of the nucleotide sequence of interest (whichever is shorter). According to this embodiment, when calculating percent identity, the FASTDB program uses the 5 ′ and 3 ′ of the subject sequence.
In order to take into account the fact that it does not take into account the truncation of
Due to the deletion (not because of an internal deletion), if shorter than the query sequence, a manual correction is made to the result. For subject sequences truncated at the 5 'or 3' end relative to the query sequence, the percent identity is the 5 'and 3' of the subject sequence that are not matched / aligned as a percentage of the total bases of the query sequence. It is corrected by calculating the number of bases in the query sequence. The determination of whether nucleotides are matched / aligned is determined by the result of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity calculated by the FASTDB program above using the specified parameters to arrive at a final percent identity score. This corrected score is used for the purpose of the present embodiment. As indicated by FASTDB alignment, only those bases outside of the 5 'and 3' bases of the subject sequence that are not matched / aligned with the query sequence are calculated for the purpose of manually adjusting the percent identity score. For example, the target sequence of 90 bases is
Aligned to a 100 base query sequence to determine percent identity. The deletion occurs at the 5 'end of the subject sequence, so the FASTDB alignment does not show a match / alignment at the first 10 bases at the 5' end. The 10 unpaired bases represent 10% of the sequence (number of bases at the unmatched 5 'and 3' ends / total number of bases in the query sequence)
So 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 bases match exactly, the final percent identity is 90%. In another example, a 90 base subject sequence is 10
Compared to a zero base query sequence. In this case, the deletion is an internal deletion, so that there are no 5 'or 3' bases of the subject sequence that do not match / align with the query. In this case, the percent identity calculated by FASTDB is not manually corrected. Again, only the 5 'and 3' bases of the subject sequence that do not match / align with the query sequence are manually corrected. Other manual corrections are not made for the purposes of this embodiment.

The present application relates to the nucleic acid sequence set forth in SEQ ID NO: 1, the nucleic acid sequence of the deposited cDNA, or a fragment thereof, whether or not they encode a polypeptide having DR4 functional activity. At least 80% identical, 85% identical, 90%
Nucleic acid molecules that are identical, 92% identical, 95% identical, 96% identical, 97% identical, 98% identical, or 99% identical. Even if a particular nucleic acid molecule does not encode a polypeptide having DR4 functional activity, one of skill in the art can use methods such as hybridization probes or the polymerase chain reaction (P
CR) as a primer). The use of a nucleic acid molecule of the invention that does not encode a polypeptide having DR4 functional activity is particularly useful for (1) cDN
Isolating the DR4 gene or an allelic variant thereof in the A library;
(2) Verma et al. (Human Chromosomes: A Manual)
of Basic Technologies, Pergamon Press,
New York (1988)), in situ hybridization to metaphase chromosome spread (eg, "FISH") to provide the exact chromosomal location of the DR4 gene; and (3)
) Northern blot analysis to detect DR4 mRNA expression in specific tissues.

However, at least 80%, 85%, 90%, 92% of the nucleic acid sequence shown in SEQ ID NO: 1, the nucleic acid sequence of the deposited cDNA, or a fragment thereof
%, 95%, 96%, 97%, 98% or 99% identical nucleic acid molecules are preferred. These in fact encode polypeptides having the functional activity of the DR4 protein. By "a polypeptide having the functional activity of a DR4 protein", a DR4 protein of the invention (full-length (ie, complete) protein or preferably mature protein, as measured in a particular functional and / or biological assay). Are contemplated that exhibit activities similar to (but need not be identical to) the functional activity of any of For example, DR4 polypeptide functional activity is the ability of a polypeptide sequence described herein to form multimers (eg, homodimers and homotrimers) with intact DR4 and to bind a DR4 ligand (eg, TRAIL). Can be measured by These functional assays can be routinely performed using the techniques described herein and other techniques known in the art.

For example, DR4 protein functional activity (eg, biological activity) can be determined essentially as previously described (AM Chinnaiyan et al., Cell 81: 5).
05-512 (1995); P. Boldin et al. Biol. Chem
. 270: 7795-8 (1995); C. Kischkel et al., EMBO
14: 5579-5588 (1995); M. Chinnaiyan et al.
J. Biol. Chem. 271: 4961-4965 (1996)), or can be routinely measured using a cell death assay performed as shown in Example 5 below. In MCF7 cells, the plasmid encoding the full-length DR4 or candidate death domain-containing receptor contains pL encoding green fluorescent protein.
Co-transfected with an intern reporter construct. The nuclei of cells transfected with DR4 show a form of apoptosis as assayed by DAPI staining. TNFR-1 and Fas / APO-1 (MM
Uzio et al., Cell 85: 817-827 (1996); P. Bold
in et al., Cell 85: 803-815 (1996); Tewari et al.
J. Biol. Chem. 270: 3255-60 (1995)).
R4-induced apoptosis is blocked by inhibitors of ICE-like proteases (CrmA and z-VAD-fmk).

Of course, due to the degeneracy of the genetic code, one skilled in the art will appreciate, for example,
At least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% of the nucleic acid sequence of NA or the nucleic acid sequence shown in SEQ ID NO: 1.
It is immediately recognized that many nucleic acid molecules having% identical sequences encode polypeptides that "have the functional activity of a DR4 protein." In fact, since all of these degenerate variants of the nucleotide sequence encode the same polypeptide, this will still be apparent to one skilled in the art without performing the above-described comparative assays. It is further recognized in the art that for such nucleic acid molecules that are not degenerate variants, a reasonable number will also encode a polypeptide having DR4 protein functional activity. This is because, as described further below, one of skill in the art will recognize that amino acid substitutions that do not or do not appear to result in significant protein function (e.g., replacing one aliphatic amino acid with a (Replace with 2 aliphatic amino acids)
Because they are fully aware of

For guidance on how to make phenotypically silent amino acid substitutions, see, for example, Bowie, J. et al. U. Et al., "Deciphering the Messa
Ge in Protein Sequences: Tolerance to
Amino Acid Substitutions "Science 24
7: 1306-1310 (1990). Here, the authors show that the protein is surprisingly tolerant of amino acid substitutions.

Polynucleotide Assay The present invention also relates to the use of a DR4 polynucleotide (eg, as a diagnostic reagent) to detect a complementary polynucleotide. DR4 associated with dysfunction
Detection of a mutant form of can add to the diagnosis of a disease (eg, a tumor or an autoimmune disease) or susceptibility to a disease resulting from reduced, over-, or altered expression of DR4 or a soluble form thereof, or Provides a diagnostic tool that can be defined.

An individual carrying a mutation in the DR4 gene can be detected at the DNA level by various techniques. Nucleic acids for diagnosis can be obtained from patient cells, such as from blood, urine, saliva, tissue biopsies, and autopsy material. Genomic DNA can be used directly for detection or can be amplified enzymatically by using PCR prior to analysis (Saiki et al., Nature, 324: 163-166 (198).
6)). RNA or cDNA can also be used for the same purpose. As an example, PCR primers complementary to the nucleic acid encoding DR4 can be used to identify and analyze DR4 expression and mutation. For example, deletions and insertions can be detected by a change in size of the amplification product when compared to a normal genotype. Point mutations are
The amplified DNA can be identified by hybridizing the amplified DNA to a radiolabeled DR4 RNA or, alternatively, to a radiolabeled DR4 antisense DNA sequence. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase A digestion or by differences in melting points.

Sequence differences between the reference gene and the mutated gene are also direct DNA
It can be shown by sequencing. In addition, cloned DNA segments can be used as probes to detect specific DNA segments. The sensitivity of such a method can be greatly enhanced by the appropriate use of PCR or another amplification method. For example, sequencing primers are used with double-stranded PCR products or single-stranded template molecules generated by modified PCR. Sequence determination is performed by conventional procedures using radiolabeled nucleotides or by automated sequencing procedures using fluorescent tags.

Genetic testing based on DNA sequence differences can be achieved by detecting changes in the electrophoretic mobility of DNA fragments in gels with or without denaturing agents. Small sequence deletions and insertions can be visualized by high resolution gel electrophoresis. DNA fragments of different sequences can be distinguished in denaturing formamide gradient gels, in which the mobility of the different DNA fragments is delayed in the gel at different locations by their specific or partial melting temperatures (eg, Myers et al., Science). , 230: 1242 (1985)).

Sequence changes at certain positions can also be caused by nuclease protection assays (eg, RNa
(eg, se and S1 protection, or chemical cleavage methods).
Cotton et al., Proc. Natl. Acad. Sci. USA, 85:43
97-4401 (1985)).

Thus, detection of a particular DNA sequence can be performed, for example, by hybridization, RN
ase protection, chemical cleavage, direct DNA sequencing or use of restriction enzymes (eg, restriction fragment length polymorphism ("RFLP")), and methods such as Southern blotting of genomic DNA.

In addition to more conventional gel electrophoresis and DNA sequencing, mutations can also be detected by in situ analysis.

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 the production of polypeptides of the present invention by recombinant techniques.

[0085] A host cell can be genetically engineered to incorporate a nucleic acid molecule and express a polypeptide of the invention. Polynucleotides can be introduced alone or with other polynucleotides. Such other polynucleotides can be introduced independently of the polypeptide of the present invention, introduced together with the polypeptide of the present invention, or introduced in association with the polypeptide of the present invention.

In accordance with 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 can be introduced into cells as polynucleotides, preferably DNA, by well known techniques for introducing DNA and RNA into cells. Viral vectors can be replication competent or replication defective. In the latter case, viral propagation generally occurs only in complementary host cells.

In certain aspects, among vectors, vectors for expression of the polynucleotides and polypeptides of the present invention are preferred. Generally, such vectors contain a cis-acting control region effective for expression in a host operably linked to the polynucleotide to be expressed. Suitable trans-acting factors are either supplied by the host, supplied by a complementing vector, or supplied by the vector itself upon introduction into the host.

[0088] A wide variety of expression vectors can be used to express the polypeptides of the present invention.
Such vectors include chromosomal vectors, episomal vectors, and virus-derived vectors (eg, bacterial plasmids, bacteriophages, yeast episomes, yeast chromosomal elements, viruses (eg, baculovirus, papovavirus (eg, SV40), vaccinia virus, Vectors derived from adenovirus, fowlpox virus, pseudorabies virus and retrovirus) and vectors derived from combinations thereof (eg, vectors derived from plasmids and bacteriophage genetic elements (eg, cosmids and phagemids);
All may be used for expression according to this aspect of the invention. Generally, any vector suitable to maintain, propagate, or express a polynucleotide in a host for expression can be used in this regard for expression.

The DNA sequence in the expression vector is operably linked to an appropriate expression control sequence, including, for example, a promoter for direct mRNA transcription. Examples of such promoters include the phage λPL promoter, E. coli, to name just a few of the well-known promoters. coli lac, trp and tac promoters, the SV40 early and late promoters and the promoter of the retroviral LTR. In general, expression constructs will contain sites for transcription initiation and termination, and, in the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcript expressed by this construct contains a translation initiation AUG at the beginning of the translated polypeptide and a stop codon (UAA, UGA, or UAG) appropriately positioned at the end of the translated polypeptide. Including.

In addition, the constructs may include control regions that regulate expression and cause expression. Generally, such regions manipulate transcription by controlling, for example, repressor binding sites and enhancers.

[0091] Vectors for propagation and expression generally include a selectable marker. Such a marker may also be suitable for propagation, or the vector may include an additional marker for this purpose. In this regard, the expression vectors preferably include one or more selectable marker genes to provide phenotypic characteristics for selection of the transformed host cells. Preferred markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, and E. coli. For culturing E. coli and other bacteria, a tetracycline or ampicillin resistance gene is included.

A vector containing a suitable DNA sequence as described elsewhere herein, as well as a suitable promoter and other suitable control sequences, is suitable for expression of a desired polypeptide therein. It can be introduced into a suitable host using a variety of well-known techniques. Representative examples of suitable hosts include bacterial cells (eg, E. coli).
, Streptomyces and Salmonella typhimuri
um cells); fungal cells (eg, yeast cells); insect cells (eg, Drosop)
Hila S2 and Spodoptera Sf9 cells); animal cells (eg, CHO, COS, and Bowes melanoma cells); and plant cells. Hosts for a wide variety of expression constructs are well known, and one of skill in the art, with the present disclosure, can readily select a host for expression of a polypeptide according to this aspect of the invention.

Among the preferred vectors for use in bacteria are pQE70, pQE60 and pQE-9 (available from Qiagen); the pBS vector, Phagecr
ipt vector, Bluescript vector, pNH8A, pNH16a,
pNH18A, pNH46A (available from Stratagene); and ptrc99a, pKK223-3, pKK233-3, pDR540, pRI
T5 (available from Pharmacia). Among preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1 and p
SG (available from Stratagene); and pSVK3, pBPV,
There are pMSG and pSVL (available from Pharmacia). These vectors are listed for the purpose of illustration only of the many commercially available vectors and well-known vectors available to those of skill in the art.

The selection of an appropriate vector and promoter for expression in a host cell is a well-known procedure, and is necessary for the expression of the vector construct, for the introduction of the vector into the host, and for expression in the host. The technology is a conventional technology in the field.

The present invention also relates to host cells containing the above-described vector constructs described herein, and further using one or more techniques known in the art for one or more heterologous control regions (eg, promoters and / or Or a host cell comprising a nucleotide sequence of the present invention operably linked to an enhancer). The host cell can be a higher eukaryotic cell (eg, a mammalian cell (eg, a human-derived cell)) or a lower eukaryotic cell (eg, a yeast cell), or the host cell can be a prokaryotic cell. It can be a biological cell, such as a bacterial cell. The host chain is, in the desired specific manner,
It may be chosen to modulate the expression of the inserted gene sequence or to modify and process the gene product. Expression from a particular promoter can be increased in the presence of a particular inducer; thus, expression of the engineered polypeptide can be controlled. In addition, different host cells have characteristics and specific mechanisms for translational and post-translational processing as well as protein modification (eg, phosphorylation, cleavage). Appropriate cell lines can be chosen to ensure the desired modification and processing of the foreign protein expressed.

The introduction of the construct into the host cell can be performed by calcium phosphate transfection, DE
It can result from AE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other methods. Such methods are described in many standard laboratory manuals (eg, Davis et al., Basic Methods in Mole).
cultural Biology (1986)).

[0097] In addition to including host cells containing the vector constructs discussed herein, the present invention also provides primar (especially mammalian) origin.
y) Including host cells, secondary host cells, and immortalized host cells. These host cells have been engineered to delete or replace endogenous genetic material (eg, a DR4 coding sequence) and / or are genetically operably linked to a DR4 polynucleotide of the invention (eg, (Eg, a heterologous polynucleotide sequence) and activate, alter, and / or amplify an endogenous DR4 polynucleotide. For example, techniques known in the art can be used to operably link heterologous control regions (eg, promoters and / or enhancers) and endogenous DR4 polynucleotide sequences via homologous recombination (eg, US Patent No. 5,641, issued June 24, 1997.
No. 670; International Publication No. WO 96/2941 published September 26, 1996.
No. 1; International Publication No. WO 94/12650 published on August 4, 1994;
See Koller et al., Proc. Natl. Acad. Sci. USA 86:89
32-8935 (1989); and Zijlstra et al., Nature 34.
2: 435-438 (1989). Each of these disclosures is incorporated by reference in their entirety).

[0098] The polypeptides of the present invention may be expressed in a modified form, and may contain not only secretion signals but also additional heterologous functional regions. Such a fusion protein is obtained by linking the polynucleotide of the present invention and a desired nucleic acid sequence encoding a desired amino acid sequence to each other in an appropriate reading frame by a method known in the art, and a method known in the art. By the way,
It can be made by expressing this fusion protein product. Alternatively, such fusion proteins can be made by protein synthesis techniques, for example, by using a peptide synthesizer. Thus, for example, additional amino acids (
In particular, the region of the charged amino acid) may improve the stability and persistence in the host cell during purification or during subsequent handling and storage, so that the N
It can be added to the end. Also, regions can be added to the polypeptide to facilitate purification. Such regions can be removed prior to final preparation of the polypeptide. For example, in one embodiment, a polynucleotide encoding a DR4 polypeptide of the invention is fused to a pelB pectate lyase signal sequence to increase the efficiency of expression and purification of such a polypeptide in Gram-negative bacteria. obtain. U.S. Patent Nos. 5,576,195 and 5,846,8
No. 18, which is incorporated herein by reference in its entirety.

In particular, the addition of peptide moieties to polypeptides to cause secretion or excretion, improve stability, and to facilitate purification is well known in the art and is well known in the art. Technology. Preferred fusion proteins contain heterologous regions from immunoglobulins useful for solubilizing the protein. For example, EP-A
-O 464 533 (Canadian application 2045869) discloses a fusion protein comprising various parts of the constant region of an immunoglobulin molecule together with another human protein or part thereof. In many cases, the Fc part in the fusion protein is sufficiently advantageous for use in therapy and diagnosis, thus resulting, for example, in improved pharmacokinetic properties (EP-A 0232 262). On the other hand, for some uses, it is desirable that the Fc portion can be deleted after the fusion protein has been expressed, detected, and purified in the advantageous manner described. This is when the Fc portion proves to be an obstacle for use in therapy and diagnosis (eg, when the fusion protein is to be used as an antigen for immunization). In drug discovery,
Human proteins, such as the hIL-5 receptor, have been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5. D. Bennett et al., Journal of
Molecular Recognition, Vol. 8: 52-58 (1995)
) And K. Johanson et al., The Journal of Biolo.
Gical Chemistry Vol. 270, No. 16, 9449-9471
(1995).

As described, DR4 polypeptides can be modified by either natural processes (eg, post-translational processing) or by chemical modification techniques well known in the art. It is clear that the same type of modification may be present at the same or varying degrees at several sites in a given DR4 polypeptide. Also,
A given DR4 polypeptide may contain many types of modifications. DR4 polypeptides can be branched (eg, as a result of ubiquitin binding), and they can be cyclic, with or without branching. Cyclic, branched, and branched cyclic DR4 polypeptides can result from post-translation natural processes or can be made by synthetic methods. Modifications include: acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of nucleotides or nucleotide derivatives, covalent attachment of lipids or lipid derivatives, Covalent bond-crosslinking, cyclization, disulfide bond formation, demethylation, covalent cross-link of phosphotidylinositol
Formation, cysteine formation, pyroglutamate formation, formylation, γ-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, PEGylation, proteolytic processing, phosphorylation , Prenylation, racemization, selenization, sulfation, transfer RNA-mediated binding of amino acids to proteins (eg, arginylation), and ubiquitin binding. (For example,
PROTEINS-STRUCTURE AND MOLECULAR PRO
PERTIES, 2nd edition, T.E. E. FIG. Creighton, W.M. H. Freema
n and Company, New York (1993); POSTTRA
NSLATIONAL COVALENT MODIFICATION OF
PROTEINS, B. C. Johnson Edition, Academic Press
, New York, pp. 1-12 (1983); Seifter et al., Meth.
Enzymol 182: 626-646 (1990); Rattan et al., An.
n NY Acad Sci 663: 48-62 (1992)).
.

DR4 can be recovered and purified from chemical synthesis and recombinant cell culture by standard methods, including but not limited to the following:
Ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography ("HPLC") is used for purification.

[0102] DR4 polynucleotides and polypeptides can be used in accordance with the present invention for various applications, particularly those that make use of the chemical and biological properties of DR4. Among them, in the treatment and prevention of tumors, parasitic infections, bacterial infections, viral infections, restenosis and graft-versus-host disease; to induce resistance to parasites, bacteria and viruses; T cells, endothelial cells and To induce proliferation of specific hematopoietic cells; to modulate antiviral responses; and DR by agonists
There are applications to treat and prevent certain autoimmune diseases after 4 stimuli. Further applications relate to the diagnosis, treatment and prevention of disorders of cells, tissues and organisms.
These aspects of the invention are discussed further below.

(DR4 Protein and Fragment) The present invention further provides a DR4 protein comprising a polypeptide sequence encoded by the polynucleotide of the present invention.

The DR4 proteins of the present invention can be monomeric or multimeric (ie, dimers, trimers, tetramers and higher multimers). Therefore,
The present invention relates to monomeric and multimeric DR4 proteins of the invention, their preparation and compositions comprising them (preferably pharmaceutical compositions). In certain embodiments, a polypeptide of the invention is a monomer, dimer, trimer or tetramer. In a further embodiment, a multimer of the invention is at least a dimer, at least a trimer, or at least a tetramer.

The multimers encompassed by the present invention may be homomers or heteromers. As used herein, the term homomer refers to a DR4 protein of the invention (
(Including DR4 fragments, variants, and fusion proteins described herein). These homomers can include DR4 proteins having the same or different polypeptide sequences. In certain embodiments, a homomer of the invention is a multimer comprising only DR4 proteins having the same polypeptide sequence. In another specific embodiment, a homomer of the invention is a multimer comprising DR4 proteins having different polypeptide sequences. In certain embodiments, the multimers of the invention are homodimers (eg, including DR4 proteins having the same or different polypeptide sequences) or homotrimers (eg, including DR4 proteins having the same or different polypeptide sequences). It is. In a further embodiment, the homomeric multimer of the invention is at least a homodimer, at least a homotrimer or at least a homotetramer.

As used herein, the term heteromer refers to a heterologous protein (ie, a protein comprising only a polypeptide sequence that does not correspond to a polypeptide sequence encoded by the DR4 gene) in addition to the DR4 protein of the present invention. )). In certain embodiments, a multimer of the invention is a heterodimer, heterotrimer or heterotetramer. In a further embodiment, the heteromeric multimer of the invention is at least a heterodimer, at least a heterotrimer or at least a heterotetramer.

The multimers of the present invention may be the result of hydrophobic, hydrophilic, ionic and / or covalent associations and / or may be indirectly linked, for example, by liposome formation. Thus, in one embodiment, a multimer of the invention, such as a homodimer or homotrimer, is formed when the proteins of the invention contact each other in solution. In another embodiment, a heteromultimer of the invention (eg, a heterotrimer or heterotetramer) is an antibody against a polypeptide of the invention in solution (a heterologous polypeptide in a fusion protein of the invention). (Including antibodies to the sequence). In other embodiments, the multimers of the invention are formed by covalent linkage to and / or between the DR4 proteins of the invention. Such covalent linkages may include one or more of the polypeptide sequences of the protein (eg, included in the polypeptide sequence set forth in SEQ ID NO: 2 or encoded by the deposited cDNA). It may include amino acid residues. In one example, the covalent bond is a bridge between cysteine residues present within the polypeptide sequence of the interacting protein in the native (ie, naturally occurring) polypeptide. In another example, the covalent bond is the result of a chemical or recombinant operation. Alternatively, such a covalent bond can include one or more amino acid residues contained in a heterologous polypeptide sequence in a DR4 fusion protein. In one example, the covalent bond is between heterologous sequences included in a fusion protein of the invention (eg,
See U.S. Patent No. 5,478,925). In certain instances, the covalent bond is between heterologous sequences contained in a DR4-Fc fusion protein of the invention (as described herein). In another particular example, the covalent attachment of the fusion protein of the invention is to another TNF family ligand /
Receptor members (eg, osteoprotegerin)
gerin) (see, eg, International Publication No. WO 98/49305, the contents of which are hereby incorporated by reference in its entirety).

The multimers of the present invention can be produced using chemical techniques known in the art.
For example, proteins desired to be included in the multimers of the invention can be chemically cross-linked using linker molecules and linker length optimization techniques known in the art (eg, US Pat. No. 478,925, which is hereby incorporated by reference in its entirety). In addition, the multimers of the present invention can be generated using techniques known in the art, with one between the cysteine residues located within the polypeptide sequence of the protein desired to be included in the multimer. These intermolecular crosslinks can be formed (see, for example, US Pat. No. 5,478,925).
No., which is hereby incorporated by reference in its entirety).
In addition, the proteins of the present invention can be conventionally modified by the addition of cysteine or biotin to the C-terminus or N-terminus of the polypeptide sequence of the protein, and techniques known in the art may be used to modify one or more of these It can be applied to generate multimers containing modified proteins (see, eg, US Pat. No. 5,478,925, which is hereby incorporated by reference in its entirety). In addition, techniques known in the art can be applied to generate liposomes containing the protein component desired to be included in the multimers of the present invention (eg, US Pat. No. 5,478,925, which is incorporated herein by reference). ), Incorporated herein by reference in its entirety).

Alternatively, the multimers of the present invention can be generated using genetic engineering techniques known in the art.

In one embodiment, the proteins comprised in the multimers of the invention are recombinantly produced using fusion protein techniques described herein or otherwise known in the art (eg, (See US Patent No. 5,478,925, which is incorporated herein by reference in its entirety). In certain embodiments, the polynucleotide encoding a homodimer of the invention comprises a polynucleotide sequence encoding a polypeptide of the invention ligated to a sequence encoding a linker polypeptide, and then further from the original C-terminal to the N-terminal. Produced by linking to a synthetic polynucleotide (lacking the leader sequence) which encodes the translation product of the polypeptide in the opposite direction to that of (e.g., U.S. Pat. See patent 5,478,925). In another embodiment, a book comprising a transmembrane domain and capable of being incorporated into liposomes by membrane reconstitution techniques, applying recombinant techniques described herein or otherwise known in the art. Produce a recombinant polypeptide of the invention (see, for example, US Pat. No. 5,478,925, which is incorporated herein by reference in its entirety).

The polypeptides of the present invention are preferably provided in an isolated form, and preferably are substantially purified. In one embodiment, the present invention provides a method for depositing c
Provided is an isolated DR4 polypeptide having the amino acid sequence encoded by DNA, or the amino acid sequence of SEQ ID NO: 2, or a peptide or polypeptide portion comprising a portion (ie, a fragment) of the polypeptide.

A polypeptide fragment of the invention comprises a polypeptide comprising or alternatively consisting of: the amino acid sequence contained in SEQ ID NO: 2, encoded by the cDNA contained in the deposited plasmid An amino acid sequence, or an amino acid sequence encoded by a nucleic acid that hybridizes (eg, under stringent hybridization conditions) to a nucleotide sequence contained in a deposited plasmid, or the amino acid sequence shown in FIG. Complementary strand of.
A protein fragment can be "free-standing" or contained within a larger polypeptide, which fragment forms a portion or region, most preferably as a single contiguous region. Can be
Representative examples of polypeptide fragments of the present invention include, for example, fragments comprising or consisting of: amino acid residues 1-23, 24-43, 44- of the coding region of SEQ ID NO: 2. 63, 64-83, 84-103
, 104-123, 124-143, 144-163, 164-183, 184
~ 203, 204 ~ 223, 224 ~ 238, 239 ~ 264, 265 ~ 284
, 285-304, 305-324, 325-345, 346-366, 367
-387, 388-418, 419-439, and / or 440-last. Further representative examples of polypeptide fragments of the present invention include, for example, fragments comprising or consisting of: amino acid residues 1-60, 11-70, 31 of the coding region of SEQ ID NO: 2. ~ 90, 41-100, 61
~ 120, 71 ~ 130, 81 ~ 140, 91 ~ 150, 101 ~ 170, 12
1-180, 131-190, 141-200, 151-210, 171-23
0, 181-240, 191-250, 201-260, 211-270, 22
1-280, 231-290, 241-300, 251-310, 261-32
0, 271-330, 281-340, 291-350, 301-360, 31
1-370, 321-380, 331-390, 341-400, 351-41
0, 361-420, 371-430, 381-440, 391-450, and / or 401-468. Isolated polynucleotides encoding these polypeptides are also included in the invention. "Approximately" in this context means
At either or both termini, some of the specifically listed ranges (5,
Includes ranges larger or smaller by 4, 3, 2 or 1) amino acids.
Further, the polypeptide fragment may have at least 10, 20, 30, 40, 5
0, 60, 70, 80, 90, 100, 110, 120, 130, 140, 15
It can be 0, 175 or 200 amino acids long. Polynucleotides encoding these polypeptides are also included in the present invention.

Preferred polypeptide fragments of the invention include members selected from the following group: include the DR4 receptor extracellular domain (estimated to comprise about amino acid residues 24 to about 238 of SEQ ID NO: 2). A polypeptide comprising or consisting of a cysteine-rich domain of DR4 (presumed to constitute amino acid residues 131 to 229 of SEQ ID NO: 2); A polypeptide comprising or consisting of (presumed to comprise about amino acid residues 239 to about 264 of SEQ ID NO: 2); a polypeptide comprising or consisting of a fragment of a putative mature DR4 polypeptide (Where the fragment is DR4 Having a functional activity (eg, having an antigenic or biological activity); D
A polypeptide comprising or consisting of the R4 receptor intracellular domain (estimated to comprise about amino acid residues 265 to about 468 of SEQ ID NO: 2); all or part of the deleted transmembrane domain A polypeptide comprising or consisting of a DR4 receptor extracellular domain and an intracellular domain; comprising a DR4 receptor death domain (estimated to comprise amino acid residues from about 379 to about 422 of SEQ ID NO: 2) Or consisting of a polypeptide; and 1, 2, 3, 4 of the DR4 receptor protein
A polypeptide comprising or consisting of more than one epitope bearing moiety. In a further embodiment, a polypeptide fragment of the invention comprises or consists of any combination of 1, 2, 3, 4, 5, 6, 7, or all 8 of the above members. As described above, the amino acid residues constituting the DR4 receptor extracellular domain, transmembrane domain and intracellular domain together with the leader sequence are estimated by computer analysis. Thus, as the skilled artisan will appreciate, the amino acid residues that make up these domains may vary slightly, depending on the criteria used to define each domain (e.g., from about 1 to about 15 amino acid residues). Polynucleotides encoding these polypeptides are also included in the present invention.

As discussed above, one or both extracellular cysteine-rich motifs of DR4 are believed to be important for the interaction between DR4 and its ligand (eg, TRAIL). Thus, in a preferred embodiment, a polypeptide fragment of the invention comprises or consists of amino acid residues 131-183 and / or 184-229 of SEQ ID NO: 2. In certain embodiments, a polypeptide of the invention comprises or consists of both extracellular cysteine-rich motifs disclosed in SEQ ID NO: 2.

Among the particularly preferred fragments of the present invention are fragments that comprise or consist of the structural or functional properties of DR4. Such a fragment contains the α-helix and α of full (ie, full-length) DR4.
Helix-forming regions (“α-regions”), β-sheets and β-sheet-forming regions (
“Β region”), turns and turn forming regions (“turn regions”), coils and coil forming regions (“coil regions”), hydrophilic regions, hydrophobic regions, α amphiphilic regions, β amphiphilic regions, A surface-forming region, and a high antigenic indicator region (ie, Jam
a region of the polypeptide comprising four or more amino acid residues having an antigenic index of 1.5 or more, as identified using the default parameters of the eson-Wolf program). Alternatively, it consists of 1, 2, 3, 4, or more of these functional domains. Particular preferred regions are those disclosed in FIG. 3 and Table 1, and include, but are not limited to, regions of the type described above identified by analysis of the amino acid sequence set forth in SEQ ID NO: 2. Such preferred regions include the Garnier-Robson estimated α, as estimated using the default parameters of these computer programs.
Region, β region, turn region, and coil region; Chou-Fasman estimated α
Region, β region, turn region and coil region; Kyte-Doolittle putative hydrophilic region and hydrophobic region; Eisenberg α amphiphilic region and β
An amphipathic region; a Karplus-Schulz flexible region; an Emini surface-forming region and a Jameson-Wolf high antigenic index region. Polynucleotides encoding these polypeptides are also included by the present invention.

The present invention relates to an epitope of a polypeptide having the amino acid sequence of SEQ ID NO: 2, or the polynucleotide sequence contained in the deposited cDNA identified as ATCC Accession No. 97853, or the complement of the sequence of SEQ ID NO: 1. An epitope of a polypeptide sequence encoded by a polynucleotide that hybridizes to, or identified as, ATCC Accession No. 97853 under stringent or lower stringency hybridization conditions as defined above. Includes a polypeptide comprising or consisting of an epitope of the polypeptide sequence encoded by the polynucleotide sequence contained in the deposited cDNA. The invention further provides a polynucleotide sequence encoding an epitope of a polypeptide sequence of the invention (eg, the sequence disclosed in SEQ ID NO: 1), a polynucleotide sequence complementary to the polynucleotide sequence encoding an epitope of the invention, And polynucleotide sequences that hybridize to complementary strands under stringent hybridization conditions or lower stringency hybridization conditions as defined above.

The term “epitope” as used herein refers to a portion of a polypeptide that has antigenic or immunogenic activity in an animal, preferably in a mammal, and most preferably in a human. Say. In a preferred embodiment,
The invention includes polypeptides that include an epitope, and polynucleotides that encode the polypeptides. An “immunogenic epitope” as used herein, as determined by any method known in the art (eg, by the methods for producing antibodies described below). , Defined as part of a protein that elicits an antibody response in an animal (eg, Geysen et al., Proc.
. Natl. Acad. Sci. USA 81: 3998-4002 (1983)
)checking). The term “antigenic epitope” as used herein refers to an antibody, as determined by any method known in the art (eg, by the immunoassays described herein). Is defined as the portion of the protein that can immunospecifically bind to its antigen. Immunospecific binding excludes non-specific binding but need not exclude cross-reactivity with other antigens. An antigenic epitope need not be immunogenic.

[0118] Fragments that function as epitopes can be produced by any conventional method. (See, for example, Houghten, Proc. Natl. Acad. Sci.
. USA 82: 5131-5135 (1985). This is further described in U.S. Pat. No. 4,631,211).

The antigenic epitope-bearing peptides and polypeptides of the invention are therefore useful for raising antibodies (including monoclonal antibodies) that specifically bind to the polypeptides of the invention. See, eg, Wilson et al., Cell 37: 767-77.
8 (1984) at 777.

In the present invention, the antigenic epitope is preferably at least 4, at least 5, at least 6, at least 7 in the amino acid sequence of the polypeptide of the present invention.
Amino acid sequences, more preferably at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, and most preferably between about 15 and about 30 amino acids. Preferred polypeptides containing an immunogenic or antigenic epitope are at least 10,
15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,
75, 80, 85, 90, 95 or 100 amino acid residues in length. Antigenic epitopes are useful (eg, for raising antibodies (including monoclonal antibodies) that specifically bind to the epitope). Antigenic epitopes can be used as target molecules in immunoassays. (See, eg, Wilson et al., Cel.
l 37: 767-778 (1984); Sutcliffe et al., Science.
e 219: 660-666 (1983)). Polynucleotides encoding these polypeptides are also included in the present invention.

Similarly, immunogenic epitopes can be used to derive antibodies, for example, according to methods well known in the art. (See, e.g., Sutcliffe et al., Supra; Wilson et al., Supra; Chow et al., Proc. Natl. Acad. Sci. USA 82:
910-914; and Bittle et al. Gen. Virol. 66:23
47-2354 (1985)). Preferred immunogenic epitopes are
Contains secreted proteins. A polypeptide comprising one or more immunogenic epitopes comprises
If the polypeptide can be presented with a carrier protein (eg, albumin) to elicit an antibody response against an animal system (eg, rabbit or mouse), or if the polypeptide is sufficiently long (at least about 25 amino acids), the polypeptide Can be presented without a carrier. However, immunogenic epitopes containing as few as 8 to 10 amino acids have been shown to be sufficient to elicit antibodies that can bind at least to linear epitopes in the modified polypeptide ( For example, in Western blotting).

[0122] The epitope-bearing polypeptides of the present invention can be used to elicit antibodies according to methods well known in the art. These well-known methods include in vivo immunization,
These include, but are not limited to, in vitro immunization, and phage display methods. See, for example, Sutcliffe et al., Supra; Wilson et al., Supra, and Bittle et al. Gen. Virol. , 66: 2347-2354 (1
985). When using in vivo immunization, animals can be immunized with the free peptide; however, anti-peptide antibody titers allow binding of the peptide to a macromolecular carrier such as keyhole limpet hemocyanin (KLH) or tetanus toxoid. Can be boosted. For example, a peptide containing a cysteine residue is a maleimidobenzoyl-N-hydroxysuccinimide ester (M
BS) can be attached to the carrier using a linker. On the other hand, other peptides can be conjugated to the carrier using more common binders such as glutaraldehyde. Animals such as rabbits, rats, and mice use either free or carrier-bound peptides, for example, about 100 μg of peptide or carrier protein and Freund's adjuvant or other known to stimulate an immune response. Is immunized by intraperitoneal injection and / or intradermal injection of an emulsion containing the adjuvant of the invention. Several booster injections may be required, for example, at intervals of about two weeks to provide useful titers of the anti-peptide antibody. This titer can be detected, for example, by an ELISA assay using the free peptide adsorbed on the solid surface. The titer of the anti-peptide antibody in the serum from the immunized animal is determined by the choice of the anti-peptide antibody (eg, by adsorption to the peptide on a solid support and elution of the selected antibody according to methods well known in the art). Can rise.

For the selection of peptides or polypeptides bearing an antigenic epitope (ie, including regions of the protein molecule to which an antibody can bind), relatively short synthetic peptides that mimic a portion of the protein sequence may be partially It is well known in the art that antisera that react with a mimicked protein can be routinely induced. For example, Su
tcliffe, J.M. G. FIG. Shinnick, T .; M. Green, N .; And Learner, R .; A. (1983) Antibodies that
react with predetermined sites on pr
oteins. Science 219: 660-666. Peptides that can elicit protein-reactive sera are often shown in the primary sequence of proteins, can be characterized by a set of simple chemical rules, and are immunodominant regions of intact proteins (ie, immunogenic epitopes). Or the amino or carboxy terminus.

Non-limiting examples of antigenic polypeptides or peptides that can be used to generate DR4-specific antibodies include: about 35 in SEQ ID NO: 2.
A polypeptide comprising or consisting of about 114 to about 160 amino acid residues in SEQ ID NO: 2; a polypeptide comprising or consisting of about 114 to about 160 amino acid residues in SEQ ID NO: 2; A polypeptide comprising or consisting of 240 amino acid residues;
A polypeptide comprising or consisting of about 298 amino acid residues; or a polypeptide comprising or consisting of about 330 to about 364 amino acid residues in SEQ ID NO: 2; about 391 to about 404 in SEQ ID NO: 2 A polypeptide comprising or consisting of the amino acid residues of SEQ ID NO: 2;
A polypeptide comprising or consisting of 18 to about 465 amino acid residues. In this context, “about” includes the specified recited range, either large or small by several (5, 4, 3, 2, or 1) amino acid residues at either or both termini. . As indicated above, we have determined that the above polypeptide fragment is an antigenic region of the DR4 protein. Polynucleotides encoding these polypeptides are also included in the present invention.

As will be appreciated by those of skill in the art and discussed above, polypeptides of the present invention that include an immunogenic or antigenic epitope may be fused to other polypeptide sequences. For example, the polypeptides of the present invention may comprise immunoglobulins (IgA, IgE, IgG)
, IgM) or portions thereof (CH1, CH2, CH3, or any combination thereof and portions thereof), which can be fused to a chimeric polypeptide. Such fusion proteins can facilitate purification and increase half-life in vivo. This is shown for a chimeric protein consisting of the first two domains of the human CD4-polypeptide and various domains of the heavy or light chain constant region of a mammalian immunoglobulin. For example, EP 394,827;
See aunecker et al., Nature 331: 84-86 (1988). It has been found that an IgG fusion protein having a disulfide-linked dimer structure due to the disulfide bond of the IgG moiety may also be more efficient at binding and neutralizing other molecules than the monomeric polypeptide or fragment thereof alone. Was issued. See, eg, Footoulakis et al. Biochem. , 270
: 3958-3964 (1995). Nucleic acids encoding the above-described epitopes can also be combined with the gene of interest as an epitope tag (eg, a hemagglutinin (“HA”) tag or a flag tag) to aid detection and purification of the expressed polypeptide. . For example, the system described by Janknecht et al. Allows for the rapid purification of native fusion proteins expressed in human cell lines (Janknecht et al., 1991, Proc. Natl. Ac).
ad. Sci. ScL USA 88: 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 six histidine residues. This tag serves as a matrix binding domain for the fusion protein. Extracts from cells infected with the recombinant vaccinia virus are loaded onto a Ni2 ⁺ nitriloacetic acid-agarose column, and histidine-tagged proteins can be selectively eluted with imidazole containing buffer.

Further fusion proteins of the invention can be made via the techniques of gene shuffling, motif shuffling, exon shuffling, and / or codon shuffling (collectively referred to as “DNA shuffling”). D
NA shuffling can be used to modulate the activity of a polypeptide of the invention, and such methods can be used to make polypeptides with altered activity, and agonists and antagonists of the polypeptide . In general, U.S. Patent Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252; and 5,837,4.
No. 58, and Patten et al., Curr. Opinion Biotech
nol. 8: 724-33 (1997); Harayama, Trends B.
iotechnol. 16 (2): 76-82 (1998); Hansson et al. Mol. Biol. 287: 265-76 (1999); and Lor.
Enzo and Blasco, Biotechniques 24 (2): 30.
8-13 (1998), each of which is hereby incorporated by reference in its entirety. In one embodiment, the alterations in the polynucleotides corresponding to SEQ ID NO: 1 and the polypeptides encoded by these polynucleotides can be achieved by DNA shuffling.
DNA shuffling involves the assembly of two or more DNA segments by homologous recombination or site-specific recombination to generate a change in a polynucleotide sequence. In another embodiment, the polynucleotide or encoded polypeptide of the present invention comprises an error-prone PCR, prior to recombination,
It can be altered by subjecting it to random nucleotide insertion or random mutagenesis by other methods. In another embodiment, one or more components, motifs, sections, portions, domains, fragments, etc., of a polynucleotide encoding a polypeptide of the invention comprise one or more components, motifs, one or more heterologous molecules.
It can be recombined in sections, parts, domains, fragments and the like.

As will be apparent to those skilled in the art, a DR4 polypeptide of the invention and an epitope-bearing fragment thereof (eg, amino acid residues 1 to 4 of SEQ ID NO: 2) described herein.
(Corresponding to a portion of the extracellular domain such as 240) are immunoglobulins (IgG
) Can be attached to portions of the constant domains, resulting in a chimeric polypeptide. These fusion proteins facilitate purification and show an increased half-life in vivo. This has been shown, for example, for chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins (EP A 394,827; Traunec
ker et al., Nature 331: 84-86 (1988)). Fusion proteins having a disulfide-linked dimer structure with an IgG moiety may also be more efficient at binding and neutralizing other molecules than monomeric DR4 proteins or protein fragments alone (Funtoulakis et al., J Bioch).
em 270: 3958-3964 (1995)). The epitope-bearing peptides and polypeptides of the present invention can be produced by any conventional means. Ho
Ughten, R.A. A. , "General method for the
rapid solid-phase synthesis of large
numbers of peptides: specificity of numbers
antigen-antibody interaction at the
level of individual amino acids, "Pro
c. Natl. Acad. Sci. USA 82: 5131-5135 (198
5). This "Simultaneous Multiple Peptide"
Synthesis (SMPS) process is described in Houghten et al. (1986).
No. 4,631,211).

[0128] Protein engineering can be used to improve or alter the characteristics of a DR4 polypeptide. Recombinant DNA techniques known to those skilled in the art can be used to generate novel mutant proteins or "muteins", including one or more amino acid substitutions, deletions, additions, or fusion proteins. Such modified polypeptides can exhibit, for example, increased activity or increased stability. In addition, they can be purified in high yields and exhibit, at least under certain purification and storage conditions, higher solubility than the corresponding native polypeptide.

For example, for many proteins, including the extracellular domain of the mature form of a membrane-associated or secreted protein, one or more amino acids have been deleted from the N-terminus or C-terminus without substantial loss of biological function It is known in the art that this can be done. See, for example, Ron et al. Biol. Chem. , 268: 2984-2.
988 (1993) reported a modified KGF protein that has heparin binding activity even without 3, 8, or 27 amino-terminal amino acid residues.
In the case of the present invention, deletion of the N-terminal amino acid up to the cysteine residue at position 132 in SEQ ID NO: 2 induces apoptosis since the protein of the present invention is a member of the death domain containing receptor (DDCR) polypeptide family It may retain some biological activity, such as ability. A polypeptide having an additional N-terminal deletion comprising a cysteine residue at position 132 (C-132) in SEQ ID NO: 2,
It is not expected to retain such biological activity. Because this residue is conserved among members of the family (see FIG. 2), it is necessary to form disulfide bridges to provide the necessary structural stability for receptor binding. This is because

However, even if the deletion of one or more amino acids from the N-terminus of the protein results in a modification or loss of one or more biological functions of the protein, other functional activities (eg, Biological activity, ability to multimerize, DR4 ligand (eg, TR
AIL) can still be retained. For example, shortened DR
The ability of a 4 mutein to induce and / or bind to an antibody that recognizes an intact or mature form of a DR4 polypeptide of the present invention (preferably, an antibody that specifically binds DR4) is determined by the ability of the intact or mature polymorph. If less than the majority of the residues in the peptide are removed from the N-terminus, they are generally retained. Whether a particular polypeptide lacking the N-terminal residue of an intact polypeptide retains such immunogenic activity is described herein and otherwise known in the art. Can be easily determined by a practical method. Possibly DR4 with multiple deleted N-terminal amino acid residues
Muteins may retain some biological or immunogenic activity. In fact, peptides consisting of six DR4 amino acid residues can often elicit an immune response.

Accordingly, the present invention relates to polypeptides having a deletion of one or more residues from the amino terminus of the DR4 amino acid sequence set forth in SEQ ID NO: 2 to the serine residue at position 463, and to such polypeptides There is further provided a polynucleotide encoding
In particular, the invention comprises the amino acid sequence of residues n ¹ -468 of SEQ ID NO: 2,
Alternatively, there is provided a polypeptide consisting of these, wherein n ¹ is an integer of 2-463 corresponding to the position of an amino acid residue in SEQ ID NO: 2.

More particularly, the present invention provides a polynucleotide encoding a polypeptide comprising or consisting of the amino acid sequence of the following residues of the sequence of DR4 shown in SEQ ID NO: 2: 2-E-468; P-3 to E-468; P
-4 to E-468; P-5 to E-468; A-6 to E-468; R-7 to E-4
68; V-8 to E-468; H-9 to E-468; L-10 to E-468; G-
11 to E-468; A-12 to E-468; F-13 to E-468; L-14 to
E-468; A-15 to E-468; V-16 to E-468; T-17 to E-4
68; P-18 to E-468; N-19 to E-468; P-20 to E-468;
G-21 to E-468; S-22 to E-468; A-23 to E-468; A-2
4-E-468; S-25-E-468; G-26-E-468; T-27-E
-468; E-28 to E-468; A-29 to E-468; A-30 to E-46.
8; A-31 to E-468; A-32 to E-468; T-33 to E-468; P
-34 to E-468; S-35 to E-468; K-36 to E-468; V-37
E-468; W-38 to E-468; G-39 to E-468; S-40 to E-.
468; S-41 to E-468; A-42 to E-468; G-43 to E-468.
R-44 to E-468; I-45 to E-468; E-46 to E-468;
47-E468; R-48-E-468; G-49-E-468; G-50-E
-468; G-51 to E-468; R-52 to E468; G-53 to E-468.
A-54 to E-468; L-55 to E-468; P-56 to E-468;
57-E468; S-58-E-468; M-59-E-468; G-60-E
-468; Q-61 to E-468; H-62 to E-468; G-63 to E-46.
8; P-64 to E-468; S-65 to E-468; A-66 to E-468; R
-67 to E-468; A-68 to E-468; R-69 to E-468; A-70
G-71 to E-468; R-72 to E-468; A-73 to E-.
468; P-74 to E-468; G-75 to E-468; P-76 to E-468
R-77 to E-468; P-78 to E-468; A-79 to E-468;
80-E-468; E-81-E-468; A-82-E-468; S-83-
E-468; P-84 to E-468; R-85 to E-468; L-86 to E-4
R-87 to E-468; V-88 to E-468; H-89 to E-468;
K-90 to E-468; T-91 to E-468; F-92 to E-468; K-9
3-E-468; F-94-E-468; V-95-E-468; V-96-E
-468; V-97 to E-468; G-98 to E-468; V-99 to E-468.
L-100 to E-468; L-101 to E-468; Q-102 to E-468;
V-103 to E-468; V-104 to E-468; P-105 to E-468;
S-106 to E-468; S-107 to E-468; A-108 to E-468;
A-109 to E-468; T-110 to E-468; I-111 to E-468;
K-112 to E-468; L-113 to E-468; H-114 to E-468;
D-115 to E-468; Q-116 to E-468; S-117 to E-468;
I-118 to E-468; G-119 to E-468; T-120 to E-468;
Q-121 to E-468; Q-122 to E-468; W-123 to E-468;
E-124 to E-468; H-125 to E-468; S-126 to E-468;
P-127 to E-468; L-128 to E-468; G-129 to E-468;
E-130 to E-468; L-131 to E-468; C-132 to E-468;
P-133 to E-468; P-134 to E-468; G-135 to E-468;
S-136 to E-468; H-137 to E-468; R-138 to E-468;
S-139 to E-468; E-140 to E-468; R-141 to E-468;
P-142 to E-468; G-143 to E-468; A-144 to E-468;
C-145 to E-468; N-146 to E-468; R-147 to E-468;
C-148 to E-468; T-149 to E-468; E-150 to E-468.
G-151 to E-468; V-152 to E-468; G-153 to E-468;
Y-154 to E-468; T-155 to E-468; N-156 to E-468;
A-157 to E-468; S-158 to E-468; N-159 to E-468;
N-160 to E-468; L-161 to E-468; F-162 to E-468;
A-163 to E-468; C-164 to E-468; L-165 to E-468;
P-166 to E-468; C-167 to E-468; T-168 to E-468;
A-169 to E-468; C-170 to E-468; K-171 to E-468;
S-172 to E-468; D-173 to E-468; E-174 to E-468;
E-175 to E-468; E-176 to E-468; R-177 to E-468;
S-178 to E-468; P-179 to E-468; C-180 to E-468;
T-181 to E-468; T-182 to E-468; T-183 to E-468;
R-184 to E-468; N-185 to E-468; T-186 to E-468;
A-187 to E-468; C-188 to E-468; Q-189 to E-468;
C-190 to E-468; K-191 to E-468; P-192 to E-468;
G-193 to E-468; T-194 to E-468; F-195 to E-468;
R-196 to E-468; N-197 to E-468; D-198 to E-468;
N-199 to E-468; S-200 to E-468; A-201 to E-468;
E-202 to E-468; M-203 to E-468; C-204 to E-468;
R-205 to E-468; K-206 to E-468; C-207 to E-468;
S-208 to E-468; T-209 to E-468; G-210 to E-468;
C-211-E-468; P-212-E-468; R-213-E-468;
G-214 to E-468; M-215 to E-468; V-216 to E-468;
K-217 to E-468; V-218 to E-468; K-219 to E-468;
D-220 to E-468; C-221 to E-468; T-222 to E-468;
P-223 to E-468; W-224 to E-468; S-225 to E-468;
D-226 to E-468; I-227 to E-468; E-228 to E-468;
C-229 to E-468; V-230 to E-468; H-231 to E-468;
K-232 to E-468; E-233 to E-468; S-234 to E-468
G-235 to E-468; N-236 to E-468; G-237 to E-468;
H-238 to E-468; N-239 to E-468; I-240 to E-468;
W-241 to E-468; V-242 to E-468; I-243 to E-468;
L-244 to E-468; V-245 to E-468; V-246 to E-468;
T-247 to E-468; L-248 to E-468; V-249 to E-468;
V-250 to E-468; P-251 to E-468; L-252 to E-468;
L-253 to E-468; L-254 to E-468; V-255 to E-468;
A-256 to E-468; V-257 to E-468; L-258 to E-468;
I-259 to E-468; V-260 to E-468; C-261 to E-468;
C-262 to E-468; C-263 to E-468; I-264 to E-468;
G-265 to E-468; S-266 to E-468; G-267 to E-468;
G-269 to E-468; G-270 to E-468;
D-271 to E-468; P-272 to E-468; K-273 to E-468;
C-274 to E-468; M-275 to E-468; D-276 to E-468;
R-277 to E-468; V-278 to E-468; C-279 to E-468;
F-280 to E-468; W-281 to E-468; R-282 to E-468;
L-283 to E-468; G-284 to E-468; L-285 to E-468;
L-286 to E-468; R-287 to E-468; G-288 to E-468;
P-289 to E-468; G-290 to E-468; A-291 to E-468;
E-292 to E-468; D-293 to E-468; N-294 to E-468;
A-295-E-468; H-296-E-468; N-297-E-468;
E-298 to E-468; I-299 to E-468; L-300 to E-468;
S-301 to E-468; N-302 to E-468; A-303 to E-468;
D-304 to E-468; S-305 to E-468; L-306 to E-468;
S-307 to E-468; T-308 to E-468; F-309 to E-468;
V-310-E-468; S-311-E-468; E-312-E-468;
Q-313 to E-468; Q-314 to E-468; M-315 to E-468;
E-316 to E-468; S-317 to E-468; Q-318 to E-468;
E-319 to E-468; P-320 to E-468; A-321 to E-468;
D-322 to E-468; L-323 to E-468; T-324 to E-468.
G-325 to E-468; V-326 to E-468; T-327 to E-468.
V-328 to E-468; Q-329 to E-468; S-330 to E-468;
P-331 to E-468; G-332 to E-468; E-333 to E-468;
A-334 to E-468; Q-335 to E-468; C-336 to E-468;
L-337 to E-468; L-338 to E-468; G-339 to E-468;
P-340 to E-468; A-341 to E-468; E-342 to E-468;
A-343 to E-468; E-344 to E-468; G-345 to E-468;
S-346 to E-468; Q-347 to E-468; R-348 to E-468;
R-349 to E-468; R-350 to E-468; L-351 to E-468;
L-352 to E-468; V-353 to E-468; P-354 to E-468;
A-355 to E-468; N-356 to E-468; G-357 to E-468;
A-358 to E-468; D-359 to E-468; P-360 to E-468;
T-361 to E-468; E-362 to E-468; T-363 to E-468.
L-364 to E-468; M-365 to E-468; L-366 to E-468;
F-368 to E-468; F-368 to E-468; D-369 to E-468;
K-370 to E-468; F-371 to E-468; A-372 to E-468;
N-373 to E-468; I-374 to E-468; V-375 to E-468;
P-376 to E-468; F-377 to E-468; D-378 to E-468;
S-379 to E-468; W-380 to E-468; D-381 to E-468;
Q-382 to E-468; L-383 to E-468; M-384 to E-468;
R-385 to E-468; Q-386 to E-468; L-387 to E-468;
D-388 to E-468; L-389 to E-468; T-390 to E-468;
K-391 to E-468; N-392 to E-468; E-393 to E-468;
I-394 to E-468; D-395 to E-468; V-396 to E-468;
V-398 to E-468; R-398 to E-468; A-399 to E-468;
G-400 to E-468; T-401 to E-468; A-402 to E-468;
G-403 to E-468; P-404 to E-468; G-405 to E-468;
D-406 to E-468; A-407 to E-468; L-408 to E-468.
Y-409 to E-468; A-410 to E-468; M-411 to E-468;
L-412 to E-468; M-413 to E-468; K-414 to E-468;
W-415 to E-468; V-416 to E-468; N-417 to E-468;
K-418 to E-468; T-419 to E-468; G-420 to E-468;
R-421 to E-468; N-422 to E-468; A-423 to E-468;
S-424 to E-468; I-425 to E-468; H-426 to E-468;
L-428 to E-468; L-429 to E-468;
D-430 to E-468; A-431 to E-468; L-432 to E-468;
E-433 to E-468; R-434 to E-468; M-435 to E-468.
E-436 to E-468; E-437 to E-468; R-438 to E-468;
H-439 to E-468; A-440 to E-468; K-441 to E-468;
E-442 to E-468; K-443 to E-468; I-444 to E-468;
Q-445 to E-468; D-446 to E-468; L-444 to E-468;
L-448 to E-468; V-449 to E-468; D-450 to E-468;
S-451 to E-468; G-452 to E-468; K-453 to E-468.
F-454 to E-468; I-455 to E-468; Y-456 to E-468.
L-457 to E-468; E-458 to E-468;
G-460 to E-468; T-461 to E-468; G-462 to E-468;
And S-463 to E-468.

The present invention also provides a polynucleotide sequence encoding at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%,
Or a nucleic acid molecule comprising or consisting of a polynucleotide sequence that is 99% identical. The invention further provides that the above polypeptide has at least 80%
, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid molecule comprising or consisting of a polynucleotide sequence. The invention also includes the above polynucleotide fused to a heterologous polynucleotide sequence. The polypeptides encoded by these polynucleotides are also included in the present invention.

[0134] In another embodiment, N-terminal deletions of the DR4 polypeptide of the general formula n ² -2
38, where n ² is a number from 2 to 238 corresponding to the amino acid sequence identified in SEQ ID NO: 2. In certain embodiments, the N-terminal deletion of the DR4 receptor of the invention encodes a polypeptide comprising or consisting of the amino acid sequence of the following residues of the DR4 extracellular domain sequence shown in SEQ ID NO: 2: Provide polynucleotides: A-2 to H-238; P-3 to H-
238; P-4 to H-238; P-5 to H-238; A-6 to H-238;
7 to H-238; V-8 to H-238; H-9 to H-238; L-10 to H-2
38; G-11 to H-238; A-12 to H-238; F-13 to H-238;
L-14 to H-238; A-15 to H-238; V-16 to H-238; T-1
7 to H-238; P-18 to H-238; N-19 to H-238; P-20 to H
-238; G-21 to H-238; S-22 to H-238; A-23 to H-23
8; A-24 to H-238; S-25 to H-238; G-26 to H-238; T
-27 to H-238; E-28 to H-238; A-29 to H-238; A-30
To H-238; A-31 to H-238; A-32 to H-238; T-33 to H-.
238; P-34 to H-238; S-35 to H-238; K-36 to H-238
V-37 to H-238; W-38 to H-238; G-39 to H-238;
40 to H-238; S-41 to H-238; A-42 to H-238; G-43 to
H-238; R-44 to H-238; I-45 to H-238; E-46 to H-2.
38; P-47 to H-238; R-48 to H-238; G-49 to H-238;
G-50 to H-238; G-51 to H-238; R-52 to H-238; G-5
3-H-238; A-54 to H-238; L-55 to H-238; P-56 to H
-238; T-57 to H-238; S-58 to H-238; M-59 to H-23.
8; G-60 to H-238; Q-61 to H-238; H-62 to H-238; G
-63 to H-238; P-64 to H-238; S-65 to H-238; A-66
-H-238; R-67 to H-238; A-68 to H-238; R-69 to H-
238; A-70 to H-238; G-71 to H-238; R-72 to H-238
A-73 to H-238; P-74 to H-238; G-75 to H-238;
76-H-238; R-77-H-238; P-78-H-238; A-79-
H-238; R-80 to H-238; E-81 to H-238; A-82 to H-2
38; S-83 to H-238; P-84 to H-238; R-85 to H-238;
L-86 to H-238; R-87 to H-238; V-88 to H-238; H-8
9-H-238; K-90-H-238; T-91-H-238; F-92-H
-238; K-93 to H-238; F-94 to H-238; V-95 to H-23
8; V-96 to H-238; V-97 to H-238; G-98 to H-238; V
-99 to H-238; L-100 to H-238; L-101 to H-238; Q-
V-103 to H-238; V-104 to H-238; P-
105-H-238; S-106-H-238; S-107-H-238; A-
108-H-238; A-109-H-238; T-110-H-238;
111-H-238; K-112-H-238; L-113-H-238; H-
114-H-238; D-115-H-238; Q-116-H-238; S-
117-H-238; 1-118-H-238; G-119-H-238; T-
120-H-238; Q-121-H-238; Q-122-H-238; W-
123-H-238; E-124-H-238; H-125-H-238; S-
126 to H-238; P-127 to H-238; L-128 to H-238; G-
129 to H-238; E-130 to H-238; L-131 to H-238; C-
132-H-238; P-133-H-238; P-134-H-238; G-
135-H-238; S-136-H-238; H-137-H-238; R-
138 to H-238; S-139 to H-238; E-140 to H-238; R-
141-H-238; P-142-H-238; G-143-H-238; A-
144-H-238; C-145-H-238; N-146-H-238; R-
147 to H-238; C-148 to H-238; T-149 to H-238; E-
150-H-238; G-151-H-238; V-152-H-238; G-
153-H-238; Y-154-H-238; T-155-H-238; N-
156 to H-238; A-157 to H-238; S-158 to H-238; N-
159 to H-238; N-160 to H-238; L-161 to H-238; F-
162 to H-238; A-163 to H-238; C-164 to H-238; L-
165 to H-238; P-166 to H-238; C-167 to H-238;
168 to H-238; A-169 to H-238; C-170 to H-238; K-
171-H-238; S-172-H-238; D-173-H-238; E-
174 to H-238; E-175 to H-238; E-176 to H-238;
177 to H-238; S-178 to H-238; P-179 to H-238; C-
180 to H-238; T-181 to H-238; T-182 to H-238;
183 to H-238; R-184 to H-238; N-185 to H-238;
186 to H-238; A-187 to H-238; C-188 to H-238; Q-
189-H-238; C-190-H-238; K-191-H-238; P-
192 to H-238; G-193 to H-238; T-194 to H-238; F-
195-H-238; R-196-H-238; N-197-H-238; D-
198 to H-238; N-199 to H-238; S-200 to H-238;
201-H-238; E-202-H-238; M-203-H-238; C-
204-H-238; R-205-H-238; K-206-H-238; C-
207 to H-238; S-208 to H-238; T-209 to H-238; G-
210-H-238; C-211-H-238; P-212-H-238; R-
213 to H-238; G-214 to H-238; M-215 to H-238; V-
216 to H-238; K-217 to H-238; V-218 to H-238;
219 to H-238; D-220 to H-238; C-221 to H-238;
222-H-238; P-223-H-238; W-224-H-238; S-
225-H-238; D-226-H-238; 1-227-H-238; E-
228 to H-238; C-229 to H-238; V-230 to H-238; H-
231 to H-238; K-232 to H-238; and E-233 to H-238
.

[0135] The present invention also provides a polynucleotide sequence encoding a polypeptide as described above comprising at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%,
Or, a nucleic acid molecule comprising or consisting of a polynucleotide sequence that is 99% identical. The invention further relates to a polypeptide as described above comprising at least 80%
, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to a nucleic acid molecule comprising or consisting of a polynucleotide sequence. The invention also includes the above polynucleotide sequences fused to a heterologous polynucleotide sequence. The polypeptides encoded by these polynucleotides are also included by the present invention.

Similarly, many examples of functional C-terminal deletion muteins are known. For example, interferon gamma exhibits a 10-fold higher activity by deleting 8-10 amino acid residues from the carboxy terminus of the protein (Doebeli et al., J. Biotechnology 7: 199-216 (1988)). In the case of the present invention, the protein of the present invention is a member of the DDCR polypeptide family and thus has a C
Deletion of the terminal amino acids can maintain some biological activity, such as receptor binding. Polypeptides further comprising a C-terminal deletion comprising C-221 of SEQ ID NO: 2, where this residue is conserved among DDCR family members and provide the structural stability required for receptor-ligand binding It is not expected to maintain such biological activity as it is required for the formation of disulfide bridges.

As mentioned above, the deletion of one or more amino acids from the C-terminus of a protein results in other functional activities (such as alterations in the loss of one or more biological functions of the protein). For example, biological activity, ability to multimerize, ability to bind DN4 ligand (eg, TRAIL) can still be retained. If less than a majority of residues of a complete or mature polypeptide are removed from the C-terminus, for example, a shortened DR4 that induces and / or binds to an antibody that recognizes the complete or mature form of the polypeptide Mutein's ability is generally retained. Whether a particular polypeptide lacking the C-terminal residue of a complete polypeptide will retain such immunological activity will depend on routine methods described herein and other methods known in the art. Can easily be determined. A DR4 mutein with a large number of deleted C-terminal amino acid residues could possibly retain some biological or immunogenic activity. In fact, peptides composed of as many as six DR4 amino acid residues can often elicit an immune response.

Accordingly, the present invention further provides a deletion of one amino acid from the carboxy terminus of the amino acid sequence of the DR4 polypeptide set forth in SEQ ID NO: 2 to the alanine residue at position 30.
Provided are polypeptides having one or more residues and polynucleotides encoding such polypeptides. In particular, the present invention comprises, or the amino acid sequence of residues 24-m ¹ of SEQ ID NO: 2, or to provide a polypeptide consisting of the amino acid,
Here, m ¹ is 30 to 46 corresponding to the position of the amino acid residue in SEQ ID NO: 2.
It is an integer up to 7.

More particularly, the present invention provides a polynucleotide encoding a polypeptide comprising or consisting of the amino acid sequence of the following residues of the DR4 sequence shown in SEQ ID NO: 2:

[0140]

Embedded image

The present invention also provides a polynucleotide sequence encoding the above-described polypeptide, comprising at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%,
Or a nucleic acid molecule comprising or consisting of a polynucleotide sequence that is 99% identical. The invention further relates to a polypeptide as described above and at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 9%
A nucleic acid molecule comprising or consisting of a polynucleotide sequence encoding an 8% or 99% identical polypeptide. The invention also includes the above polynucleotide sequences fused to a heterologous polynucleotide sequence. The polypeptides encoded by these polynucleotides are also included by the present invention.

[0142] In another embodiment, the C-terminal deletion of the DR4 polypeptide has the general formula 24-m ^Two Where m^TwoCorresponds to the amino acid sequence identified in SEQ ID NO: 2
30 to 238. In certain embodiments, the present invention relates to SEQ ID NO: 2.
Contains the amino acid sequence of the following residues of the DR4 extracellular domain sequence shown in
Or a polynucleotide encoding a polypeptide consisting of that sequence.
To serve:

[0143]

Embedded image

The present invention also provides a polynucleotide sequence encoding a polypeptide as described above, comprising at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%,
Or a nucleic acid molecule comprising or consisting of a polynucleotide sequence that is 99% identical. The present invention further provides a polypeptide as described above comprising at least 8
0%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99%
A nucleic acid molecule comprising or consisting of a polynucleotide sequence that encodes a polypeptide that is% identical. The invention also includes the above polynucleotide sequences fused to a heterologous polynucleotide sequence. The polypeptides encoded by these polynucleotides are also included by the present invention.

The present invention further relates to polypeptides having one or more residues from the carboxy terminus of the amino acid sequence of DR4 set forth in SEQ ID NO: 2 to C-221 of SEQ ID NO: 2, and to encoding such polypeptides. A polynucleotide is provided. In particular,
The present invention provides a polypeptide having the amino acid sequence of residues 1-m ² of the amino acid sequence of SEQ ID NO: 2, where m ² is any integer in the range of 221 to 468, and residue C -221 is the position of the first residue from the C-terminus of the complete DR4 polypeptide (shown in SEQ ID NO: 2) which is believed to require receptor binding activity of the DR4 protein. Polynucleotides encoding these polypeptides are also provided.

The present invention also provides a polypeptide having one or more amino acids deleted from both the amino and carboxy termini of a DR4 polypeptide, wherein the polypeptide comprises residues n ^1-of SEQ ID NO: 2. It may be generally described as having m ¹ and / or n ² -m ² , where n ¹ , n ² , m ¹ , and m ² are integers as described above.

Also included is a nucleotide sequence that encodes a polypeptide consisting of a portion of the complete DR4 amino acid sequence encoded by the cDNA clone contained in ATCC Accession No. 97853, wherein this portion is a cDNA contained in ATCC Accession No. 97853. 1 to about 108 from the amino terminus of the complete amino acid sequence encoded by
Or any combination of the above amino-terminal and carboxyl-terminal deletions of the complete amino acid sequence encoded by the amino acid or carboxyl-terminal 1 to about 247 amino acids, or the cDNA contained in ATCC Accession No. 97853 . Polynucleotides encoding all of the above deletion mutant polypeptide forms are also provided.

Preferred among the N-terminal deletion mutants and C-terminal deletion mutants are those that contain only a portion of the extracellular domain; that is, residues 24-238. Because any part of it is expected to be soluble.

It is recognized in the art that some amino acid sequences of DR4 can be changed without significant effect on protein structure or function. When such differences in sequence are contemplated, it should be noted that there are critical regions of the protein that determine activity. Such regions usually contain residues that either make up the ligand binding site, or death domains, or form tertiary structures that affect these domains.

Accordingly, the present invention further provides a variant of a DR4 protein that exhibits substantial DR4 protein activity, or a DR4 protein, such as a protein fragment discussed below.
And a DR4 protein variant containing the region of Such variants include deletions, insertions, inversions, repeats, and type substitutions. As indicated above, guidance on which amino acid changes appear to be phenotypically silent can be found in Bowie.
, J. et al. U. Et al., Science 247: 1306-1310 (1990).

Thus, a fragment, derivative or analog of the polypeptide of SEQ ID NO: 2,
Or a fragment of the polypeptide encoded by the deposited cDNA,
Derivatives or analogs include (i) at least one or more amino acid residues are conserved or non-conserved amino acid residues (preferably conserved amino acid residues, and more preferably at least one but less than ten conserved amino acid residues Amino acid residues) and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) one or more amino acid residues Contains a substituent, or (iii) the mature polypeptide is fused to another compound such as a compound (eg, polyethylene glycol) to increase the half-life of the polypeptide, or (iv) an additional amino acid. Is a mature polypeptide (eg, an IgG Fc fusion region peptide sequence), or a leader sequence or secretory sequence. Or fused to a sequence used for purification of a mature polypeptide or proprotein sequence. Such fragments, derivatives and analogs are deemed to be within the skill of the art in light of the teachings herein. Polynucleotides encoding these fragments, derivatives or analogs are also encompassed by the present invention.

Of particular interest is the replacement of a charged amino acid with another charged amino acid and a neutral or negatively charged amino acid. The latter results in a protein with a reduced positive charge and improves the characteristics of the DR4 protein. Prevention of agglomeration is highly desirable. Aggregation of proteins not only reduces activity, but can also be problematic when preparing pharmaceutical formulations because aggregates can be immunogenic (Pinkard et al., Clin. Exp. Immunol. 2: 331-3
40 (1967); Robbins et al., Diabetes 36: 838-84.
5 (1987); Cleland et al., Crit. Rev .. Therapeuti
c Drug Carrier Systems 10: 307-377 (19
93)).

Amino acid substitutions may also alter the selectivity of binding to cell surface receptors. Ostade et al., Nature 361: 266-268 (1993).
Describe certain mutations that result in the selective binding of TNF-α to only one of the two known types of TNF-receptors. Thus, the DR4 receptor of the present invention is a method for replacing one or more amino acids derived from either natural mutations or man-made manipulations,
It may include deletions or additions.

As indicated, changes in insignificant properties are preferred, such as conservative amino acid substitutions that do not significantly affect the folding or activity of the protein (see Table II).

[0155]

[Table 2]

In certain embodiments, substitutions, additions or deletions in the amino acid sequence of SEQ ID NO: 2 and / or any of the polypeptide fragments (eg, extracellular or intracellular domains) described herein. Are 75, 70, 6
0, 50, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5,
4, 3, 2, 1 or 30 to 20, 20 to 15, 20 to 10, 15 to 10, 1
0, 1, 5, 10, 1-5, 1-3, or 1-2.

The amino acids of the DR4 protein of the invention that are essential for function can be determined by methods known in the art (eg, site-directed mutagenesis or alanine scan mutagenesis).
Cunningham and Wells, Science 244: 1081-
1085 (1989)). The latter procedure introduces one alanine mutation at each residue in the molecule. The resulting mutant molecules are then tested for biological activity (eg, receptor binding, or in vitro or in vivo proliferative activity). Sites important for ligand-receptor binding can also be determined by structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith et al., JM).
ol. Biol. 224: 899-904 (1992) and de Vos et al.
Science 255: 306-312 (1992)).

In addition, protein engineering can be used to improve or alter the characteristics of a DR4 polypeptide. Novel mutant proteins or muteins, including single or multiple amino acid substitutions, deletions, additions or fusions, can be made using recombinant DNA techniques known to those of skill in the art. Such modified polypeptides can exhibit, for example, enhanced activity or increased stability. In addition, they can be produced in higher yields, at least under certain purification and storage conditions, and can exhibit increased solubility over the corresponding native polypeptide.

[0159] Non-naturally occurring variants can be generated using mutagenesis techniques known in the art. These techniques include oligonucleotide-mediated mutagenesis, alanine scanning, PCR mutagenesis, site-directed mutagenesis (eg, Carter et al., N.
ucl. Acids Res. 13: 4331 (1986); and Zolle
r et al, Nucl. Acids Res. 10: 6487 (1982)), cassette mutagenesis (e.g., Wells et al., Gene 34: 315 (1
985)), restriction selection mutagenesis (eg, Wells et al., Phil).
os. Trans. R. Soc. London SerA 317: 415 (1
986)), but is not limited thereto.

[0160] Accordingly, the present invention also provides for the deletion, addition or substitution of one or more amino acid residues in a selected host cell to produce a DR4 polypeptide that is more suitable for expression, scale-up, etc. And derivatives and analogs of DR4. For example, a cysteine residue can be deleted to remove a disulfide bridge, or replaced with another amino acid residue; an N-linked glycosylation site, for example, hyperglycosylates the N-linked site ( hyperglycosyla
te) can be modified or removed to achieve expression of a homogeneous product that is more easily recovered and purified from yeast hosts. To this end, various amino acid substitutions at one or both of the first or third amino acid positions on any one or more of the glycosylation recognition sequences of a DR4 polypeptide of the invention, and / or any one of Such amino acid deletion at the second position of the recognition sequence prevents glycosylation of DR4 at the modified tripeptide sequence (eg, Miyaja
See imo et al., EMBO J 5 (6): 1193-1197).

[0161] The polypeptides of the present invention may also comprise a deposited cDNA containing a leader (ATCC
A polypeptide comprising or consisting of the polypeptide encoded by the deposit having accession number 97853); comprising the mature polypeptide encoded by the deposited cDNA without a leader (ie, the mature protein). Or a polypeptide consisting of this mature polypeptide; comprising a polypeptide of SEQ ID NO: 2 including a leader; or a polypeptide consisting of this polypeptide; comprising a polypeptide of SEQ ID NO: 2 having no amino-terminal methionine Or a polypeptide consisting of this polypeptide; a polypeptide comprising or consisting of the polypeptide of SEQ ID NO: 2 without a leader; a polypeptide comprising or consisting of this domain of DR4 extracellular; DR4 A peptide comprising or consisting of a stain-rich domain; a polypeptide comprising or consisting of a DR4 transmembrane domain; a polypeptide comprising or consisting of a DR4 intracellular domain; A polypeptide comprising or consisting of a soluble polypeptide comprising all or part of the extracellular and intracellular domains but lacking the transmembrane domain And the above polypeptides (eg, the polypeptide encoded by the deposited cDNA, the polypeptide of SEQ ID NO: 2, and such polypeptides having at least 30 amino acids and more preferably at least 50 amino acids) At least 80% for some de)
Polypeptides that are identical, more preferably those that are at least 90% or 95% identical, and even more preferably those that are 96%, 97%, 98% or 99% identical. Polynucleotides encoding these polypeptides are also encompassed by the present invention.

A polypeptide having an amino acid sequence that is at least 95% “identical” to the reference amino acid sequence of a DR4 polypeptide, such that the amino acid sequence of the polypeptide is 5 per 100 amino acids of each of the reference amino acids of the DR4 polypeptide. Identical to the reference sequence, except for the polypeptide sequence, which may contain up to amino acid mutations,
The amino acid sequence of this polypeptide is contemplated. In other words, to obtain a polynucleotide having an amino acid sequence that is at least 95% identical to the reference amino acid sequence, up to 5% of the amino acid residues in the reference sequence can be deleted or replaced with another amino acid Or 5% of all amino acid residues in the reference sequence
Up to as many amino acids as can be inserted into the reference sequence. These variations of the reference sequence may be individually scattered, either in residues in the reference sequence or in one or more contiguous groups in the reference sequence, at the amino-terminal position of the reference amino acid sequence or in the carboxy group. It can occur at terminal positions or somewhere between these terminal positions.

In practice, for example, any particular polypeptide may have at least 80%, 85%, 90%, 92%, 80%, 85%, 90%, 92% 95%, 96%, 97%, 98% or 9
Whether it is 9% identical is determined by a known computer program (Wisconsin Sequence Analysis) such as the Bestfit program.
Package, Version 8 for Unix (registered trademark), Ge
netics Computer Group, University Research
Park, 575 Science Drive, Madison, WI 53
711) can be determined conventionally. For example, if Bestfit or any other sequence alignment program is used to determine whether a particular sequence is 95% identical to a reference sequence according to the present invention, the parameters will, of course, have a percentage identity of It is calculated relative to the total length of the reference amino acid sequence and is set to allow for gaps in homology of up to 5% of the total number of amino acid residues in the reference sequence.

In certain embodiments, the identity between a reference (query) sequence (a sequence of the invention) and a subject sequence, also referred to as an overall sequence alignment, is determined by the method of Brutlag et al.
Comp. App. Biosci. 6: 237-245 (1990)). F
Preferred parameters used in the ASTDB amino acid alignment are: Matrix = PAM 0, k-tuple = 2, Mismatch
Penalty = 1, Joining Penalty = 20, Random
Ization Group Length = 0, Cutoff Score =
1, Window Size = length of array, Gap Penalty = 5, Ga
p Size Penalty = 0.05, Window Size = 500 or the length of the amino acid sequence of interest (whichever is shorter). According to this embodiment, when calculating the overall percent identity, if the subject sequence is shorter than the query sequence due to N- or C-terminal deletions, not due to internal deletions, FAS
Taking into account the fact that the TDB program does not account for the N-terminal and C-terminal truncations of the subject sequence, manual corrections are made to the results. For subject sequences truncated at the N- and C-termini to the query sequence, the percent identity is the number of residues in the query sequence that are N- and C-terminal to the subject sequence that do not match / align with the corresponding subject residue Is calculated as a percentage of the total bases of the query sequence. The determination of whether residues are matched / aligned is determined by the result of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity calculated by the above FASTDB program using the specified parameters, leading to a final percent identity score. This final percent identity score is what is used for the purposes of this embodiment. Only N-terminal and C-terminal residues of the subject sequence that are not matched / aligned with the query sequence are considered for the purpose of manually adjusting the percent identity score. That is, only the query residue positions outside the farthest N- and C-terminal residues of the subject sequence. For example, a subject sequence of 90 amino acid residues may have 1% to determine percent identity.
Aligned with a query sequence of 00 residues. This deletion occurs at the N-terminus of the subject sequence,
Thus, the FASTDB alignment shows no match / alignment for the first 10 residues at the N-terminus. The 10 unpaired residues represent 10% of the sequence (number of unmatched N- and C-terminal residues / total number of residues in query sequence). As a result, 10%
It is subtracted from the percent identity score calculated by the TDB program. 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. This deletion is now an internal deletion and there is no residue at the N- or C-terminus of the subject sequence that does not match / align with this query. In this case, FASTD
The percent identity calculated by B is not manually corrected. Again, FA
Only residue positions outside the N- and C-termini of the subject sequence that do not match / align with the query sequence, as shown in the STDB alignment, are manually corrected.
No other manual corrections are made for the purposes of this embodiment.

The present application also provides that at least 80%, 85%, 90%, 92%, at least 80%, 85%, 90%, 92%, or 48% of the DR4 polypeptide sequence shown herein as n ¹ -m ¹ and / or n ² -m ² .
A protein comprising polypeptides that are 95%, 96%, 97%, 98%, or 99% identical. In a preferred embodiment, the present application provides for at least 80%, 85%, 90%, 92% of the polypeptides having the specific DR4 N-terminal and C-terminal deletion amino acid sequences described herein. , 95%, 96%, 9
A protein comprising polypeptides that are 7%, 98%, or 99% identical. Polynucleotides encoding these polypeptides are also encompassed by the present invention.

In certain preferred embodiments, a DR4 protein of the invention comprises a fusion protein as described above, wherein the DR4 polypeptide is herein defined as n ¹ -m ¹ ,
And a polypeptide described as / or n ² -m ^2. In a preferred embodiment, the present application provides at least 80%, 85%, 90% relative to a nucleic acid sequence encoding a polypeptide having the specific N-terminal and C-terminal deletion amino acid sequences described herein. , 92%, 95%, 96%, 97%, 98%, or 9
For nucleic acid molecules that are 9% identical. Polynucleotides encoding these polypeptides are also encompassed by the present invention.

[0167] We have discovered that the DR4 polypeptide is a 468 residue protein that exhibits three major structural domains. First, the ligand binding domain (
The extracellular domain) was identified to be within about 24 to about 238 residues of SEQ ID NO: 2. Second, the transmembrane domain was identified to be within residues from about 239 to about 264 of SEQ ID NO: 2. Third, the intracellular domain comprises about 265 to about 468 of SEQ ID NO: 2.
Within the residue. Importantly, this intracellular domain comprises a death domain of from about 379 to about 422 residues of SEQ ID NO: 2. Further preferred fragments of this polypeptide set forth in SEQ ID NO: 2 include about 24 to about 468
And soluble polypeptides containing all or part of the extracellular or intracellular domain, but lacking the transmembrane domain. In this context, "about" (approximately) means only a few (5, 4, 3, 2, or 1) amino acids, at either or both termini, of a particular enumerated size. Larger or smaller ones are included.

In another aspect, the invention provides a peptide or polypeptide comprising an epitope-bearing portion of a polypeptide described herein. The epitope of the polypeptide portion is an immunogenic or antigenic epitope of the polypeptide of the present invention. The number of immunogenic epitopes on a protein is generally less than the number of antigenic epitopes. See, for example, Geysen et al., Proc. Natl. Acad. S
ci. USA 81: 3998-4002 (1983).

For the selection of peptides or polypeptides that carry an antigenic epitope (ie, include a region of a protein molecule to which an antibody can bind), a relatively short synthetic peptide that mimics a portion of the protein sequence will partially mimic It is well known in the art that antisera reactive with a given protein can be routinely induced. For example, Sut
Cliffe, J.M. G. FIG. , Shinnick, T .; M. Green, N .; And Learner, R .; A. (1983) Antibodies that
react with predetermined sites on pr
oteins, Science 219: 660-666. Peptides that can elicit protein-reactive sera are frequently represented in the primary sequence of the protein, can be characterized by a set of simple chemical rules, and can be characterized by immunodominant regions of the intact protein (ie, immunogenic epitopes). ) Or amino or carboxyl terminus.

Non-limiting examples of antigenic polypeptides or peptides that can be used to raise DR4-specific antibodies include the following: from about 35 to about 92 amino acid residues of SEQ ID NO: 2. A polypeptide comprising or consisting of about 114 to about 160 amino acid residues of SEQ ID NO: 2; a polypeptide comprising or consisting of about 114 to about 160 amino acid residues of SEQ ID NO: 2; A polypeptide comprising or consisting of an amino acid residue; or a polypeptide comprising or consisting of about 267 to about 298 amino acid residues of SEQ ID NO: 2; A polypeptide comprising or consisting of about 364 to about 364 amino acid residues; about 391 to about 404 of SEQ ID NO: 2
Or a polypeptide comprising or consisting of the amino acid residues of SEQ ID NO: 2 from about 418 to about 465. In this context, “about” means at the or at both termini, in particular the recited size, several (5, 5,
Only 4, 3, 2, or 1) amino acids, larger or smaller, are included. As noted above, the inventors have determined that the above polypeptide fragments are:
It was determined to be the antigenic region of the DR4 protein. Polynucleotides encoding these polypeptides are also encompassed by the present invention.

[0171] The epitope-bearing peptides and polypeptides of the present invention can be produced by any conventional means. Houghten, R .; A "General method"
for the rapid solid-phase synthesis
of large numbers of peptides: specif
icity of antigen-antibody interaction
n at the level of individual amino a
cids. Proc. Natl. Acad. Sci. USA 82: 5131
-5135 (1985). This "Simultaneous Multiple"
The Peptide Synthesis (SMPS) process is described in Hough
No. 4,631,211 (1986).
As will be appreciated by one of skill in the art, a DR4 polypeptide of the present invention and an epitope-bearing fragment thereof described herein (eg, a portion of an extracellular domain (eg,
Corresponding to amino acid residues 1-240 of SEQ ID NO: 2) is an immunoglobulin (
IgG) and can bind to portions of the constant domains, resulting in chimeric polypeptides. These fusion proteins facilitate purification and show an increased half-life in vivo. This has been shown, for example, for chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins (EPA 394,827; Traun
ecker et al., Nature 331: 84-86 (1988)). Fusion proteins having a disulfide-linked dimer structure with an IgG moiety also produce monomeric DR
4 may be more efficient at binding and neutralizing other molecules than proteins or protein fragments alone (Funtoulakis et al., J Bioch)
em 270: 3958-3964 (1995)).

The polypeptides of the invention include the following: naturally purified products, products of chemical synthesis procedures, and prokaryotic or eukaryotic hosts (eg, bacterial cells, yeast cells,
(Including higher plant cells, insect cells, and mammalian cells) by recombinant techniques. Depending on the host used in the recombinant production procedure, the polypeptide of the invention may be glycosylated or non-glycosylated. In addition, the polypeptides of the invention may also contain an altered starting methionine residue, in some cases as a result of a host-mediated process.

Further, the proteins of the present invention can be chemically synthesized using techniques known in the art (eg, Creighton, 1983, Proteins: Struc).
tures and Molecular Principles, W.C. H. F
Leeman & Co. , N .; Y. And Hunkapiller, M et al., N
atature 310: 105-111 (1984)). For example, a peptide corresponding to a fragment of a DR4 polypeptide of the present invention can be synthesized by use of a peptide synthesizer. In addition, if desired, analogs of non-classical amino acids or chemical amino acids can be introduced as substitutions or additions into the DR4 polypeptide sequence. Non-classical amino acids include, but are not limited to, the D isomer of a common amino acid, 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, designer amino acid (eg, α-methyl amino acid), Ca-methyl amino acid, Na-methyl Amino acids, and common amino acid analogs. further,
The amino acid can be D (dextrorotary) or L (levorotary).

Non-naturally occurring variants can be made using mutagenesis techniques known in the art, including oligonucleotide-mediated mutagenesis, alanine scanning, PCR mutagenesis, site-directed mutagenesis. Induction (eg, Carter et al., N
ucl. Acids Res. 13: 4331 (1986); and Zolle
r et al, Nucl. Acids Res. 10: 6487 (1982)), cassette mutagenesis (e.g., Wells et al., Gene 34: 315 (1
985)), restriction selection mutagenesis (eg, Well et al., Philo).
s. Trans. R. Soc. London SerA 317: 415 (19
86)), but is not limited thereto.

The present invention further provides methods for, for example, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, antibody molecules or other cells during or after translation. DR4 polypeptides that are differentially modified, such as by binding to a sex ligand. Any of numerous chemical modifications, including the following may be carried out by known, but not limited to techniques: cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH ₄
Chemical cleavage, acetylation, formylation, oxidation, reduction; metabolic synthesis in the presence of tunicamycin;

Additional post-translational modifications encompassed by the present invention include, for example: N-linked or O-linked sugar chains (N- or C-terminal processing), chemical moieties to the amino acid backbone. Modification, chemical modification of N-linked or O-linked sugar chains, and addition or deletion of N-terminal methionine residues as a result of prokaryotic host cell expression. The polypeptide may also be modified with a detectable label (eg, an enzymatic, fluorescent, isotopic or affinity label) to allow for detection and isolation of the protein.

The present invention also provides chemically modified derivatives of DR4 that can provide additional benefits, such as increased solubility, stability and circulation time, or reduced immunogenicity of the polypeptide (US See patent 4,179,337).
The chemical moiety for derivatization can be selected from water-soluble polymers such as polyethylene glycol, ethylene glycol / propylene glycol copolymer, carboxymethyl cellulose, dextran, polyvinyl alcohol, and the like. The polypeptide can be modified at random positions within the molecule or at predetermined positions within the molecule, and can include one, two, three or more attached chemical moieties.

The polymer can be of any molecular weight and can be branched or unbranched. For polyethylene glycol, the preferred molecular weight is between about 1 kDa and about 100 kDa for ease of handling and manufacture (the term "about" means that some molecules have been referred to in the preparation of polyethylene glycol). Heavier than the molecular weight, and some are lighter than the indicated molecular weight). The desired treatment profile (e.g., the desired time of sustained release, (
(If present) effects on biological activity, ease of handling, degree of antigenicity or lack of antigenicity, and other known effects of polyethylene glycol on therapeutic proteins or analogs). Size can be used. For example, polyethylene glycol is about 200, 500, 1000, 1500, 2
000, 2500, 3000, 3500, 4000, 4500, 5000, 55
00, 6000, 6500, 7000, 7500, 8000, 8500, 900
0, 9500, 10,000, 10,500, 11,000, 11,500, 1
2,000, 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, 19,5
00, 20,000, 25,000, 30,000, 35,000, 40,00
0, 50,000, 55,000, 60,000, 65,000, 70,000
, 75,000, 80,000, 85,000, 90,000, 95,000,
Or it may have an average molecular weight of 100,000 kDa.

As described above, polyethylene glycol can have a branched structure. Branched polyethylene glycols are described, for example, in US Pat. No. 5,643,575; Morpur.
go et al., Appl. Biochem. Biotechnol. 56: 59-72
(1996); Vorovjev et al., Nucleosides Nucleot.
ids 18: 2745-2750 (1999); and Caliceti et al., Bioconjug. Chem. 10: 638-646 (1999), the disclosure of each of which is incorporated herein by reference.

A polyethylene glycol molecule (or other chemical moiety) should be attached to the protein in view of its effect on the functional or antigenic domain of the protein. There are a number of conjugation methods available to those of skill in the art (eg, EP 0 401 384 (G-CSF, which is incorporated herein by reference).
To PEG), Malik et al., Exp. Hematol. 20: 102
8-1035 (1992) (tresyl chloride)
(See also report PEGylation of GM-CSF). For example, polyethylene glycol can be covalently linked by an amino acid residue via a reactive group (eg, a free amino or carboxyl group). Reactive groups are groups to which activated polyethylene glycol molecules can bind. Amino acid residues having a free amino group can include, for example, lysine residues and N-terminal amino acid residues;
Amino acid residues having a free carboxyl group may include aspartic acid residues, glutamic acid residues and C-terminal amino acid residues. Sulfhydryl groups can also be used as a reactive group for attaching polyethylene glycol molecules. Preferred for therapeutic purposes is attachment at an amino group, such as attachment at the N-terminus or lysine group.

[0181] As suggested above, polyethylene glycol can be linked to proteins via linkage to any of a number of amino acid residues. For example, polyethylene glycol can be linked to a protein via a covalent bond to a lysine residue, a histidine residue, an aspartic acid residue, a glutamic acid residue, or a cysteine residue.
One or more reaction chemistries may be used to bind specific amino acid residues (eg,
Lysine, histidine, aspartic acid, glutamic acid, or cysteine) or amino acid residues of more than one type of protein (eg, lysine, histidine, aspartic acid, glutamic acid, or cysteine, and combinations thereof)
To polyethylene glycol.

[0182] Proteins that are chemically modified at the N-terminus may be particularly desirable. Using polyethylene glycol as an example of a composition of the present invention, the ratio of polyethylene glycol to protein (or polypeptide) molecules in a reaction mixture from various polyethylene glycol molecules (by molecular weight, branching, etc.), The type of PEGylation reaction to be performed and the method of obtaining the selected N-terminal PEGylated protein can be selected. The method of obtaining an N-terminal PEGylated preparation (ie, if necessary, separating this portion from other mono-PEGylated portions) is accomplished by purification of the N-terminal PEGylated material from a population of PEGylated protein molecules. obtain. Select proteins chemically modified with an N-terminal modification are the first of a different type available for derivatization in a particular protein.
It can be achieved by reductive alkylation utilizing the differential reactivity of the primary amino group (lysine versus the N-terminus). Selective proteins chemically modified with N-terminal modifications can be used for reductive alkylation (lysine versus N-terminus) utilizing different reactivities of different types of primary amine groups available for derivatization in certain proteins Can be achieved by: Under the appropriate reaction conditions, substantially selective derivatization of the protein at the N-terminus with a carbonyl group containing polymer is achieved.

As indicated above, pegylation of the proteins of the present invention can be achieved by any number of means. For example, polyethylene glycol can be attached to the protein, either directly or by an intervening linker. Linkerless systems for attaching polyethylene glycol to proteins are described in Delgado et al., Crit. Rev .. Thera. Drug Carri
er Sys. 9: 249-304 (1992); Francis et al., Inte.
rn. J. of Hematol. 68: 1-18 (1998); U.S. Pat.
U.S. Pat. No. 5,349,052; WO 95/06058.
And WO 98/32466, the disclosure of each of which is incorporated herein by reference.

[0184] Polyethylene is directly added to amino acid residues of the protein without intervening linkers.
One system for attaching renglycol is tresylated.
MPEG (this is tresylchloride (ClSO _Two CH_TwoCF_Three) Using monomethoxy (polyethylene)
Glycol (MPEG)). G
Upon reaction of the protein with resylated MPEG, polyethylene glycol
Directly attached to the amine group of the protein. Therefore, the present invention relates to the protein of the present invention.
Quality and 2,2,2-trifluoroethane sul
Produced by reacting with a polyethylene glycol molecule having a honyl group
Protein-polyethylene glycol conjugate.

[0185] Polyethylene glycol can also be attached to proteins using a number of different intervening linkers. For example, US Pat. No. 5,612,460, the entire disclosure of which is incorporated herein by reference, discloses a urethane linker for attaching polyethylene glycol to proteins. The protein-polyethylene glycol conjugate, where polyethylene glycol is linked to the protein by a linker, is also known as MPEG-succinimidyl succinate (succin
imidylsuccinate), MPEG activated with 1,1'-carbonyldiimidazole, MPEG-2,4,5-trichlorophenylcarbonate, MPEG-p-nitrophenol carbonate, and various MPEG-succinate derivatives Can be produced by the reaction of a protein with a compound such as A number of additional polyethylene glycol derivatives and reaction chemistries for attaching polyethylene glycol to proteins are described in WO 98/32466, the entire disclosure of which is incorporated herein by reference. PEGylated protein products produced using the reaction chemistry set forth 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, a PEGylated protein of the invention
On average 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 2,
Zero or more polyethylene glycol molecules may be linked. Similarly, the average degree of substitution is 1-3, 2-4, 3-5, 4-6, 5 per protein molecule.
-7, 6-8, 7-9, 8-10, 9-11, 10-12, 11-13, 12-
14, 13-15, 14-16, 15-17, 16-18, 17-19, or 18-20 within such ranges as polyethylene glycol moieties. Methods for determining the degree of substitution are described, for example, in Delgado et al., Crit. Rev .. Thera. D
rug Carrier Sys. 9: 249-304 (1992).

Antibodies In addition, the present invention provides for immunospecifically a polypeptide of the invention, preferably an epitope (as determined by immunoassays well known in the art for assaying specific antibody-antigen binding). Antibodies and T cell antigen receptor (
TCR). The antibodies of the present invention may be polyclonal, monoclonal, multispecific, human, humanized or chimeric, single chain, Fab
Fragments, F (ab ') fragments, fragments produced by Fab expression libraries, anti-idiotype (anti-Id) antibodies (including, for example, anti-Id antibodies to the antibodies of the invention), and epitope binding of any of the above Including but not limited to fragments. As used herein, the term "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that include an antigen binding site that immunospecifically binds to an antigen. Say. The immunoglobulin molecules of the present invention can be of any type (eg, IgG, IgE) of immunoglobulin molecules.
, IgM, IgD, IgA, and IgY), any class (eg, IgG1
, IgG2, IgG3, IgG4, IgA1 and IgA2) or immunoglobulin molecules.

Most preferably, the antibody is a human antigen binding antibody fragment of the invention, including Fab, Fab ′ and F (ab ′) 2, Fd, single chain Fvs (sc
Fv), single chain antibodies, disulfide-linked Fvs (sdFv), and fragments containing either the _VL or _VH domains.
Antigen binding antibody fragments, including single chain antibodies, can include the variable region alone or in combination with all or part of the following: hinge region, CH1 domain, CH1
2 domain and CH3 domain. Also included in the invention are antigen binding fragments that also include any combination of a variable region with a hinge region, a CH1, a CH2 domain, and a CH3 domain. The antibodies of the invention can be from any animal origin, including birds and mammals. Preferably, the antibody is human, murine, donkey, ship rabbit, goat,
Guinea pig, camel, horse, or chicken. As used herein, a "human" antibody includes an antibody having the amino acid sequence of a human immunoglobulin,
And antibodies isolated from animals that are transgenic for a human immunoglobulin library or one or more human immunoglobulins and do not express endogenous immunoglobulins (as described below, and, for example, Kucherlapa).
ti et al., as described in US Pat. No. 5,939,598).

The antibodies of the present invention can be monospecific, bispecific, trispecific, or more multispecific. Multispecific antibodies can be specific for different epitopes of a polypeptide of the invention, or both a polypeptide of the invention and a heterologous epitope (eg, a heterologous polypeptide or solid support material). May be specific to For example, PCT publication WO 93/17715; WO 9/17715
WO08 / 00802; WO 91/00360; WO 92/05793; Tu
tt et al. Immunol. 147: 60-69 (1991); U.S. Pat.
No. 4,474,893, No. 4,714,681, No. 4,925,648,
Nos. 5,573,920 and 5,601,819; Kostelny et al.
J. Immunol. 148: 1547-1553 (1992).

The antibodies of the present invention may be described or specified in terms of the epitopes or portions of the polypeptides of the present invention to which they recognize or specifically bind. The portion of the epitope or polypeptide may be specified as described herein, for example, by N-terminal and C-terminal positions, by size in contiguous amino acid residues, or as listed in the tables and figures. Can be Antibodies that specifically bind to any epitope or polypeptide of the present invention can also be excluded. Accordingly, the present invention includes antibodies that specifically bind a polypeptide of the present invention and allow for exclusion of a polypeptide of the present invention.

The antibodies of the present invention may also be described or specified for their cross-reactivity. Antibodies that do not bind any other analog, ortholog or homolog of a polypeptide of the present 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% and at least 50% identity to the polypeptides of the invention Antibodies that bind a polypeptide having (as calculated using methods known in the art and described herein) are also
Included in the present invention. Less than 95%, less than 90%, 8 relative to the polypeptide of the invention
Less than 5%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, 5
Antibodies that do not bind polypeptides having an identity of less than 5% and less than 50% (as calculated using methods known in the art and described herein) are also included in the invention. It is. Furthermore, antibodies that bind a polypeptide encoded by a polynucleotide that hybridizes to a polynucleotide of the invention under stringent hybridization conditions (as described herein) are included in the invention. . Antibodies of the invention can also be described or specified for their binding affinity to a polypeptide of the invention. Preferred binding affinity is less than 5 × 10 ⁻² M, less than 10 ⁻² M, less than 5 × 10 ⁻³ M, less than 10 ⁻³ M, less than 5 × 10 ⁻⁴ M, less than 10 ⁻⁴ M, 5 × 10 ⁻⁵ M or less, 10 ⁻⁵ M or less, 5 × 1
Less than 0 ^-6 M, less than 10 ^-6 M, less than 5 × 10 ^-7 M, less than 10 ^-7 M, less than 5 × 10 ^-8 M, less than 10 ^-8 M, less than 5 × 10 ^-9 M, 10 ⁻ Less than ⁹ M, less than 5 × 10 ^-10 M, 1
0 less than ^-10 M, less than 5 × 10 ^-11 M, less than 10 ^-11 M, 5 × 10 ^-12 less than M, 10 ^-1 of less than ² M, 5 × less than 10 ^-13 M, less than 10 ^-13 M, 5 Less than × 10 ^-14 M, 10 ^-14 M
And an affinity with a dissociation constant or Kd of less than 5 × 10 ⁻¹⁵ M or less than 10 ⁻¹⁵ M.

The present invention also provides antibodies to the epitopes of the invention, as determined by any method known in the art for determining competitive binding (eg, the immunoassays described herein). Antibodies that competitively inhibit the binding of. 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 antibodies of the present invention may act as agonists or antagonists of the polypeptide of the present invention. For example, the invention includes antibodies that disrupt receptor / ligand interactions with the polypeptides of the invention, either partially or completely. The invention has the characteristics of both receptor-specific and ligand-specific antibodies. The invention also features receptor-specific antibodies that do not interfere with ligand binding but do interfere with receptor activation. Receptor activation (ie, signal transduction) can be determined by techniques described herein or otherwise known in the art. For example, receptor activation can be determined by phosphorylation of the receptor (eg, tyrosine or serine / threonine), or by detecting its substrate by immunoprecipitation followed by Western blot analysis (eg, as described above). In certain embodiments, an antibody is provided that inhibits ligand activity or receptor activity in the absence of the antibody by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%. You.

The present invention also relates to receptor-specific antibodies that prevent both ligand binding and receptor activation, as well as to recognize receptor-ligand complexes and, preferably, to bind unbound receptors or unbound ligands. Have the characteristics of an antibody that does not recognize Similarly, the present invention relates to neutralizing antibodies that bind to a ligand and prevent the binding of the ligand to the receptor, and bind to the ligand, thereby preventing receptor activation but not preventing the ligand from binding the receptor. Including antibodies. Furthermore, the present invention includes antibodies that activate the receptor. These antibodies can act as receptor agonists, ie, enhance or activate any or all of the biological activations of ligand-mediated receptor activation. The antibody can be specified as an agonist, antagonist or inverse agonist for a biological activity, including the specific biological activity of a peptide of the invention disclosed herein. Accordingly, the present invention further relates to antibodies that act as agonists or antagonists of the polypeptides of the present invention. The antibody agonist can be made using methods known in the art. For example, P
U.S. Pat. No. 5,811,097; Deng et al., Blood 92 (6): 1981-1988 (1998); Chen et al., C.
cancel Res. 58 (16): 3668-3678 (1998); Har.
rop et al. Immunol. 161 (4): 1786-1794 (1998)
Zhu et al., Cancer Res: 58 (15): 3209-3214 (1).
998); Yoon et al. Immunol. 160 (7): 3170-317
9 (1998); Prat et al. Cell. Sci. 111 (Pt2): 23
7-247 (1998); Pitard et al. Immunol. Method
s 205 (2): 177-190 (1997); Lioutard et al., Cyt.
okine 9 (4): 233-241 (1997); Carlson et al.
Biol. Chem. 272 (17): 11295-11301 (1997);
Taryman et al., Neuron 14 (4): 755-762 (1995);
Muller et al., Structure 6 (9): 1153-1167 (199
8); Bartunek et al., Cytokine 8 (1): 14-20 (199
6), all of which are incorporated herein by reference in their entirety.

The antibodies of the present invention can be used, for example, but not limited to, purify, detect and target a polypeptide of the present invention. These include diagnostic and therapeutic methods both in vitro and in vivo. For example, the antibodies have use in immunoassays for qualitatively and quantitatively measuring levels of a polypeptide of the invention in biological samples. For example, Harl
ow et al., Antibodies: A Laboratory Manual, (
Cold Spring Harbor Laboratory Press,
Second Edition, 1988), which is incorporated herein by reference in its entirety.

As discussed in further detail below, the antibodies of the invention can be used either alone or in combination with other compounds. The antibody can be further recombinantly fused to the polypeptide or other compound at the N-terminus or C-terminus to a heterologous polypeptide, or chemically conjugated (including covalent and non-covalent bonds). For example, the antibodies of the present invention can be recombinantly fused or conjugated to molecules useful as labels in detection assays and to effector molecules such as heterologous polypeptides, drugs, radionuclides, or toxins. For example, PCT Publication WO92 / 0849
5; WO 91/14438; WO 89/12624; U.S. Pat. No. 5,314,9.
95; and EP 396,387.

The antibodies of the present invention may be modified (ie, covalently attached).
Attachment) includes derivatives (by covalent attachment of any type of molecule to the antibody) that do not prevent the antibody from producing an anti-idiotypic response. For example, without limitation, antibody derivatives include, for example, glycosylation, acetylation, pegylation, phosphorylation, and the like.
n), amidation, known protecting / blocking group
Antibodies that have been modified by derivatization, proteolytic cleavage, ligation to cellular ligands or other proteins, and the like. Numerous optional chemical modifications can be performed by known techniques, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like. Further, the derivative may contain one or more non-classical amino acids.

[0198] Antibodies of the present invention can be produced by any suitable method known in the art.
Polyclonal antibodies to an antigen of interest can be produced by various procedures well known in the art. For example, the polypeptides of the present invention can be used in various host animals (rabbits,
(Including but not limited to mice, rats, etc.) to induce the production of serum containing polyclonal antibodies specific for the antigen. Various adjuvants can be used to increase the immunological response, depending on the host species, including Freund (complete and incomplete), mineral gels such as aluminum hydroxide, mineral gels, Surfactants such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and BCG (bacillus Calmette-Guerin) and corynebacterium p
Including but not limited to potentially useful human adjuvants such as arvum. Such adjuvants are also well-known in the art.

[0199] Monoclonal antibodies can be prepared using a wide variety of techniques known in the art, including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced using the following hybridoma techniques known in the art and include, for example, Harr
low et al., Antibodies: A Laboratory Manual,
(Cold Spring Harbor Laboratory Press
Hammerling et al., Monoclonal An.
tibodies and T-Cell Hybridomas 563-6
81 (Elsevier, NY 1981), the above references being incorporated by reference in their entirety. As used herein, the term "monoclonal antibody" is not limited to antibodies produced through hybridoma technology. The term "monoclonal antibody" refers to an antibody derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it was generated. Thus, the term "monoclonal antibody" is not limited to antibodies produced through hybridoma technology. Monoclonal antibodies can be prepared using a wide variety of techniques known in the art, including the use of hybridoma and recombinant and phage display technologies.

Methods for producing and screening for specific antibodies using hybridoma technology are routine and well known in the art, and are discussed in detail in Example 21.
Briefly, mice can be immunized with a polypeptide of the invention or cells expressing such a peptide. Once an immune response is detected (eg, an antibody specific for the antigen is detected in the serum of the mouse), the spleen of the mouse is collected and splenocytes are isolated. The splenocytes are then fused by well known techniques to any suitable myeloma cells (eg, cells from cell line SP20 available from the ATCC). Hybridomas are selected and cloned by limiting dilution. Next, the hybridoma clones are assayed for cells secreting antibodies capable of binding the polypeptide of the invention by methods known in the art. Ascites fluid, which generally contains high levels of antibody, can be produced by immunizing mice with a positive hybridoma clone.

Accordingly, the present invention provides methods for producing monoclonal antibodies as well as antibodies produced by methods comprising culturing hybridoma cells secreting the antibodies of the present invention, wherein preferably Hybridomas are obtained by fusing myeloma cells with splenocytes isolated from mice immunized with an antigen of the invention, and then hybridoma clones secreting antibodies capable of binding a polypeptide of the invention. Is produced by screening.

[0202] Antibody fragments that recognize a particular epitope can be generated by known techniques. For example, Fab and F (ab ') 2 fragments of the present invention comprise (Fa
It can be produced by proteolytic cleavage of immunoglobulin molecules using an enzyme such as papain (to produce the b-fragment) or pepsin (to produce the F (ab ') 2 fragment). The F (ab ') 2 fragment contains the variable region, the light chain constant region and the CH1 domain of the heavy chain.

For example, the antibodies of the present invention can also be produced using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles that carry the polynucleotide sequences that encode them. In particular, such phages have a repertoire (reperto).
ire) or antigen binding domains expressed from combinatorial antibody libraries (eg, human or mouse). Phage expressing an antigen binding domain that binds the antigen of interest can be selected or identified using the antigen (eg, using a labeled antigen or an antigen bound or captured to a solid surface or beads). The phage used in these methods is typically a phage having the antibody domain of a Fab, Fv or disulfide stabilized Fv recombinantly fused to either the phage gene III protein or the phage gene VIII protein. Fd and M13 expressed from
Filamentous phage containing a binding domain. Examples of phage display methods that can be used to make the antibodies of the invention include the methods disclosed below: Brinkman et al. Im
munol. Methods 182: 41-50 (1995); Ames et al.,
J. 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 / GB91 / 01134;
PCT Publication WO 90/02809; WO 91/10737; WO 92/0
1047; WO 92/18619; WO 93/11236; WO 95/1
WO 95/20401; and U.S. Patent Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,7.
No. 17, No. 5,427,908; No. 5,750, 753; No. 5,82
No. 1,047; No. 5,571,698; No. 5,427,908; No. 5
, 516,637; 5,780,225; 5,658,727;
Nos. 5,733,743 and 5,969,108 (each of which is
Which is hereby incorporated by reference in its entirety).

As described in the above references, after phage selection, the regions encoding phage-derived antibodies may be used to generate whole antibodies, including human antibodies, or any other desired antigen-binding fragment. Can be isolated and used, and can be expressed in any desired host, including, for example, mammalian cells, insect cells, plant cells, yeast and bacteria, as described in detail below. For example, techniques for recombinantly producing Fab, Fab ', and F (ab') 2 fragments can also be used using methods known in the art, and such methods are disclosed below. : PC
T-Publication WO 92/22324; Mullinax et al., BioTechnique.
es 12 (6): 864-869 (1992); and Sawai et al., AJR.
I 34: 26-34 (1995); and Better et al., Science.
240: 1041-1043 (1988) (these references are incorporated by reference in their entirety).

Examples of techniques 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., Methods in Enzymology 203: 46-88 (19
91); Shu et al., PNAS 90: 7995-7999 (1993); and Skerra et al., Science 240: 1038-1040 (1988). For some uses, including in vivo use of antibodies in humans and in vitro detection assays, the use of chimeric, humanized or human antibodies may be preferred. Chimeric antibodies are different parts of an antibody,
Molecules from different animal species (eg, antibodies having a variable region derived from a mouse monoclonal antibody and a human immunoglobulin constant region). Methods for producing chimeric antibodies are known in the art. For example, Morrison,
Science 229: 1202 (1985); Oi et al., BioTechni.
ques 4: 214 (1986); Gillies et al., (1989) J. Am. Im
munol. Methods 125: 191-202; U.S. Pat. No. 5,807.
Nos. 4,715,567; and 4,816,397, which are incorporated herein by reference in their entirety. A humanized antibody is an antibody molecule from a non-human species antibody that binds a desired antigen having one or more complementarity determining regions (CDRs) from a non-human species and framework regions from a human immunoglobulin molecule. . Often, framework residues within the human framework regions are replaced with corresponding residues from a CDR donor antibody to alter (preferably improve) antigen binding. These framework substitutions are identified by methods well known in the art, for example, modeling the interaction of CDR and framework residues to identify framework residues important for antigen binding, and at specific positions. By sequence comparison to identify abnormal framework residues. (For example, Que
en et al., US Patent No. 5,585,089; Riechmann et al., Natur.
e 332: 323 (1988), which are incorporated herein by reference in their entirety. ) Antibodies can be humanized using various techniques known in the art, including: CDR-grafting (EP 239,40)
No. 0; PCT Publication WO 91/09967; US Pat. No. 5,225,539;
Nos. 5,530,101 and 5,585,089), veneer rings (
Vening or resurfacing (
EP 592,106; EP 519,596; Padlan, Mole.
cultural Immunology 28 (4/5): 489-498 (199)
1); Studnicka et al., Protein Engineering 7
(6): 805-814 (1994); Roguska et al., PNAS 91: 9.
69-973 (1994)), and chain shuffling (chains).
huffling) (U.S. Pat. No. 5,565,332).

[0206] Fully human antibodies are particularly desirable for therapeutic treatment of human patients. Human antibodies can be produced by various methods known in the art, including the phage display method described above using an antibody library derived from human immunoglobulin sequences. U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCT
Published WO 98/46645, WO 98/50433, WO 98/2489
3, WO 98/16654, WO 96/34096, WO 96/3373
5, and WO 91/10741; each of which is incorporated herein by reference in its entirety.

[0207] Human antibodies can also be produced using transgenic mice, which are unable to express functional endogenous immunoglobulin, but are capable of expressing human immunoglobulin genes. For example, a complex of a human heavy chain immunoglobulin gene and a human light chain immunoglobulin gene can be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, the human variable region, constant region, and diversity region (diversity)
region) can be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes. The mouse heavy and light chain immunoglobulin genes can be rendered non-functional separately or simultaneously with the introduction of the human immunoglobulin locus by homologous recombination. In particular, homozygous deletion of the JH region prevents endogenous antibody production. The modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. Then, the chimeric mouse was
Breed to produce homozygous offspring that express human antibodies. The transgenic mice are immunized in the normal fashion with a selected antigen (eg, all or a portion of a polypeptide of the invention). Monoclonal antibodies against the antigen
It can be obtained from immunized transgenic mice using conventional hybridoma technology. The human immunoglobulin transgenes retained in the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation. Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg and Huszar.
, 1995, Int. Rev .. Immunol. 13: 65-93. For a detailed discussion of this technology for producing human and human monoclonal antibodies and protocols for producing such antibodies, see, e.g.,
PCT Publication WO98 / 24893; WO96 / 34096; WO96 / 3373
5, U.S. Patent Nos. 5,413,923; 5,625,126; 5,6.
Nos. 33,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; and 5,939,5.
No. 98, which are incorporated herein by reference in their entirety.
. Further, Abgenix, Inc. (Freemont, CA) and Gen
Companies such as Pharm (San Jose, CA) may be engaged in providing human antibodies against the selected antigen using techniques similar to those described above.

A fully human antibody recognizing the selected epitope was identified as “guided”
ed) Selection "techniques. In this approach, selected non-human monoclonal antibodies (eg, mouse antibodies) are used to guide the selection of human antibodies that fully recognize the same epitope (Jespers.
Et al., Bio / technology 12: 899-903 (1988)).

In addition, antibodies to the polypeptides of the invention may in turn be utilized to produce anti-idiotypic antibodies that "mimic" the polypeptides of the invention using techniques well known to those of skill in the art. (Eg, Greenspan and Bona, FASEB
J. 7 (5): 437-444; (1989) and Nissinoff, J.
. Immunol. 147 (8): 2429-2438 (1991). E.g., bind and multimerize polypeptides
) and / or antibodies that competitively inhibit the binding of the polypeptide of the invention to its ligand are used to produce anti-idiotypes that "mimic" the multimerization and / or binding domains of the polypeptide. And thus binds and neutralizes the polypeptide and / or its ligand.
Neutralization of such anti-idiotypes or Fab fragments of such anti-idiotypes can be used in therapeutic regimes to neutralize polypeptide ligand. For example, such anti-idiotype antibodies can be used to bind a polypeptide of the invention and / or bind its ligand / receptor, thereby blocking its biological activity.

A. Polynucleotides Encoding Antibodies The present invention further provides polynucleotides comprising nucleotide sequences encoding the antibodies of the present invention and fragments thereof. The present invention also provides antibodies (preferably as specifically defined above) under stringent or lower stringency hybridization conditions (preferably, which specifically bind to a polypeptide of the present invention, preferably , An antibody that binds to a polypeptide having the amino acid sequence of SEQ ID NO: 2).

[0211] A polynucleotide can be obtained by any method known in the art, and the nucleotide sequence of the polynucleotide is determined. For example, if the nucleotide sequence of an antibody is known, the polynucleotide encoding the antibody can be constructed from chemically synthesized oligonucleotides (eg, Kutmeie).
et al., BioTechniques 17: 242 (1994), which, in brief, involves the following steps: Synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody. Annealing and ligation of these oligonucleotides, followed by amplification of the ligated oligonucleotides by PCR.

Alternatively, a polynucleotide encoding an antibody can be produced from nucleic acid from a suitable source. If no clone is available containing the nucleic acid encoding the particular antibody, but 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 an antibody cDNA library). From any tissue or cell that expresses (eg, a hybridoma cell selected for expression of an antibody of the invention), or a nucleic acid (preferably poly A + RNA) isolated therefrom. For example, to identify a cDNA clone from a cDNA library that encodes the antibody, by PCR amplification using synthetic primers that can hybridize to the 3 'and 5' ends of the sequence, or specific to a particular gene sequence Can be obtained by cloning using a unique oligonucleotide probe The amplified nucleic acid generated by PCR can then be cloned into a replicable cloning vector using any method known in the art.

Once the nucleotide sequence and the corresponding amino acid sequence of the antibody have been determined,
The nucleotide sequence of an antibody can be determined by methods known in the art for manipulating nucleotide sequences (eg, recombinant DNA technology, site-directed mutagenesis, PCR, etc. (eg,
Sambrook et al., 1990, Molecular Cloning, AL.
laboratory Manual, 2nd edition, Cold Spring Har
bor Laboratory, Cold Spring Harbor, NY
And Ausubel et al., Eds. 1998, Current Protocols.
in Molecular Biology, John Wiley & Sons
, NY. These are both incorporated herein by reference in their entirety. )) Can be used to generate antibodies with different amino acid sequences to make, for example, amino acid substitutions, deletions, and / or insertions.

In certain embodiments, the amino acid sequences of the heavy and / or light chain variable domains are identified by methods well known in the art for identification of complementarity determining region (CDR) sequences (eg, sequences To determine the region of hypervariability, other heavy chains,
And by comparing the variable region of the light chain with the known amino acid sequence). Using conventional recombinant DNA techniques, one or more CDRs can be inserted into a framework region as described above (eg, into a human framework region to humanize a non-human antibody). . The framework region can be naturally occurring or a consensus framework region, and can preferably be a human framework region (eg, for the listed human framework regions, see Chothia et al., J. Mol. Biol.278: 457-479 (1
998)). Preferably, the polynucleotide generated by the combination of the framework regions and the CDRs encodes an antibody that specifically binds a polypeptide of the invention. Preferably, as discussed above, one or more amino acid substitutions can be made within the framework regions, and preferably, the amino acid substitutions improve the binding of the antibody to its antigen. Further, such methods can produce antibody molecules that lack one or more intrachain disulfide bonds,
It can be used to make amino acid substitutions or deletions of cysteine residues in one or more of the variable regions involved in intrachain disulfide bonds. Other changes to polynucleotides are encompassed by the present invention and in the art.

Further, by splicing genes from mouse antibody molecules of appropriate antigen specificity with genes from human antibody molecules of appropriate biological activity,
Techniques developed to produce "chimeric antibodies" (Morrison et al., 1984)
Proc. Natl. Acad. Sci. 81: 851-855; Neube
rger et al., 1984, Nature 312: 604-608; Takeda.
Et al., 1985, Nature 314: 452-454) may be used. As described above, chimeric antibodies are molecules in which different portions are derived from different animal species, and such molecules have variable regions derived from the constant regions of mouse monoclonal antibodies and human immunoglobulins (eg, humanized antibodies). ).

Alternatively, techniques described for the production of single chain antibodies (US Pat. No. 4,694,941)
No. 778; Bird, 1988, Science 242: 423-42; Hu
Stone et al., 1988, Proc. Natl. Acad. Sci. USA 85
: 5879-5883; and Ward et al., 1989, Nature 334:
544-554) can be adapted for the production of single chain antibodies. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide. E. FIG. Techniques for the assembly of functional Fv fragments in E. coli can also be used (Skerr
a, et al., 1988, Science 242: 1038-1041).

B. Methods for Producing Antibodies The antibodies of the present invention can be produced by any method known in the art for synthesizing antibodies, particularly by chemical synthesis, or preferably, by recombinant expression techniques. Can be produced.

An antibody of the invention, or a fragment, derivative or analog thereof (eg,
Recombinant expression of the antibody (heavy or light chain) of the present invention requires the construction of an expression vector containing a polynucleotide encoding the antibody. Once a polynucleotide encoding the antibody molecule or antibody heavy or light chain, or portions thereof, (preferably containing the heavy or light chain variable domains) of the invention is obtained, the production of the antibody molecule is Can be produced by recombinant DNA technology using techniques well known in the art. Accordingly, methods for preparing a protein by expression of a polynucleotide containing a nucleotide sequence encoding an antibody are described herein. Methods well known to those skilled in the art can be used for the construction of expression vectors containing antibody-encoding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant D
NA techniques, synthetic techniques, and in vivo genetic recombination. Accordingly, the present invention provides a replicable vector comprising a nucleotide sequence encoding an antibody molecule of the present invention, or a heavy or light chain thereof, or a variable domain of a heavy or light chain, operably linked to a promoter. I will provide a. Such a vector is
And may comprise a nucleotide sequence encoding the constant region of an antibody molecule (see, eg, PCT Publication WO 86/05807; PCT Publication WO 89/01036; and US Pat. No. 5,122,464), and the variable domain of the antibody. Is
It can be cloned into such a vector for expression of the entire heavy or light chain.

[0219] The expression vector is transferred into a host cell by conventional techniques, and the transfected cells are then cultured by conventional techniques to produce an antibody of the present invention. Accordingly, the invention includes a host cell comprising 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 a double-chain antibody, the vector encoding both the heavy and light chains is, as detailed below,
It may be co-expressed in a host cell for total expression of the immunoglobulin molecule.

[0220] A variety of host expression vector systems can be utilized to express the antibody molecules of the present invention. Such host expression systems represent vehicles in which the coding sequence of interest can be produced and subsequently purified, but also when transformed or transfected with sequences encoding the appropriate nucleotides, in situ. 1 represents a cell capable of expressing the antibody molecule of the present invention. These include bacteria (eg, E. coli, B. subtilis) transformed with a recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vector containing the sequence encoding the antibody.
Such as microorganisms; yeast transformed with a recombinant yeast expression vector containing a sequence encoding an antibody (eg, Saccharomyces, Pichia); and a recombinant virus expression vector containing a sequence encoding an antibody (eg, baculovirus). Infected insect cell lines; plant cell lines infected with a recombinant viral expression vector (eg, cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or a recombinant plasmid expression vector containing a sequence encoding an antibody (eg, a Ti plasmid )); Or a promoter derived from the genome of a mammalian cell (eg, a metallothionein promoter).
Alternatively, a mammalian cell line (eg, COS, CHO, BHK, 29) carrying a recombinant expression construct containing a promoter derived from a mammalian virus (eg, adenovirus late promoter; vaccinia virus 7.5K promoter).
3, 3T3 cells). For expression of recombinant antibody molecules, preferably bacterial cells such as Escherichia coli, and more preferably, eukaryotic cells are used, especially for expression of whole recombinant antibody molecules. For example, mammalian cells such as Chinese hamster ovary cells (CHO) in combination with vectors such as the major intermediate early gene promoter element from human cytomegalovirus are an effective expression system for antibodies. (Foecking et al., 1986, Gene 45: 101; Coc.
kett et al., 1990, Bio / Technology 8: 2).

In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the expression of the antibody molecule. For example, where large quantities of such proteins are produced, vectors that direct high levels of expression of fusion protein products that are readily purified may be desirable for the production of pharmaceutical compositions of antibody molecules. In such a vector, the region encoding the antibody can be separately linked in frame with the vector along with the region encoding lacZ, resulting in the production of a fusion protein. co
li expression vector pUR278 (Ruther et al., 1983, EMBO J.2
: 1791); pIN vector (Inouye & Inouye, 1985, Nu)
cleic Acids Res. 13: 3101-3109; Van Hee
ke & Schuster, 1989, J. Am. Biol. Chem. 24: 5503
-5509) and the like, but are not limited thereto. The pGEX vector can also be used as a fusion protein with glutathione S-transferase (GST) to express foreign polypeptides. 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 designed to include 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 polynucleosis virus (Aut)
Ographa California nuclear polyhedr
Osis virus (AcNPV) is used as a vector for expressing a foreign gene. The virus is Spodoptera frugi
Proliferate in perda cells. Sequences encoding this antibody can be cloned individually into non-essential regions of the virus, such as the polyhedrin gene, and placed under the control of an AcNPV promoter, such as the polyhedrin promoter.

[0223] In mammalian host cells, a number of viral-based expression systems may be utilized. If an adenovirus is used as the expression vector, the sequence of interest encoding the antibody may be ligated to an adenovirus transcription / translation control complex (eg, the late promoter and tripartite leader sequence). This chimeric gene can then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion into a non-essential region of the viral genome (eg, region E1 or E3) results in a recombinant virus that survives in the infected host and can express the antibody molecule. (See, e.g., Logan & Shenk, 1984, Proc. Nat.
l. Acad. Sci. ScL USA 81: 355-359). Certain initiation signals may also be required for efficient translation of the inserted antibody-encoding sequence. These signals include the ATG start codon and adjacent sequences. In addition, this initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of various origins, both natural and synthetic. The efficiency of expression can be increased by including appropriate transcription enhancer elements, transcription terminators, etc. (Bittner et al., 1987, M
methods 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. Such modifications (eg, glycosylation) and processing (eg, cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of protein and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells that have the cellular machinery for proper processing of the primary transcript, glycosylation of the gene product, and phosphorylation can be used. Such mammalian host cells include CHO, VERY, BHK, Hela, COS, M
DCK, 293, 3T3, WI38, and especially breast cancer cell lines (eg, BT
483, Hs578T, HTB2, BT20 and T47D) and normal mammary gland cell lines (such as CRL7030 and Hs578Bst).

For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines that stably express the antibody molecule can be engineered. Rather than using an expression vector containing a viral origin of replication, the host cells are transfected with appropriate expression control elements (eg, promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.), and DNA controlled by a selectable marker. Can be converted. Following the introduction of the exogenous DNA, the engineered cells can be grown for 1-2 days in a concentrated medium and then switched to a selective medium. The selectable marker in the recombinant plasmid confers resistance to selection and allows cells to stably integrate the plasmid into their chromosomes, and grow, forming foci that can then be cloned and propagated into cell lines. . This method can be advantageously used to engineer cell lines that express the antibody molecule. Such engineered cell lines may be particularly useful in screening and evaluating compounds that interact directly or indirectly with the antibody molecule.

A number of selection systems can be used, including herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11: 223), hypo, which can be used in tk-, hgprt-, or aprt- cells, respectively. Xanthine-guanine phosphoribosyltransferase (Szybalska and Sz
ybalski, 192, Proc. Natl. Acad. Sci. USA 4
8: 202), and the adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22: 817) gene. Also, antimetabolite resistance can be used as a basis for selection of the following genes: dhfr (Wigler et al., 1980), which confers resistance to methotrexate.
Natl. Acad. Sci. ScL USA 77: 357); O'Hare et al., 1
981, Proc. Natl. Acad. Sci. USA 78: 1527; gpt conferring resistance to mycophenolic acid (Mulligan and Berg, 1
981, Proc. Natl. Acad. Sci. ScL USA 78: 2072);
Neo (Clinical Ph) which confers resistance to aminoglycoside G-418
armacy 12: 488-505; Wu and Wu, 1991, Bioth.
erapy 3: 87-95; Tolstoshev, 1993, Ann. Re
v. Pharmacol. Toxicol. 32: 573-596; Mulli
gan, 1993, Science 260: 926-932; and Mor
gan and Anderson, 1993, Ann. Rev .. Biochem.
62: 191-217; May 1993, TIB TECH 11 (5): 15.
5-215); and hygro (San) which confers resistance to hygromycin.
Terre et al., Gene 1984, 30: 147 ()). Methods of recombinant DNA technology generally known in the art can be routinely applied to the selection of desired recombinant clones, and such methods are described, for example, in Ausubel et al.
3, Current Protocols in Molecular Bio
Kriegler, 199, J. Logy, John Wiley & Sons, NY;
0, Gene Transfer and Expression, A Lab
laboratory Manual, Stockton Press, NY; and Dracopoli et al. (eds.), 1994, Current Protocol.
s in Human Genetics, John Wiley & Sons,
NY Chapters 12 and 13; Colberre-Garapin et al., 1981, J.
. Mol. Biol. 150: 1, which are incorporated herein by reference in their entirety.

The expression level of an antibody molecule can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of
vectors based on gene amplification
for the expression of cloned genes i
n mammarian cells in DNA cloning, Volume 3 (Academic Press, New York, 1987). If the marker is amplifiable in a vector system expressing the antibody, increasing the level of inhibitor present in the culture of the host cell will increase the number of copies of the marker gene. Because the amplified region is linked to the antibody gene, production of the antibody is also increased (Crouse et al., 1983, Mol. Cell. Bio.
l. 3: 257).

The host cells can be co-expressed with two expression vectors of the invention, a first vector encoding a polypeptide derived from the heavy chain and a second vector encoding a polypeptide derived from the light chain. Can be transfected. The two vectors may contain the same selectable marker that allows for equal expression of the heavy and light chain polypeptides. Alternatively, a single vector encoding both heavy and light chain polypeptides can be used. In such situations, the light chain should be placed before the heavy chain to avoid excess toxic free heavy chains (Proudf
oot, Nature 1986, 322: 52; Kohler, 1980, P.
rc. Natl. Acad. Sci. ScL USA 77: 2197). The heavy and light chain coding sequences can include cDNA or genomic DNA.

Once the antibody molecule of the present invention is expressed in animals, chemically synthesized, or recombinantly, the antibody molecule can be purified by any method known in the art for purification of immunoglobulin molecules (eg, , Chromatography (eg, ion exchange chromatography, affinity (especially by protein A followed by affinity to a specific antigen), and sizing column chromatography), centrifugation, differential solubility, or protein purification. Can be purified (by any other standard technique).

C. Antibody Conjugates The present invention relates to polypeptides of the present invention (or a portion thereof, preferably at least 10, 20 or 50 of a polypeptide) for making fusion proteins.
Amino acids), or antibodies that are chemically fused (including both covalent and non-covalent bonds). The fusion need not be direct, but can occur through a linker sequence. The antibody may comprise a polypeptide of the invention (or a portion thereof, preferably at least 10,
(20 or 50 amino acids). For example, an antibody can be used to fuse a polypeptide of the invention, either in vitro or in vivo, to an antibody specific for a particular cell surface receptor, thereby binding the polypeptide of the invention to a particular cell type. Can be used to target Antibodies fused or conjugated to a polypeptide of the invention can also be used in in vitro immunoassays and purification methods using methods known in the art. See, for example, Harbor et al., Supra and PCT Publication WO 93/21232;
P439, 095; Naramura et al., Immunol. Lett. 39: 9
1-99 (1994); U.S. Patent No. 5,474,981; Gillies et al.
PNAS 89: 1428-1432 (1992); Fell et al. Immu
nol. 146: 2446-2452 (1991), which are incorporated by reference in their entirety.

The present invention further includes compositions comprising a polypeptide of the present invention fused or conjugated to a domain of the antibody other than the variable regions. For example, a polypeptide of the invention can be fused or conjugated to the Fc region of an antibody, or a portion thereof. The antibody portion fused to the polypeptide of the present invention comprises a constant region, a hinge region, a CH1 domain,
It may comprise a CH2 domain, and a CH3 domain, or any combination of all or part of those domains. The polypeptide can also be fused or conjugated to the antibody portion described above to form a multimer. For example, an Fc portion fused to a polypeptide of the invention can form a dimer via a disulfide bond between the Fc portions. Higher order multimeric forms can be made by fusing the polypeptide to portions of IgA and IgM. Methods for fusing or linking a polypeptide of the invention to an antibody moiety are known in the art. For example, U.S. Patent Nos. 5,336,603; 5,622,929; 5,359,046; 5,349,053; 5,447,851; 5,112,9
No. 46; EP 307, 434; EP 367, 166; PCT Publication WO 96
WO 91/06570; Ashkenazi et al., Proc. N
atl. Acad. Sci. USA 88: 10535-10538 (1991)
); Zheng et al. Immunol. 154: 5590-5600 (199
5); and Vil et al., Proc. Natl. Acad. Sci. USA 89
11337-11341 (1992) (the above references are incorporated by reference in their entirety).

As discussed above, the polypeptides of the invention may increase the in vivo half-life of the polypeptide or may be used as described above for use in immunoassays using methods known in the art. It can be fused or conjugated to an antibody moiety. Further, the polypeptides of the present invention can be fused or conjugated to the antibody portions described above to facilitate purification. One reported example is the first human CD4 polypeptide.
And a chimeric protein consisting of two domains of various regions of the heavy or light chain constant region of a mammalian immunoglobulin. (EP 394, 82
7; Traunecker et al., Nature 331: 84-86 (1988).
). Polynucleotides of the invention fused or conjugated to an antibody having a disulfide-linked dimeric structure (due to IgG) also bind and neutralize other molecules than monomeric secreted proteins or protein fragments alone. May be more efficient. (Funtoulakis et al., J. Biochem. 27
0: 3958-3964 (1995)). In many cases, the Fc portion of the fusion protein is advantageous in therapy and diagnosis, and thus may result, for example, in improved pharmacokinetic properties. (EP A 232,262). Or,
Deletion of the Fc portion after the fusion protein has been expressed, detected, and purified is preferred. For example, if this fusion protein is used as an antigen for immunization,
This Fc protein can interfere with therapy and diagnosis. In drug development, for example, human proteins (eg, hIL-5) have been fused with Fc proteins for the purpose of high-throughput screening assays to identify antagonists of hIL-5. (D. Bennett et al., J. Molecular R.
Economy 8: 52-58 (1995); Johanson et al. Biol. Chem. 270: 9449-9471 (1995)).

Further, the antibodies or fragments thereof of the present invention can be fused to a marker sequence, such as a peptide to facilitate purification. In a preferred embodiment, the marker amino acid sequence is, inter alia, a pQE vector (QIAGEN, Inc.).
, 9259 Eton Avenue, Chatsworth, CA, 9131.
Hex-histidine peptides such as the tags provided in 1), many of which are commercially available. See, for example, Gentz et al., Proc. Natl. Ac
ad. Sci. USA 86: 821-824 (1984), hexa-histidine provides for convenient purification of the fusion protein. Other peptide tags useful for purification include the “HA” tag corresponding to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell 37: 767 (19).
84)) and the "flag" tag.

The present invention further includes antibodies or fragments thereof that bind to a diagnostic or therapeutic agent. The antibodies can be used diagnostically to monitor tumor development or progression, for example, as part of a clinical trial procedure (eg, to determine the efficacy of a given therapeutic and / or prophylactic regime). Can be done. Detection can be facilitated by coupling the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron-emitting metals using various positron emission tomography, and non-radioactive paramagnetic metals Ions. See, for example, US Pat. No. 4,741,900 for metal ions that can be conjugated to antibodies for use as diagnostics according to the present invention. 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 fluorescent materials include umbelliferone,
Fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; examples of luminescent materials include luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin Examples of suitable radioactive materials include ¹²⁵ I, ¹³¹ I, ¹¹¹ In or ⁹⁹ Tc.

In addition, the antibody or fragment thereof can be conjugated to a therapeutic moiety, such as a cytotoxin (eg, a cytostatic or cytocidal agent), a therapeutic or a radiometal ion. Cytotoxic or cytotoxic agents include any agents that are harmful to cells. Examples include paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetin, mitomycin, etoposide, tenoposid.
de), vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracindione, mitozantrone, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoid,
Procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof are included. Therapeutic agents include antimetabolites (eg, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (eg, mechlorethamine, thioepa)
pa) chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide
, Busulfan, dibromomannitol, streptozotocin (strepto)
zotocin), mitomycin C, and cis-dichlorodiamineplatinum (I
I) (DDP) cisplatin), anthracycline
ne) (eg, daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (eg, dactinomycin (formerly actinomycin)),
Bleomycin, mithramycin, and anthramycin
cin) (AMC)), as well as antimitotic agents (eg, vincristine and vinblastine).

The conjugates of the present invention can be used for modifying a given biological response, and the therapeutic or drug moiety should not be construed as being limited to classical chemotherapeutic agents. . For example, the drug moiety can be a protein or polypeptide that possesses the desired biological activity. Such proteins include, for example, 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 plasminol. Gene activators, thrombotics or anti-angiogenic agents (eg, angiostatin or endostatin (en)
or a biological response modifier (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, etc.).

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

Techniques for coupling such therapeutic moieties to antibodies are well known and include, for example, Arnon
Et al., "Monoclonal Antibodies For Immunot."
arranging Of Drugs In Cancer Therapy "
Monoclonal Antibodies And Cancer The The
rapy, Reisfeld et al. (eds.), pages 243-56 (Alan R. Lis.
s, Inc. 1985); Hellstrom et al., "Antibodies F.
or Drug Delivery ", Controlled Drug De
livery (second edition), Robinson et al. (eds.), pp. 623-53 (Mar
cel Dekker, Inc. 1987); Thorpe, "Antibod
y Carriers Of Cytotoxic Agents In Ca
nc Therapy: A Review ", Monoclonal An
tibodies'84: Biological And Clinical
Applications, Pinchera et al. (Eds.), Pages 475-506 (1
985); “Analysis, Results, And Future Pr.
Ospective Of The Therapeutic Use Of
Radiolabeled Antibody In Cancer Ther
apy ", Monoclonal Antibodies For Cance
r Detection And Therapy, Baldwin et al.
Pages 303-16 (Academic Press 1985), and Thor
Pe et al., "The Preparation And Cytotoxic P
rightsies of Antibody-Toxin Conjugator
es ", Immunol. Rev .. 62: 119-58 (1982).

Alternatively, the antibody is conjugated to a secondary antibody and antibody heterologous binding as described by Segal in US Pat. No. 4,676,980, which is incorporated herein by reference in its entirety. Heterojugates may be formed.

Antibodies with or without a therapeutic moiety attached to the antibody, administered alone or in combination with cytotoxic factors and / or cytokines, can be used as therapeutics.

D. Assay for Antibody Binding The antibodies of the present invention can be assayed for immunospecific binding by any method known in the art. Immunoassays that can be used include (only a few named Western blot, radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), "sandwich" immunoassay, immunoprecipitation assay, sedimentation reaction, gel diffusion sedimentation. Competitive and non-competitive assay systems using techniques such as reactions, immunodiffusion assays, agglutination assays, complement fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays. Such assays are conventional and well known in the art (eg, Ausubel et al., Eds., 1994, Current Pro, which is incorporated herein by reference in its entirety).
tocols in Molecular Biology, Volume 1, John
Wiley & Sons, Inc. , New York). Exemplary immunoassays are briefly described below (although these are not intended to be limiting).

Immunoprecipitation protocols generally involve RIPA buffer (1% NP-40 or Trito) supplemented with protein phosphatase and / or protease inhibitors (eg, EDTA, PMSF, aprotinin, sodium vanadate).
nX-100, 1% sodium deoxycholate, 0.1% SDS, 0.1%
0.01 M sodium phosphate at 15 M NaCl, pH 7.2, 1% Tr
lysing a population of cells in a lysis buffer, such as asylol, adding the antibody of interest to the cell lysate, incubating at 4 ° C. for a period of time (eg, 1-4 hours), protein A and Adding protein G Sepharose beads to the cell lysate, incubating at 4 ° C. for at least about 1 hour, washing the beads in lysis buffer, and resuspending the beads in SDS / sample buffer The step of performing The ability of an antibody of interest to immunoprecipitate a particular antigen
For example, it can be assayed by Western blot analysis. One skilled in the art is familiar with parameters that can be modified to increase the binding of the antibody to the antigen and reduce the background (eg, pre-clearing cell lysate using Sepharose beads). . For further discussion of immunoprecipitation protocols, see, for example, Ausubel et al., Eds., 1994, Current P.
rotocols in Molecular Biology, Volume 1, Jo
hn Wiley & Sons, Inc. , New York, 10.16.
See 1.

[0243] Western blot analysis generally involves preparing a protein sample, subjecting the protein sample to a polyacrylamide gel (for example, an 8 depending on the molecular weight of the antigen).
% In 20% SDS-PAGE), transferring the protein sample from the polyacrylamide gel to a membrane such as nitrocellulose, PVDF or nylon, having a blocking solution (eg, 3% BSA or skim milk) Blocking the membrane in PBS), washing the membrane in a wash buffer (eg, PBS-Tween 20), membrane with primary antibody (antibody of interest) diluted in blocking buffer Blocking, washing the membrane in a washing buffer, adding an enzyme substrate (eg, horseradish peroxidase or alkaline phosphatase) or a radioactive molecule (eg, ³² P or ¹²⁵ I) diluted in the blocking buffer. Bound secondary antibody (recognizing primary antibody, eg, anti-human Blocking the membrane with an antibody), washing the membrane in a wash buffer, and detecting the presence of the antigen. One skilled in the art is familiar with parameters that can be modified to increase the signal detected and reduce background noise. For further discussion on Western blot protocols, see, for example, Ausubel
Ed., 1994, Current Protocols in Molecul.
ar Biology, Volume 1, John Wiley & Sons, Inc.
. , New York, 10.8.1.

[0244] ELISA involves preparing an antigen, coating the wells of a 96-well microtiter plate with the antigen, binding to a detectable compound such as an enzyme substrate (eg, horseradish peroxidase or alkaline phosphatase). Adding the antibodies to the wells and incubating for a period of time, and detecting the presence of the antigen. In an ELISA, the antibody of interest need not be conjugated to a detectable compound; instead, a secondary antibody (recognizing the antibody of interest) conjugated to the detectable compound can be added to the well. Further, instead of coating the well with the antigen, the antibody can be coated on the well. In this case, a secondary antibody conjugated to the detectable compound can be added following addition of the antigen of interest to the coated wells. One skilled in the art is familiar with parameters that can be modified to increase the signal detected, and other variations of ELISAs known in the art. For further discussion on ELISA, see, for example, Ausubel et al., Ed., 1994, Cu.
rent Protocols in Molecular Biology
, Volume 1, John Wiley & Sons, Inc. , New York
, 11.2.1.

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

E. Antibody-Based Therapy The present invention further relates to antibody-based therapies, wherein the therapy comprises treating the antibody of the invention with one or more disorders or disorders described herein. Administering to an animal, preferably a mammal, and most preferably a human patient, to treat and / or prevent the condition. Therapeutic compounds of the invention include antibodies of the invention (including fragments, analogs and derivatives thereof, as described herein) and nucleic acids encoding the antibodies of the invention (described herein). As well as fragments, analogs and derivatives thereof).

[0247] Without intending to be bound by theory, it is believed that the DR4 receptor induces programmed cell death by a process involving the association / crosslinking of death domains between different receptor molecules. In addition, DR4 ligands (eg, TRAIL) that induce DR4-mediated programmed cell death are associated with DR4 death domain association /
It is thought to work by causing crosslinking. Thus, agents that prevent DR4 death domain association / crosslinking (eg, antibodies) prevent DR4-mediated programmed cell death and agents that promote DR4 death domain association / crosslinking (eg,
Antibody) induces DR4-mediated programmed cell death.

As noted above, the DR4 receptor has been shown to bind to TRAIL. DR4 receptors are also known to be present in many tissues and on the surface of many cell types. These tissues and cell types include amniotic cells, heart,
Liver cancer cells, kidney, leukocytes, activated T cells, K562 cells (erythroleukemia cell line) plus PMA, W138 cells (human lung fibroblast cell line), Th2 cells (lymphocytes), human tonsils, and cord blood CD34 deficient buffy coat cells. Furthermore, as described in more detail below, TRAIL has been shown to induce apoptosis and inhibit tumor cell growth in vivo. Furthermore, TRAIL activity is thought to be modulated, at least in part, by interaction with DR4 and DR5 receptors.

TRAIL is a member of the TNF family of cytokines, which has been shown to induce apoptotic cell death in many tumor cell lines, and
It appears to mediate apoptosis which induces effects by interaction with R4 and DR5 receptors. These death domain containing receptors are thought to form a membrane-bound self-activating signaling complex that initiates apoptosis by caspase cleavage.

In addition to the DR4 and DR5 receptors, TRAIL also
coy) ", a number of receptors proposed to be receptors, DcR2
(Receptor with a truncated death domain), DcR1 (GPI anchoring domain), and OPG (RANKL, a secreted protein that binds to another member of the TNF family).

In addition, recent studies have shown that TRAIs for recombinant soluble forms of the receptor
The order of affinity of L was shown to be very temperature dependent. In particular, at 37 ° C., DR5 has the highest affinity for TRAIL and OPG has the lowest affinity.

The DR4 and DR5 receptor genes, and the genes encoding the two decoy receptors, have been shown to be located on human chromosome 8p21-22. In addition, the human genome in this region divides frequently in head and neck cancer.

The FaDu nasopharyngeal carcinoma cell line was recently found to contain an abnormal chromosome 8p21-22 region (Ozoren et al., Int. J. Oncol. 16: 917-925).
(2000)). In particular, homozygous deletions involving DR4 but not DR5 were found in these cells (Ozoren et al., Int. J. Oncol. 16: 9).
17-925 (2000)). The homozygous deletion in the DR4 receptor gene in these FaDu cells contains the DR4 receptor death domain. This disruption of the DR4 receptor death domain is associated with resistance to TRAIL-mediated cytotoxicity. In addition, reintroduction of the wild-type DR4 receptor gene can reduce apoptosis and F
It has been shown to cause both TRAIL-sensitive repair of aDu cells (Oz
oren et al., Int. J. Oncol. 16: 917-925 (2000)).
These data indicate that the DR4 receptor gene can be inactivated in human cancers and that DR4 receptor gene splitting can contribute to resistance to TRAIL treatment. It is expected that similar results will be found in cells with a similar deletion in the DR5 gene.

Overexpression of the cytoplasmic domain of the DR4 receptor in human breast, lung, and colon cancer cell lines has also been shown to cause p53-independent apoptotic cell death, including caspase cleavage (Xu et al., Biochem). .Biophy
s. Res. Commun. 269: 179-190 (2000)). further,
Overexpression of the DR4 cytoplasmic domain has also been shown to cause cleavage of both poly (ADP-ribose) polymerase (PARP) and a DNA-cleaving factor (ie, ICAD-DFF45) (Xu et al., Biochem. Biophy).
s. Res. Commun. 269: 179-190 (2000)). further,
Despite similar levels of DR4 cytoplasmic domain protein compared to cancer cells tested, normal lung fibroblasts were shown to be resistant to overexpression of DR4 cytoplasmic domain, indicating that caspase-cleaved No evidence was shown (Xu et al., Biochem. Biophys. Res. Commun. 269:
179-190 (2000)). Similar results are expected when cells that overexpress the DR5 cytoplasmic domain are used. Thus, the cytoplasmic domains of DR4 and DR5 receptors are useful as agents for inducing apoptosis, for example, in cancer cells.

Furthermore, the cyclin-dependent kinase inhibitor p21 (WAF1 / CIP1
), As well as overexpression of the N-terminal 91 amino acids of this protein has cell cycle inhibitory activity and inhibits DR4 cytoplasmic domain-dependent caspase cleavage. Thus, the DR4 receptor is also involved in regulating cell cycle progression. As described above, similar results are expected when using the DR5 receptor. Thus, the DR4 and DR5 receptors, and agonists and antagonists of those receptors, are useful for regulating cell cycle progression.

Antibodies that bind to the DR4 receptor are useful for treating and / or preventing diseases and conditions associated with increased or decreased DR4-induced apoptotic cell death. Furthermore, these antibodies differ in the effects they have on the DR4 receptor. These effects differ based on the particular portion of the DR4 receptor to which the antibody binds, the three-dimensional structure of the antibody molecule itself, and / or the manner in which they interact with the DR4 receptor. Thus, antibodies that bind to the extracellular domain of the DR4 receptor can either stimulate or inhibit DR4 activity (eg, induction of apoptosis). Stimulates DR4 receptor activity (eg,
Antibodies (by facilitating association between the DR4 receptor death domains)
Are agonists and inhibit DR4 receptor activity (eg, TRAI
Antibodies are DR4 antagonists (by blocking L binding and / or preventing association between DR4 receptor death domains).

Antibodies of the present invention that function as agonists and antagonists of the DR4 receptor include antigen-binding antibody fragments such as Fab and F (ab ') ₂ fragments, Fd, single-chain Fvs (scFv), disulfide-bonded Fvs (sdF
v), and fragments containing either the _VL or _VH domains, as well as polyclonal, monoclonal, and humanized antibodies. Bivalent antibodies are preferred as agonists. Each of these antigen-binding antibody fragments and antibodies is described in more detail elsewhere herein.

In view of the above, the antibodies of the present invention, as well as other agonists, are useful for stimulating DR4 death domain activity to promote apoptosis in cells expressing DR4 receptor (eg, cancer cells). . Antibodies of this type are useful for diseases and conditions associated with increased cell survival and / or insensitivity to apoptosis inducers (eg, TRAIL), such as solid tissue cancers (eg, skin cancer, head and neck tumors, Breast cancer, endothelioma, lung cancer, osteoblastoma, osteoclastoma, and Kaposi's sarcoma), and leukemia).

The antagonists of the present invention (eg, anti-DR4 antibodies) function by preventing DR4-mediated apoptosis and are useful for preventing and / or treating diseases associated with increased apoptotic cell death. Examples of such diseases include diabetes, AIDS, neurodegenerative disorders, myelodysplasia, ischemic trauma, toxin-induced liver disease, septic shock, cachexia and anorexia.

As mentioned above, the DR4 receptor is on the surface of T cells. Thus, the agonists of the invention (eg, anti-DR4 receptor antibodies) are also useful for inhibiting T cell mediated immune responses and preventing and / or treating diseases and conditions associated with increased T cell proliferation. is there. Diseases and conditions associated with T cell-mediated immune responses and increased T cell proliferation include anti-host reactions and diseases, osteoarthritis, psoriasis, sepsis, inflammatory bowel disease, general inflammation, autoimmune diseases , And T-cell leukemia.

When an agonist of the invention is administered to an individual for the treatment and / or prevention of a disease and condition associated with an increased T cell population or increased cell proliferation (eg, cancer), the antagonist will undergo apoptosis. (Eg, T
RAIL), or an agent that otherwise inhibits cell proliferation (eg, an anticancer agent)
It can be administered simultaneously. Combination treatments for this feature, as well as other combination treatments, are discussed in more detail below.

In addition, antagonists (eg, anti-DR4 receptor antibodies) of the present invention also enhance T cell-mediated immune responses and to prevent and / or treat diseases and conditions associated with decreased T cell proliferation. Useful for Antibodies of the invention that block the binding of the DR4 receptor ligand to the DR4 receptor or prevent DR4 receptor conformational changes associated with membrane signaling can inhibit DR4-mediated T cell apoptosis. Inhibition of DR4-mediated apoptosis, for example, can either result in an increase in the rate of proliferation of the T cell population in vivo, or can prevent a decrease in the size of such a population. Thus, the antagonists of the present invention can be used to prevent and / or treat a disease or condition associated with a reduced T cell population or reduced T cell population. Examples of such diseases and conditions include acquired immunodeficiency syndrome (AIDS) and related distress (eg, AIDS-related complex), T-cell immunodeficiency, radiation sickness, and T-cell deficiency due to radiation therapy and chemotherapy Is mentioned.

When an antagonist of the present invention is administered to an individual for the treatment and / or prevention of a disease or condition associated with a decrease in the T cell population, the antagonist activates and / or induces lymphocyte proliferation. It can be co-administered with an agent (eg, a cytokine). Combination treatments of this nature, as well as other combination treatments, are described in more detail below.

Accordingly, anti-DR4 antibodies are useful for treating and / or preventing malignancies, abnormalities, diseases, and / or conditions involving tissues and cell types that express the DR4 receptor. In addition, malignancies, abnormalities, diseases, and / or conditions that can be treated and / or prevented by the induction of programmed cell death in cells expressing the DR4 receptor are treated and / or treated using the DR4 receptor agonists of the present invention. Or can be prevented. Similarly, malignancies, abnormalities, diseases, and / or conditions that can be treated and / or prevented by inhibiting programmed cell death in cells that express the DR4 receptor are identified using the DR4 receptor antagonists of the present invention. It can be treated and / or prevented.

A number of additional malignancies, abnormalities, diseases, and / or conditions that can be treated using the agonists and antagonists of the invention are described elsewhere herein (eg, with “therapeutic agents” below). Entitled section).

The antibodies of the present invention can be used therapeutically in a number of ways. For example, an antibody that binds a polynucleotide or polypeptide of the invention can be administered to an individual (eg, a human) either locally or systemically. Further, these antibodies can be administered alone, in combination with another therapeutic agent, or associated with or conjugated to a toxin.

Anti-DR4 antibodies can be other monoclonal or chimeric antibodies, or lymphokines, tumor necrosis factors, or TNF-related molecules (eg, TNF-α, TNF-
β, TNF-γ, TNF-γ-α, TNF-γ-β, and TRAIL), or in combination with hematopoietic growth factors (eg, IL-2, IL-3 and IL-7). For example, an agonistic anti-DR4 antibody may be required to induce DR4-mediated cell death in cells that express the DR4 receptor of the present invention.
It can be co-administered with RAIL. Combination treatments of this nature and other combination treatments may be discussed in more detail below.

The antibodies of the present invention can be administered alone or in combination with other types of treatment, such as radiation therapy, chemotherapy, hormonal therapy, immunotherapy and antitumor agents.
Generally, administration of a species-derived or species-reactive product that is the same species as the patient (in the case of antibodies) is preferred. Thus, in a preferred embodiment, a human antibody, fragment derivative, analog, or nucleic acid is administered to a human patient for treatment or prevention.

For both immunoassays for the polynucleotides or polypeptides (including fragments thereof) of the invention and the treatment of disorders related thereto, high affinity and / or high affinity for the polypeptides or polynucleotides of the invention are provided. Or potent, in vivo inhibitory and / or neutralizing antibodies, fragments thereof,
Alternatively, it is preferable to use those regions. Such an antibody, fragment or region is preferably a polynucleotide or polypeptide (
(Including its fragments). Preferred binding affinities include 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, 10 ⁻¹⁰ M, 5 × 10 ⁻¹¹ M, 1
0 ⁻¹¹ M, 5 × 10 ⁻¹² M, 10 ⁻¹² M, 5 × 10 ⁻¹³ M, 10 ⁻¹³ M, 5 × 10 ⁻¹⁴ M, 10 ⁻¹⁴ M, 5 × 10 ⁻¹⁵ M, and 10 Binding affinities with a dissociation constant less than ^-15 M, ie, Kd.

Polypeptide Assays The present invention also provides quantitative and diagnostic assays for detecting levels of DR4 protein or its soluble form in cells and tissues, including determining normal and abnormal levels. Diagnostic assays. Thus, for example, a diagnostic assay according to the invention for detecting overexpression of DR4 or a soluble form thereof, as compared to a normal control tissue sample, can be used, for example, to detect the presence of a tumor. Assay techniques that can be used to determine levels of a protein, such as a DR4 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 radioimmunoassay, competitive binding assay, Western blot analysis and E
LISA assay.

Assaying DR4 protein levels in a biological sample can be performed using any method known in the art. By "biological sample" is intended any biological sample obtained from an individual, cell line, tissue culture, or other source that contains a DR4 receptor protein or mRNA. Antibody-based techniques are preferred for assaying DR4 protein levels in a biological sample. For example, DR4 protein expression in tissues can be studied with classical immunohistological methods. (Jalkanen, M. et al., J. Cell. Biol. 10
1: 976-985 (1985); Jalkanen, M .; J. et al. Cell.
Biol. 105: 3087-3096 (1987)). Other antibody-based methods useful for detecting DR4 protein gene expression include immunoassays, such as enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA).

Suitable labels are known in the art and include enzyme labels (eg,
Glucose oxidase) and radioisotopes (eg, iodine ( ¹²⁵ I, ¹²¹ I
), Carbon ( ¹⁴ C), sulfur ( ³⁵ S), tritium ( ³ H), indium ( ¹¹² In)
And technetium ( ^99mTc )); and fluorescent labels (eg, fluorescein and rhodamine), and biotin.

Transgenic and “Knockout” DR4 polypeptides of the invention can also be expressed in transgenic animals. Any species (mouse, rat, rabbit, hamster, guinea pig, pig, micro-pig, goat, sheep, cow, and non-human primate (
(Eg, but not limited to, baboons, monkeys, and chimpanzees) can be used to generate transgenic animals. In certain embodiments, techniques described herein or otherwise known in the art are 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 (ie, a nucleic acid of the present invention) into an animal to create a founder line of the transgenic animal. Such techniques include pronuclear microinjection (Paterso).
n et al., Appl. Microbiol. Biotechnol. 40: 691-
698 (1994); Carver et al., Biotechnology (NY) 1
1: 1263-1270 (1993); Wright et al., Biotechnol.
ogy (NY) 9: 830-834 (1991); and Hoppe et al., U.S. Patent No. 4,873,191 (1989)); retroviral-mediated gene transfer into the germline (Vander Putten et al., Proc. Natl. Acad.
Sci. USA 82: 6148-6152 (1985)), retrovirus-mediated gene transfer into blastocysts or embryos; gene targeting in embryonic stem cells (Thom
Pson et al., Cell 56: 313-321 (1989)); Electroporation of cells or embryos (Lo, 1983, Mol Cell. Biol. 3:
1803-1818 (1983)); introduction of a polynucleotide of the present invention using a gene gun (see, for example, Ulmer et al., Science 259: 1745 (1993)); transfer of a nucleic acid construct into embryonic pluripotent stem cells Transfection and retransfer of this stem cell into blastocysts; and sperm-mediated gene transfer (Lavitrano et al., C
ell 57: 717-723 (1989)); and the like. For a review of such techniques, Gordon, "Transgenic Animals", Int., Incorporated herein by reference in its entirety.
l. Rev .. Cytol. 115: 171-229 (1989).
Further, the contents of each of the documents listed in this paragraph are incorporated herein by reference in their entirety. No. 5,464,764 (Capecc).
hi et al., the Positive-Nagative Selection Meth.
ods and Vectors); U.S. Patent No. 5,631,153 (Cap
ecchi et al., Cells and Non-Human Organisms.
Containing Predetermined Genomic Mo
modifications and Positive-Negative Se
Selection Methods and Vectors for Maki
ng Same); U.S. Patent No. 4,736,866 (Leder et al., Tran).
generic Non-Human Animals); and U.S. Pat.
No., 873,191 (Wagner et al., Genetic Transforma).
See Tion of Zygotes, each of which is incorporated herein by reference in its entirety.

[0275] Any technique known in the art can be used to generate transgenic clones containing the polynucleotides of the present invention, including, for example, Embryonic (embryonic)
) Nuclear import of nuclei from cells, nuclei from fetal cells or nuclei from adult cells (
Campell et al., Nature 380: 64-66 (1996); Wilm.
ut et al., Nature 385: 810-813 (1997), each of which is incorporated herein by reference).

The present invention provides transgenic animals that have the transgene in all of their cells, as well as animals that have the transgene in some (but not all) of the cells (ie, mosaic or chimeric animals). I do. The transgene can be a single transgene or a concatamer (eg, head-to-head tandem or head-to-head).
Tail tandem). This transgene is also described, for example, in the teachings of Lasko et al. (Proc. Natl. Acad. Sci.
USA 89: 6322-2236 (1992)) and can be selectively introduced into specific cell types and activated. The regulatory sequences required for such cell type-specific activation will depend on the particular cell type of interest, and they will be apparent to those skilled in the art. If it is desired that the polynucleotide transgene be integrated into the chromosomal site of the endogenous gene, gene targeting is preferred. Briefly, when such a technique is used, a vector containing several nucleotide sequences homologous to the endogenous gene is transformed into a nucleotide sequence of the endogenous gene through homologous recombination with a chromosomal sequence. And designed to disrupt the function of the nucleotide sequence. The transgene may also be selectively introduced into a particular cell type, and thus may be, for example, Gu et al. (Science 265: 103-106 (1
994)), the endogenous gene is inactivated only in the introduced cell type. The regulatory sequences required for such cell type-specific inactivation will depend on the particular cell type of interest, and they will be apparent to those skilled in the art. The contents of each of the sentences listed in this paragraph are incorporated herein by reference in their entirety.

Once the transgenic animal has been produced, the expression of the recombinant gene can be assayed using standard techniques. Initial screening can be accomplished by Southern blot analysis or PCR techniques, and analysis of animal tissue can confirm that transgene integration has occurred. Transgene mRNA expression levels in the tissues of transgenic animals can also be determined by techniques including, but not limited to, Northern blot analysis, in situ hybridization analysis, and reverse transcriptase PCR (rt-PCR) of tissue samples obtained from the animals. Can be evaluated using Samples of the transgenic gene-expressing tissue can also be assessed immunocytochemically or immunohistochemically using antibodies specific for the transgene product. Once founders are generated, they can be crossed, inbred, outbred or crossed to produce colonies of a particular animal. Examples of such mating strategies include, but are not limited to: outcrossing founders with more than one integration site to establish another lineage; additive expression of each transgene By effect, inbreeding of separate strains to produce complex transgenics expressing higher levels of the transgene; certain routines for both enhancing expression and eliminating the need to screen animals by DNA analysis. A cross of a heterozygous transgenic animal that produces an animal homozygous for the integration site;
Crosses of separate homozygous lines to generate complex heterozygous or homozygous lines; and crosses to place the transgene on a different background appropriate to the experimental model of interest.

The transgenic and “knockout” animals of the present invention can be used to elaborate the biological function of DR4 polypeptides, study conditions and / or disorders associated with aberrant DR4 expression, and such conditions and / or disorders. It has uses, including but not limited to animal model systems, useful for screening for compounds that are effective in ameliorating the disorder.

In a further embodiment of the present invention, cells engineered to express a protein of the present invention or cells that have been engineered not to express a protein of the present invention (eg, knockouts) can be used in vivo. Administer to patients. Such cells can be obtained from patients (ie, animals including humans) or MHC compatible donors, and include fibroblasts, bone marrow cells, blood cells (eg, lymphocytes), adipocytes, muscle cells, endothelial cells, and the like. But not limited to them. This cell,
For example, transduction (using viral vectors and preferably vectors that incorporate the transgene into the cell genome) or transfection procedures (including the use of plasmids, cosmids, YACs, naked DNA, electroporation, liposomes, etc. , But not limited to, for introducing a coding sequence of a polypeptide of the present invention into a cell, or a coding sequence linked to this coding sequence and / or a polypeptide of the present invention and / or endogenous It is engineered in vitro using recombinant DNA technology to destroy regulatory sequences. The coding sequence for a polypeptide of the present invention may be placed under the control of a strong constitutive or inducible promoter or promoter / enhancer to achieve expression and preferably secretion of the polypeptide of the present invention. Engineered cells that express and preferably secrete a polypeptide of the present invention can be introduced systemically into a patient, for example, in the circulation or intraperitoneally. Alternatively, the cells can be incorporated into a matrix and implanted into the body (eg, engineered fibroblasts can be implanted as part of a skin graft); Can be implanted as part of an implant (eg, Anderson et al., US Pat. No. 5,399,349; and Mulligan and Wilson,
See U.S. Patent No. 5,460,959. Each of these is hereby incorporated by reference in its entirety).

If the cells to be administered are non-autologous or non-MHC compatible cells, they may be administered using well-known techniques that would prevent the generation of a host immune response against the transduced cells. For example, the cells can be introduced in a capsular form, which does not allow the introduced cells to be recognized by the host immune system, while allowing the components to exchange with the immediate extracellular environment.

Therapeutic Agents Tumor Necrosis Factor (TNF) Family Ligands Induce Many Cellular Responses, Including Cytotoxicity, Antiviral Activity, Immunomodulatory Activity, and Transcriptional Regulation of Several Genes Are known to belong to specific cytokines (Goeddel
, D. V. Et al., “Tumor Necrosis Factors: Gene.
Structure and Biological Activities "
, Symp. Quant. Biol. 51: 597-609 (1986), Co
ld Spring Harbor; Beutler, B .; And Cerami
, A. , Annu. Rev .. Biochem. 57: 505-518 (1988)
Old, L .; J. , Sci. Am. 258: 59-75 (1988); Fi.
ers, W.S. , FEBS Lett. 285: 199-224 (1991)).
The TNF-family ligand is a TNF-family receptor (DR of the present invention).
4 to induce such various cellular responses.

The DR4 polynucleotides and polypeptides of the present invention can be used to treat and / or prevent any disorder mediated (directly or indirectly) by a missing or insufficient amount of DR4. Can be used to develop DR
The 4 polypeptides 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 treat and / or prevent such disorders. D in this specification
Disclosure of the R4 nucleotide sequence allows detection of the defective DR4 gene and replacement of the defective DR4 gene with a gene encoding normal DR4. The defective gene is
It can be detected in in vitro diagnostic assays and by comparing the DR4 nucleotide sequence disclosed herein to the nucleotide sequence of a DR4 gene from a patient suspected of having a deficiency in this gene.

In another embodiment, the polypeptides of the invention are TRAIs in different cell types.
It can be used as a research tool to study biological effects resulting from inhibiting the L / DR4 interaction. DR4 polypeptides can also be used in in vitro assays to detect TRAIL or DR4 or their interactions.

In another embodiment, purified DR4 polypeptides or DR antagonists of the invention are used to inhibit TRAIL binding to endogenous cell surface TRAIL receptors. Certain ligands of the TNF family (of which TRAIL is a member) have been reported to bind to more than one distinct cell surface receptor protein. TRAIL is also believed to bind multiple cell surface proteins. By binding TRAIL, the soluble DR4 polypeptide and / or DR4 antagonist of the present invention can
A TRAI separate from DR4, not only to inhibit binding to cell surface DR4
It can also be used to inhibit binding to the L receptor protein. Thus, in another embodiment, the DR4 polypeptide and / or antagonist is used to inhibit the biological activity of TRAIL in an in vitro or in vivo procedure. By inhibiting TRAIL binding to cell surface receptors, DR4 polypeptides and antagonists also inhibit biological effects resulting from TRAIL binding to endogenous receptors. Various forms of the DR4 polypeptide may be utilized, including, for example, the above-described DR4 fragments, derivatives, and variants capable of binding TRAIL. In a preferred embodiment, soluble DR4 is used to inhibit the biological activity of TRAIL (
For example, to inhibit apoptosis mediated by TRAIL that is susceptible to such apoptosis).

In a further embodiment, a DR4 composition of the invention (eg, a DR4 polynucleotide, polypeptide, agonist and / or antagonist) comprises a TR4
Mammals (eg, humans) for treating and / or preventing AIL-mediated disorders
To be administered. Such TRAIL-mediated disorders include conditions caused or worsened (directly or indirectly) by TRAIL.

Cells expressing a DR4 polypeptide and believed to have an effective cellular response to a DR4 ligand include amnion cells, heart, liver cancer, kidney, peripheral blood leukocytes, activated T cells, Th2 cells Tissue, human tonsils, and CD34 depleted buffy coat (umbilical cord blood). By "cell response to TNF family ligand", cells, cell lines,
Genotypic, phenotypic, and / or morphological changes to a tissue, tissue culture, or patient are contemplated. As shown, such cellular responses include normal physiological responses to TNF family ligands as well as diseases associated with increased apoptosis or inhibition of apoptosis. Apoptosis (programmed cell death) is a physiological mechanism involved in the loss of peripheral T lymphocytes of the immune system, and its dysregulation can lead to many different pathogenic processes (Ame
isen, J .; C. , AIDS 8: 1197-1213 (1994); Kra.
mmer, P .; H. Et al., Curr. Opin. Immunol. 6: 279-2
89 (1994)).

Diseases associated with increased inhibition of cell survival or apoptosis include the following: cancer (eg, follicular lymphoma, cancer with p53 mutation, and hormone-dependent tumors (colorectal cancer, heart tumor, pancreas) Cancer, melanoma, retinoblastoma, glioblastoma, lung cancer, intestinal cancer, testicular cancer, gastric cancer, neuroblastoma, myxoma, myoma, lymphoma, endothelioma, osteoblastoma, giant cell tumor Autoimmune disorders (eg, multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, bile) Cirrhosis, Behcet disease, Crohn's disease, polymyositis, systemic lupus erythematosus and immune-associated glomerulonephritis and rheumatoid arthritis); viral infection (
For example, herpes virus, poxvirus and adenovirus); inflammation;
Graft-versus-host disease; acute and chronic graft rejection. In a preferred embodiment, the DR4 polynucleotides, polypeptides, and / or antagonists of the invention are used in the growth,
Used to inhibit progression and / or metastasis.

Additional diseases or conditions associated with increased cell survival include, but are not limited to, malignancies and the progression and / or metastasis of malignancies and related disorders such as: leukemia (acute leukemia) (Including, for example, acute lymphocytic leukemia, acute myeloid leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia)) and chronic leukemia (eg, chronic myeloid ( Granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphomas (eg,
Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors (sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenesis) Sarcoma, chordoma, hemangiosarcoma, endotheliosarcoma, lymphatic sarcoma, lymphatic endothelioma, periosteoma, mesothelioma, Ewing tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,
Breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland cancer, sebaceous gland cancer,
Papillary carcinoma, papillary adenocarcinoma, cystic adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonic carcinoma, Wilms tumor, cervical carcinoma, testicular tumor , Lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, auditory nerve Tumors, oligodendrogliomas, meningiomas, melanomas, neuroblastomas, and retinoblastomas).

Diseases associated with increased apoptosis include: AIDS; neurodegenerative disorders (eg, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis,
Autoimmune disorders (eg, multiple sclerosis, Sjögren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease) Polymyositis, systemic lupus erythematosus and immune-associated glomerulonephritis and rheumatoid arthritis), myelodysplastic syndrome (eg, aplastic anemia), graft-versus-host disease,
Ischemic injuries (such as those caused by myocardial infarction, stroke and reperfusion injury), liver injuries (eg, hepatitis-related liver injury, cirrhosis, ischemia / reperfusion injury, cholestasis)
cholestosis (bile duct injury) and liver cancer); toxic-induced liver disease (
Alcohol-like), septic shock, cachexia and anorexia. In a preferred embodiment, a DR4 polynucleotide, polypeptide,
And / or agonists are used to treat the above diseases and disorders.

Many pathologies associated with HIV are mediated by apoptosis, including HIV-induced nephropathy and HIV encephalitis. Thus, in a preferred embodiment, a DR4 polynucleotide, polypeptide and / or D4 polynucleotide of the invention
R4 agonists are used to treat AIDS and pathologies associated with AIDS. Another embodiment of the present invention relates to the use of DR4 for reducing TRAIL-mediated death of T cells in HIV infected patients.

The immunodeficient condition that defines AIDS is secondary to a decrease in the number and function of CD4 ⁺ T lymphocytes. Recent reports indicate that daily CD4 ⁺ T cell loss
. Estimate to be between 5 × 10 ⁷ and 2 × 10 ⁹ cells (Wei X et al., Nat
ure 373: 117-122 (1995)). One cause of CD4 ⁺ T cell depletion in the setting of HIV infection is thought to be HIV-induced apoptosis (eg, Meyaard et al., Science 257: 217-2).
19, (1992); Groux et al., J Exp. Med. 175: 331,
(1992); and Oyaizu et al., Cell Activation an.
d Apoptosis in HIV Infection Andrieu
And Lu, Ed., Plenum Press, New York, 1995, 1
01-114). Indeed, HIV-induced apoptotic cell death is
Not only demonstrated in vitro, but more importantly, in infected individuals (Ameisen, JC, AIDS 8: 1197-1213).
(1994); Finkel, T .; H. And Banda, N .; K. , Curr
. Opin. Immunol. 6: 605-615 (1995); Muro-C.
acho, C .; A. J. et al. Immunol. 154: 5555-5566 (1
995)). In addition, apoptosis and depletion of CD4 ⁺ T lymphocytes are associated with AID
Closely correlates with different animal models of S. (Brunner, T. et al., Nature
373: 441-444 (1995); Gougeon, M .; L. AID
S Res. Hum. Retroviruses 9: 553-563 (199
3)), and apoptosis is not observed in these animal models where viral replication does not result in AIDS (Gougeon, ML et al., AIDS R).
es. Hum. Retroviruses 9: 553-563 (1993))
. Further data shows that uninfected T lymphocytes from HIV infected individuals, but primed or activated T lymphocytes undergo apoptosis after encountering a TNF family ligand (FasL). Show. Using a monocyte cell line that dies after HIV infection, infection of U937 cells with HIV was
L, and FasL has been demonstrated to mediate HIV-induced apoptosis (Badley, AD, et al., J. Virol. 70:19).
9-206 (1996)). In addition, TNF family ligands are detectable in uninfected macrophages, and their expression is up-regulated, after which HIV infection results in selective killing of uninfected CD4 ⁺ T lymphocytes (Badley J. Virol. 70: 199-206.
(1996)). In addition, further studies indicate that Fas-mediated apoptosis is
Implies T cell loss in V individuals (Katsikis et al., J. Exp. M.
ed. 181: 2029-2036, 1995). T cell apoptosis also appears to occur through multiple mechanisms. In addition, at least some of the death of T cells as seen in HIV can be mediated by TRAIL.

Thus, the present invention provides a method for treating and / or preventing an HIV ⁺ individual, comprising administering a DR4, DR4 peptide, polynucleotide, antagonist and / or agonist of the invention. And CD
4 ⁺ T reduce selective killing of lymphocytes. The modes of administration and dosing are discussed in detail below. Without wishing to be bound by theory, activated human T cells undergo programmed cell death (apoptosis) (activation-induced cell death (AICD) upon induction through the CD3 / T cell receptor complex. )). C isolated from asymptomatic individuals infected with HIV
AICD of D4 ⁺ T cells has been reported (Groux et al., Supra). Therefore,
AICD may play a role in CD4 ⁺ T cell depletion and in the development of AIDS in HIV-infected individuals. Thus, the present invention relates to TRA in HIV patients.
Provided is a method of inhibiting IL-mediated T cell death, the method comprising administering to the patient a DR4 polynucleotide (preferably a soluble DR4 polypeptide) and / or a DR4 antagonist of the invention. The modes of administration and dosing are discussed in detail below. In one embodiment, when treatment with DR4 is initiated, the patient is asymptomatic. If desired, prior to treatment, peripheral blood T cells may be
V can be extracted from patients and tested for susceptibility to TRAIR-mediated cell death by means known in the art. In one embodiment, the blood or plasma of a patient is contacted ex vivo with a DR4 polypeptide of the present invention. The DR4 polypeptide can be bound to a suitable chromatography matrix by means known in the art. The patient's blood or plasma flows through a chromatography column containing DR4 bound to the matrix and is then returned to the patient. The immobilized DR4 polypeptide binds to TRAIL and thus removes TRAIL protein from the patient's blood.

In a further embodiment, a DR4 polypeptide and / or antagonist of the invention is administered in combination with other inhibitors of T cell apoptosis. For example, as described above, Fas-mediated apoptosis has also been associated with T cell loss in HIV individuals (Katsikis et al., J. Exp. Med. 1).
81: 2029-2036 (1995)). Thus, patients susceptible to both Fas ligand mediated and DR4 mediated T cell death will have TRAIL / TRAIL
The agent can be treated with both an agent that blocks the receptor interaction and an agent that blocks the Fas ligand / Fas interaction. Suitable agents for blocking the binding of Fasi ligand to Fas include soluble Fas polypeptides; multimeric forms of soluble Fas polypeptides (eg, sFas / Fc dimers)
Binds Fas without the transmission of a biological signal that causes apoptosis;
Anti-Fas antibodies; anti-Fas ligand antibodies that block binding of Fas ligand to Fas; and muteins of Fas ligand that bind Fas but do not transmit biological signals that cause apoptosis. Not done. Preferably, the antibodies used according to this method are monoclonal antibodies. Examples of suitable agents for blocking the Fas ligand / Fas interaction (including blocking anti-Fas monoclonal antibodies) are described in International Patent Application No. WO95 /
10540, which is hereby incorporated by reference.

A suitable agent that blocks TRAIL binding to the TRAIL receptor is administered with a DR4 polynucleotide and / or polypeptide of the invention.
Such agents include, but are not limited to: soluble TRAIL receptor polypeptides (eg, solubilized form of OPG, TR5 (International Patent Application No. WO98 / 30693); DR5 (International Patent Application No. WO98). /
41629); and TR10 (International Patent Application No. WO 98/54202));
Multimeric forms of soluble TRAIL receptor polypeptides; as well as TRAIL receptor antibodies, TRAIL to one or more TRAIL receptors, which bind to the TRAIL receptor without transmitting a biological signal that causes apoptosis
Anti-TRAIL antibodies, and muteins of TRAIL that bind to TRAIL receptors but do not transmit biological signals that cause apoptosis. Preferably, the antibodies used in this method are monoclonal antibodies.

In allograft rejection, the immune system of the recipient animal has not been previously sensitized to respond, since in most cases the immune system is sensitized only by surrounding antigens. Tissues from other members of the same species are not presented in the same way that, for example, viruses and bacteria are presented. In the case of allograft rejection, immunosuppressive regimens are designed to prevent the immune system from reaching the effector phase. However, the immune profile of xenograft rejection may be more similar to disease recurrence than to allograft rejection. In the case of disease recurrence, the immune system is already activated, as indicated by the destruction of natural islet cells. Thus, in disease recurrence, the immune system is already in the effector phase. Activated and differentiated lymphocytes into effector cells express the DR4 polypeptide, so that the DR4 polynucleotides, polypeptides and / or agonists of the present invention can elicit an immune response against both allografts and xenografts. Can be suppressed, thereby being sensitive to compounds that enhance apoptosis. Accordingly, the present invention further provides a method for producing an immunologically tolerant tissue. The DR4 antagonists of the present invention can be further used for treating and / or preventing inflammatory bowel disease.

A DR4 antagonist or agonist of the present invention can be used for the treatment and / or prevention of inflammatory diseases, such as rheumatoid arthritis, osteoarthritis, psoriasis, sepsis, and inflammatory bowel disease.

In addition, due to the expression of DR4 in lymphoblasts, soluble DR4 agonist or antagonist monoclonal antibodies can be used to treat and / or prevent this form of cancer. In addition, soluble DR4 or neutralizing monoclonal antibodies
It can 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.

In one embodiment, the DR4 polypeptides, polynucleotides and / or antagonists of the invention can be used to treat or prevent a cardiovascular disorder, including peripheral artery disease such as limb ischemia.

[0299] Cardiovascular disorders include arterio-arterial fistulas.
stula), arteriovenous fistula, cerebral arteriovenous congenital anomaly, congenital heart defect (congeni)
tal heart defects), cardiovascular abnormalities such as pulmonary valve atresia, and scimitar syndrome. Congenital heart defects include aortic constriction, triatrial heart, coronary vessel anomalies.
, Cross heart, right thoracic heart, patent ductus arteri
osus), Ebstein malformation, Eisenmenger complex, left ventricular dysgenesis syndrome, left thoracic heart, tetralogy of Fallot, transposition of great arteries, right ventricle of both great vessels, tricuspid regurgitation, remnant ductus arteriosus , And heart septal defect
ts) (eg, aortopulmonary septum defect)
eptal defect, endocardial bed deficiency, Ryutanbache syndrome, trilogy of Fallot, ventricular heart septum (ventricular heart sep)
tal defects))).

Cardiovascular disorders also include atherosclerosis, high cardiac output (high)
cardiac output, low cardio output
output), cardiac tamponade, endocarditis (including bacterial), cardiac aneurysm, cardiac arrest, congestive heart failure, congestive cardiomyopathy, paroxysmal dyspnea, cardiac edema, cardiac hypertrophy, congestive cardiomyopathy, left Ventricular hypertrophy, right ventricular hypertrophy, post-infarct cardiac rupture (post-infar)
ction heart rupture, ventricular septum rupture, heart valve disease, myocardial disease, myocardial ischemia, endocardial effusion, pericarditis (including infarct and tuberculosis), pneumocardiosis, postpericardiotomy syndrome , Pulmonary fibrosis
, Right heart disease, rheumatic heart disease, ventricular dysfunction, hyperemia, cardiovascular pregnancy complications (c
cardiovascular pregnancy complication
s), scimitar syndrome, cardiovascular syphilis, and cardiovascular tuberculosis (cardiovasc)
heart disease, such as, for example, the occurrence of a large tuberculosis.

Arrhythmias include sinus arrhythmias, atrial fibrillation, atrial flutter, bradycardia, extrasystole, Adams-Stokes syndrome, leg block, sinoatrial block, long QT syndrome (long
QT syndrome, accessory contractions, Lone-Gangong-Levine syndrome, Mahaim-type pre-excita syndrome
sion syndrome), Wolf-Parkinson-White syndrome, sinus dysfunction syndrome, tachycardia, and ventricular fibrillation. Tachycardia includes paroxysmal tachycardia,
Supraventricular tachycardia, ventricular specific rhythm promotion, atrioventricular nodal reentrant tachycardia (atrioventri
Circular nodal reentry tachycardia), ectopic atrial tachycardia, ectopic junctional tachycardia, sinoatrial nodal reentry tachycardia (sinoatrial no)
dal reentry tachycardia), sinus tachycardia, torsade
De pointes, and ventricular tachycardia.

[0302] Heart valve diseases include aortic valve dysfunction, aortic stenosis, heart murmur (hea).
murmurs), aortic valve prolapse, mitral valve prolapse, tricuspid valve prolapse, mitral dysfunction, mitral stenosis, pulmonary valve regurgitation, pulmonary valve dysfunction, pulmonary valve stenosis, tricuspid Examples include atresia, tricuspid dysfunction, and tricuspid stenosis.

[0303] Cardiomyopathy includes alcoholic cardiomyopathy, congestive cardiomyopathy, hypertrophic cardiomyopathy, hypovalvular aortic stenosis, hypovalvular pulmonary stenosis, restricted cardiomyopathy, Chagas cardiomyopathy, intracardiac Membrane fibroelastosis, endocardial myocardial fibrosis, Keynes syndrome, myocardial reperfusion injury, and myocarditis.

Myocardial ischemia includes angina, coronary aneurysms, coronary atherosclerosis, coronary thrombosis, coronary artery vasospasm, myocardial infarction, and myocardial stunnin
g) coronary artery disease.

Cardiovascular diseases also include aneurysms, angiogenesis, hemangiomatosis, bacterial hemangioma symptoms, Hippel-Lindau disease (Hippel-Lindau disease).
), Klipel-Tornonnay-Weber Syndrome, Sturge-Weber Syndrome,
Vasomotor edema, aortic disease, Takayasu arteritis, aortitis, Lurish syndrome,
Arterial occlusive disease, arteritis, arteritis, arteritis nodosa, cerebrovascular disorder, diabetic vascular disorder, diabetic retinopathy, embolism, thrombosis, acrolysis, hemorrhoid, liver Venous obstruction disorder, hypertension, hypotension, ischemia, peripheral vascular disorder, phlebitis, pulmonary vein obstruction disease, Raynaud's disease, CREST syndrome, retinal vein occlusion, scimitar syndrome, superior vena cava syndrome, telangiectasia, telangiectasia Ataxia (ataci)
a telangiectasia), hereditary hemorrhagic telangiectasia, varicocele, varicose veins, varicose ulcers, vasculitis, and vascular diseases such as venous insufficiency.

The aneurysms include dissecting aneurysms, pseudo-aneurysms, infected aneurysms, ruptured aneurysms, aortic aneurysms, cerebral aneurysms, coronary aneurysms, cardiac aneurysms, and iliac aneurysms Is mentioned.

Arterial occlusive diseases include arteriosclerosis, intermittent claudication, carotid stenosis, fibromuscular dysplasia, mesenteric vascular occlusion, moyamoya disease, renal artery occlusion, retinal artery occlusion, and obstructive thrombus Vasculitis.

Cerebrovascular disorders include carotid artery disease, cerebral amyloid angiopathy, cerebral aneurysm, cerebral anoxia, cerebral atherosclerosis, cerebral arteriovenous malformations, cerebral artery disease, cerebral obstruction and thrombosis, Carotid thrombosis, sinus thrombosis, Wallenberg syndrome, cerebral hemorrhage, epidural hematoma, subdural hematoma, subarachnoid hemorrhage (subaraxhnoid hemorr)
hage), cerebral infarction, cerebral ischemia (including transient), subclavian artery stealing syndrome, periventricular leukomalacia,
Vascular headache, cluster headache, migraine, and vertebrobasi
lar) dysfunction.

Embolisms include air embolism, amniotic fluid embolism, cholesterol embolism, toe cyanosis syndrome, fat embolism, pulmonary embolism, and thromboembolism. Thrombosis includes coronary thrombosis, hepatic vein thrombosis, retinal vein occlusion, carotid thrombosis,
Sinus thrombosis, Wallenberg syndrome, and thrombophlebitis.

[0310] Ischemia includes cerebral ischemia, ischemic colitis, compartment syndrome, precompartment syndrome, myocardial ischemia, reperfusion injury, and peripheral limb ischemia. As vasculitis, aoritis, arteritis, Behcet syndrome, Churg-Strauss syndrome, mucocutaneous lymph node syndrome, obstructive thrombotic vasculitis, irritable vasculitis, Schoenlein-Henoch purpura. ), Allergic cutaneous vasculitis and Wegener's granulomatosis.

[0311] In one embodiment, the DR4 polypeptides, polynucleotides and / or antagonists of the invention are used for the treatment and / or prevention of thrombotic microangiopathy. One such disorder is thrombotic thrombocytopenic purpura (
TTP) (Kwaan, HC, Semin. Hematol. 24:
71 (1987); Thompson et al., Blood 80: 1890 (199
2)). Increasing TTP-related mortality was reported in US S. Centers for Dise
case Control (Torok et al., Am. JH).
ematol. 50:84 (1995)). Plasma from patients affected by TTP (including HIV + and HIV- patients) induces apoptosis of human endothelial cells of microvascular origin in the skin but not human endothelial cells of macrovascular origin (Laurence Et al., Blood 87: 3245 (1996)). Thus, the plasma of a TTP patient is thought to contain one or more factors that directly or indirectly induce apoptosis. As described in International Patent Application No. WO 97/01633, which is incorporated herein by reference, TRAIL comprises TTP
It is present in the serum of patients and appears to play a role in inducing apoptosis of microvascular endothelial cells. 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 embodiment,
The present invention relates to the use of DR4 for treating or preventing a condition often referred to as "adult HUS" (although children can be affected as well). The disorder known as HUS associated with childhood / diarrhea differs in etiology from adult HUS. In another embodiment, conditions characterized by small vessel coagulation can be treated using DR4 of the present invention. 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 coagulation of small blood vessels. Disruption of microvessels in the heart has been reported in several sclerosis patients. As a further example, the treatment of systemic lupus erythematosus (SLE) is contemplated. In one embodiment, the patient's blood or plasma is contacted ex vivo with a DR4 polynucleotide and / or polypeptide of the invention. DR4 polynucleotides and / or polypeptides of the invention can be bound to a suitable chromatography matrix using techniques known in the art. According to this embodiment, the patient's blood or plasma flows through a chromatography column comprising a DR4 polynucleotide and / or polypeptide of the invention bound to a matrix before being returned to the patient. The immobilized DR4 binds TRAIL, thus removing TRAIL protein from the patient's blood. Alternatively, a DR4 polynucleotide and / or polypeptide of the invention can be administered in vivo to a patient affected by thrombotic microangiopathy. In one embodiment, a soluble form of a DR4 polypeptide of the invention is administered to a patient. Accordingly, the present invention provides a method for treating and preventing thrombotic microangiopathy, comprising using an effective amount of the DR4 of the present invention. The DR4 polypeptide is used in an in vivo or ex vivo procedure,
It can inhibit TRAIL-mediated damage to microvascular endothelial cells (eg, apoptotic).

[0312] The DR4 polynucleotides and / or polypeptides of the invention can be used with other agents useful in treating and preventing certain disorders. For example, La
In an in vitro study reported by urence et al. (Blood 87: 3245, 1996), some reduction in TTP plasma-mediated apoptosis of microvascular endothelial cells depleted anti-Fas blocking antibodies, aurin tricarboxylic acid, or cryoprecipitates. Achieved by using normal plasma. Therefore,
Patients will receive additional agents (inhibiting Fas ligand-mediated apoptosis of endothelial cells)
For example, it can be treated in combination with the agents described above. In one embodiment, a DR4 polynucleotide and / or DR4 polypeptide of the invention and an anti-FAS blocking antibody are administered to a patient suffering from a disorder characterized by thrombotic microangiopathy such as TTP or HUS. Fas antigen (C
Examples of blocking monoclonal antibodies directed to D95) are described in International Application Publication No. WO95 / 10540, which is hereby incorporated by reference.

The naturally occurring equilibrium between endogenous stimulators of angiogenesis and inhibitors is such that inhibitory effects predominate (Rasti
Nejad et al., Cell 56: 345-355 (1989)). In the rare cases where neovascularization occurs under normal physiological conditions (eg, wound healing, organ regeneration, embryonic development and female reproductive processes), angiogenesis is tightly regulated and spatially and temporally Stipulated. Under conditions of pathological angiogenesis (eg, those that characterize solid tumor growth), these regulatory controls fail. Unregulated angiogenesis becomes pathological and sustains the progression of many neoplastic and non-neoplastic diseases.
A number of serious diseases are associated with abnormal neovascularization (solid tumor growth and metastasis, arthritis,
(Including several types of ocular disorders, as well as psoriasis). For example,
Moses et al., Biotech. 9: 630-634 (1991); Folkm.
an et al. Engl. J. Med. , 333: 1757-1763 (1995).
); Auerbach et al. Microvasc. Res. 29: 401-4
11 (1985); Folkman, Advances in Cancer.
Academic, edited by Research, Klein and Weinhouse.
Press, New York, pp. 175-203 (1985); Patz,
Am. J. Opthalmol. 94: 715-743 (1982); and F
See review by Olkman et al., Science 221: 719-729 (1983). In many pathological conditions, the process of angiogenesis contributes to the disease state. For example, significant data has been accumulated that indicates that solid tumor growth is dependent on angiogenesis. Folkman and Klagsbrunn, Sc
issue 235: 442-447 (1987).

The present invention provides a DR4 polynucleotide and / or polypeptide (D
(Including R4 agonists and / or antagonists) for the treatment and / or prevention of diseases or disorders associated with neovascularization. Malignant and metastatic conditions that can be treated and / or prevented with the polynucleotides and polypeptides of the present invention include those described herein and otherwise known in the art, such as malignancies, solid tumors, and the like. (For a review of such disorders, see Fishman et al., Medici.
ne (2nd edition) B. Lippincott Co. , Philadelph
ia (1985)).

In addition, ocular disorders associated with neovascularization that can be treated and / or prevented with DR4 polynucleotides and polypeptides of the invention (including DR4 agonists and antagonists) include neovascular glaucoma, diabetic retinopathy , Retinoblastoma, post lens fibroplasia, uveitis, precocious plaques
y macular) degenerative retinopathy, corneal transplant neovascularization, and other ocular inflammatory diseases, ocular tumors and diseases associated with choroidal or iris neovascularization. See, for example, Waltman et al., Am. J. Ophth
al. 85: 704-710 (1978) and Gartner et al., Surv.
Ophthal. 22: 291- 312 (1978).

In addition, disorders that can be treated and / or prevented with DR4 polynucleotides and polypeptides of the invention (including DR4 agonists and antagonists) include hemangiomas, arthritis, psoriasis, hemangiofibromas, atherosclerosis Sex group,
Delayed wound healing, granulation, hemophilic joints, hyperplastic scars, pseudoarticular damage, Osler
-Weber syndrome, purulent granulomas, scleroderma, trachoma and vascular adhesions, but are not limited to these.

The polynucleotides and / or polypeptides of the invention and / or their agonists and / or antagonists may be used in a wide range of diseases and / or
Or it is useful in diagnosis, prognosis, treatment and / or prevention of a condition. Such diseases and conditions include cancers (eg, immune cell-related cancers, breast cancer, prostate cancer, ovarian cancer, follicular lymphoma, cancers associated with mutations or alterations in p53, brain tumors, bladder cancers,
Cervical cancer, colon cancer, colorectal cancer, non-small cell lung cancer, small cell lung cancer, gastric cancer)
, Lymphoproliferative disorders (eg, lymphadenopathy), microbial (eg, viruses, bacteria, etc.) infections (eg, HIV-infection, HIV-2 infection, herpes virus infection (
HSV-1, HSV-2, CMV, VZV, HHV-6, HHV-7, EBV), adenovirus infection, poxvirus infection,
Human papillomavirus infection, hepatitis infection (eg, HAV, HBV, HCV, etc.), Helicobacter pylori infection, invasive staphylococcal infection, etc., parasite infection, nephritis, bone disease (eg, osteoporosis), atherosclerosis Disease, pain, cardiovascular disorders (eg, neovascularization, hypovascularization or decreased circulation (eg, ischemic disease (eg, myocardial infarction, stroke, etc.)), AIDS,
Allergies, inflammation, neurodegenerative diseases (eg, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, pigmentary retinopathy, cerebellar degeneration, etc.), transplant rejection (acute and 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), autoimmune diseases (eg, multiple sclerosis) Disease, rheumatoid arthritis, systemic lupus erythematosus, immune complex glomerulonephritis, autoimmune diabetes, autoimmune thrombocytopenic purpura, Graves' disease, Hashimoto's thyroiditis, etc., cardiomyopathy (eg, dilated cardiomyopathy) ), Diabetes, diabetic complications (eg, diabetic nephropathy, diabetic neuropathy, diabetic retinopathy), influenza, asthma,
These include, but are not limited to, psoriasis, glomerulonephritis, septic shock, and ulcerative colitis.

The polynucleotides and / or polypeptides of the invention and / or agonists and / or antagonists thereof are useful in promoting angiogenesis, wound healing (eg, wounds, burns, and fractures).

The polynucleotides and / or polypeptides of the present invention and / or agonists and / or antagonists thereof are useful as adjuvants for enhancing immune responsiveness to specific antigens and antiviral immune responses.

More generally, the polynucleotides and / or polypeptides of the invention and / or agonists and / or antagonists thereof are useful in modulating (ie, enhancing or reducing) an immune response. For example, the polynucleotides and / or polypeptides of the invention may be useful in preparing or recovering from surgery, trauma, radiation therapy, chemotherapy, and transplantation, or may be useful for preparing immune responses and the immune response in elderly and immunocompromised individuals. // can be used to boost recovery. Alternatively, the polynucleotides and / or polypeptides of the invention and / or agonists and / or antagonists thereof are useful, for example, as immunosuppressants in the treatment and / or prevention of autoimmune disorders. In certain embodiments, the polynucleotides and / or polypeptides of the invention have a chronic inflammatory, allergic or autoimmune condition (eg, those described herein or others known in the art). Used to treat and / or prevent.

In one aspect, the invention relates to a method for enhancing TNF-family ligand-induced apoptosis, the method comprising increasing DR4-mediated signaling to a cell expressing a DR4 polypeptide. And administering an effective amount of the resulting DR4 ligand, analog, or agonist. Preferably,
DR4-mediated signaling reduces apoptosis, or decreases cytokines,
And expression of adhesion molecules is increased to treat and / or prevent the diseases indicated. Agonists can include soluble forms of DR4, and monoclonal antibodies directed against the DR4 polypeptide.

In a further aspect, the present invention inhibits TNF-family ligand-induced apoptosis, comprising administering to a cell expressing a DR4 polypeptide an effective amount of an antagonist capable of reducing DR4-mediated signaling. For how to. Preferably, DR4-mediated signaling is reduced for the treatment and / or prevention of diseases that exhibit apoptosis or increased NF-KB expression. Antagonists can include soluble forms of DR4 (eg, a polypeptide comprising all or part of the DR4 extracellular domain), and monoclonal antibodies directed against the DR4 polypeptide.

An “agonist” may enhance or enable apoptosis (eg,
Stimulation of DR4 activity), naturally occurring and synthetic compounds are contemplated. "
By "antagonist" is intended naturally occurring and synthetic compounds that can inhibit apoptosis (eg, inhibit DR4 activity). Whether any candidate "agonist" or "antagonist" of the present invention can enhance or inhibit apoptosis is known in the art, including the assays described in more detail below, in TNF-family ligand / receptor cells. It can be determined using a response assay.

[0324] One such screening procedure involves the use of transfected melanophore cells to express the receptor of the invention. Such screening techniques are described in PCT WO 92/0 published February 6, 1992.
1810. Such assays inhibit (or enhance) activation of a receptor polypeptide of the invention, for example, by contacting a melanophore cell encoding the receptor with both a TNF family ligand and a candidate antagonist (or agonist). It can be used for screening for compounds. Inhibition or enhancement of the signal produced by the ligand indicates that the compound is an antagonist or agonist of the ligand / receptor signaling pathway.

[0325] Other screening techniques are described, for example, in Science 246: 181-29.
Use of cells expressing a receptor (eg, transfected CHO cells) in a system for measuring extracellular pH changes caused by receptor activation, as described in US Pat. Is included. For example, a compound can be contacted with a cell that expresses a receptor polypeptide of the invention, and a second messenger response (eg, signal transduction or pH change) is measured and the potential compound activates the receptor Or to inhibit.

Another such screening technique involves introducing RNA encoding the receptor into Xenopus oocytes to transiently express the receptor. The receptor oocyte is then contacted with the receptor ligand and the compound to be screened, and then, in the case of screening for a compound that would inhibit receptor activation, inhibition or activation of the calcium signal is Can be detected.

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

Another method is to screen for compounds that inhibit the activation of the receptor polypeptide, which is an antagonist of the invention, by determining the inhibition of binding of the labeled ligand to cells having the receptor on the cell surface. Is included. Such methods include transfecting a DNA encoding the receptor into a eukaryotic cell so that the cell expresses the receptor on its surface, and treating the cell with a compound in the presence of a known form of a labeled ligand. Contacting. 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. As determined by the decrease in labeled ligand binding to the receptor,
When the compound binds to the receptor, binding of the labeled ligand to the receptor is inhibited.

Further screening assays for agonists and antagonists of the present invention are described in Tartaglia, L .; A. Goeddel, D .; V.
, J. et al. Biol. Chem. 267 (7): 4304-4307 (1992).

Accordingly, in a further aspect, the candidate agonist or antagonist is TN
Screening methods are provided for determining whether a cell response to an F-family ligand can be enhanced or inhibited. The method includes contacting a cell expressing the DR4 polypeptide with a candidate compound and a TNF-family ligand, assaying a cellular response, and comparing the cellular response to a standard cellular response. Wherein the standard is assayed when contact with the ligand is performed in the absence of the candidate compound, such that an increased cellular response above the standard indicates that the candidate compound has a ligand / receptor signaling pathway. A cell response that is indicative of an agonist and reduced compared to a standard indicates that the candidate compound is an antagonist of the ligand / receptor signaling pathway. "
Qualitatively or quantitatively measuring a cellular response to a candidate compound and / or a TNF-family ligand by assaying the cellular response (eg, determining or assessing an increase or decrease in T cell proliferation or tritiated thymidine labeling). ) Is intended. According to the present invention, a cell expressing a DR4 polypeptide comprises:
It can be contacted with either an endogenously or exogenously administered TNF-family ligand.

As agonists according to the present invention, for example, TNF family ligand peptide fragments, transforming growth factors, neurotransmitters (eg, glutamic acid, dopamine, N-methyl-D-aspartic acid), tumor suppressors (
Naturally occurring and synthetic compounds, such as p53), cytolytic T cells, and antimetabolites. Preferred agonists include, for example,
Chemotherapeutic agents such as cisplatin, doxorubicin, bleomycin, cytosine arabinoside, nitrogen mustard, methotrexate, and vincristine. Others include ethanol and β-amyloid peptide (Science 267: 1457-1458 (1995)).
. More preferred agonists include polyclonal and monoclonal antibodies raised against the DR4 polypeptide, or fragments thereof. Such agonistic antibodies raised against the TNF family receptor are described in Tartaglia, L .; A. Proc. Natl. Aca
d. Sci. USA 88: 9292-9296 (1991): and Tar.
taglia, L .; A. Goeddel, D .; V. J. Biol. C
hem. 267 (7): 4304-4307 (1992). P
See also CT application WO 94/09137.

[0332] Antagonists according to the present 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-Barr virus BHRF1, LMP-1, African swine fever virus LMW5-HL, and herpesvirus yl 34.5), calpain inhibitors, cysteine protease inhibitors, and tumor promoters (eg, PMA, phenobarbital, and hexachlorocyclohexane (eg, , Α-, β-, or γ-hexachlorocyclohexane)) and naturally occurring compounds and synthetic compounds.

[0333] Other potential antagonists include antisense molecules. Antisense technology can be used to control gene expression through antisense DNA or RNA, or through triple helix formation. Antisense technology is described, for example, in Okano, J .; Neurochem. 56: 560 (1991)
); Oligodeoxynucleotides as Antisense
Inhibitors of Gene Expression, CRCP
res, Boca Raton, FL (1988). Triple helix formation is described, for example, by Lee et al., Nucleic Acids Research.
rch 6: 3073 (1979); Cooney et al., Science 24.
1: 456 (1988); and Dervan et al., Science 251: 1.
360 (1991). The method is based on the binding of the polynucleotide to complementary DNA or RNA.

For example, the 5 'coding portion of a polynucleotide encoding a mature polypeptide of the present invention can be used to design antisense RNA oligonucleotides of about 10 to 40 base pairs in length. DNA oligonucleotides are designed to be complementary to regions of the gene involved in transcription, thereby preventing transcription and receptor production. Antisense RNA oligonucleotides hybridize to mRNA in vivo and block translation of the mRNA molecule into receptor polypeptide. The oligonucleotides described above may also include antisense R
NA or DNA can be delivered to cells such that they can be expressed in vivo to inhibit receptor production.

In one embodiment, a DR4 antisense nucleic acid of the invention is produced intracellularly by transcription from an exogenous sequence. For example, the vector or a portion thereof is transcribed,
Produce the antisense nucleic acid (RNA) of the present invention. Such vectors are DR
4 includes a sequence encoding an antisense nucleic acid. Such vectors can remain episomal or become chromosomally integrated as long as they can be transcribed to produce the desired antisense RNA. Such vectors can be constructed by recombinant DNA technology techniques standard in the art. Vectors can be plasmids, viral, or others known in the art, used for replication and expression in vertebrate cells. Expression of the sequence encoding DR4, or a fragment thereof, can be driven by any promoter known in the art to work in vertebrate, preferably human cells. Such a promoter can be inducible or constitutive. Such promoters include the SV40 early promoter region (Bernoist and Chambon, Nature 2).
9: 304-310 (1981)), a promoter contained in the 3 'long terminal repeat of Rous sarcoma virus (Yamamoto et al., Cell 22: 787-
797 (1980)) herpes thymidine promoter (Wagner et al., Pro.
c. Natl. Acad. Sci. U. S. A. 78: 1441-1445 (1
981)), the regulatory sequence of the metallothionein gene (Brinster et al., Nat).
296: 39-42 (1982)), but are not limited thereto.

The antisense nucleic acids of the present invention include a sequence that is complementary to at least a portion of a DR4 gene RNA transcript. However, although preferred, absolute complementarity is not required. A “complementary to at least a portion of an RNA” sequence as referred to herein is:
Means a sequence having sufficient complementarity to hybridize to RNA to form a stable duplex; in the case of a double-stranded DR4 antisense nucleic acid, can a single strand of the double-stranded DNA be tested? , Or triple helix formation can be assayed. The ability to hybridize depends on both the degree of complementarity and the length of the antisense nucleic acid. In general, the larger the hybridizing nucleic acid, the more bases in DR4
It is a mismatch with the RNA, which can include a stable duplex and still form a stable duplex (or possibly a triple helix). One of skill in the art can ascertain the acceptable degree of mismatch by using standard procedures to determine the melting point of the hybridized complex.

[0337] Oligonucleotides that are complementary to the 5 'end of the message (eg, a 5' untranslated sequence up to and including the AUG start codon) should work most efficiently at inhibiting translation. is there. However, sequences complementary to the 3 'untranslated sequence of mRNA have been shown to be effective in inhibiting translation of mRNA as well. Generally, Wagner, R.A. , Nature 372: 333-335 (199).
See 4). Therefore, the 5'- or 3'- of DR4 shown in SEQ ID NO: 1
Oligonucleotides complementary to any of the untranslated noncoding regions of the endogenous DR
4 Can be used in antisense approaches to inhibit translation of mRNA. mRN
Oligonucleotides complementary to the 5 'untranslated region of A should include the complement of the AUG start codon. Antisense oligonucleotides complementary to the mRNA coding region are less efficient translation inhibitors, but can be used in accordance with the present invention. Whether designed to hybridize to the 5 ', 3' or coding region of the DR4 mRNA, the antisense nucleic acid should be at least 6 nucleotides in length, and preferably between 6 and about Oligonucleotides that are 50 nucleotides long. In certain aspects, the oligonucleotide is at least 10 nucleotides, at least 17 nucleotides, at least 25 nucleotides or at least 50 nucleotides.

A polynucleotide of the invention can be DNA or RNA, or a chimeric mixture, or a derivative or modified version thereof, single-stranded or double-stranded. The oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone to improve, for example, molecular stability, hybridization, and the like.
The oligonucleotide may be linked to other additional groups such as peptides (eg, to target host cell receptors in vivo), or factors that enhance transport across cell membranes (eg, Letsinger et al., Proc. Natl. Acad.
. Sci. U. S. A. 86: 6553-6556 (1989); Lemait.
Re et al., Proc. Natl. Acad. Sci. 84: 648-652 (19
87); PCT Publication No. WO 88/09810 (Released on December 15, 1988)
Or the blood-brain barrier (see, eg, PCT Publication No. WO 89/101).
34 (published April 25, 1988)), hybridization-triggered cleaavag.
e. agent) (eg, Kroll et al., BioTechniques, 6: 9).
58-976 (1988)), or intercalating agents (see, for example, Zon, Pharm. Res. 5: 539-549 (1988)). For this purpose, the oligonucleotide is a separate molecule (eg,
Peptide, hybridization induced cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc.).

An antisense oligonucleotide can include at least one modified base moiety, wherein the base moiety is selected from the group including, but not limited to: 5-fluorouracil, 5-bromouracil, 5 -Chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2
-Thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosylcuosin, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D- Mannosylcuosin, 5-methoxycarboxymethyluracil, 5-methoxyuracil, 2-
Methylthio-N6-isopentenyl adenine, uracil-5-oxyacetic acid (v)
, Wybutoxine, pseudouracil, cuosine, 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 2,6-diaminopurine.

An antisense oligonucleotide can 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 oligonucleotide comprises at least one modified phosphate backbone selected from the group including, but not limited to: phosphorothioate, phosphorodithioate, phosphorothioate Amidothioates, phosphoramidates, phosphorodiamidates, methylphosphonates, alkyl phosphotriesters and formacetals or analogs thereof.

In yet another embodiment, the antisense oligonucleotide is an α-anomeric oligonucleotide. α-anomeric oligonucleotides form specific double-stranded hybrids with complementary RNA, whose strands are parallel to each other, as opposed to the normal β-unit (Gautier et al., Nucl. Acids Re.
s. 15: 6625-6641 (1987)). This oligonucleotide has 2
'-0-methylribonucleotide (Inoue et al., Nucl. Acid)
s Res. 15: 6131-6148 (1987)), or chimeric RNA-
DNA analogs (Inoue et al., FEBS Lett. 215: 327-
330 (1987)).

The polynucleotides of the present invention can be prepared by standard methods known in the art (eg, automated D
With NA synthesizer (such devices are available from Biosearch, Applied B
(commercially available from iosystems, etc.). For example, phosphorothioate oligonucleotides can be prepared using the method of Stein et al. (Nuc).
l. Acids Res. 16: 3209 (1988)), and the methylphosphonate oligonucleotide is a controlled-pore glass (cont.
rolled pore glass polymer support (Sarin et al., Pro)
c. Natl. Acad. Sci. U. S. A. 85: 7448-7451 (1
988)).

On the other hand, antisense nucleotides complementary to the DR4 coding region sequence can be used, with antisense nucleotides complementary to the transcribed untranslated region being most preferred.

Potential antagonists according to the present invention also include catalytic RNAs, ie, ribozymes (see, eg, PCT International Publication WO 90/11364, October 4, 1990).
Sunver et al., Science, 247: 1222-1225 (19
90)). On the other hand, a ribozyme that cleaves mRNA at a site-specific recognition sequence can be used to destroy DR4 mRNA, but the use of a hammerhead ribozyme is preferred. Hammerhead ribozymes cleave mRNAs at locations determined by flanking regions that form complementary base pairs with the target mRNA. The only requirement is that the target mRNA has the following two base sequences: 5'-UG-3 '. The construction and production of hammerhead ribozymes is well known in the art, and is disclosed in Haseloff and Gerlach, Nature,
334: 585-591 (1988). There are many potential hammerhead ribozyme cleavage sites within the nucleotide sequence of DR4 (SEQ ID NO: 2). Preferably, the ribozyme has a cleavage recognition site of DR4 mR.
It is engineered to be located near the 5 'end of the NA; that is, to increase efficiency and minimize intracellular accumulation of non-functional mRNA transcripts.

In the case of the antisense approach, the ribozymes of the present invention may be comprised of modified oligonucleotides (eg, improved in stability, targeting, etc.) and delivered to cells expressing DR4 in vivo. Should be. A ribozyme-encoding DNA construct can be introduced into cells in the same manner as described above for the introduction of antisense encoding DNA. The preferred method of delivery is
This involves using a DNA construct that "encodes" the ribozyme under the control of a strong constitutive promoter (such as, for example, the pol III or pl II promoter), so that the transfected cells can express the endogenous DR4 message. It produces enough ribozyme to disrupt and inhibit translation. Because ribozymes are catalytic, unlike antisense molecules, lower intracellular concentrations are required for efficiency.

[0347] Endogenous gene expression can also be reduced by inactivating or "knocking out" the DR4 gene and / or its promoter using targeted homologous recombination (eg, Smithies et al., Nature 317). : 23
0-234 (1985); Thomas and Capecchi, Cell 5.
1: 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 variant, non-functional polynucleotide (or completely unrelated DNA) of the present invention flanked by DNA homologous to an endogenous polynucleotide sequence (either the coding or regulatory region of the gene). Array)
Can be used with or without a selectable and / or negative selectable marker to transfect cells that express 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 this DNA construct via targeted homologous recombination results in inactivation of the targeted gene. Such an approach is particularly suitable for the research and agricultural fields that can be used to generate progeny of animals with targeted genes in which modifications to the embryonic stem cell are inactive (eg, Thomas and Capecchi (1987)
) And Thompson (1989), supra). However, this approach uses recombinant viral vectors, using appropriate viral vectors apparent to those of skill in the art.
If the construct is administered directly to the required site in vivo or is targeted, it may be routinely adapted for use in humans. The contents of each of the documents cited in this paragraph are hereby incorporated by reference in their entirety.

Further antagonists according to the present invention are soluble forms of DR4 (ie, a DR4 fragment containing the ligand binding domain from the extracellular region of the full length receptor).
including. Such soluble forms of the receptor, which can be naturally occurring or synthetic, antagonize DR4-mediated signaling by competing with cell surface DR4 for binding to TNF-family ligands. Thus, the soluble form of the receptor containing the ligand binding domain is a novel cytokine that can inhibit apoptosis induced by TNF-family ligands. They are,
Preferably, it is expressed as a dimer or trimer. Because these are monomeric forms of soluble receptors as antagonists (eg, IgG Fc-TN
(F receptor family fusion). Other such cytokines are known in the art and include Fas B (a soluble form of the mouse Fas receptor), which acts physiologically to limit apoptosis induced by Fas ligand (Hughes, DP). And C
rispe, I .; N. , J. et al. Exp. Med. 182: 1395-1401 (1
995)).

The experiments set forth in Example 5 show that DR4 is a death domain-containing molecule that can trigger apoptosis that is important in regulating the immune system. In addition, the experiments presented below show that DR4-induced apoptosis is blocked by inhibitors of ICE-like proteases (CrmA, and z-VAD-fmk). Thus, inhibitors of ICE-like proteases (FADD-DN and FLICE
-DN / MACHalC360S) can also be used as an antagonist for DR4 activity.

As discussed above, as used herein, the term “antibody” (Ab) or “Ab”
“Monoclonal antibodies” (mAbs) are intact molecules and fragments thereof (eg, Fab and F (ab ′) ₂ fragments) that can bind antigen.
Is included. Fab and F (ab ') ₂ fragments lack the Fc fragment of intact antibody, are cleared more rapidly from the circulation, and may have less binding of the intact antibody to non-specific tissues (Wahl et al.) , J
. Nucl. Med. 24: 316-325 (1983)).

[0351] Antibodies according to the present invention can be prepared by any of a variety of methods using the DR4 immunogens of the present invention. As indicated, such DR4 immunogens include full-length (complete) DR4 polypeptide (with or without a leader sequence) and DR4 polypeptide fragments (eg, ligand binding domain, transmembrane domain, cell Domain and death domain).

Proteins and other compounds that bind to the DR4 domain are also candidate agonists and antagonists of the present invention. Such binding compounds can be "captured" using the yeast two-hybrid system (Fields and Son
g, Nature 340: 245-246 (1989)). A modified version of the yeast two-hybrid system has been described by Roger Brent and his co-workers (Gyuris, J. et al., Cell 75: 791-803).
(1993); S. Et al., Cell 72: 223-232 (
1993)). Preferably, the yeast two-hybrid system is used in accordance with the present invention to capture compounds that bind to either the DR4 ligand binding domain or the DR4 intracellular domain. Such compounds are good candidate agonists and good antagonists of the present invention.

By “TNF-family ligands”, naturally occurring ligands, recombinant ligands, which are capable of binding to members of the TNF receptor family and inducing and / or blocking the ligand / receptor signaling pathway, Synthetic ligands are contemplated. Members of the TNF ligand family include, but are not limited to: DR4 ligand, TRAIL, TNF-α, T
NF-β-α, Lymphotoxin-α (also known as LT-α, TNF-β), LT-β (LT-α2-β found as a heterotrimeric complex), Fas
L, VEGI (International Publication Number WO 96/14328), AIM-I (International Publication Number WO 97/33899), AIM-II (International Publication Number WO 97/34)
911), APRIL (J. Exp. Med. 188 (6): 1185-119).
0), endokine-α (International Publication No. WO 98/07)
880), Neutrokine-α (International Publication Number WO
98/18921) CD40L, CD27L, CD30L, 4-1BBL, O
X40L, and nerve growth factor (NGF).

Gene Therapy In certain embodiments, a nucleic acid comprising a sequence encoding an antibody or a functional derivative thereof treats a disease or disorder associated with aberrant expression and / or activity of a polypeptide of the invention. Administered for gene therapy purposes to inhibit and / or prevent. Gene therapy refers to therapy performed by the administration of an expressed or expressible nucleic acid to a subject. In this embodiment of the invention, the nucleic acids produce their encoded protein, which mediates a therapeutic effect.

[0355] Any of the methods for gene therapy available in the art can be used according to the present invention. An exemplary method is described below.

For a general review of methods for gene therapy, see Goldspiel et al., Cli.
natural Pharmacy 12: 488-505 (1993); Wu and Wu, Biotherapy 3: 87-95 (1991); Tolstos.
hev, Ann. Rev .. Pharmacol. Toxicol. 32: 573
-596 (1993); Mulligan, Science 260: 926-.
932 (1993); and Morgan and Anderson, Ann.
Rev .. Biochem. 62: 191-217 (1993); May, TIB.
TECH 11 (5): 155-215 (1993). Methods generally known in the recombinant DNA art that can be used are Ausubel
Et al. (Ed.), Current Protocols in Molecular
Biology, John Wiley & Sons, NY (1993); and Kriegler, Gene Transfer and Expressio.
n, A Laboratory Manual, Stockton Press
, NY (1990).

In a preferred aspect, the compound comprises a nucleic acid sequence encoding an antibody, wherein the nucleic acid sequence is an expression vector that expresses the antibody, or a fragment or chimeric protein thereof, or a heavy or light chain thereof, in a suitable host. Part of. In particular, such nucleic acid sequences have a promoter operably linked to the antibody coding region, wherein the promoter is inducible or constitutive, and optionally tissue-specific. In another particular embodiment, a nucleic acid molecule is used in which the sequence encoding the antibody and any other desired sequences are flanked by regions that promote homologous recombination at the desired site in the genome, thus rendering the antibody Provides intrachromosomal expression of the encoding nucleic acid (Koller and Smithies, Proc. N
atl. Acad. Sci. USA 86: 8932-8935 (1989);
Zijlstra et al., Nature 342: 435-438 (1989)).
In certain embodiments, the expressed antibody molecule is a single-chain antibody; or the nucleic acid sequence comprises a sequence encoding both the heavy and light chains of the antibody or a fragment thereof.

Delivery of nucleic acid to a patient can be direct (where the patient is directly exposed to the nucleic acid or nucleic acid-bearing vector) or indirect (where the cells are first transduced with the nucleic acid in vitro). Converted and then implanted into the patient). These two approaches are known, respectively, as in vivo gene therapy or as ex vivo gene therapy.

In certain embodiments, a nucleic acid sequence is administered directly in vivo, where the nucleic acid sequence is expressed to produce an encoded product. This involves a number of methods known in the art, such as constructing them as part of a suitable nucleic acid expression vector, and then constructing it (eg, a defective or attenuated retrovirus or other viral vector (US Pat. No. 4,980,286), by intracellular administration, or by direct injection of naked DNA, or by microparticle bombardment (eg, a gene gun; Biol.
istic, Dupont), or by coating with lipids or cell surface receptors, or by transfecting the drug, or by encapsulation in liposomes, microparticles, or microcapsules, or by nucleating them. By conjugation with a peptide known to enter into the cell, and by conjugation with a ligand that undergoes receptor-mediated endocytosis (eg, Wu and Wu, J. Biol. Chem.
. 262: 4429-4432 (1987)), which can be used to target cell types that specifically express the receptor. In another embodiment, a nucleic acid-ligand complex may be formed, wherein the ligand is a fusogen that disrupts endosomes.
ic) a viral peptide, which enables this nucleic acid to avoid lysosomal degradation. In yet another embodiment, nucleic acids can be targeted in vivo for cell-specific uptake and expression by targeting specific receptors (eg, PCT Publication No. WO 92/06180 on April 16, 1992). No. (Wu et al.); WO 92/22635, Dec. 23, 1992 (Wils
on et al.); WO92 / 20316, Nov. 26, 1992 (Finde);
WO93 / 14188, Jul. 22, 1993 (Clarke).
Et al., WO 93/20221 (Young), Oct. 14, 1993). Alternatively, the nucleic acid can be introduced inside the cell and integrated into host cell DNA for expression by homologous recombination (Koller and S.A.
mities, Proc. Natl. Acad. Sci. USA 86:89
32-8935 (1989); Zijlstra et al., Nature 342: 4.
35-438 (1989)).

In certain embodiments, a viral vector is used that contains a nucleic acid sequence encoding an antibody of the invention. For example, retroviral vectors can be used (Mi
ller et al., Meth. Enzymol. 217: 581-599 (1993)
checking). These retroviral vectors lack retroviral sequences that are not required for packaging of the viral genome and integration into host cell DNA. Nucleic acid sequences encoding antibodies used in gene therapy are cloned into one or more vectors, which facilitates delivery of the gene into a patient. Further details on retroviral vectors can be found in Boese
n et al., Biotherapy 6: 291-302 (1994) (this describes the use of retroviral vectors to deliver the mdr1 gene to hematopoietic stem cells to make them more resistant to chemotherapy. ) Can be found.
Other references describing the use of retroviral vectors in gene therapy include:
The following are: Crowes et al. Clin. Invest. 93: 644-6
51 (1994); Kiem et al., Blood 83: 1467-1473 (19).
94); Salmons and Gunzberg, Human Gene Th.
erapy 4: 129-141 (1993); and Grossman and Wilson, Curr. Opin. in Genetics and De
vel. 3: 110-114 (1993).

Adenoviruses are other viral vectors that can be used in gene therapy. Adenoviruses are particularly attractive vehicles for delivering genes to the respiratory epithelium. Adenoviruses naturally infect respiratory epithelia where they cause a mild disease. Other targets for adenovirus-based delivery systems are the liver, central nervous system, endothelial cells, and muscle. Adenovirus has the advantage that it can infect non-dividing cells. Kozarsky and Wilson, Curr
ent Opinion in Genetics and Development
ent 3: 499-503 (1993) provides an overview of adenovirus-based gene therapy. Bout et al., Human Gene Therapy 5: 3.
-10 (1994) showed the use of adenovirus vectors to transfer genes to the airway epithelium of rhesus monkeys. Other examples of the use of adenoviruses in gene therapy include Rosenfeld et al., Science 252: 431-434 (19).
91); Rosenfeld et al., Cell 68: 143-155 (1992).
Mastranelli et al., J .; Clin. Invest. 91: 225-2
34 (1993); PCT Publication No. WO 94/12649; and Wang et al.,
Gene Therapy 2: 775-783 (1995).
In a preferred embodiment, an adenovirus vector is used.

Adeno-associated virus (AAV) has also been proposed for use in gene therapy (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:
289-300 (1993); U.S. Patent No. 5,436,146).

Another approach to gene therapy involves transferring the gene to cells in tissue culture by such methods as electroporation, lipofection, calcium phosphate mediated transfection, or viral infection. Usually, the method of transfer involves the transfer of a selectable marker to the cells. The cells are then placed under selection to isolate cells that take up and express the transferred gene. The cells are then delivered to the patient.

In this embodiment, the nucleic acid is introduced into the cells prior to in vivo administration of the resulting recombinant cells. Such introduction can be performed by any method known in the art, including, but not limited to: transfection, electroporation, microinjection, viruses including nucleic acid sequences. Vector or bacteriophage vector infection, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, and the like. Numerous techniques are known in the art for introducing foreign genes into cells (eg, Loeffler and Behr, Met).
h. Enzymol. 217: 599-618 (1993); Cohen et al., M.
eth. Enzymol. 217: 618-644 (1993); Cline,
Pharmac. Ther. 29: 69-92 (1985)) and can be used in accordance with the present invention provided that the necessary developmental and physiological functions of the recipient cells are not disrupted. The technique should provide for a stable transfer of the nucleic acid into the cell, so that the nucleic acid is expressible by the cell, and preferably is hereditary and expressible by the progeny of the cell.

[0365] 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 hematopoietic progenitor cells) are preferably administered intravenously. The amount of cells contemplated for use depends on the desired effect, patient condition, etc., and can be determined by one skilled in the art.

Cells into which nucleic acids can be introduced for purposes of gene therapy include any desired available cell type, including but not limited to: epithelial cells, endothelial cells,
Keratinocytes, fibroblasts, myocytes, hepatocytes; blood cells such as T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or progenitor cells; In particular, hematopoietic stem cells or hematopoietic progenitor cells (eg, cells obtained from bone marrow, umbilical cord blood, peripheral blood, fetal liver, etc.).

In a preferred embodiment, the cells used for gene therapy are autologous to the patient.

In embodiments where recombinant cells are used in gene therapy, the nucleic acid sequence encoding the antibody is introduced into the cell such that the nucleic acid sequence is expressible by the cell or its progeny, and then the recombinant cell is Administered in vivo for therapeutic effect. In a specific embodiment, stem cells or progenitor cells are used. Any stem and / or progenitor cells that can be isolated in vitro and maintained in vitro can potentially be used according to this embodiment of the invention (
For example, PCT Publication No. WO 94/08598 on April 28, 1994: Ste
Mple and Anderson, Cell 71: 973-985 (1992).
Rheinwald, Meth. Cell Bio. 21A: 229 (19
80); and Pittelkow and Scott, Mayo Cini.
c Proc. 61: 771 (1986)).

In certain embodiments, the nucleic acid to be introduced for gene therapy purposes contains an inducible promoter operably linked to the coding region so that expression of the nucleic acid is controlled by a suitable transcription inducing factor Can be controlled by controlling the presence or absence of.

Modes of Administration The present invention provides methods for treatment, inhibition and prevention by administering to a subject an effective amount of a compound or pharmaceutical composition of the invention, preferably an antibody of the invention. In a preferred aspect, the compound is substantially purified (eg, substantially free of substances that limit its effect or produce undesired side effects). The subject is preferably
Animals including but not limited to animals such as cows, pigs, horses, chickens, cats, dogs, and the like, and are preferably mammals, and most preferably humans.

Formulations and methods of administration that can be used when the compound comprises a nucleic acid or immunoglobulin are described above; further suitable formulations and routes of administration are selected from those described herein below. Can be done.

An agonist or antagonist described herein can be administered to cells in vitro, ex vivo, or in vivo. They express the receptor of the invention. Administration of an "effective amount" of an agonist or antagonist intends the amount of the compound. It sufficiently enhances or inhibits the cellular response of TNF family ligands and includes polypeptides. In particular, administration of an “effective amount” of an agonist or antagonist contemplates an effective amount that enhances or inhibits DR4-mediated apoptosis. Of course, if apoptosis is desired to be enhanced, an agonist according to the invention may be administered with a TNF family ligand. Those skilled in the art will understand the following. An effective amount of an agonist or antagonist can be determined empirically, and can be in pure or pharmaceutically acceptable salt form;
It is understood that it can be used in the form of an ester or in the form of a prodrug.
The agonist or antagonist may be administered in a composition in combination with one or more pharmaceutically acceptable excipients.

The following is understood. When administered to a human patient, the overall daily use of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The dosage level for a particular therapeutically effective amount for any particular patient will depend on factors well known in the medical arts.

As a general suggestion, the total pharmaceutically effective amount of a DR4 polypeptide administered parenterally per dose will range from about 1 μg / kg / day to 10 mg / kg / day of the patient's body weight, As noted above, this is subject to therapeutic discretion. More preferably,
This dose is at least 0.01 mg / kg / day, and most preferably for humans, between about 0.01 mg / kg / day and 1 mg / kg / day for hormones. When given continuously, a DR4 agonist or antagonist is
Typically, at dose rates of about 1 μg / kg / hr to about 50 μg / kg / hr,
It is administered either by 1-4 injections per day or by continuous subcutaneous infusion using, for example, a minipump. An intravenous bag solution may also be used.

Administration is also arranged in a patient-specific manner, providing a determined concentration of agonist or antagonist to the blood (as determined by RIA techniques). Thus, dosing to the patient can be adjusted to achieve regular continuation with blood levels measured by RIA. The order is 50-10
00 ng / ml, preferably 150 to 500 ng / ml.

The pharmaceutical compositions of the invention for parenteral injection may be sterile pharmaceutically acceptable aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, and sterile injectables immediately before use. It may include a sterile powder for reconstitution into a solution or dispersion.

In addition to soluble DR4 polypeptides, DR4 polypeptides containing the enteric and extra-intestinal regions can also be appropriately solubilized by including a surfactant such as CHAPS or NP-40 with a buffer, Can be used.

Prior to use in humans, compounds or pharmaceutical compositions of the invention are tested for the desired therapeutic or prophylactic activity, preferably in vitro and then in vivo. For example, in vitro assays to demonstrate the therapeutic or prophylactic utility of a compound or pharmaceutical composition include the effect of the compound on a cell line or patient tissue sample. The effect of a compound or composition on a cell line and / or tissue sample can be determined using techniques known to those of skill in the art, including, but not limited to, rosette formation assays and cell lysis assays. In vitro assays that can be used to determine whether administration of a particular compound is indicated in accordance with the present invention include in vitro cell culture assays, wherein a patient's tissue sample is grown in culture, The compound is exposed or otherwise administered, and the effect of such compound on the tissue sample is observed.

[0379] The present invention also provides pharmaceutical compositions. Such compositions comprise a therapeutically effective amount of the compound, and a pharmaceutically acceptable carrier. In certain embodiments,
Pharmaceutical compositions are provided that include an agonist or antagonist, and a pharmaceutically acceptable carrier or excipient, which can be administered orally, rectally, parenterally, intracistemally, intrathecally. It can be administered intraperitoneally, topically (by powder, ointment, drop or transdermal patch), buccally or as an oral or nasal spray. Importantly, by administering the agonist and the TNF-family ligand together,
Clinical side effects can be reduced by lower doses of both ligand and agonist. It is understood that depending on the urgency of the particular therapeutic application, the agonist may be "co-administered" before, after, or simultaneously with the TNF-family ligand. “Pharmaceutically acceptable carrier” refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or any type of formulation auxiliary. In a specific embodiment, the term "pharmaceutically acceptable" has been approved by the Federal regulatory authority or the U.S. Government for use in animals, and more particularly in humans, or Means listed in the recognized pharmacopeias. The term "parenteral" as used herein refers to modes of administration, including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers include sterile liquids such as water and oils, including oils of petroleum, animal, plant, or synthetic origin (eg, peanut oil, soybean oil, mineral oil, sesame oil, and the like). Can be Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene , Glycol, water, ethanol and the like. The composition may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral 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 described in W. "Remington's Pharmaceutical Science" by Martin
es ". Such compositions comprise a therapeutically effective amount of the compound, preferably in purified form, together with a suitable amount of carrier to provide a form for proper administration to the patient. The formulation should suit the mode of administration.

In a preferred embodiment, the compositions are formulated according to conventional procedures, as a pharmaceutical composition adapted for intravenous administration to humans. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the components are either separated or mixed together in a single dosage form, for example, in an ampoule or sachet (s) representing a fixed amount of active agent.
Supplied as a lyophilized powder or water-free concentrate in a sealed container such as an acetette. If the composition is to be administered by infusion, the composition can be dispensed into infusion bottles containing sterile pharmaceutical grade water or saline. When the composition is administered by injection, the components can be mixed prior to administration.
An ampoule of sterile water for injection or saline can be provided.

The compounds of the present invention may be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids and the like, and sodium, potassium, ammonium, calcium, ferric hydroxide, Isopropylamine, triethylamine,
Salts formed with cations such as those derived from 2-ethylaminoethanol, histidine, procaine, and the like.

As indicated above, the compositions of the present invention may be administered alone or in combination with other therapeutic agents. Therapeutic agents that can be administered with the compositions of the present invention include, but are not limited to: other members of the TNF family, chemotherapeutic agents, antibiotics, steroidal and non-steroidal anti-inflammatory agents Conventional immunotherapeutic agents, cytokines, chemokines and / or growth factors. The combination may be administered, for example, either simultaneously, as a mixture, separately but simultaneously or concurrently; or sequentially. This includes the indication that the combined agents are administered together as a therapeutic mixture, and that the combined agents are separate but simultaneously (eg, through separate intravenous lines to the same individual).
The procedure to be administered is also included. "Combination" administration further includes separately administering one of the compounds or agents given first, followed by the second.

In one embodiment, the compositions of the invention are administered in combination with a member of the TNF family. A TNF molecule, T, which can be administered with a composition of the present invention.
NF-related or TNF-like molecules include, but are not limited to: soluble forms of TNF-α, lymphotoxin-α, (LT-α, TN
F-β), LT-β (found in the complex heterotrimer LT-α2-β), OPGL, FasL, CD27L, CD30L, CD40L,
-1BBL, DcR3, OX40L, TNF-γ (International publication number WO96 / 14
328), TNF-γ-α, TNF-γ-β (WO 00/0813)
9), TRAIL, AIM-II, (International Publication No. WO 97/34911), A
PRIL (J. Exp. Med. 188 (6): 1185-1190) endokine-α (International Publication No. WO98 / 07880), TR6
(International Publication No. WO 98/30694), OPG, and Neutrokine (n
eutrokine) -α (International Publication No. WO 98/18921), OX40,
And nerve growth factor (NGF), and soluble form of Fas, soluble form of C
D30, soluble form of CD27, soluble form of CD40 and soluble form of 4-
IBB, TR2 (International Publication No. WO96 / 34095), DR3 (International Publication No. WO97 / 33904), TR5 (International Publication No. WO98 / 30693), TR
6 (International publication number WO98 / 30694), TR7 (International publication number WO98 / 4)
1629), TRANS, TR9 (International Publication No. WO 98/56892), TR
10 (International Publication Number WO98 / 54202), 312C2 (International Publication Number WO9
8/06842), and TR12, AIM-I (International Publication No. WO 97/33).
899) and soluble forms of CD154, soluble forms of CD70, and soluble forms of CD153.

In another embodiment, a composition of the invention comprises a CD40 ligand (CD40L)
, A soluble form of CD40L (eg, AVREND ^™ ), a biologically active fragment, variant, or derivative of CD40L, an anti-CD40L antibody (eg, an agonist or antagonist antibody), and / or an anti-CD40 antibody (eg, (Agonist or antagonist antibodies).

In still another embodiment, the composition of the present invention comprises 1, 2, 3, 4, 5
Administered in combination with one or more of the following compositions: tacrolimus (
Fujisawa), thalidomide (eg, Celgene), anti-Tac (Fv
) -PE40 (e.g., Protein Design Labs), inolimomab (Biotest), MAK-195F (Kno)
11), ASM-981 (Novartis), interleukin-1 receptor (e.g. Immunex), interleukin-4 receptor (e.g.
mmunex), ICM3 (ICOS), BMS-188667 (Bristo)
l-Myers Squibb), anti-TNF Ab (eg, Therapeu
tic antibody), CG-1088 (Celgene), anti-B7 monoclonal antibody (e.g., Innotics), MEDI-507 (BioTrans)
plant), ABX-CBL (Abgenix).

According to the present invention, Fas ligand (Fas-L) mediated cell death and TRAIL
Patients who are susceptible to both mediated cell death can be treated with both agents that inhibit TRAIL / TRAIL-R interaction and agents that inhibit Fas-L / Fas interaction. Suitable agents for blocking the binding of Fas-L to Fas include, but are not limited to: soluble Fas polypeptide; oligomeric forms of soluble Fas polypeptide (eg, sFas / Fc dimer) ;
Anti-Fas binds to Fas without transducing the biological signal resulting in cell death
An anti-Fas-L antibody that blocks binding of Fas-L to Fas; and Fas.
Fas-L binds to but does not transduce the biological signal that causes apoptosis
Mutein. Preferably, the antibodies used according to this method are monoclonal antibodies. Suitable agents for blocking the Fas-L / Fas interaction (anti-Fa
(including monoclonal antibodies) are described in WO 95/10540, which is incorporated herein by reference.

In certain embodiments, the compositions of the invention 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 R
ETROVIR ^™ (Zidovudine / AZT), VIDEX ^™ (Zidanocine / ddl)
), HIVID ^™ (Zalcitabine / ddC), ZERIT ^™ (Stavudine (st
avudine) / d4T), EPIVIR ^™ (lamivudi)
ne) / 3TC), and COMBIVIR ^™ (Zidovudine / Lamivudine). Non-nucleoside reverse transcriptase inhibitors that can be administered in combination with the compositions of the present invention include VIRAMUNE ^™ (nevirapine), RESCRIPTOR ^™ (delavidin)
virdine)), and SUSTIVA ^™ (efavirene).
nz)), but are not limited thereto. Protease inhibitors that may be administered in combination with the compositions of the present invention include CRIXIVAN ^™ (indinavir), NORVIR ^™ (ritonavir)
ir)), INVIRASE ^™ (saquinavir), and VIRACEPT ^™ (nelfinavir). In certain embodiments, an antiretroviral agent,
A nucleoside reverse transcriptase inhibitor, a non-nucleoside reverse transcriptase inhibitor, and / or a protease inhibitor is used in any combination with the compositions of the invention to treat AIDS and / or prevent or treat HIV infection. obtain.

In other embodiments, the compositions of the present invention are combined with an anti-opportunistic infectious agent.
Can be administered. Anti-opportunistic infectious agents that can be administered in combination with the compositions of the present invention are
, TRIMETHOPRIM-SULFAMETHOXAZOLE^TM, DAPS
ONE^TM, PENTAMIDINE^TM, ATOVAQONE^TM, ISONIA
ZID^TM, RIFAMPIN^TM, PYRAZINAMIDE^TM, ETHAMBU
TOL^TM, RIFABUTIN^TM, CLARATHROMYCIN^TM, AZIT
HROMYCIN^TM, GANICLOVIR^TM, FOSCARNET^TM, CI
DOFOVIR^TM, FLUCONAZOLE^TM, ITRACONAZOLE^TM,
KETOCONAZOLE^TM, ACYCLOVIR^TM, FAMCICOLVIR ^TM , PYRIMETHAMINE^TM, LEUCOVORIN^TM, NEUPAGE
N^TM(Filgrastim / G-CSF), and LE
UKINE^TM(Sargramostim / GM-CSF
), But is not limited thereto. In certain embodiments, the present invention
Adults preventively treat or prevent opportunistic Pneumocystis pneumonia infection
TRIMETHOPRIM-SULFAMETHOXAZOLE^TM, D
APSONE^TM, PENTAMIDINE^TM, And / or ATOVAQUA
NE^TMUsed in any combination with In another specific embodiment, the present invention
The composition may be used to prophylactically treat or prevent opportunistic avian infections.
NIAZID^TM, RIFAMPIN^TM, PYRAZINAMIDE^TM,and/
Or ETHAMBUTOL^TMUsed in any combination with Another specific
In embodiments, the present invention provides for the treatment or prevention of an opportunistic M. tuberculosis infection.
The composition of the invention comprises RIFABTIN^TM, CLARATHROMYCIN^TM, And
And / or AZITHROMYCIN^TMUsed in any combination with Another
In certain embodiments, the composition of the present invention comprises an opportunistic cytomegaloyl
GANICLOVIR to prevent or prevent prophylactic infections^TM, FO
SCARNET^TMAnd / or CIDOFOVIR^TMAny combination with
Used in. In another specific embodiment, the composition of the present invention is an opportunistic drug.
FLUCONAZOLE for the prophylactic treatment or prevention of mycosis^TM, I
TRACONAZOLE^TMAnd / or KETOCONAZOLE^TMAnd responsibilities
Used in any combination. In another specific embodiment, the composition of the invention comprises
Treatment of opportunistic herpes simplex virus type I and / or type II infection
Or to prevent, ACYCLOVIR^TMAnd / or FAMCICOL
VIR^TMUsed in any combination with In another specific embodiment, the book
The compositions of the invention prevent opportunistic Toxoplasma gondii infection
PYRIMETHAMINE to treat or prevent^TMAnd / or L
EUCOVORIN^TMUsed in any combination with Another specific embodiment
Wherein the composition of the invention prophylactically treats or prevents opportunistic bacterial infections.
LEUCOVORIN^TMAnd / or NEUPOGEN^TMOptional with
Used in combination.

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

In a further embodiment, a composition of the present invention is administered in combination with an antibiotic drug. Antibiotic agents that can be administered with the compositions of the present invention include amoxicillin, aminoglycosides, β-lactams (glycopeptides), β-lactamases, clindamycin, chloramphenicol, cephalosporins, ciprofloxacin, ciprofl. Examples include, but are not limited to, loxacin, erythromycin, fluoroquinolone, macrolide, metronidazole, penicillin, quinolone, rifampin, streptomycin, sulfonamide, tetracycline, trimtoprim, trimtoprim-sulfamethoxazole and vancomycin.

Conventional non-specific immunosuppressants that can be administered in combination with the compositions of the present invention include steroids, cyclosporins, cyclosporin analogs, cyclophosphamide, methylprednisone, prednisone, azathioprine, FK-506, 15 −
Deoxyspergualin (T-deoxyspergualin), and T
Other immunosuppressive agents that act by suppressing the ability to respond to cells include, but are not limited to.

In certain embodiments, the compositions of the present invention are combined with an immunosuppressant preparation.
Is administered. Immunosuppressant preparations that can be administered in combination with the compositions of the present invention include:
Orthoclone^TM(OKT3), SANDIMMUNE^TM/ NEORAL ^TM / SANGDYA^TM(Cyclosporine), PROGRAF^TM(Tacrolimus)
, CELLCEPT^TM(Mycophenolate), azathioprine, glucomicos
Glucomicosteroids, and RAPAMUNE^TM(
Sirolimus). Special
In certain embodiments, the immunosuppressive drug prevents organ rejection or bone marrow transplant rejection
Can be used to

In a further embodiment, a composition of the present invention is administered alone or in combination with one or more intravenous immunoglobulin preparations. Intravenous immunoglobulin preparations that can be administered with the compositions of the present invention include GAMMER ^™ , IVEEG
AM ^™ , SANDOGLOBULIN ^™ , GAMMAGARD S / D ^™ and GAMIMUNE ^™, but are not limited thereto. In certain embodiments, the compositions of the invention are administered in combination with an intravenous immunoglobulin preparation in a transplantation therapy (eg, a bone marrow transplant).

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

In a further embodiment, a composition of the present invention is administered alone or in combination with an anti-inflammatory agent. Anti-inflammatory agents that can be administered with the compositions of the present invention include glucocorticoid and non-steroidal anti-inflammatory agents, aminoarylcarboxylic acid derivatives, arylacetic acid derivatives, arylbutyric acid derivatives, arylcarboxylic acids, arylpropionic acid derivatives, pyrazoles, Pyrazolones, salicylic acid derivatives, thiazinecarboxamides, e-acetamidocaproic acid, S-adenosylmethionine, 3-amino-4-hydroxybutyric acid, amixetrine
e), bendazac, benzidamine, bucolome, difenpyramide, ditazole, emorfazone, guaiazulene, nabumetone, nimesulide, orgothein, oxaseprol, paranyline, perisoxal,
Pifoxime, procazone, proxazole, and tenidap,
It is not limited to these.

In one embodiment, the compositions of the invention are administered in combination with steroid therapy. Steroids that can be administered in combination with the compositions of the present invention include:
Oral (oral) corticosteroids, prednisone, and methylprednisolone (eg, IV methylprednisolone). In certain embodiments, the compositions of the present invention are administered in combination with prednisone. In a more particular embodiment, the compositions of the invention are administered in combination with prednisone and an immunosuppressant. Immunosuppressants that can be administered with the compositions and prednisone of the present invention are the immunosuppressants 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 methylprednisolone. In a more particular embodiment, the compositions of the invention are administered in combination with methylprednisolone and an immunosuppressant. Immunosuppressive agents that can be administered with the compositions of the present invention and methylprednisolone are the immunosuppressive agents described herein, and include azathioprine, cyclophosphamide, and cyclophosphamide IV It is not limited to.

In another embodiment, a composition of the present invention is administered in combination with an antimalarial agent. Antimalarials that can be administered with the compositions of the present invention include, but are not limited to, hydroxychloroquine and / or quinacrine.

In another embodiment, a composition of the invention is administered in combination with an NSAID.

In a non-exclusive embodiment, the composition of the invention comprises 1,2,3,4,5,10
, Or more, in combination with the following drugs: NRD-101 (
Hoechst Marion Roussel), Diclofenac (Dime)
thid), potassium oxaprozin (Monsanto), mecamermin (
mecasermin) (Chiron), T-614 (Toyama), pemetrexed disodium (Eli)
Lilly), atreluton (Abbott), valdecoxib (Monsanto), Eltenac (
Byk Gulden), campath, AGM-1470 (
Takeda), CDP-571 (Celltech Chiroscience)
e), CM-101 (CarboMed), ML-3000 (Merckle)
, CB-2431 (KS Biomedix), CBF-BS2 (KS Biomedix)
medix), IL-1Ra gene therapy (Val)
entis), JTE-522 (Japan Tobacco), paclitaxel (Angiotech), DW-166HC (Dong Wha), darbuferone mesylate (Warne)
r-Lambert), soluble TNF receptor 1 (syngen; Amg)
en), IPR-6001 (Institute for Pharmaceu)
physical Research), trocade (Hoffm)
an-La Roche), EF-5 (Scotia Pharmaceuti)
cals), BIIL-284 (Boehringer Ingelheim)
, BIIF-1149 (Boehringer Ingelheim), Leu
koVax (Inflammatics), MK-663 (Merck), ST
-1482 (Sigma-Tau) and butisocoat propionate (but
ixocort proposal (WarnerLambert).

In still another embodiment, the composition of the present invention comprises 1, 2, 3, 4, 5, 10,
Or more, in combination with the following drugs: methotrexate,
Sulfasalazine, sodium gold thiomalate, auranofin, cyclosporine, penicillamine, azathioprine, antimalarial drugs (eg, as described above),
Cyclophosphamide, chlorambucil, gold, ENBREL ^™ (Etanerce
pt), anti-TNF antibodies, and prednisolone. In a more preferred embodiment, the composition of the invention comprises an antimalarial drug, methotrexate, anti-TNF antibody, ENB
It is administered in combination with REL ^™ and / or sulfasalazine.

In one embodiment, a composition of the present invention is administered in combination with methotrexate. In another embodiment, a composition of the present invention is administered in combination with an anti-TNF antibody. In another embodiment, a composition of the present invention is administered in combination with methotrexate and an anti-TNF antibody. In another embodiment, a composition of the present invention is administered in combination with sulfasalazine. In another specific embodiment, a composition of the invention is administered in combination with methotrexate, an anti-TNF antibody, and sulfasalazine. In another embodiment, a composition of the present invention is administered in combination with ENBREL ^™ . In another embodiment, a composition of the present invention is administered in combination with ENBREL ^™ and methotrexate. In another embodiment, a composition of the present invention is administered in combination with ENBREL ^™ , methotrexate and sulfasalazine. In another embodiment, a composition of the present invention is administered in combination with ENBREL ^™ , methotrexate and sulfasalazine. In other embodiments, one or more antimalarials are combined with one of the combinations listed above. In certain embodiments, the compositions of the invention are administered in combination with an antimalarial drug (eg, hydroxychloroquine), ENBREL ^™ , methotrexate and sulfasalazine. In another specific embodiment, the composition of the present invention comprises an antimalarial drug (
(Eg, hydroxychloroquine), sulfasalazine, anti-TNF antibodies, and methotrexate.

[0402] In another embodiment, a composition of the present invention is administered in combination with a chemotherapeutic agent. Chemotherapeutic agents that can be administered with the compositions of the present invention include antibiotic derivatives (eg, doxorubicin, bleomycin, daunorubicin, and dactinomycin); anti-estrogens (eg, tamoxifen); antimetabolites (eg, fluorouracil, 5 -FU, methotrexate, floxuridine, 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, cis-platin) And vincristine sulfate); hormones (eg, medroxyprogesterone, estramustine sodium phosphate, ethinyl estradiol, estradiol, megestrol acetate, methyltestosterone, diethylstilbestrol diphosphate, chlorotrianisene, and test lactone); Nitrogen mustard derivatives (eg, mephalen, chlorambucil, mechlorethamine (ni Roger emissions mustard) and thiotepa); steroids and combinations (e.g., betamethasone sodium); and others (
Examples include, but are not limited to, dicarbazine, asparaginase, mitotene, vincristine sulfate, vinblastine sulfate, and etoposide.

[0403] In a further embodiment, a composition of the present invention is administered in combination with a cytokine. Cytokines that can be administered with the compositions of the present invention include IL
2, IL3, IL4, IL5, IL6, IL7, IL10, IL12, IL13
, IL15, anti-CD40, CD40L, IFN-γ, and TNF-α.

In a further embodiment, a composition of the present invention is 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 (GDGF) as disclosed in European Patent No. EP-399816; platelet-derived as disclosed in European Patent No. EP-682110. Growth Factor A (PDGF-A); Platelet-Derived Growth Factor B (PDGF-B) as disclosed in European Patent No. EP-282317; International Publication No. WO 92/0.
Placental Growth Factor (PIGF) as disclosed in US Pat.
Orthh Factors, 4: 259-268 (1993); placental growth factor 2 (PIGF-2); vascular endothelial growth factor (VEGF) as disclosed in International Publication No. WO 90/13649; European Patent No.EP-5064
Vascular endothelial growth factor A (VEGF-A) as disclosed in International Publication No. W;
Vascular endothelial growth factor 2 (VEGF-2) as disclosed in O96 / 39515
Vascular endothelial growth factor B as disclosed in International Publication No. WO 96/26736
-186 (VEGF-B186); vascular endothelial growth factor D (VEGF-D) as disclosed in International Publication No. WO 98/02543; International Publication No. WO 98/0.
Vascular endothelial growth factor D (VEGF-D) as disclosed in US Pat. No. 7832; and vascular endothelial growth factor E as disclosed in German Patent No. DE19639601 (
VEGF-E). The above references are incorporated herein by reference.

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

In a further embodiment, the compositions of the present invention are administered in combination with other therapeutic or prophylactic regimes (eg, radiation therapy).

In one embodiment, a composition of the present invention is administered in combination with one or more chemokines. In certain embodiments, the composition of the present invention comprises gamma interferon-inducible protein-10 (γIP-10), interleukin 8 (IL-8),
Platelet factor-4 (PF4), neutrophil activating protein (NAP-2), GRO-
α, GRO-β, GRO-γ, neutrophil active compound peptide (ENA-78), granulocyte chemoattractant protein-2 (GCP-2), and stromal cell-derived factor-1 (
Α (CxC) chemokines selected from the group consisting of: SDF-I, or pre-B cell stimulating factor (PBSF)); and / or RANTES (regulated, expressed and secreted normal T in activation) , Macrophage inflammatory protein-1α (MIP-1α), macrophage inflammatory protein-1β (MIP-
1β), monocyte chemotactic protein-1 (MCP-1), monocyte chemotactic protein-2
(MCP-2), monocyte chemotactic protein-3 (MCP-3), monocyte chemotactic protein-4 (MCP-4), macrophage inflammatory protein-1γ (MIP-1
γ), macrophage inflammatory protein-3 (MIP-3α), macrophage inflammatory protein-3β (MIP-3β), macrophage inflammatory protein-4 (MIP-4 / DC-CK-1 / PARC), eotaxin ( eotaxi
β) selected from the group consisting of: n), Exodus, and I-309; and / or γ (C) chemokines, lymphotactin (lym
(photoactin).

A variety of delivery systems are known and can be used to administer the compounds of the invention (eg, encapsulation in liposomes, microparticles, microcapsules,
Recombinant cells capable of expressing this compound, receptor-mediated endocytosis (eg, Wu and Wu, 1987, J. Biol. Chem. 262: 4429).
-4432), construction of nucleic acids as part of retroviruses or other vectors, etc.). 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 convenient route (eg, by infusion).
Alternatively, they can be administered by bolus injection, by absorption through the epithelial or mucosal lining (eg, oral, rectal and intestinal mucosa, etc.), and can be administered together with other biologically active agents. Administration can be systemic or local. In addition, the pharmaceutical compounds or compositions of the present invention can be administered by any suitable route, including intraventricular and intrathecal injections; intraventricular injection can be, for example, an intraventricularly attached reservoir such as an Ommaya reservoir. It may be desirable to introduce it into the central nervous system (which can be facilitated by a catheter). Pulmonary administration may also be employed, for example, with the use of an inhaler or nebulizer (nebulizer), and formulation with an aerosolizing agent.

In a specific embodiment, it may be desirable to administer the pharmaceutical compounds or compositions of the invention locally to the area in need of treatment; this is not a limitation, but includes, for example, By local injection during surgery, topical application (eg, in combination with post-surgical wound dressing), by injection, by catheter, by suppository, or by implant (the implant may be a membrane such as a sialastic membrane). Or a porous, non-porous, or glue-like material comprising fibers). Preferably, when administering a protein of the invention, including an antibody, care must be taken to use materials to which the protein does not absorb.

In another embodiment, the compound or composition can be delivered into vesicles, especially liposomes (Langer, Science 249: 1527-1533 (
1990); Treat et al., Liposomes in the Therap.
y of Infectious Disease and Cancer, L
opez-Berstein and Fidler (eds.), Liss, New
York, pp. 353-365 (1989); Lopez-Berstein,
See pages 317-327 of the same book; see broadly the same book).

[0411] In yet another embodiment, the compound or composition can be delivered in a controlled release system. In one embodiment, a pump may be used (Lang
er, (supra); Sefton, CRC Crit. Ref. Biomed. E
ng. 14: 201 (1987); Buchwald et al., Surgery 88.
: 507 (1980); Saudek et al. Engl. J. Med. 321:
574 (1989)). In another embodiment, a polymeric material may be used (Medical Applications of Control).
led Release, Langer and Wise (eds.), CRC Pre
s. , Boca Raton, Florida (1974); Control.
ed Drug Bioavailability, Drug Product
Design and Performance, Smolen and Bal
1 (eds), Wiley, New York (1984); Ranger and P.
eppas, J .; Macromol. Sci. Rev .. Macromol. Ch
em. 23:61 (1983); Levy et al., Science 2
28: 190 (1985); During et al., Ann. Neurol. 25: 3
51 (1989); Howard et al. Neurosurg. 71: 105 (
1989)). In yet another embodiment, the controlled release system can be placed near the therapeutic target, ie, the brain, and thus requires only a portion of the systemic dose (eg, Goodson, Medical Applicat).
ions of Controlled Release, (supra), Volume 2,
Pp. 115-138 (1984)).

Other controlled release systems are discussed in a review by Langer (1990, Science 249: 1527-1533).

In a specific embodiment, where the compound of the invention is a nucleic acid encoding a protein, the nucleic acid comprises it as part of a suitable nucleic acid expression vector,
And by administration such that it is intracellular (eg, by use of a retroviral vector (see US Pat. No. 4,980,286)), or by direct injection, or by microparticle bombardment (eg, Gene gun; Bio
administration, by coating with lipids or cell surface receptors or transfectants, or in conjunction with a homeobox-like peptide known to enter the nucleus (eg, 1991, Joliot). Proc. Natl. Acad. Sci. U.
SA 88: 1864-1868) and the like, to enhance the expression of the encoded protein in vivo. Alternatively, the nucleic acid can be introduced intracellularly and the host cell DNA homologously recombined for expression.
May be incorporated within.

The amount of a compound of the invention that is effective in this treatment (suppression and prevention of a disease or disorder associated with abnormal expression and / or activity of a polypeptide of the invention) is determined by standard clinical techniques. Can be done. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be used in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses can be extrapolated from dose-response curves obtained from in vitro or animal model test systems.

For antibodies, the dosage administered to a patient is typically 0.1 mg / kg to 100 mg / kg of the patient's body weight. Preferably, the dosage administered to a patient is between 0.1 mg / kg and 20 mg / kg of the patient's body weight, more preferably between 1 mg / kg and 10 mg / kg of the patient's body weight. Generally, human antibodies have a longer half-life in the human body than antibodies from other species, due to the immune response to the foreign polypeptide. Thus, low dosages and infrequent administration of human antibodies are often possible. Further, the dosage and frequency of administration of the antibodies of the invention can be modified (eg,
It can be reduced by enhancing antibody uptake by lipidation (such as lipidation) and tissue penetration (eg, into the brain).

In one embodiment, the present invention relates to a composition comprising a polypeptide of the present invention (eg, a heterologous polypeptide, a heterologous nucleic acid, a D toxin or a prodrug associated with a prodrug).
(Including R4 polypeptides or anti-DR4 antibodies) to target cells that express a membrane-bound form of DR4 on the cell surface. The DR4 polypeptide or anti-DR4 antibody of the present invention is associated with a heterologous polypeptide, a heterologous nucleic acid, a toxin or a prodrug via hydrophobic, hydrophilic, ionic and / or covalent interactions. I can do it.

In one embodiment, the invention provides a method of specific delivery of a composition of the invention (eg, a DR4 or anti-DE4 antibody) to a cell by administering a polypeptide of the invention. I do. The compositions of the invention are associated with a heterologous polypeptide or nucleic acid. In one example, the invention provides a method of therapeutic protein delivery to a target cell. In another example, the invention relates to single-stranded nucleic acids (eg, antisense or ribozymes) or double-stranded nucleic acids (eg, DNA that can integrate into the genome of a cell or replicate as an episome and can be transcribed). Provided to a target cell.

In another embodiment, the present invention provides for the identification of cells by administering a polypeptide of the present invention (eg, a DR4 polypeptide or an anti-DR4 antibody) in association with a toxin or cytotoxic prodrug. Methods of targeted destruction (eg, destruction of tumor cells).

In certain embodiments, the present invention relates to cells expressing a DR4 receptor on a surface thereof (eg, activated T cells, cancer cells) by administering a DR4 polypeptide in association with a toxin or cytotoxic prodrug. Cells, or leukemia cells).

In another particular embodiment, the invention provides a method for administering a cell that expresses a membrane-bound form of DR4 (eg, spleen) by administering an anti-DR4 antibody in association with a toxin or cytotoxic prodrug. , Bone marrow, kidney and PBL).

A “toxin” is an endogenous cytotoxic effector system, radioisotope, holotoxin (holotoxin), modified toxin, catalytic subunit of toxin, cytotoxin (cytotoxic factor), or any molecule. Or a compound that binds and activates an enzyme (which is not normally present on or in the cell under defined conditions that results in cell death). 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 an intrinsic endogenous cytotoxic effector system or an induced endogenous cytotoxic effector system (or Such as thymidine kinase, endobuclease, RNAse
, Α-toxin, ricin, abrin, Pseudomonas exotoxin A, diphtheria toxin, saporin, momordin, gelonin, pokeweed antiviral protein, α-sarcin (sa)
rcin) and cholera toxin. “Toxin” also includes cytostatic or cytocidal agents, therapeutic agents or radiometal ions, such as α-emitters (eg, ²¹³ Bi), or other radioisotopes (eg, 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 Cr, ₅₄ Mn, ₇₅ Se, ₁₁₃ Sn
, ₉₀ yttrium, ₁₁₇ Tin, ₁₈₆ rhenium, ₁₆₆ holmium, and ₁₈₈ rhenium); luminescent labels (eg, luminol); and fluorescent labels (eg, fluorescein and rhodamine, and biotin).

[0422] Techniques known in the art can be applied to the labeled proteins (including antibodies) of the present invention. Such techniques include, but are not limited to, the use of bifunctional (bifunctional) binders (eg, US Pat. Nos. 5,756,065; 5,714,631; 5,696,239; 5,652,361; 5,505,931; 5,489,425; 5,435,9.
No. 90; No. 5,428,139; No. 5,342,604; No. 5,27
4,119; 4,994,560 and 5,808,003 (the contents of each of which are incorporated herein by reference in their entirety). A cytotoxin or cytotoxic factor includes any drug that is harmful to cells. Examples include paclitaxel, cytochalasin B, gramicidin D
, Ethidium bromide, methine, mitomycin, etoposide, tenoposide (
tenoposide), vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracindione, mitoxantrone, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoid, procaine, tetracaine, lidocaine, propranolol, and puromycin; Analogs or homologs thereof.
Therapeutic agents include antimetabolites (eg, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine),
Alkylating agents (eg, mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU)
, Cyclotosfamide, busulfan,
Dibromomannitol, streptozotocin
, Mitomycin C, and cis-dichlorodiamineplatinum (II) (DDP) cisplatin), anthracycline (eg,
Daunorubicin (formerly daunomycin), and doxorubicin), antibiotics (eg, dactinomycin (formerly actinomycin), bleomycin,
Mithramycin and anthramycin (AMC
)), And antimitotic agents (eg, vincristine and vinblastine)
But not limited thereto.

“Cytotoxic prodrug” refers to a non-toxic compound that is converted to a cytotoxic compound by an enzyme not normally present in the cell. Cytotoxic prodrugs that can be used in accordance with the methods of the present invention include glutamyl derivatives of benzoic acid mustard alkylating agents, phosphate derivatives of etoposide or mitomycin C, cytosine arabinoside, daunorubicin, and phenoxyacetamide derivatives of daunorubicin. But not limited thereto.

The present invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more components of the pharmaceutical composition of the present invention. A notice in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceutical or biological products may optionally accompany such containers. This notice reflects the agency's approval of manufacture, use or sale for human administration.

Diagnostics and Imaging Labeled antibodies that specifically bind to a polypeptide of interest, and derivatives and analogs thereof, are used for diagnostic purposes to detect abnormal expression and expression of a polypeptide of the invention. Diseases, disorders and / or conditions associated with / or activity may be detected, diagnosed or monitored. The present invention provides for the detection of abnormal expression of a polypeptide of interest, comprising the steps of (a) using one or more antibodies specific for the polypeptide of interest in a cell or body fluid of an individual. Assaying the expression of the polypeptide of the invention, and (b) comparing the gene expression level to the level of standard gene expression, whereby the assayed assay is compared to the standard expression level. An increase or decrease in the level of polypeptide gene expression indicates abnormal expression.

The present invention provides a diagnostic assay for diagnosing a disorder, the assay comprising:
a) assaying the expression of the polypeptide of interest in cells or body fluids of an individual using one or more antibodies specific for the polypeptide of interest; and (b) Comparing the level of gene expression, whereby an increase or decrease in the assayed polypeptide gene expression level compared to its standard expression level is indicative of a particular disorder. For cancer, the presence of relatively high amounts of transcripts in biopsy tissue from an individual may indicate a predisposition for the development of the disease, or provide a means for detecting the disease before the actual clinical symptoms appear. Can provide. A more conclusive diagnosis of this type may allow health professionals to use preventive measures, or allow early aggressive treatment, thereby preventing the development or further progression of cancer.

The antibodies of the present invention can be used to assay protein levels in biological samples using classical immunohistological methods known to those of skill in the art (eg, Jalk
anen et al. Cell. Biol. 101: 976-985 (1985);
Jalkanen et al. Cell. Biol. 105: 3087-3096 (
1987)). Other antibody-based methods useful for detecting protein gene expression include immunoassays, such as enzyme-linked immunosorbent assays (ELISAs) and radioimmunoassays (RIAs). Suitable antibody assay labels are known in the art, and are enzymatic labels (eg, glucose oxidase); radioisotopes (eg, iodine (125I,
21I), carbon (14C), sulfur (35S), tritium (3H), indium (112In), and technetium (99Tc)); luminescent labels (eg, luminol); and fluorescent labels (eg, fluorescein and rhodamine); Biotin.

One aspect of the present invention is the detection and diagnosis of a disease or disorder associated with abnormal expression of a polypeptide of interest in an animal, preferably a mammal, and most preferably a human. In one embodiment, the diagnosis comprises: a) administering to the subject (eg, parenterally, subcutaneously, or intraperitoneally) an effective amount of a labeled molecule that specifically binds the polypeptide of interest. B) to allow the labeled molecule to preferentially concentrate at the site in the subject where the polypeptide is expressed (and to remove unbound labeled molecule to background levels); A) waiting for a time interval after administration; c) determining a background level; and d) detecting a labeled molecule in the subject, such that the label exceeds the background level. Detection of an activating molecule indicates that the subject has a particular disease or disorder associated with abnormal expression of the polypeptide of interest. Background levels can be determined by a variety of methods, including comparing the amount of labeled molecule detected with standard values previously determined for a particular system.

It is understood in the art that the size of the subject and the imaging system used will determine the amount of imaging moiety needed to generate a diagnostic image. In the case of a radioisotope moiety, for a human subject, the amount of radioactivity injected will usually be about 5-
It is in the range of 99 mTc of 20 millicuries. The labeled antibody or antibody fragment is then preferentially accumulated at locations in the cell that contain the particular protein.
In vivo tumor imaging is described by S.D. W. Burchiel et al., "Immunophar.
mackinetics of Radiolabeled Antibod
is and Their Fragments "(Tumor Image
ng: The Radiochemical Detection of Ca
Chapter 13, S.Ncer. W. Burchiel and B.A. A. Rhodes, Masson Publishing Inc. (1982).

Depending on several variables, including the type of label used and the mode of administration, the labeled molecule will preferentially concentrate at the site of the subject and the unbound labeled molecule Is between 6 and 48 hours or between 6 and 24 hours or between 6 and 12 hours after which administration can be cleared to background levels.
In another embodiment, the time interval after administration is 5-20 days or 5-10 days.

In one embodiment, monitoring a disease or disorder comprises repeating the method for diagnosing the disease or disorder (eg, 1 month after first diagnosis, 6 months after initial diagnosis, first diagnosis One year later).

[0432] The presence of the labeled molecule can be detected from the patient using methods known in the art for in vivo scanning. These methods depend on the type of label used. One of skill in the art can determine the appropriate method for detecting a particular label. Methods and devices that can be used in the diagnostic methods of the invention include, but are not limited to, computerized tomography (CT), whole body scanning such as proton emission tomography (PET), magnetic resonance imaging (MRI). , And ultrasound diagnostics.

In certain embodiments, the molecule is labeled with a radioisotope and detected from the patient using a radioresponsive surgical instrument (Thurston et al., US Pat. No. 5,441,050). . In another embodiment, the molecule is labeled with a fluorescent compound and detected from the patient using a fluorescence-responsive scanning device. In another embodiment, the molecule is labeled with a positron emitting metal and is detected from the patient using proton emission tomography. In yet another embodiment, the molecule is labeled with a paramagnetic label and detected from the patient using magnetic resonance imaging (MRI).

(Kit) The present invention provides a kit that can be used in the above method. In one embodiment, the kit comprises an antibody (preferably a purified antibody) of the invention in one or more containers. In certain embodiments, a kit of the invention comprises a substantially isolated polypeptide comprising an epitope that is specifically immunoreactive with the antibodies 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 kits of the present invention provide for the binding of an antibody to a polypeptide of interest (eg, the antibody is a detectable substrate (eg, the antibody is a fluorescent compound, an enzyme substrate, a radioactive compound or (A second antibody that can be conjugated with a luminescent compound) or that recognizes the first antibody can be conjugated to a detectable substrate).

In another particular embodiment of the invention, the kit is used for screening diagnostic sera containing antibodies specific for proliferative and / or cancerous polynucleotides and polypeptides. It is a kit. Such a kit can include a control antibody that does not react with the polypeptide of interest. Such a kit may comprise a substantially isolated polypeptide antigen comprising an epitope specifically immunoreactive with at least one anti-polypeptide antigen antibody. In addition, such kits can be used to detect binding of the antibody to an antigen (eg, the antibody can be conjugated to a fluorescent compound such as fluorescein or rhodamine, which can be detected by flow cytometry). Means. In certain embodiments, the kit may comprise a recombinantly produced polypeptide antigen or a chemically synthesized polypeptide antigen. The polypeptide antigens of the kit can also be attached to a solid support.

In a more specific embodiment, the detection means of the above kit comprises a solid support to which the polypeptide antigen is attached. Such kits also include a non-attached reporter-labeled anti-human antibody. In this embodiment, binding of the antibody to the polypeptide antigen can be detected by binding of the reporter-labeled antibody.

In a further embodiment, the present invention includes a diagnostic kit for use in screening serum containing an antigen of a polypeptide of the present invention. The diagnostic kit comprises a substantially isolated antibody that is specifically immunoreactive with a polypeptide or polynucleotide antigen, and means for detecting binding of the polynucleotide or polypeptide antigen to the antibody. . In one embodiment, the antibody is:
Attached to a solid support. In certain embodiments, the antibodies can be monoclonal antibodies. The detection means of the kit may include a second labeled monoclonal antibody. Alternatively or additionally, the detection means may include a labeled, competitor antigen.

In one diagnostic configuration, a test serum is reacted with a solid phase reagent having a surface-bound antigen obtained by a method of the invention. After binding specific antigen-antibody to the reagent and removing unbound serum components by washing, depending on the amount of anti-antigen antibody bound on the solid support, to bind the reporter to the reagent, This reagent is reacted with a reporter-labeled anti-human antibody. The reagent is washed again to remove unbound labeled antibody and the amount of reporter associated with the reagent is determined. Typically, a reporter is an enzyme that is detected by incubating this solid phase in the presence of a suitable fluorometric, luminescent or colorimetric substrate (Sigma, St. Louis, MO).

[0439] The solid surface reagents in the assays described above can be used to convert protein material to solid support material (
For example, prepared by known techniques for attachment to polymeric beads, dipsticks, 96-well plates or filtration materials). These methods of attachment generally include non-specific adsorption of proteins to a support or chemically active groups (eg, activated carboxyl, hydroxyl, or aldehyde groups) on a solid support. Covalent attachment of the protein (typically via a free amine group). Alternatively, streptavidin-coated plates can be used with biotinylated antigen.

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

Chromosome Assays The sequences of the present invention are also useful for chromosome identification. This sequence is specifically targeted to specific locations on individual human chromosomes and is capable of hybridizing. Mapping of DNA to chromosomes according to the present invention is an important first step in correlating those sequences with disease-related genes.

In certain preferred embodiments in this regard, c disclosed herein.
The DNA is used to clone the genomic DNA of the DR4 gene.
This can be accomplished using a variety of well-known techniques and libraries that are commonly available. The genomic DNA is then used for in situ chromosome mapping, using techniques well known for this purpose.

In addition, sequences can be used to convert cDNA to PCR primers (preferably 15-25 b
By preparing p), it can be mapped to a chromosome. Computer analysis of the 3 'untranslated region of the gene is used to rapidly select primers that do not span more than one exon in genomic DNA, thus complicating the amplification process. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes.

[0444] Fluorescence in situ hybridization ("FISH") of cDNA to a metaphase chromosomal spread can be used to provide accurate chromosomal location in one step. This technique uses cDNAs as short as 50 or 60 bp.
It can be used with probes from origin. For a review of this technology, see Verma et al., Human Chromosomes: A Manual Of Basic.
Techniques, Pergamon Press, New York (
1988).

Once a sequence has been mapped to a precise chromosomal location, the physical location of the sequence on the chromosome can be correlated with genetic map data. Such data,
For example, V. McKusick, Mendelian Inheritance
In Man (Johns Hopkins University, Wel)
ch, available online from the Medical Library). The association between the gene and the disease mapped to the same chromosomal region is then identified 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, then the mutation is likely a causative agent of the disease.

Example 1 Expression and Purification in E. coli Deposited cDNA encoding mature DR4 protein (ATCC No. 9785)
3) is amplified using PCR oligonucleotide primers specific for the amino terminal sequence of the DR4 protein and the vector sequence 3 'to the gene. Additional nucleotides containing restriction sites to facilitate cloning are added to the 5 'and 3' sequences, respectively.

The following primers were used For expression of the E. coli DR4 extracellular domain:
5 'primer

[0449]

Embedded image

Contains an underlined SphI site: 3 ′ primer

[0451]

Embedded image

Contains an underlined HindIII site. The vector is pQE60.

The restriction sites are convenient for restriction enzyme sites in the bacterial expression vector pQE60,
These are used for bacterial expression in this example. (Qiagen, Inc.
9259 Eton Avenue, Chatsworth, CA, 91311.
). pQE60 encodes ampicillin antibiotic resistance ("Amp ^r ") and contains a bacterial origin of replication ("ori"), an IPTG-inducible promoter, and a ribosome binding site ("RBS").

[0454] Both the amplified DR4 DNA and the vector pQE60 were digested with SphI and HindIII, and the digested DNAs were then ligated together. DDC
Insertion of the R protein DNA into the restricted pQE60 vector is performed by IPT of the vector.
The DR4 protein coding region is placed in frame with the starting AUG, which is operably linked downstream of the G-inducible promoter and is appropriately positioned for translation of the DR4 protein.

The ligation mixture was transformed into competent E. coli using standard procedures. Transform E. coli cells. Such a procedure is described in Sambrook et al., Molecular.
Cloning: a Laboratory Manual, 2nd edition; Cold
Spring Harbor Laboratory Press, Cold
Spring Harbor, N.M. Y. (1989). E. FIG.
The E. coli strain M15 / rep4, which contains multiple copies of the plasmid pREP4, expresses the lac repressor, and confers kanamycin resistance ("Kan ^r "), has been described in the illustrative examples described herein. Used in implementing. This strain is one of many suitable for expressing the DR4 protein and is commercially available from Qiagen.

Transformants are identified by their ability to grow on LB plates in the presence of ampicillin and kanamycin. Plasmid DNA is isolated from resistant colonies and the identity of the cloned DNA is confirmed by restriction analysis.

Clones containing the desired construct are grown overnight (“O / N”) in liquid culture in LB medium supplemented with both ampicillin (100 μg / ml) and kanamycin (25 μg / ml). .

This O / N culture is used to inoculate many cultures at a dilution of about 1: 100 to 1: 250. Cells were treated with an optical density at 600 nm ("OD600") of 0.
. Grow to between 4 and 0.6. Then, isopropyl-β-D-thiogalactopyranoside (“IPTG”) was added to a final concentration of 1 mM and lacI
Inactivating the repressor induces transcription from the lac repressor-sensitive promoter. The cells are subsequently incubated for a further 3-4 hours. The cells are then harvested by centrifugation and disrupted using standard methods. The inclusion bodies are purified from the disrupted cells using conventional harvesting techniques, and the protein is solubilized from the inclusion bodies in 8M urea. The 8M urea solution containing the solubilized protein is passed over a PD-10 column in 2x phosphate buffered saline ("PBS"), thereby removing the urea, replacing the buffer, and removing the protein. Let it refold. The protein is purified by a further step of chromatography,
Remove endotoxin. This is then sterile filtered. The sterile filtered protein preparation is stored in 2 × PBS at a concentration of 95 μg / ml.

Example 2 Expression in Mammalian Cells Most vectors used for the transient expression of certain gene sequences in mammalian cells carry the SV40 origin of replication. This means that the virus DN
It allows the replication of vectors up to high copy numbers in cells (eg, COS cells) that express the T antigen required for initiation of A synthesis. Any other mammalian cell lines can also be used for this purpose.

A typical mammalian expression vector contains a promoter element (which mediates the initiation of transcription of mRNA), a protein coding sequence, and signals necessary for termination of transcription and polyadenylation of the transcript. Additional elements include enhancers, Kozak sequences, and intervening sequences flanking donor and acceptor sites for RNA splicing. Highly efficient transcription was achieved using the early and late promoters from SV40, the long terminal repeat (LTR) from retroviruses (eg, RSV, HTLVI, HIVI) and the early promoter of cytomegalovirus (CMV). obtain. However, cellular elements can also be used (eg, the human actin promoter). Expression vectors suitable for use in the practice of the present invention include, for example, pSVL and pSVL.
MSG (Pharmacia, Uppsala, Sweden), pRSV
cat (ATCC 37152), pSV2dhfr (ATCC 37146)
And vectors such as pBC12MI (ATCC 67109). Mammalian host cells that can be used include human Hela cells, 283 cells, H9 cells and Jurkat cells, mouse NIH3T3 cells and C127 cells, Cos1 cells, Cos7 cells and CV1 cells, quail QC1-3 cells, mouse L cells, and Chinese hamster ovary (CHO) -cells.

Alternatively, the gene of interest can be expressed in a stable cell line that contains the gene for integration into the chromosome. Co-transfection with a selectable marker (eg, dhfr, gpt, neomycin, hygromycin) allows identification and isolation of the transfected cells.

The transfected gene can also be amplified to express large amounts of the encoded protein. DHFR (dihydrofolate reductase) markers are useful for developing cell lines with hundreds or thousands of copies of the gene of interest.
Using these markers, mammalian cells are grown in selective media and cells with the highest resistance are selected. These cell lines contain the amplified gene integrated into the chromosome. Chinese hamster ovary (CHO) cells are often used for protein production.

The expression vectors pC1 and pC4 are compatible with the Rous sarcoma virus strong promoter (LTR) (Cullen et al., Molecular and Cellula).
r Biology, 438-47 (1985)) and fragments of the CMV-enhancer (Boshart et al., Cell 41: 521-530 (1)
985)). Multiple cloning sites (eg, restriction enzyme Bam
HI, XbaI, and Asp718) facilitate cloning of the gene of interest. The vector further contains the 3 ′ of the rat preproinsulin gene.
Includes intron, polyadenylation, and termination signals.

Cloning and Expression in CHO Cells The vector pC4 is used for expression of the DR4 polypeptide. Plasmid pC4 is a derivative of plasmid pSV2-dhfr (ATCC Accession No. 37146). This plasmid contains the mouse DHFR gene under the control of the SV40 early promoter. Chinese hamster ovary cells or other cells lacking dihydrofolate activity that are transfected with these plasmids are selected from selective media (α minus MEM, Li) supplemented with the chemotherapeutic agent methotrexate (MTX).
fe Technologies). Amplification of the DHFR gene in cells that are resistant to methotrexate (MTX) is well documented (eg, Alt, FW et al., J. Biol. C).
hem. 253: 1357-1370 (1978); Hamlin, J. Mol. L. And Ma, C.I. , Biochem. et Biophys. Acta, 1097
: 107-143 (1990); Page, M .; J. And Sydenham,
M. A. 1991, Biotechnology 9: 64-68 (1991).
checking). Cell growth at increasing concentrations of MTX results in resistance to the drug by overproducing the target enzyme DHFR as a result of amplification of the DHFR gene. When the second gene is linked to the DHFR gene, it is usually co-amplified,
And it is overexpressed. It is known in the art that this approach can be used to develop cell lines with more than 1,000 copies of the amplified gene. Subsequently, if the methotrexate is removed, a cell line is obtained that contains the amplified gene integrated into one or more chromosomes of the host cell.

Plasmid pC4 contains a strong promoter for the Rous sarcoma virus long terminal repeat (LTR) (Cullen et al., Molecula) for expression of the gene of interest.
r and Cellular Biology 5: 438-47 (19
March 1985)), and a fragment isolated from an enhancer of the immediate early gene of human cytomegalovirus (CMV) (Boshart et al., Cell 41).
: 521-530 (1985)). Downstream of the promoter are the following single restriction enzyme cleavage sites, BamHI, XbaI and Asp718, which allow for gene integration. Behind these cloning sites, the plasmid contains the 3 'intron and polyadenylation site of the rat preproinsulin gene. Other high efficiency promoters can also be used for expression (eg,
Human β-actin promoter, SV40 early or late promoter, or long terminal repeats from other retroviruses (eg, HIV and HTLVI).
Clontech's Tet-Off and Tet-On gene expression systems and similar systems can be used to express DR4 polypeptides in a regulated manner in mammalian cells (Gossen, M., and Bujard, H., et al. Pr
oc. Natl. Acad. Sci. ScL USA 89: 5547-5551 (19
92)). For polyadenylation of mRNA, other signals, such as from human growth hormone or from the globin gene, can be used as well. Stable cell lines that carry the gene of interest integrated into the chromosome can also be selected for co-transfection with a selectable marker (eg, gpt, G418, or hygromycin). It is advantageous to use more than one selection marker at the start (eg G418 and methotrexate).

The plasmid pC4 is digested with the restriction enzyme BamHI and then dephosphorylated using bovine intestinal phosphate by procedures known in the art. The vector is then isolated from a 1% agarose gel.

The DNA sequence encoding the complete polypeptide is amplified using PCR oligonucleotide primers corresponding to the 5 ′ and 3 ′ sequences of the desired portion of the gene. The 5 'primer containing the underlined BamHI site, Kozak sequence, and AUG start codon has the following sequence: 5' GCG GGATCC GCCATCATGGCGCCACCACACCAGCTA
GA3 '(SEQ ID NO: 10). The 3 'primer containing the underlined BamHI site has the following sequence:
'GCG GGATCC TCACTCCAAGGACACGGCAGAGCC3'
(SEQ ID NO: 11).

The amplified fragment is digested with the endonuclease BamHI and then purified again on a 1% agarose gel. The isolated fragment and the dephosphorylated vector are then ligated with T4 DNA ligase. Then, E. c
Oli HB101 or XL-1 Blue cells are transformed and bacteria containing the fragment inserted into plasmid pC4 are identified using, for example, restriction enzyme analysis.

[0469] Chinese hamster ovary cells lacking the active DHFR gene are used for transfection. 5 μg of the expression plasmid pC4 was combined with 0.5 μg of the plasmid pSVn using the lipofectin method (Felgner et al., Supra).
Co-transfect with eo. Plasmid pSV2-neo contains a dominant selectable marker (the neo gene from Tn5 encoding an enzyme that confers resistance to a group of antibiotics, including G418). Cells are seeded in α-min MEM supplemented with 1 mg / ml G418. Two days later, the cells are trypsinized and
0, 25 or 50 ng / ml methotrexate and 1 mg / ml G4
Hybridoma cloning plate (G
reiner, Germany). After about 10-14 days, a single clone was trypsinized and then different concentrations of methotrexate (50 nM, 10
(0 nM, 200 nM, 400 nM, 800 nM) into 6-well petri dishes or 10 ml flasks. The clones growing at the highest concentration of methotrexate were then replaced with higher concentrations of methotrexate (1 μM, 2 μM, 5 μM,
(10 mM, 20 mM). Do the same for 10
Repeat until a clone is obtained that grows at a concentration of 0-200 μM. The expression of the desired gene product is analyzed, for example, by SDS-PAGE and Western blot or by reverse phase HPLC analysis.

(Example 3) (DR4 protein fusion) The DR4 fusion protein of the present invention can be fused to another protein, if necessary. These fusion proteins may be useful for various applications. For example, fusion of a DR4 polypeptide to a His tag, HA tag, Protein A, IgG domain, and maltose binding protein facilitates purification. (EP A
394,827; Traunecker et al., Nature 331: 84-8.
6 (1988)). Similarly, fusion to IgG-1, IgG-3, and albumin increases half-life in vivo. A nuclear localization signal fused to a DR4 polypeptide can target the protein to a specific subcellular location, while a covalent heterodimer or homodimer can increase or decrease the activity of the fusion protein . Fusion proteins can also produce chimeric molecules with more than one function. Finally, the fusion protein may increase the solubility and / or stability of the fusion protein as compared to the unfused protein. All of the above types of fusion proteins can be obtained using techniques known in the art, or using the following protocol, which outlines the fusion of polypeptides to IgG molecules, or It can be made by modification.

[0471] Briefly, the human Fc portion of an IgG molecule is 5 'of the sequence set forth in SEQ ID NO: 13.
PCR can be performed using primers that span the ends and the 3 'end. These primers also preferably contain convenient restriction enzyme sites to facilitate cloning into expression vectors, preferably mammalian expression vectors.

For example, if a pC4 (Accession No. 209646) expression vector is used, the human Fc portion can be ligated into a BamHI cloning site. Note that the 3 'BamHI site should be destroyed. The vector containing the human Fc portion is then re-restricted with BamHI, linearizing the vector, and
A DR4 polynucleotide isolated by the PCR protocol described in Example 1 is ligated to this BamHI site. It should be noted that the polynucleotide is cloned without a stop codon, otherwise no fusion protein will be produced.

When using a native signal sequence to produce a secreted protein, pC4
No second signal peptide is required. Alternatively, if a native signal sequence is not used, the vector can be modified to include a heterologous signal sequence. (See, for example, WO 96/34891).

Example 4 Cloning and Expression of Soluble Extracellular Domain of DR4 in Baculovirus Expression System Deposited cDNA encoding the soluble extracellular domain of DR4 protein
The sequence (ATCC No. 97853) was added to the P's corresponding to the 5 'and 3' sequences of the gene.
Amplify using CR oligonucleotide primers: The 5 'primer for DR4 is the sequence 5' GCG GGATCC GCCATCATGGCGCCACCACCA containing the underlined BamHI restriction enzyme site.
It has GCTAGA 3 '(SEQ ID NO: 10). The 5 'end of the amplified fragment encoding DR4, inserted into an expression vector as described below, provides an efficient cleavage signal peptide. Efficient signals for initiation of translation in eukaryotic cells are described in Kozak. M. , J. et al. Mol. Biol. 196: 947-950
(1987) is appropriately located in the vector portion of the construct.

The 3 ′ primer for both DR4s has an underlined BamHI restriction enzyme site followed by nucleotides complementary to the DR4 nucleotide sequence shown in FIG.
Followed by the sequence 5 ′ GCG GGATCC TCAATTATGTCC including the stop codon
ATTGCCTG 3 '(SEQ ID NO: 12).

The amplified fragment was prepared using a commercially available kit (“Geneclean” BIO
101 Inc. , La Jolla, Ca. ) To isolate from a 1% agarose gel. The fragment is then digested with BamHI and Asp718 and purified again on a 1% agarose gel.

The vector pA2 is prepared using standard methods (eg, Summers et al., A Manu).
al of Methods for Baculovirus Vector
s and Insect Cell Culture Procedures
, Texas Agricultural Experimental Sta
Tion Bulletin No. 1555 (1987)) to express the DR4 protein in a baculovirus expression system. This expression vector is available from Autograph California.
Includes the strong polyhedron promoter of nucleopolyhedrovirus (ACMNPV), followed by convenient restriction sites. For easy selection of recombinant viruses, see E.C. col
The β-galactosidase gene from i is inserted in the same orientation as the polyhedron promoter, followed by a polyadenylation signal of the polyhedron gene. On either side of this polyhedral sequence is flanked by viral sequences for cell-mediated homologous recombination with wild-type viral DNA to produce a live virus expressing the cloned polynucleotide.

As one of skill in the art will readily appreciate, many other baculovirus vectors (eg, pAc373, pVL941, and pAcIM1) are used to construct,
It can be used in place of pA2 as long as it provides appropriately arranged signals for translation, intracellular transport, etc. (eg, in-frame AUG, and signal peptide, if needed). Such vectors are described, inter alia, in Luckow et al., Vir.
policy 170: 31-39, and others.

This plasmid is digested with the restriction enzyme BamHI and then dephosphorylated using bovine intestinal phosphatase using routine procedures known in the art. The DNA was then transferred to a commercially available kit ("Geneclean" BIO 101 In
c. , La Jolla, Ca. ) To isolate from a 1% agarose gel.

The fragment and the dephosphorylated plasmid were ligated with T4 DNA ligase and E. coli HB101 cells are transformed with the ligation mixture and spread on culture plates. Bacteria containing a plasmid having the human DDCR gene,
Identify by digesting DNA from individual colonies using BamHI and then analyze the digest by gel electrophoresis. The sequence of the cloned fragment is determined by DNA sequencing. This plasmid is referred to herein as pBacDR4.

5 μg of plasmid pBac DR4 was replaced with 1.0 μg of commercially available linear baculovirus DNA (“BaculoGold ^™ baculovirus DNA”).
, Pharmingen, San Diego, CA. Lipofection method (Felgner et al., Proc. Natl. Acad. Sci. USA).
84: 7413-7417 (1987)). 1 μg of BaculoGold ^™ viral DNA and 5 μg of plasmid pBac DR4 were added to 50 μl of serum-free Grace's medium (Life Te
channels Inc. , Gaithersburg, MD) in a sterile well of a microliter plate. Thereafter, 10 μl Lipofectin + 90 μl Grace's medium is added, mixed and incubated for 15 minutes at room temperature. The transfection mixture was then transferred to Sf9 insect cells (see below) plated in 35 mm tissue culture plates with 1 ml serum-free Grace's medium.
ATCC CRL 1711). The plate is shaken and the newly added solution is mixed. The plate is then incubated for 5 hours at 27 ° C. After 5 hours, the transfection solution was removed from the plate,
Then, 1 ml of Grace's insect medium supernatant supplemented with 10% fetal calf serum is added. Return the plate to the incubator and continue the culture at 27 ° C. for 4 days.

After 4 days, supernatants are collected and plaque assays are performed as described by Summers and Smith et al. (Cited above). "Blue Gal"
(Life Technologies Inc., Gaithersburg)
) Is used to allow easy identification and isolation of gal-expressing clones, which produce plaques that stain blue. (A detailed description of this type of “plaque assay” is also available at Life Technology
ies Inc. , Distributed by Gaithersburg, user's
guide for insect cell culture and ba
culovirology, pages 9-10).

After 4 days of serial dilution, the virus is added to the cells. After appropriate incubation, the plaque stained blue is picked up with the tip of an Eppendorf pipette. The agar containing the recombinant virus is then resuspended in an Eppendorf tube containing 200 μl Grace's medium. The agar is removed by simple centrifugation and the supernatant containing the recombinant baculovirus is used to infect Sf9 cells seeded in 35 mm culture dishes. Four days later, the supernatants of these culture dishes are collected, and then they are stored at 4 ° C. Clones reliably containing the properly inserted DR4 are identified by DNA analysis, including restriction mapping and sequencing. This is referred to as V-DR4 in this specification.

Sf9 cells are cultured in Grace's medium supplemented with 10% heat-inactivated FBS. The cells are infected with the recombinant baculovirus V-DR4 at a multiplicity of infection ("MOI") of about 2 (about 1 to about 3). After 6 hours, the medium was removed and SF900 II medium lacking methionine and cysteine (LifeTechnol)
offices Inc. , Available from Gaithersburg).
After 42 hours, 5 μCi of ³⁵ S-methionine and 5 μCi of ³⁵ S cysteine (A
(available from Mersham). The cells are incubated for a further 16 hours, then they are collected by centrifugation, lysed, and the labeled proteins are visualized by SDS-PAGE and autoradiography.

Example 5 DR4 Induced Apoptosis in Mammalian Cells Overexpression of Fas / APO-1 and TNFR-1 in mammalian cells mimics receptor activity (M. Muzio et al., Cell 85, 817-827 (
1996); P. Boldin et al., Cell 85, 803-815 (19
96)). Therefore, this system was used to study the functional role of DR4. M
Transient expression of DR4 in CF7 human breast cancer cells and 293 human embryonic kidney cells induced rapid apoptosis.

The cell death assay is performed essentially as described previously (AMC
Hinnaiyan et al., Cell 81,505-12 (1995); P.
Boldin et al., J Biol Chem 270, 7795-8 (1995).
F .; C. Kischkel et al., EMBO 14, 5579-5588 (199).
5); M. Chinnaiyan et al., J Biol Chem 271, 4
961-4965 (1996)). Briefly, vectors alone or CrmA expression constructs (M. Tewari et al., J Biol Chem 270,3255-
60 (1995)), a 10-fold excess of the MCF-7 human breast cancer clonal cell line stably transfected with the pcDNA3 expression construct encoding the indicated protein using lipofectamine (GIBCO-BRL). Below, it is transiently transfected with pCMV-DR4-galactosidase (or pCMV-DR4-galactosidase (lacking the death domain)). 2
93 cells are similarly transfected using the CaPO ₄ method. ICE Family Inhibitor z-VAD-fmk (Enzyme Systems Pr
products, Dublin, CA).
Add to cells at a concentration of 0 μM. 32 hours after transfection, cells are fixed and stained with X-Gal as previously described (AM Chinna).
iyan et al., Cell 81.505-12 (1995); P. Boldi
J. Biol Chem 270, 7795-8 (1995); C.
Kischkel et al., EMBO 14, 5579-5588 (1995)).

The cells show morphological changes typical of cells undergoing apoptosis,
It became round, condensed, and detached from the dish. TNFR-1 and Fa
s / APO-1 (M. Muzio et al., Cell 85,817-827 (199)
6); P. Boldin et al., Cell 85, 803-815 (1996).
M .; Tewari et al., J Biol Chem 270, 3255-60 (1
As in 995)), DR4-induced apoptosis was blocked by inhibitors of the ICE-like protease, CrmA and z-VAD-fmk.

Example 6 Extracellular Domain of DR4 Binds Cytotoxic Ligand TRIL and Blocks TRIL-Induced Apoptosis This example demonstrates that the receptor DR4 of the present invention binds TRAIL. Show. The soluble extracellular ligand-binding domain of DR4 is converted to human immunoglobulin (IgG
) Was expressed as a fusion to the Fc portion. The cDNA encoding the extracellular domain of DR4 (amino acids 110-239) was obtained by polymerase chain reaction and cloned into a modified pCMV1FLAG vector that allows in-frame fusion with the Fc portion of human IgG. .

As shown in FIGS. 6A and 6B, DR4-Fc specifically binds TRAIL but did not bind to the related cytotoxic ligand TNFα. In this experiment, we transfected 293 cells with constructs encoding the extracellular domain of DR4, TNFR-1 or Fas-Fc and the corresponding ligand, and harvested conditioned media after 72-80 hours And clarified by centrifugation, split and stored at -80 ° C. For the binding assay, equal volumes of conditioned medium containing receptor-Fc and conditioned medium containing ligand were added to 50 mM Hep.
es, pH 7.0, 150 mM NaCl, 1 mM EDTA, 0.5% NP
Mixed in a buffer containing the -40 and protease inhibitor mixture, and the samples were incubated at 4 ° C with continuous rotation for 4 hours. The receptor-Fc-ligand complex was precipitated with protein G-Sepharose, washed extensively with the above buffer, boiled in SDS sample buffer, and
% SDS-polyacrylamide gel separated and co-precipitated soluble ligand was detected using anti-Flag (Babco) or anti-myc-HRP (BMB) or by immunoblotting against FasL (Pharmingen).

Further, DR4-Fc blocked the ability of TRAIL to induce apoptosis (
(FIG. 6A). MCF7 cells were treated with soluble TRAIL (400 ng / ml) in the presence of equal amounts of Fc fusion or Fc alone. After 5 hours, the cells were fixed with formaldehyde and the nuclei were 4 ', 6'-diamidino-2-phenylindole (
DAPI) and examined by fluorescence microscopy using barrier filter cubes in the range of fluorescein isothiocyanate. The data shown in FIG. 6A (mean σ ± SD) is the percentage of apoptotic nuclei of total nuclei counted (
n = 3).

Finally, DR4-Fc had no effect on apoptotic TNFα-induced cell death under conditions where TNFR1-Fc completely abolished TNFα killing (FIG. 6B). MCF7 cells were isolated from TNF in the presence of an equal amount of Fc fusion or Fc alone.
Treated with α (40 ng / ml; Genentech, Inc.). Nuclei were stained and examined 15-18 hours later.

Example 7 Assay to Detect Stimulation or Inhibition of B Cell Proliferation and Differentiation The generation of a functional humoral immune response depends on the interaction between B-lineage cells and their microenvironment.
It requires both soluble and cognate signaling. The signal is a positive stimulus (which causes B-lineage cells to sustain their programmed development)
, Or a negative stimulus (instructing the cells to stop the current developmental pathway). To date, IL-2, IL-4, IL-5, IL-6, IL-7, IL
A number of stimulatory and inhibitory signals have been found to affect B cell responsiveness, including -10, IL-13, IL-14 and IL-15. Interestingly, these signals are weak effectors themselves, but can be combined with various costimulatory proteins to induce activation, proliferation, differentiation, homing, tolerance, and death in B cell populations. One of the most studied classes of B cell costimulatory proteins is the TNF superfamily. Within this family,
CD40, CD27 and CD30 are their respective ligands, CD1
54, together with CD70 and CD153, have been found to modulate various immune responses. Assays that allow detection and / or observation of the proliferation and differentiation of these B cell populations and their progenitor cells determine the effects that various proteins may have on these B cell populations with respect to proliferation and differentiation. This is a valuable tool. Listed below are the differentiation of B cell populations and their precursors,
Two assays designed to allow detection of proliferation, or inhibition.

Experimental Procedures (In Vitro Assay)-Purified DR4 protein or truncated form thereof was
The ability to induce activation, proliferation, differentiation or inhibition and / or death in cell populations and their precursors is assessed. DR to purified human tonsillar B cells
The activity of the four proteins (qualitatively measured over a dose range of 0.1 to 10,000 ng / mL) is assessed in a standard B lymphocyte costimulation assay. In this assay, purified tonsillar B cells were used as priming factors in formalin-fixed Staphylococcus aureus Cowan I (SAC).
, Or in the presence of immobilized anti-human IgM antibodies. IL-2
And a second signal, such as IL-15, synergizes with SAC and IgM crosslinking to induce B cell proliferation as measured by tritiated thymidine incorporation. New synergists can be easily identified using this assay. This assay involves isolating human tonsils B cells by magnetic bead (MACS) depletion of CD3-positive cells. The resulting cell population is greater than 95% B cells as assessed by CD45R (B220) expression. Samples of each of the various dilutions were placed in individual wells of a 96-well plate, and the culture medium (1
0% FBS, 5 × 10 ⁻⁵ M β-ME, 100 U / ml penicillin, 10 μg /
RPMI 16 containing ml streptomycin, and 10 ^-5 dilution of SAC
Add 10 ⁵ B cells in a total volume of 150 μl suspended in 40). Growth or inhibition started 72 hours after addition of the factor and was measured using ³ H-thymidine (6.7 C
i / mM) and a 20 h pulse (1 μCi / well). Positive and negative controls are IL2 and medium, respectively.

In Vivo Assay-BALB / c mice are injected (ip) twice daily with buffer alone or 2 mg / Kg of the DR4 protein of the invention or a shortened form thereof. Mice were given this treatment for 4 consecutive days, at which point they were sacrificed and various tissues and sera were collected for analysis. Normal spleen and DR
Comparison of H & E sections from spleen treated with the 4 protein results in marked activity of the DR4 protein on spleen cells, eg, marked spread of peri-arterial lymphatic sheath and / or nucleated cellularity of the red pulp region An increase is confirmed, which may indicate activation of differentiation and proliferation of the B cell population. Anti-CD, a B cell marker
Using immunohistochemical studies with 45R (B220), any physiological changes to spleen cells (eg, spleen tissue disruption) are within a vaguely defined B cell band that infiltrates established T cell areas. To determine if this is due to increased B cell presentation.

Using flow cytometric analysis of spleens from mice treated with DR4 protein, the DR4 protein was identified as ThB +, CD45R (B220) du.
Indicates whether the ratio of 11 B cells is specifically increased above that observed in control mice.

Similarly, the possible consequences of in vivo presentation of increased mature B cells is that serum I
g is a relative increase in titer. Therefore, IgM and IgA levels in serum are compared between buffer-treated and DR4 protein-treated mice.

The test described in this example tests activity on the DR4 protein. However, those skilled in the art will recognize that DR4 polynucleotides (eg, gene therapy), DR4
The exemplary studies can be readily modified to test the activity of agonists and / or antagonists of.

Example 8 T Cell Proliferation Assay A CD3-induced proliferation assay is performed on PBMC and measured by ³ H-thymidine incorporation. This assay is performed as follows. A 96-well plate was prepared using a monoclonal antibody against CD3 (HIT3a, Pharminge).
n) in 100 μl / well, or isotype matched control monoclonal antibody (B33.1) (0.05M bicarbonate buffer, pH 9.5)
(1 μg / ml) at 4 ° C. overnight, then wash three times with PBS. PBM
C was isolated from human peripheral blood by F / H gradient centrifugation and various concentrations of D
In RPMI containing 10% FCS and P / S in the presence of R4 protein,
Add to quadruplicate wells (5 × 10 ⁴ / well) of plate coated with monoclonal antibody (200 μl total volume). The relevant protein buffer and medium alone are controls. After 48 hours at 37 ° C., the plate is spun at 1000 rpm for 2 minutes, and 100 μl of the supernatant is removed, and if an effect on growth is observed, for measurement of IL-2 (or other cytokines) Stored at -20 ° C. The wells are supplemented with 100 μl of medium containing 0.5 μCi of ³ H-thymidine and incubated at 37 ° C. for 18-24 hours. Collect the wells, and ³ H-
Thymidine incorporation was used as an indicator of proliferation. Anti-CD3 alone is a positive control for proliferation. IL-2 (100 U / ml) is also used as a control to enhance proliferation. A control antibody that does not induce T cell proliferation is used as a negative control for the effect of the DR4 protein.

The study described in this example tests the activity of DR4 protein. However, one of skill in the art can readily modify the illustrated studies to test the activity of DR4 polynucleotides (eg, gene therapy), DR4 agonists and / or antagonists.

Example 9: Effect of DR4 on MHC Class II, costimulatory and adhesion molecule expression, and cell differentiation of monocytes and monocyte-derived human dendritic cells Dendritic cells are found in peripheral blood Produced by expansion of proliferative progenitors: adherent PBMC or isolated monocyte fractions are cultured for 7-10 days with GM-CSF (50 ng / ml) and IL-4 (20 ng / ml). These dendritic cells have the characteristic phenotype of immature cells (expression of CD1, CD80, CD86, CD40 and MHC class II antigens). Treatment with activators such as TNF-α rapidly changes the surface phenotype (MHC class I and II, increased expression of costimulatory and adhesion molecules, down-regulation of FCγRII, up-regulation of CD83) Is generated. These changes correlate with increased antigen-presenting capacity and functional maturation of dendritic cells.

[0501] FACS analysis of surface antigens is performed as follows. Cells were treated with increasing concentrations of DR4 or LPS (positive control) for 1-3 days, 1% BSA and 0.02
Wash with PBS containing mM sodium azide, then with a 1:20 dilution of the appropriate FITC- or PE-labeled monoclonal antibody,
Incubate at 4 ° C for 30 minutes. After further washing, the labeled cells were
Analyze by flow cytometry on a can (Becton Dickinson).

Effect on Cytokine Production Cytokines produced by dendritic cells, particularly IL-12, are important in initiating a T-cell dependent immune response. IL-12 strongly influences the development of a Th1 helper T cell immune response and induces cytotoxic and NK cell functions. The IL-12 release is measured using an ELISA as follows. Dendritic cells (10 ⁶ / ml) are treated with increasing concentrations of DR4 for 24 hours. LPS (10
0 ng / ml) is added to the cell culture as a positive control. The supernatant from the cell culture is then collected and a commercially available ELISA kit (eg, R & D)
Analyze for IL-12 content using Systems (Minneapolis, MN). Use the standard protocol provided in the kit.

Effects on the Expression of MHC Class II, Co-stimulatory and Adhesion Molecules Three major families of cell surface antigens (adhesion molecules, molecules involved in antigen presentation,
And Fc receptors) can be identified on monocytes. Modulation of the expression of MHC class II antigens and other costimulatory molecules (eg, B7 and ICAM-1) can result in changes in the ability of monocytes to present antigen and to induce T cell activation. Increased expression of the Fc receptor may correlate with improved monocyte cytotoxic activity, cytokine release and phagocytosis.

[0504] FACS analysis is used to test for surface antigens as follows. Monocytes were treated with increasing concentrations of DR4 or LPS (positive control) for 1-5 days, 1%
Wash with PBS containing BSA and 0.02 mM sodium azide, then incubate with the appropriate FITC- or PE-labeled monoclonal antibody at a 1:20 dilution for 30 minutes at 4 ° C. After a further wash, the labeled cells are analyzed by flow cytometry on a FACScan (Becton Dickinson).

(Monocyte Activation and / or Increase in Survival) Molecules that activate (or inactivate) monocytes and / or increase the survival of monocytes (or decrease the survival of monocytes) Assays for are known in the art and can be routinely applied to determine whether a molecule of the present invention functions as a monocyte inhibitor or activator. DR4, D
R4 agonists or antagonists can be screened using the three assays described below. For each of these assays, peripheral blood mononuclear cells (PBMCs) were centrifuged through Histopaque gradients (Sigma) by single donor leukocyte packs (America).
n Red Cross, Baltimore, MD). Monocytes are counterflowed by centrifugal elutriation (counterflow centrifuga).
Isolation from PBMCs by elution.

1. Monocyte Survival Assay Human peripheral blood monocytes gradually lose viability when cultured in the absence of serum or other stimuli. Their death results from an internally regulated process (apoptosis). Addition of an activator, such as TNF-α, to the culture dramatically improves cell survival and prevents DNA fragmentation. Apoptosis is measured using propidium iodide (PI) staining as follows. Monocytes are cultured for 48 hours in serum-free medium (positive control) in polypropylene tubes in the presence of 100 ng / ml TNF-α (negative control) and various concentrations of DR4. Cells are suspended at a concentration of 2 × 10 ⁶ / ml in PBS containing PI at a final concentration of 5 μg / ml, and then incubated for 5 minutes at room temperature before FACScan analysis. PI incorporation has been shown to correlate with DNA fragmentation in this experimental paradigm.

(2. Effect on cytokine release) The important functions of monocytes / macrophages are
Their regulatory activity on other cell populations of the immune system through the release of cytokines after stimulation. An ELISA for measuring cytokine release is performed as follows. Human monocytes are incubated at a density of 5 × 10 ⁵ cells / ml with increasing concentrations of DR4 and under the same conditions but without DR4. IL-
For the production of 12, the cells were transformed with IFN-γ (100 U /
primed overnight with 1 ml). Then LPS (10 ng / ml) is added. Conditioned media is collected after 24 hours and stored frozen until use. The measurement of TNF-α, IL-10, MCP-1 and IL-8 was then performed by a commercial ELISA.
A kit (eg, R & D Systems Minneapolis, MN)
And applying standard protocols provided in the kit.

(3. Oxidative burst) Purified monocytes are plated in a 96-well plate at 2-1 × 10 ⁵ cells / well. Increasing concentrations of DR4 were added to the wells in a total volume of 0.2 ml of culture medium (RPMI 1640 + 1).
0% FCS, glutamine and antibiotics). After 3 days of incubation, the plate is centrifuged and the medium is removed from the wells. In a monolayer of macrophages, 0.2 ml of a phenol red solution (140 m
M NaCl, 10 mM potassium phosphate buffer pH 7.0, 5.5 mM dextrose, 0.56 mM phenol red and 19 U / ml HRPO)
Add with stimulant (200 nM PMA). Plate at 37 ° C for 2
Incubate for hours and stop the reaction by adding 20 μl per well of 1 N NaOH. The absorbance is read at 610 nm. To calculate the amount of H ₂ O ₂ produced by the macrophages, a standard curve of a known molar concentration of the H ₂ O ₂ solution is performed for each experiment.

The study described in this example tests activity on the DR4 protein. However, those skilled in the art will appreciate that DR4 polynucleotides (eg, gene therapy), DR4
The exemplified studies can be readily modified to test the activity of the four agonists and / or antagonists.

Example 10: Effect of DR4 on Proliferation of Vascular Endothelial Cells On day 1, human umbilical vein endothelial cells (HUVEC) were treated with 4% fetal bovine serum (FBS).
), 16 units / ml heparin, and 50 units / ml endothelial cell growth supplement (ECGS, Biotechnique, Inc.) in M199 medium at a density of 2-5 x 10 ⁴ cells per 35 mm dish. On day 2, the medium is replaced with M199 containing 10% FBS, 8 units / ml heparin. The DR4 protein of SEQ ID NO: 2 and a positive control (eg, V
EGF and basic FGF (bFGF)) are added at various concentrations. On days 4 and 6, the medium is changed. On day 8, cell numbers are determined using a Coulter Counter.

The increase in HUVEC cell numbers after treatment with DR4 indicates that DR4 can proliferate vascular endothelial cells.

The study described in this example tests activity on the DR4 protein. However, those skilled in the art will appreciate the activity of DR4 polynucleotides (eg, gene therapy).
The illustrated studies can be readily modified to test agonists and / or antagonists of DR4.

Example 11 Stimulating Effect of DR4 on Proliferation of Vascular Endothelial Cells To evaluate the mitogenic activity of growth factors, a colorimetric MTS (3 -(4,5-Dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) 2H-tetrazolium) assay was performed (CellTiter).
96 AQ, Promega). Cells are seeded in 96-well plates (5,000 cells / well) in 0.1 ml serum-supplemented medium and allowed to attach overnight. 0.
After 12 hours of serum starvation in 5% FBS, conditions (bFGF, VEGF ₁₆₅ or DR4 in 0.5% FBS) with or without heparin (8 U / ml) are added to the wells for 48 hours. 20 mg of MTS / PMS mixture per well (
1: 0.05) and allowed to incubate at 37 ° C. for 1 hour, after which E
Measure absorbance at 490 nm with a LISA plate reader. The background absorbance of the control wells (with medium, no cells) is subtracted and 7 wells are run in parallel for each condition. Leak et al., In V.
in vitro Cell. Dev. Biol. 30A: 512-518 (1994)
checking ...

The study described in this example tested the activity of the DR4 protein. However, one of skill in the art can readily modify the illustrated studies to test the activity of DR4 polynucleotides (eg, gene therapy), DR4 agonists, and / or antagonists.

Example 12 Inhibition of PDGF-Induced Vascular Smooth Muscle Cell Growth Stimulating Effect HAoSMC proliferation can be measured, for example, by BrdUrd incorporation. Briefly, subconfluent stationary phase cells growing on 4-chamber slides were converted to C
Transfect with RP or FITC-labeled AT2-3LP. The cells are then pulsed with 10% bovine serum and 6 mg / ml BrdUrd. 24
After time, immunocytochemistry is performed by using the BrdUrd staining kit (Zymed Laboratories). Briefly, the cells are incubated with a biotinylated mouse anti-BrdUrd antibody for 2 hours at 4 ° C. after exposure to a denaturing solution, and then with streptavidin-peroxidase and diaminobenzidine. After counterstaining with hematoxylin, the cells are mounted for microscopic examination and BrdUrd-positive cells are counted. BrdU
The rd index is calculated as the ratio of BrdUrd-positive cells to the total cell number. In addition, simultaneous detection of BrdUrd staining (nuclei) and FITC incorporation (cytoplasm) is performed for individual cells by the simultaneous use of light and dark UV fluorescent illumination. Hayashida et al. Biol. Chem. 6: 271 (36
): 21985-19992 (1996).

The study described in this example tested the activity of the DR4 protein. However, one of skill in the art can readily modify the illustrated studies to test the activity of DR4 polynucleotides (eg, gene therapy), DR4 agonists, and / or antagonists.

Example 13 Stimulation of Endothelial Migration This example is used to explore the potential of DR4 to stimulate lymphatic endothelial cell migration.

The endothelial cell migration assay is performed using a 48-well microchemotaxis chamber (Neuroprobe Inc., Cabin John, MD; Falk).
, W et al., "A48 well microchemotaxis asse."
mbly for rapid and accurate measure
ent of leukocyte migration " Immunol
official Methods 33: 239-247 (1980)). Polycarbonate filters without polyvinylpyrrolidone, which have a pore size of 8 μm (Nucleopore Corp. Cambridge, Mass.) Are coated with 0.1% gelatin for at least 6 hours at room temperature and dried under sterile air. The test substance is diluted to the appropriate concentration in M199 supplemented with 0.25% bovine serum albumin (BSA) and a final dilution of 25 μl is placed in the bottom chamber of the modified Boyden apparatus. HUVE at early passage (2-6), less than confluent
The C or BMEC culture is washed and trypsinized for the minimum time necessary to achieve cell detachment. After placing the filter between the bottom and top chambers, 2.5 × suspended in 50 μl M199 containing 1% FBS.
Seed 10 ⁵ cells in the upper compartment. The device is then switched to 5% CO2
Incubate at 37 ° C. for 5 hours in a humidified chamber containing to allow the cells to migrate.
After an incubation period, the filter is removed and the top side of the filter containing non-migrating cells is scraped with a rubber policeman. The filter was fixed with methanol and the Giemsa solution (Diff-
Quick, Baxter, McGraw Park, IL). Migration is quantified by counting cells in three random high power fields (40 ×) in each well. Then, all groups are executed in quadruplicate.

The study described in this example tests the activity of DR4 protein. However, those skilled in the art will recognize that the activity of DR4 polynucleotides (eg, gene therapy), DR4
The illustrated studies can be readily modified to test for agonists and / or antagonists of.

Example 14: Stimulation of Nitric Oxide Production by Endothelial Cells Nitric oxide released by vascular endothelium is believed to be a mediator of vascular endothelial relaxation. Thus, DR4 activity can be assayed by measuring nitric oxide production by endothelial cells in response to DR4.

Nitric oxide was starved for 24 hours, followed by various levels of positive control (
For example, confluent microvascular endothelial cells are measured in 96-well plates after 4 hours of exposure to VEGF-1) and DR4. Nitric oxide in the medium,
The total amount of nitrous acid after the reduction of nitric acid derived from nitric oxide by nitrate reductase is measured by using the Griess reagent. The effect of DR4 on nitric oxide release is tested in HUVEC.

Briefly, NO release from cultured HUVEC monolayers was measured using a NO meter (Iso
-NO, World Precision Instruments Inc.
) Is measured using a NO-specific polarographic electrode connected to the above. Calibration of the NO element is performed according to the following equation: 2KNO ₂ + 2KI + 2H ₂ SO ₄ 62NO + I ₂ + 2H ₂ O + 2K ₂ SO _{4 A} standard calibration curve is prepared with graded concentrations of KN in a calibration solution containing KI and H ₂ SO _4.
O ₂ (0, 5, 10, 25, 50, 100, 250, and 500 nmol / L
). The specificity of the Iso-NO electrode for NO is determined in advance by measuring NO from a sample NO gas. The culture medium is removed and HUVECs are washed with Dulbecco's phosphate buffered saline for 2 hours.
Wash twice. The cells were then plated in a 6-well plate with 5 ml of filtered Kreb.
Batch with s-Henseleit solution and plate the cells at 37 ° C.
The slide warmer (Lab Line Instru
uments Inc. ). The NO sensor probe is inserted vertically into the well and the tip of the electrode is held 2 mm below the surface of the solution before adding different conditions. S-Nitrosoacetylpenicillamine (SNAP) is used as a positive control. The amount of NO released is expressed as picomoles per 1 × 10 ⁶ endothelial cells. All values reported are the average of 4-6 measurements in each group (number of cell culture wells). Leak et al., Biochem. and Biophys.
Res. Comm. 217: 96-105 (1995).

The study described in this example tests activity on the DR4 protein. However, one of skill in the art can readily modify the illustrated studies to test the activity of DR4 polynucleotides (eg, gene therapy), DR4 agonists and / or antagonists.

Example 15: Effect of DR4 on Cord Formation in Angiogenesis Another step in angiogenesis is the formation of cords (c) characterized by endothelial cell differentiation.
ord) formation. This bioassay measures the ability of microvascular endothelial cells to form capillary-like structures (hollow structures) when cultured in vitro.

[0525] CADME (microvascular endothelial cells) was used as a proliferating cell (passage 2) as Cell
Applications Inc. Purchased from Cell Applica
cultivated in ADEME Growth Medium, and 5
Use at the passage. For the in vitro angiogenesis assay, the wells of a 48-well cell culture plate were prepared using Cell Applications' Attachme.
Coat using nt Factor Medium (200 ml / well) at 37 ° C. for 30 minutes. CADME was added to the coated wells at 7,500 cells /
Seed in wells and culture overnight in Growth Medium. The Growth Medium was then added to control buffer or DR4 (0.1-1
100 ng / ml) of Cell Applications'C
Replace with the medium, and culture the cells for an additional 48 hours. The number and length of capillary-like cords were determined using Boeckeler VI
Quantification is through the use of an A-170 video image analyzer. All assays are performed in triplicate.

[0526] Commercially available (R & D) VEGF (50 ng / ml) is used as a positive control. β-estradiol (1 ng / ml) is used as a negative control. An appropriate buffer (no protein) is also used as a control.

The study described in this example tests the activity of DR4 protein. However, one skilled in the art can readily modify the illustrated studies to test the activity of DR4 polynucleotides (eg, gene therapy), DR4 agonists, and / or antagonists.

Example 16 Angiogenesis Effect on Chick Chorioallantoic Membrane Chick chorioallantoic membrane (CAM) is a well-established system for testing angiogenesis. Angiogenesis in the CAM can be easily visualized and quantified. D
R4 may be tested for its ability to stimulate angiogenesis in the CAM.

[0527] White Leghorn chick (Gallus gallus) fertilized eggs and Japanese quail (Coturnix coturnix) are incubated at 37.8 ° C and 80% humidity. 16 day old chicken differentiated CAM and 13 day old quail embryos are studied in the following manner.

On the fourth day of development, a window is made in the eggshell of the chicken egg. The embryo is examined for normal development, and the eggs are sealed with Cellotape®. Add one more
Incubate until day 3. Thermanox cover glass (Nunc,
Naperville, IL) into disks about 5 mm in diameter. Dissolve the sterile and salt-free growth factor and DR4 in distilled water, and pipet approximately 3.3 mg / 5 ml to a disk. After air drying, apply the inverted disc to the CAM. Three days later, the specimens are fixed in 3% glutaraldehyde and 2% formaldehyde and rinsed with 0.12 M sodium cacodylate buffer. These were converted to a stereoscopic microscope [W
ild M8] and embedded for semi-thin and ultrathin sectioning as described above. Control is performed only with the carrier disk.

The study described in this example tests activity on the DR4 protein. However, one skilled in the art can readily modify the illustrated studies to test the activity of DR4 polynucleotides (eg, gene therapy), DR4 agonists, and / or antagonists.

Example 17 Angiogenesis Assay Using Matrigel Implants in Mice To establish an in vivo model for angiogenesis to test DR4 protein activity, mice and rats were given 20 mg BSA. (Negative control) A methylcellulose disc containing either 1 mg of DR4 or 0.5 mg of VEGF-1 (positive control) is implanted subcutaneously. The negative control disc should contain little angiogenesis, while the positive control disc should show signs of angiogenesis.

The study described in this example tests activity on the DR4 protein. However, one skilled in the art can readily modify the illustrated studies to test the activity of DR4 polynucleotides (eg, gene therapy), DR4 agonists, and / or antagonists.

Example 18: Rescue of ischemia in a rabbit lower limb model To study the effect of DR4 on ischemia in vivo, a rabbit hind limb ischemia model was described previously (Takeshita, S et al., Am J. Pathol
147: 1649-1660 (1995)) by surgical removal of one femoral artery. Resection of the femoral artery results in retrograde propagation of thrombus and occlusion of the external iliac artery. As a result, blood flow to the ischemic limb is dependent on collateral vessels arising from the internal iliac artery (Takeshita, S. et al., Am J. Pathol 147: 1).
649-1660 (1995)). At 10-day intervals, allow post-operative recovery of rabbits and development of endogenous collateral vessels. Ten days after surgery (day 0), after performing baseline angiography, the internal iliac artery of the ischemic limb was removed (Riessen, R. et al., Hum Gene Ther. 4: 749-758 (1993)). ; Lecle
rc, G .; J. et al. Clin. Invest. 90: 936-944 (1992)
Transfect using 500 mg of the naked DR4 expression plasmid by the arterial gene transfer technique using a hydrogel-coated balloon catheter as described in)). When DR4 is used for treatment, a single bolus of 500 mg DR4 protein or control is delivered through the infusion catheter for 1 minute into the internal iliac artery of the ischemic limb. On day 30, various parameters are measured in these rabbits: (a) BP ratio-systolic blood pressure of ischemic limb versus systolic blood pressure of normal limb; (b) blood flow and reflux-arrest. FL (blood flow during the non-dilated state) and max FL (blood flow during the fully dilated state (also an indirect measure of vascular volume)) and regurgitation are reflected in the ratio of max FL: stop FL. (C) angiographic values (angiog);
raphic Score)-measured by collateral angiography. Values are determined by the percentage of circles in the overlaid grid (divided by the total number m of rabbit thighs, using transversal opacifying arteries); (d) capillary density-light microscopy sections from hind limbs Number of collateral capillaries determined in.

The study described in this example tests activity on the DR4 protein. However, one of skill in the art can readily modify the illustrated studies to test the activity of DR4 polynucleotides (gene therapy), DR4 agonists and / or antagonists.

Example 19 Diabetic Mice and Glucocorticoid-Injured Wound Healing Model A. Diabetic db + / db + Mouse Model To demonstrate that DR4 promotes the healing process, genetically diabetic mice with wound healing Use the model. Full thickness (full) in db + / db + mice
(thickness) wound healing model is a well-characterized, clinically relevant and reproducible model of wound healing. Healing of diabetic wounds depends on granulation tissue formation and re-epithelialization rather than contraction (Gartn
er, M .; H. J. et al. Surg. Res. 52: 389 (1992); Gre.
enhalgh, D .; G. FIG. Et al., Am. J. Pathol. 136: 1235 (1
990)).

This diabetic animal has many of the characteristic features observed in type II diabetes mellitus. Homozygous (db + / db +) mice have their normal heterozygous (
db + / + m) Obese compared to littermates. Mutant diabetic (db + / db +) mice have a single autosomal recessive mutation (db +) on chromosome 4 (Co
Leman et al., Proc. Natl. Acad. Sci. USA 77: 283
-293 (1982)). Animals exhibit polyphagia, polydipsia, and polyuria. Mutant diabetic mice (db + / db +) have elevated blood glucose, increased or normal insulin levels, and suppressed cell-mediated immunity (Man
del et al. Immunol. 120: 1375 (1978); Debray
-Sachs, M .; Et al., Clin. Exp. Immunol. 51 (1): 1-
7 (1983); Leiter et al., Am. J. of Pathol. 114: 4
6-55 (1985)). Peripheral neuropathy, myocardial complications, and microvascular injury, basement membrane thickening, and glomerular filtration abnormalities have been described in these animals (N
orido, F .; Et al., Exp. Neurol. 83 (2): 221-232 (1
984); Robertson et al., Diabetes 29 (1): 60-67.
(1980); Giacomelli et al., Lab Invest. 40 (4):
460-473 (1979); Coleman, D .; L. , Diabetes
31 (supplement): 1-6 (1982)). These homozygous diabetic mice develop hyperglycemia, which is similar to human type II diabetes and is resistant to insulin (Mandel et al., J. Immunol. 120: 1375-137).
7 (1978)).

The characteristics observed in these animals indicate that the healing in this model may be similar to the healing observed in human diabetes (Greenhalgh et al., Am. J. of Pathol. 136: 1235-1246 ( 1990))
.

[0539] Genetically diabetic female C57BL / KsJ (db + / db +) mice, and their non-diabetic (db + / + m) heterozygous littermates are used in this study (J
ackson Laboratories). These animals were purchased at the age of 6 weeks,
And these animals are 8 weeks old at the start of the study. Animals are housed individually and are given food and water ad libitum. All operations are performed using aseptic techniques. This experiment was performed as described in Human Genome Sciences, Inc. Insit
universal Animal Care and Use Committe
e and the Guidelines for the Care an
d. Perform according to the rules and guidelines of Use of Laboratory Animals.

The wound protocol was performed using previously reported methods (Tsuboi, R. and Rif).
Kin, D.C. B. , J. et al. Exp. Med. 172: 245-251 (1990).
). Briefly, on the day of wounding, animals were treated with A dissolved in deionized water.
Anesthetize with intraperitoneal injection of vertin (0.01 mg / mL), 2,2,2-tribromoethanol and 2-methyl-2-butanol. The dorsal area of the animal is shaved and the skin is washed with a 70% ethanol solution and iodine. The surgical area is dried with sterile gauze before wounding. The 8 mm full-thickness wound is then
Prepared using a Keyes tissue punch. Immediately after wounding, the surrounding skin is gently stretched to remove wound enlargement. The wound is allowed to open during the experiment. Application of treatment is given topically for 5 consecutive days starting from the day of wounding. Prior to treatment, the wound is gently washed with sterile saline and gauze sponge.

The wounds are visually inspected and photographed at a fixed distance on the day of surgery and two days thereafter. Wound closure is determined by daily measurements on days 1-5 and day 8. Wounds are measured horizontally and vertically using a Calibrated Jameson caliper. A wound is considered healed when granulation tissue is no longer visible and continuous epithelium covers the wound.

DR4 was administered in a vehicle at different dose ranges (4 per wound per day) for 8 days.
mg-500 mg) of DR4. Vehicle control groups include
50 mL of vehicle solution was given.

The animals are euthanized on day 8 with an intraperitoneal injection of sodium pentobarbital (300 mg / kg). The wound and surrounding skin are then collected for histology and immunohistochemistry. Tissue specimens are placed in 10% neutral buffered formalin in a tissue cassette between biopsy sponges for further processing.

Three groups of 10 animals each (5 diabetic and 5 non-diabetic controls) are evaluated: 1) vehicle placebo control, 2) DR4, and 3) positive control.

[0545] Wound closure is analyzed by measuring the area on the vertical and horizontal axes and obtaining the sum of the areas of the wound. Contraction is then assessed by establishing the difference between the area of the original wound (day 0) and the area after treatment (day 8). The wound area on day 1 was 64 mm ² (size corresponding to a skin punch). The calculation was performed using the following formula: ([open area on day 8]-[open area on day 1]) / [open area on day 1].

Specimens are fixed in 10% buffered formalin and paraffin embedded masses are cut perpendicular to the wound surface (5 mm) and cut using a Reichert-Jung microtome. Routine hematoxylin-eosin (H & E) staining is performed on transverse sections of bisected wounds. Histological examination of the wound is used to assess whether the healing process and morphological appearance of the repaired skin has been altered by treatment with DR4. This assessment includes cell accumulation, inflammatory cells, capillaries, fibroblasts,
Including re-epithelialization, and verification of the presence of epidermal maturation (Greenhalgh, D .;
G. FIG. Et al., Am. J. Pathol. 136: 1235 (1990)). Blind observer with graduated lens micrometer
Is used.

[0547] Tissue sections are also immunohistochemically stained with a polyclonal rabbit anti-human keratin antibody using the ABC Elite detection system. Human skin is used as a positive tissue control, while non-immune IgG is used as a negative control.
Keratinocyte proliferation is determined by assessing the extent of wound re-epithelialization using a graduated lens micrometer.

Proliferating cell nuclear antigen / cyclin (PCNA) in skin specimens was analyzed by ABC El
Demonstrated by using anti-PCNA antibody (1:50) with an item detection system. Human colon cancer can serve as a positive tissue control, and human brain tissue can be used as a negative tissue control. Each specimen contains sections with primary antibody shedding and replacement with non-immune mouse IgG. The ranking of these sections is based on the degree of growth on a scale of 0-8 (lower scale reflecting slight growth to higher reflecting strong growth).

The experimental data is analyzed using a one-tailed t-test. A p-value of <0.05 is considered significant.

B. Steroid Impaired Rat Model Inhibition of wound healing by steroids has been well documented in various in vitro and in vivo systems (Wahl, SM, Glucocorticoi).
ds and Wound hearing: Anti-Inflammato
ry Steroid Action: Basic and Clinical
Aspects. 280-302 (1989); Wahl, S .; M. J
. Immunol. 115: 476-481 (1975); Werb et al. E
xp. Med. 147: 1684-1694 (1978)). Glucocorticoids inhibit angiogenesis, vascular permeability (Ebert, RH, et al., An.
Intern. Med. 37: 701-705 (1952)), fibroblast proliferation, and collagen synthesis (Beck, L.S., et al., Gross Factors).
. 5: 295-304 (1991); Haynes, B .; F. J. et al. Clin
. Invest. 61: 703-797 (1978)), as well as producing a transient decrease in circulating monocytes (Haynes, BF et al., J.
. Clin. Invest. 61: 703-797 (1978); Wahl, S.
. M. , Glucocorticoids and wound healin
g: Antiinflammatory Steroid Action: Ba
sic and Clinical Aspects, Academic Pr
Ess, New York, pp. 280-302 (1989)). Systemic administration of steroids for impaired wound healing is a well-established phenomenon in rats (Beck, LS et al., Growth Fa.
ctors. 5: 295-304 (1991); Haynes, B .; F. Et al.,
J. Clin. Invest. 61: 703-797 (1978); Wahl.
, S .; M. , Glucocorticoids and wound sound
ing: Antiinflammatory Steroid Action:
Basic and Clinical Aspects, Academic
Press, New York, pp. 280-302 (1989); Pierce.
Proc. Natl. Acad. Sci. USA 86: 2229-223
3 (1989)).

To demonstrate that DR4 can promote the healing process, evaluate the effects of multiple topical applications of DR4 on full thickness excised skin wounds in rats where healing is impaired by systemic administration of methylprednisolone .

Young adult male Sprague Dawley rats weighing 250-300 g
) (Charles River Laboratories) is used in this experiment. The animals are purchased at the age of 8 weeks. At the start of the study, he is 9 weeks old. The healing response of rats was determined at the time of wounding by systemic administration of methylprednisolone (17 mg / k
g / rat, intramuscular). Animals are housed individually and are given food and water ad libitum. All operations are performed using aseptic techniques. Huma
n Institutional of Genome Sciences, Inc
Animal Care and Use Committee and th
e Guidelines for the Care and Use of
Perform according to Laboratory Animal Rules and guidelines.

The wound protocol follows Section A above. On the day of wounding, the animals are treated with ketamine (5 m
g / kg) and xylazine (5 mg / kg) by intramuscular injection. The dorsal area of the animal is shaved and the skin is washed with a 70% ethanol and iodine solution. The surgical area is dried with sterile gauze before wounding. An 8 mm full thickness wound is created using a Keyes tissue punch. During the experiment, open the left side of the wound. Application of the test substance is given topically once daily for 7 consecutive days, starting from the day of wounding and then methylmethylprednisolone administration. Prior to treatment, the wound is gently washed with sterile saline and gauze sponge.

The wounds are visually inspected and photographed at a fixed distance on the day of wounding and at the end of treatment. Wound closure is determined by daily measurements on days 1-5 and measurements on day 8. Wounds are measured horizontally and vertically using a Calibrated Jameson caliper. A wound is considered healed when granulation tissue is no longer visible and the wound is covered by continuous epithelium.

[0555] DR4 was administered in a vehicle at different dose ranges (4 wounds per day per day) for 8 days.
mg-500 mg) of DR4. Vehicle control group is 5
Received 0 mL of vehicle solution.

The animals are euthanized on day 8 with an intraperitoneal injection of sodium pentobarbital (300 mg / kg). The wound and surrounding skin are then collected for histology. Tissue specimens are placed in 10% neutral buffered formalin in a tissue cassette between biopsy sponges for further processing.

Four groups of 10 animals each (5 with methylprednisolone and 5 without glucocorticoid) are evaluated: 1) untreated group, 2) vehicle placebo control, and 3) DR4 treated. group.

The wound closure is analyzed by measuring the area on the vertical and horizontal axes and obtaining the sum of the wound areas. Closure is then assessed by establishing the difference between the area of the original wound (day 0) and the area after treatment (day 8). The wound area on day 1 is 64 mm ² (size corresponding to a skin punch). The calculation is performed using the following formula: [open area on day 8]-[open area on day 1] / [open area on day 1].

The specimen is fixed in 10% buffered formalin, and the paraffin-embedded mass is cut perpendicular to the wound surface (5 mm) and cut using an Olympus microtome. Routine hematoxylin-eosin (H & E) staining is performed on transverse sections of bisected wounds. Histological examination of the wound makes it possible to assess whether the healing process and morphological appearance of the repaired skin has been improved by treatment with DR4. A blinded observer (b)
used to determine the distance of the wound gap.

The experimental data is analyzed using a one-tailed t-test. A p-value of <0.05 is considered significant.

The study described in this example tests activity on the DR4 protein. However, one of ordinary skill in the art can readily modify the illustrated studies to test the activity of DR4 polynucleotides (eg, gene therapy), agonists, and / or antagonists of DR4.

Example 20 Lymphedema Animal Model The purpose of this experimental approach was to create lymphatic vessels (lymph
a suitable and consistent model of lymphedema to test the therapeutic effect of DR4 in reestablishment of the lymphatic circulatory system. Efficacy is measured by swelling volume of the affected limb, quantification of the amount of lymphatic vessels, amount of total plasma protein, and histopathology. Acute lymphedema is 7-10
Observed for days. Perhaps more importantly, the chronic progression of edema lasts up to 3-4 weeks.

Before starting surgery, blood samples are drawn for protein concentration analysis.
Male rats (weighing about 350 g) are dosed with pentobarbital. Next, the right limb is shaved from the knee to the hip joint. The shaved area is wiped with gauze soaked in 70% EtOH. Blood is drawn for serum total protein test. After measuring the circumference and volume, two measurement levels (0.5 cm above the heel, midpoint of the back of the foot) are marked, and the foot is injected with dye. On both right and left dorsal endothelium,
Inject 0.05 ml of 1% Evan's Blue. Circumferential and volume measurements are then made after injection of the dye into the paw.

Using the knee joint as a landmark, a mid-limb inguinal incision is made annularly so that the position of the femoral vessels can be confirmed. The flap is dissected and separated using forceps and hemostat. After locating the femoral vessels, locate the lymph vessels that run alongside and below the vessels. The major lymphatic vessels in this area are then electrocoagulated or sutured.

Using a microscope, the muscles in the back of the limb (semitendinosis)
And near the adductor muscle). The location of the popliteal lymph nodes is then confirmed. The two proximal and two distal lymph vessels and the distal blood supply of the popliteal node are then positioned and ligated with sutures. The popliteal lymph nodes and any accompanying fat tissue are then removed by cutting connective tissue.

Care should be taken to control any minor bleeding that occurs in this procedure. After occlusion of the lymphatic vessels, the skin flap was replaced with liquid skin (Vetbond) (AJ Buck).
Block by using. The separate skin margins are sealed to the underlying muscle tissue, leaving a gap of about 0.5 cm around the leg. The skin may also be anchored when necessary by suturing to the underlying muscle.

Animals are housed individually using mesh (no bedding) to avoid infection. The recovered animals are checked daily through the optimal edema peak. This peak typically occurs for up to 5-7 days. The plateau edema peak is then observed. To assess the intensity of lymphedema, the circumference and volume of two designated locations on each limb are measured before manipulation and daily for 7 days. The effect of plasma proteins on lymphedema is determined and it is also investigated whether protein analysis is a useful test perimeter. The weights of both the control limb and the edema limb are evaluated in two places. The analysis is performed in a blind manner.

[0568] Circumference measurement: Under a short-term gas anesthesia to prevent limb movement, the limb circumference is measured using a cloth tape measure. Measurements are taken by two different persons at the talus and dorsal foot, and then these two readings are averaged. Readings are obtained from both the control and edema paw.

[0566] Volume measurement: On the day of surgery, animals are anesthetized with pentobarbital and tested prior to surgery. For daily volume measurements, animals are placed under short-term halothane anesthesia (rapid fixation and quick recovery), both legs are shaved, and legs are marked equally with a water-resistant marker . The legs are first dipped in water, then dipped into the device to each marked level, and then the Buxco edema software (Chen / Vic
tor). The data is recorded by one person, while the other
Dip the leg down to the marked area.

Blood-plasma protein measurement: Blood is removed, centrifuged, and serum separated before surgery, and then concluded for total protein and Ca 2+ comparison.

[0571] Limb Weight Comparison: After blood is drawn, the animals are prepared for tissue collection.
The limb is cut with quiltine, then both the experimental and control legs are ligated and cut and weighed. A second weighing is performed by removing the tibio-cacaneal joint and weighing the foot.

Histological Preparation: Transverse muscle located behind the knee (popliteal) area was cut out and placed in a metal mold, filled with freezeGel, immersed in cold methyl butane and at -80EC until cut into labeled sample bags. To place. At the time of the cut, the muscle is observed for lymphatic vessels under a fluorescent microscope. Other immuno / histological methods are currently being evaluated.

The study described in this example tested activity on the DR4 protein. However, one of ordinary skill in the art can readily modify the illustrated studies to test the activity of DR4 polynucleotides (eg, gene therapy), agonists, and / or antagonists of DR4.

Example 21 Production of Antibodies A. Hybridoma Technology Antibodies of the invention can be prepared by various methods (Current Pro
tocols, see Chapter 2). As one example of such a method, DR
Cells expressing 4 are administered to animals to induce the production of serum containing polyclonal antibodies. In a preferred method, a preparation of the DR4 protein is prepared and purified to be substantially free of native contaminants. Such a preparation is then introduced into an animal to produce a higher specific activity polyclonal antiserum.

[0575] Monoclonal antibodies specific for protein DR4 can be prepared using hybridoma technology (Koehler et al., Nature 256: 495 (19
75); Koehler et al., Eur. J. Immunol. 6: 511 (197)
6); Koehler et al., Eur. J. Immunol. 6: 292 (1976)
); Hammerling et al .: Monoclonal Antibodies.
and T-Cell Hybridomas, Elsevier, N.W. Y. ,
563-681 (1981)). Generally, animals (preferably mice) are
Immunize with R4 polypeptide, or more preferably, with secreted DR4 polypeptide expressing cells. Such polypeptide expressing cells are supplemented with any suitable tissue culture medium, preferably 10% fetal calf serum (inactivated at about 56 ° C.), and contain about 10 g / l of non-essential amino acids, about 1 2,000 U / ml penicillin,
And Earle's modified Eagle's medium supplemented with about 100 μg / ml streptomycin).

The splenocytes of such mice are extracted and fused with a suitable myeloma cell line. Any suitable myeloma cell line can be used according to the present invention;
It is preferred to use the parental myeloma cell line (SP2O) available from C. After fusion, the resulting hybridoma cells are selectively maintained in HAT medium and then expanded to Wands et al. (Gastroenterology 80: 225-232 (1
Cloning by limiting dilution as described in 981)). The hybridoma cells resulting from such a selection are then assayed to identify clones that secrete antibodies capable of binding the DR4 polypeptide.

Alternatively, additional antibodies capable of binding the DR4 polypeptide can be generated in a two-step procedure using anti-idiotypic antibodies. Such methods take advantage of the fact that the antibody itself is an antigen, and thus it is possible to obtain an antibody that binds to a second antibody. According to this method, an animal, preferably a mouse, is immunized using a protein-specific antibody. The spleen cells of such animals are then used to produce hybridoma cells. The hybridoma cells are then screened to identify a clone that produces an antibody that can be blocked by DR4 for its ability to bind to a DR4 protein-specific antibody. Such an antibody is D
It includes anti-idiotypic antibodies to the R4 protein-specific antibody and can be used to immunize animals to induce the formation of additional DR4 protein-specific antibodies.

For in vivo use of antibodies in humans, the antibodies are “humanized”. Such antibodies can be produced by using a genetic construct derived from a hybridoma cell producing the monoclonal antibody described above. Methods for producing chimeric and humanized antibodies are known in the art (for a review, see Mor)
rison, Science 229: 1202 (1985); Oi et al., Bio.
Techniques 4: 214 (1986); Cabilly et al., U.S. Patent No. 4,816,567; Taniguchi et al., EP 171496; Mor.
Rison et al., EP 173494; Neuberger et al., WO 86015.
33; Robinson et al., WO 8702671; Boulianne et al., N.
Nature 312: 643 (1984); Neuberger et al., Natu.
re 314: 268 (1985)).

B. Isolation of Antibody Fragments Against DR4-V1 and DR4 from a Library of scFvs The naturally-occurring V genes isolated from human PBLs were isolated from exposed and unexposed donors. May be constructed into large libraries of antibody fragments reactive with the polypeptides of the invention (see, eg, US Pat. No. 5,885,7).
No. 93, which is hereby incorporated by reference in its entirety).

(Library Rescue) As described in WO 92/01047, scFvs was synthesized from human PBL RNA.
Build a library for To rescue phage displaying antibody fragments, approximately 10 ⁹ E. coli harboring phagemids were rescued. E. coli, 50m
1 × 2 × TY (containing 1% glucose and 100 μg / ml ampicillin) (2 × TY-AMP-GLU) and inoculate 0.8 O.L. with shaking.
D. Propagate to growth. Using 5 ml of this culture, 50 ml of 2 × TY-AMP
It was inoculated into -GLU, 2 × 10 ⁸ TU of Δ gene 3 helper (M13 gene III
, WO92 / 01047) and the cultures are shaken without shaking for 3 hours.
Incubate at 7 ° C for 45 minutes and then at 37 ° C with shaking for 45 minutes. The culture is incubated at 4000 r. p. m. And pellet the pellet in 2 liters of 2 × TY (100 μg / ml ampicillin and 50 μl).
(containing μg / ml kanamycin) and grow overnight. Phage is prepared as described in WO 92/01047.

The M13 gene III is prepared as follows: M13 gene III helper phage does not encode gene III protein. Therefore, phage displaying antibody fragments (phagemids) have a greater binding avidity for the antigen. During phage morphogenesis, infectious M13 gene III particles are generated by growing helper phage in cells carrying a pUC19 derivative that supplies the wild-type gene III protein. Cultures were grown 3 times without shaking.
Incubate at 7 ° C. for 1 hour, then incubate at 37 ° C. for another hour with shaking. The cells are pelleted (IEC-Centra 8, 400r.
p. m. / Min for 10 minutes), 100 μg / ml ampicillin and 25 μg / ml
2 x TY broth containing 2 ml of kanamycin (2 x TY-AMP-KAN) 3
Resuspended in 00 ml and grown overnight at 37 ° C. with shaking. The phage particles were purified and concentrated from the culture medium by two PEG precipitations (Sambrook et al., 1990), resuspended in 2 ml PBS, and passed through a 0.45 μm filter (Minisart NML; Sartorius) to about 10 ¹³ A final concentration of transduction units / ml (ampicillin resistant clone) is obtained.

(Panning of Library) Immunotubes (Nunc) was prepared by using 100 mg of the polypeptide of the present invention.
Coat overnight in PBS with 4 ml of either 10 / ml or 10 mg / ml. Tubes are blocked with 2% Marvel-PBS at 37 ° C. for 2 hours, then washed three times in PBS. Approximately 10 ¹³ TU of phage is applied to the tube and incubated at room temperature for 30 minutes while tilting up and down on a rotating wheel and then allowed to stand for an additional 1.5 hours. Tube is PBS 0.1% Twe
Wash 10 times with en-20 and 10 times with PBS. The phage was eluted by adding 1 ml of 100 mM triethylamine and spinning up and down on a turntable for 15 minutes, after which 0.5 ml of 1.0 M Tris-HCl was added to the solution.
Neutralize immediately at pH 7.4. The eluted phages were then combined with bacteria with 37
C. for 30 minutes, using phage with 10 ml of mid-log phase E. coli. E. coli TG1. The E.C. E. coli is plated on a TYE plate containing 1% glucose and 100 μg / ml ampicillin. The resulting bacterial library is then rescued with M13 gene III helper phage as described above to prepare phage for subsequent rounds of selection. This process was then repeated for all four times of affinity purification, with the third and fourth times washing the tubes with PBS, 0.1% Tw.
Increase to 20 times with een-20 and 20 times with PBS.

Binder Characterization Using phage eluted from the third and fourth rounds of selection, E. coli coli
HB 2151 is infected and soluble scFv is generated from a single colony for the assay (Marks et al., 1991). 50 mM bicarbonate, pH 9.
ELISA is performed using microtiter plates coated with any of the polypeptides of the present invention in 6 at 10 pg / ml. Positive clones in the ELISA are further characterized by PCR fingerprinting (see, eg, WO 92/01047), followed by sequencing.

Example 22: Tissue distribution of DR4 gene expression Northern blot analysis was performed, especially using the method described by Sambrook et al., Supra, using the DR4 gene in human tissues (ATCC No. 97).
853) Check expression. Complete nucleotide sequence of DR4 protein (SEQ ID NO: 1)
And a cDNA probe containing rediprime ^™ DNA labeling system (Am
labeling with ³² P using an ELISA kit according to the manufacturer's instructions. After labeling, the probe is purified using a CHROMA SPIN-100 ^™ column (Clontech Laboratories, Inc.) according to the manufacturer's protocol number PT1200-1. Various human tissues are then examined for DR4 mRNA using the purified labeled probe.

Multiple (MTN) blots of multiple tissues, including various human tissues (H) or human immune system tissues (IM), were obtained from Clontech and Express.
The Hyb ^™ hybridization solution (Clontech) is probed with a labeled probe according to the manufacturer's protocol number PT1190-1. After hybridization and washing, the blot is mounted and exposed to film overnight at -70 ° C, and the film is developed according to standard procedures. DR4
Expression of amniotic cells, heart, liver cancer, kidney, peripheral leukocytes, activated T cells, K562
And PMA, W138 cells, Th2 cells, human tonsil, and CD34 depleted buffy coat (umbilical cord blood). DR4 is predicted to play a role in lymphocyte homeostasis.

Example 23 Method for Determining Changes in the DR4 Gene RNs from whole family or individual patients presenting the phenotype of interest (eg, disease)
A is isolated. CDNA is then generated from these RNA samples using protocols known in the art (see Sambrook). Then
This cDNA is used as a template for PCR using primers surrounding the region of interest in SEQ ID NO: 1. Suggested PCR conditions are Sidrans
ky, D.S. Et al., Science 252: 706 (1991) using a buffer solution at 95 ° C. for 30 seconds; 52-58 ° C. for 60-120 seconds; and 70
Consists of 35 cycles at 60C for 60-120 seconds.

The PCR products are then sequenced using SequiTherm Polymerase using primers labeled at their 5 ′ end with T4 polynucleotide kinase. (Epicentre Technologies). D
The intron-exon boundaries of the selected exon of R4 are also determined, and the genomic PCR products are analyzed to confirm the results. The PCR product with the suspected mutation in DR4 is then cloned and sequenced, confirming the results of direct sequencing.

The DR4 PCR product was prepared as described in Holton, T .; A and Graham, M .; W.
Nucleic Acids Research, 19: 1156 (1991).
) And cloned into a T-tail vector as described in T7 polymerase (Uni).
Sequenced by ted States Biochemical. Affected individuals are identified by mutations in DR4 that are not present in unaffected individuals.

[0589] Genomic rearrangement is also observed as a way to determine changes in the DR4 gene. Genomic clones isolated using techniques known in the art can be isolated from digoxigenin deoxy-uridine 5'-triphosphate (Boehringer Manheii).
nick translation using Johnson, C.M. M
methods Cell Biol. FISH is performed as described in 35: 73-99 (1991). Hybridization with a labeled probe is performed using a large excess of human cot-1 DNA for specific hybridization to the DR4 genome locus.

The chromosomes are counterstained with 4,6-diamino-2-phenylidol and propidium iodide to generate a combination of C and R bands. Aligned images for accurate mapping were obtained using a triple band filter set (Chroma T).
technology, Brattleboro, VT) and a cooled charge-coupled device camera (Photometrics, Tucson, AZ) and a variable excitation wavelength filter. (Johnson, C. et al., Genet.
Anal. Tech. Appl. , 8:75 (1991)). Isee Gra
Image collection, analysis, and chromosome segment length measurements are performed using the physical Program System. (Invision Corporatio
n, Durham, NC). Chromosomal changes in the genomic region of DR4 (hybridized by the probe) are identified as insertions, deletions and translocations. These DR4 changes are used as diagnostic markers for related diseases.

Example 24 Method for Detecting Abnormal Levels of DR4 in a Biological Sample A DR4 polypeptide can be detected in a biological sample, and if an increase or decrease in DR4 is detected, A polypeptide is a marker for a particular phenotype. It is understood that there are a number of detection methods and, therefore, those skilled in the art can modify the following assays to suit their particular needs.

For example, using an antibody sandwich ELISA, preferably in a sample,
Detect DR4 in a biological sample. The wells of the microtiter plate are coated with an antibody specific for DR4 at a final concentration of 0.2-10 μg / ml. The antibodies are either monoclonal or polyclonal, and are produced using techniques known in the art. This well is blocked so that non-specific binding of DR4 to the well is reduced.

Next, the coated wells are incubated with the DR4 containing sample for more than 2 hours at room temperature. Preferably, serial dilutions of the sample should be used to confirm the results. Next, the plate is washed three times with deionized or distilled water,
Remove unbound DR4.

Next, 50 μl of the specific antibody-alkaline phosphatase conjugate was
g and incubate for 2 hours at room temperature. The plate is again washed three times with deionized or distilled water to remove unbound conjugate.

Add 75 μl of 4-methylumbelliferyl phosphate (MUP) or p-nitrophenyl phosphate (NPP) substrate solution to each well and incubate at room temperature for 1 hour to cleave the substrate and the fluorescence. Fluorescence is measured with a microtiter plate reader. A standard curve is generated using experimental results from serial dilutions of control samples, and plots sample concentration on the X-axis (log scale) and fluorescence or absorbance on the Y-axis (linear scale). The DR4 polypeptide concentration in the sample is then interpolated using a standard curve based on the measured fluorescence of the sample.

Example 25 Method of Treating Reduced Levels of DR4 The present invention relates to a method for treating an individual in need of increasing the level of biological activity of DR4 in the body, comprising: Administering to such an individual a composition comprising a therapeutically effective amount of a DR4 agonist. Preferred antagonists for use in the present invention are DR4 specific antibodies.

It is further understood that conditions caused by reduced levels of normal or normal expression of DR4 in an individual can be treated by administering DR4, preferably in a soluble and / or secreted form. Accordingly, the present invention also provides a method of treating an individual in need of increasing levels of a DR4 polypeptide. The method comprises administering to such an individual a pharmaceutical composition comprising DR4 in an amount that increases the level of biological activity of DR4 in such individual.

For example, a patient with a reduced level of DR4 polypeptide will receive 1 polypeptide.
Take a daily dose of 0.1-100 μg / kg for 6 consecutive days. Preferably, the polypeptide is in a soluble and / or secreted form.

Example 26: Method of Treating Elevated DR4 Levels The present invention also relates to a method for treating an individual in need of decreasing the level of DR4 biological activity in the body, the method comprising: Administering to such an individual a composition comprising a therapeutically effective amount of DR4 or an antagonist thereof.

Use antisense technology to inhibit production of DR4. This technique is one example of a method for reducing the level of a DR4 polypeptide, preferably in soluble and / or secreted form, resulting from various etiologies, such as cancer.

For example, patients diagnosed with abnormally elevated levels of DR4 may receive antisense polynucleotides at 0.5, 1.0, 1.5, 2.0 and 3.0 mg / kg.
/ Day intravenously for 21 days. If there is sufficient resistance to the treatment, the treatment is repeated after a 7 day drug holiday.

Example 27 Treatment Methods Using Gene Therapy-Ex Vivo One method of gene therapy is to transplant fibroblasts capable of expressing a soluble and / or mature DR4 polypeptide into a patient. Generally, fibroblasts are obtained from a subject by skin biopsy. The resulting tissue is placed in a tissue culture medium and divided into small pieces. A small chunk of tissue is placed on the wet surface of a tissue culture flask and approximately 10 pieces are placed in each flask. The flask is inverted, closed tightly, and left at room temperature overnight. After 24 hours at room temperature, the flask is inverted, the tissue mass remains fixed at the bottom of the flask, and fresh medium (eg, Ham's F12 medium containing 10% FBS, penicillin and streptomycin) is added. The flask is then incubated at 37 ° C. for about one week.

At this point, fresh medium is added and then changed every few days. After another two weeks of culture, a monolayer of fibroblasts appears. The monolayer is trypsinized and scaled up to a larger flask.

PMV-7 (Ki) flanked by Moloney murine sarcoma virus long terminal repeats
rschmeier, P .; T. DNA, 7: 219-25 (1988)) is digested with EcoRI and HindIII and treated with calf intestinal phosphatase. The linear vector is fractionated on an agarose gel and purified using glass beads.

The cDNA encoding DR4 can be amplified using PCR primers corresponding to the 5 ′ and 3 ′ coding end sequences, respectively. Preferably, the 5 'primer contains an EcoRI site and the 3' primer contains a HindIII site. Equal amounts of the Moloney murine sarcoma virus linear skeleton and amplified EcoRI
And the HindIII fragment are added together in the presence of T4 DNA ligase. The resulting mixture is maintained under conditions appropriate for ligation of the two fragments. The ligation mixture was then used and E.I. E. coli HB101. It is then plated on agar containing kanamycin to confirm that the vector has the DR4 properly inserted.

The amphotropic pA317 or GP + am12 packaging cells were
Grow to confluent density in tissue culture in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% calf serum (CS), penicillin and streptomycin. Next, the MSV vector containing the DR4 gene is added to the medium and the packaging cells are transduced with the vector. At this time, the packaging cells are D
Produce infectious virus particles containing the R4 gene (where packaging cells are referred to as producer cells).

Add fresh medium to the transduced producer cells and then add 10 c medium
Collect from m-plate confluent producer cells. Spent media containing infectious virus particles is filtered through a Millipore filter to remove detached producer cells, and the media is then used to infect fibroblasts. Remove the medium from the subconfluent plate of fibroblasts and immediately replace with the medium from the producer cells. The medium is removed and replaced with fresh medium. If the titer of the virus is high, virtually all fibroblasts will be infected and no selection is necessary. If the titer is very low, neo or h
It is necessary to use a retroviral vector with a selectable marker such as is. Once the fibroblasts are efficiently infected, the fibroblasts are analyzed and the DR
4. Determine if 4 proteins are being produced.

The engineered fibroblasts are then implanted into a host, either alone or after being grown to confluence on cytodex 3 microcarrier beads.

Example 28: Methods of Treatment Using Gene Therapy-In Vivo Another aspect of the invention is the use of in vivo gene therapy methods to treat disorders, diseases, and conditions. This method of gene therapy involves the introduction of a naked nucleic acid (DNA, RNA, and antisense DNA or RNA) DR4 sequence into an animal to increase or decrease the expression of a DR4 polypeptide. A DR4 polynucleotide can be operably linked to a promoter, or any other genetic element required for expression of a DR4 polypeptide by a target tissue. Techniques and methods for such gene therapy and delivery are known in the art, for example,
O90 / 11092, WO98 / 11779; U.S. Pat. Nos. 5,693,622, 5,705,151 and 5,580,859; Et al., 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 (
1997), Schwartz B. et al. Et al., Gene Ther. 3: 405-4
11 (1996); Et al., Circulation 94:
3281-3290 (1996), incorporated herein by reference.

The DR4 polynucleotide construct is delivered by any method that delivers an injectable substance to the cells of an animal, such as injection into the interstitial space of tissue (heart, muscle, skin, lung, liver, intestine, etc.). Can be done. DR4 polynucleotide constructs can be delivered in a pharmaceutically acceptable liquid or aqueous carrier.

The term “naked” polynucleotide, DNA or RNA, refers to any delivery vehicle (virus sequence, virus particle, liposome formulation, lipofectin, or lipofectin) that acts to assist, facilitate, or facilitate entry into cells. (Including a precipitant and the like). However, DR4 polynucleotides can also be prepared in liposome formulations (eg, Felgner P), which can be prepared by methods well known to those skilled in the art.
. L. (1995) Ann. NY Acad. Sci. 772: 126-13
9 and Abdallah B. (1995) Biol. Cell 85 (1
): 1-7).

The DR4 polynucleotide vector construct used in the gene therapy method is preferably a construct that does not integrate into the host genome and does not contain sequences that allow replication. Any strong promoter known to those of skill in the art can be used to drive expression of the DNA. Unlike other gene therapy techniques, one major advantage of introducing a naked nucleic acid sequence into a target cell is the transient nature of polynucleotide synthesis in that cell. Studies have shown that non-replicating DNA sequences can be introduced into cells to provide production of the desired polypeptide for up to six months.

The DR4 polynucleotide construct is used in tissues (muscle, skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gallbladder, stomach,
(Including the intestine, testis, ovary, uterus, rectum, nervous system, eyes, glands, and connective tissue). The interstitial space of the tissue is within the intercellular fluid, the mucopolysaccharide matrix between the reticulum fibers of the organ tissue, the elastic fibers in the walls of the blood vessels or chambers, the collagen fibers of the fibrous tissue, or the connective tissue surrounded by the muscle cell sheath The same matrix (t
and the same matrix in connective tissue in a bone hiatus. This is also the space occupied by the circulating plasma and the lymph of the lymph channels. Delivery of muscle tissue to the interstitial space is preferred for reasons discussed below. They can be conveniently delivered by injection into the tissue containing these cells. They are preferably delivered to and expressed in differentiated, persistent, non-dividing cells, but are delivered and expressed in non-differentiated cells or in fully differentiated cells (eg, blood stem cells). Or dermal fibroblasts). In vivo, muscle cells are particularly competent in their ability to take up and express polynucleotides.

For naked DR4 polynucleotide injections, effective dosages of DNA or RNA range from about 0.05 μg / kg body weight to about 50 mg / kg body weight. Preferably, the dosage will be from about 0.005 mg / kg to about 20 mg / kg, and more preferably, from about 0.05 mg / kg to about 5 mg / kg. Of course, as the skilled artisan will appreciate, this dosage will vary according to the tissue site of the injection. Appropriate and effective dosages of nucleic acid sequences can be readily determined by one skilled in the art, and will depend on the condition being treated and the route of administration. The preferred route of administration is by parenteral injection into the interstitial space of the tissue. However, other parenteral routes (eg,
Inhalation of aerosol formulations, particularly for delivery to the lung or bronchial tissue, throat, or nasal mucosa) may also be used. In addition, a naked DR4 polynucleotide construct can be delivered to an artery during angioplasty by the catheter used in this procedure.

The dose response effect of DR4 polynucleotide injected into muscle in vivo is determined as follows. An appropriate DR4 template DNA for the production of an mRNA encoding a DR4 polypeptide is prepared according to standard recombinant DNA methodology.
The template DNA, which can be either circular or linear, is either used as naked DNA or complexed with liposomes. The quadriceps of the mouse are then injected with various amounts of template DNA.

[0617] Female and male Balb / C mice, 5-6 weeks old, were given 0.3 ml of 2.5%
Anesthetize by injecting Avertin intraperitoneally. A 1.5 cm incision is made in the anterior thigh and the quadriceps are visualized directly. 0.1 ml of DR4 template DNA
In a 1cc syringe through a 27 gauge needle for 1 minute
The knee is injected at a depth of about 0.2 cm about 0.5 cm from the distal insertion site of the muscle. Sutures are made over the injection site for future positioning and the skin is closed with a stainless steel clip.

After an appropriate incubation time (eg, 7 days), a muscle extract is prepared by cutting out all four animals. A 15 μm section of every five quadriceps was placed on D
Histochemically stain for R4 protein expression. A time course for DR4 protein expression can be performed in a similar manner, except that four animals from different mice are harvested at different times. The persistence of DR4 DNA in muscle after injection can be determined by Southern blot analysis after preparing total cellular DNA and HIRT supernatant from injected and control mice. The results of the above experiments in mice can be used to extrapolate appropriate dosages and other treatment parameters in humans and other animals using naked DR4 DNA.

It is clear that the invention can be carried out in other ways than those described in detail in the foregoing description and examples. Numerous modifications and variations of the present invention are possible in light of the above teachings, and so are within the scope of the appended claims.

The complete disclosure of each document cited in the background, detailed description and examples of the invention (including patents, patent applications, academic literature, abstracts, experimental manuals, books or other disclosures)
Is incorporated herein by reference.

In addition, the Sequence Listing submitted in paper form with this specification is incorporated herein by reference in its entirety.

[0621]

[Table 3]

[Sequence list]

[Brief description of the drawings]

FIG. 1 shows the nucleotide sequence and the deduced amino acid sequence of DR4. Amino acids from about 1 to about 23 constitute a signal peptide, amino acids from about 24 to about 238 constitute an extracellular domain, amino acids from about 131 to about 229 constitute a cysteine-rich domain, and amino acids from about 239 to about 264 transmembrane. The amino acids from about 265 to about 468 of the intracellular domain (of which amino acids from about 379 to about 42
2 make up the death domain).

FIG. 2 shows DR4, human tumor necrosis factor receptor 1 (SEQ ID NO: 3), human Fas
Protein (SEQ ID NO: 4) and receptor 3 (DR3) containing the death domain (
The region of similarity between the amino acid sequences of SEQ ID NO: 5) is shown. Residues that match the consensus are shaded.

FIG. 3 shows an analysis of the DR4 amino acid sequence. α, β, turn, and coil regions; hydrophilicity and hydrophobicity; amphipathic regions; flexible regions; antigenicity index and surface probability are described for the amino acid sequence shown in FIG. 1 (SEQ ID NO: 2). As expected using the default parameters of the computer program used.
In the graph of “antigenicity index-Jameson-Wolf”, FIG. 1 (SEQ ID NO: 2)
Amino acid residues 35-92, 114-160, 169-240, 267-29
8, 330-364, 391-404, and 418-465 show the indicated DR
It corresponds to the high antigenicity region of the four proteins.

FIG. 4 shows the related nucleic acid fragments HTOIY07R (SEQ ID NO: 6) and H
Figure 6 shows the nucleotide sequence of TXEY80R (SEQ ID NO: 7).

FIGS. 5A and 5B show the ability of DR4 to induce apoptosis in MCF7 and 293 cell lines. FIG. 5C shows the ability of the death protease inhibitors z-VAD-fmk and CrmA to inhibit the apoptotic effects of DR4.

FIG. 6A shows soluble extracellular D blocking TRAIL's ability to induce apoptosis.
9 shows the ability of the R4-Fc fusion. FIG. 6B shows that the soluble extracellular DR4-Fc fusion has no ability to block the ability of TNF-α to induce apoptosis.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification code FI Theme coat ゛ (Reference) A61K 47/48 A61P 35/00 4H045 A61P 31/12 35/02 35/00 37/02 35/02 C07K 16 / 28 37/02 19/00 C07K 16/28 C12P 21/08 19/00 G01N 33/574 A C12N 15/02 A61K 37/02 15/09 ZNA C12N 15/00 ZNAA C12P 21/08 A61K 37/66 G G01N 33/574 C12N 15/00 C (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, W, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, 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 Reg nts of the University of Michi gan United States Michigan 48109-1280, Ann Arbor, es. State Street 3003, Room 2071, Wolverine Tower Wolverine Tower, Room 2071, 3003 State Street, Ann Arbor, MI 48109-1280, U.S. Pat. S. A. (72) Inventor D, Jian United States of America 20853, Maryland, Rockville, Manorfield Road 5502 (72) Inventor Rosen, Craig A. United States Maryland 20882, Laytonsville, Rolling Hill Road 22400 (72) Inventor Pan, James G. United States Michigan 48197, Ypsilanti, Green Meadows Boulevard 4124, Apartment Number 11 (72) Inventors Gentz, Reiner L. United States Maryland 20850, Rockville, Mount Prospect Drive 13608 (72) Inventor Dikit, Bishba Em. United States California 94022, Los Altos Hills, Shady Oaks Court 26750 F-term (reference) 4B024 AA01 AA11 BA63 CA04 CA10 DA02 DA06 EA02 EA04 FA02 GA11 HA01 4B064 AG20 AG26 CA10 CA19 CA20 CC24 DA01 4C076 CC07 CC27 CC35 EE59 4C08BA A08 AA BA01A DC50 MA17 MA21 MA22 MA23 MA28 MA43 MA52 MA55 MA56 MA60 MA63 MA66 NA14 ZB072 ZB262 ZB272 ZB332 4C085 AA13 AA14 AA16 BA99 BB44 CC22 CC23 GG02 GG06 GG08 4H045 AA10 AA30 BA10 BA41 BA57 CA40 DA51 EA28 FA74

Claims (25)

[Claims] 1. A method for treating graft versus host disease, viral infection, cancer, leukemia, immunodeficiency or autoimmune disorder, the method comprising the steps of: (a) SEQ ID NO: A first therapeutic agent comprising an antibody that binds to a polypeptide consisting of two amino acids 1 to 468; and (b) a second therapeutic agent selected from the group consisting of: (i) TRAIL; (ii) And (iii) a tumor necrosis factor diagnostic agent; (iv) an immunosuppressant; (v) an antibiotic; (vi) an anti-inflammatory agent; (vii) a chemotherapeutic agent; and (viii) a cytokine. A method comprising the step of: 2. The method of claim 1, wherein said first therapeutic agent comprises an antibody that binds to a polypeptide consisting of amino acids 24-238 of SEQ ID NO: 2. 3. The method of claim 1, wherein said antibody is a monoclonal antibody. 4. The method of claim 1, wherein said antibody is a polyclonal antibody. 5. The method of claim 1, wherein said antibody is a chimeric antibody. 6. The method of claim 1, wherein said antibody is a humanized antibody. 7. The method of claim 1, wherein said antibody is a single-chain Fv.
The method, which is an antibody. 8. The method of claim 1, wherein said antibody is a Fab antibody fragment. 9. The method of claim 1, wherein the first therapeutic agent and the second therapeutic agent are administered to the individual at the same time. 10. The method of claim 1, wherein the first therapeutic agent and the second therapeutic agent are administered to the individual at different times. 11. The method of claim 1, wherein said second therapeutic agent is TRAIL. 12. The method according to claim 1, wherein the diagnostic agent for tumor necrosis factor comprises: (a) TNF-α; (b) TNF-β; (c) TNF-γ; (d) C) an antibody that binds to a protein selected from the group consisting of: TNF-γ-α; and (e) TNF-γ-β. 13. The method of claim 1, wherein the immunosuppressant comprises: (a) cyclosporin; (b) cyclophosphamide; (c) methylprednisone; (d) prednisone; (E) azathioprine; (f) FK-506; and (g) 15-deoxyspergualin. 14. The method of claim 1, wherein the cytokine is: (a) IL-2; (b) IL-3; (c) IL-4; (d) IL- (E) IL-6; (f) IL-7; (g) IL-10; (h) IL-12; (i) IL-13; (j) IL-15; and (k) IFN-. A method selected from the group consisting of γ. 15. A composition comprising: (a) a first therapeutic agent comprising an antibody that binds to a polypeptide consisting of amino acids 1-468 of SEQ ID NO: 2; and (b) a group consisting of: (Ii) TRAIL; (ii) tumor necrosis factor; (iii) diagnostic agent for tumor necrosis factor; (iv) immunosuppressant; (v) antibiotic; A composition comprising: (vii) a chemotherapeutic agent; and (viii) a cytokine. 16. The composition according to claim 15, further comprising a pharmaceutically acceptable carrier or excipient. 17. An isolated polypeptide comprising an amino acid sequence at least 90% identical to amino acids 24-238 of SEQ ID NO: 2, wherein said polypeptide is covalently linked to polyethylene glycol, Glycol is 2000, 3000, 4000, 5000, 6000, 7
000, 8000, 9000, 10,000, 15,000 and 20,000
An isolated polypeptide having an average molecular weight selected from the group consisting of: 18. The polypeptide of claim 17, which comprises an amino acid sequence that is at least 95% identical to amino acids 24-238 of SEQ ID NO: 2. 19. The polypeptide of claim 18, wherein said amino acid sequence comprises amino acids 24-238 of SEQ ID NO: 2. 20. The polypeptide according to claim 17, wherein the polypeptide comprises polyethylene glycol, 1-3, 2-4, 3-5, 4-6,
A polypeptide having an average degree of substitution within a range selected from the group consisting of 5-7, 6-8, 7-9, 8-10, 9-11 and 10-12. 21. The polypeptide of claim 17, produced by a recombinant host cell. 22. The recombinant host cell of claim 2, wherein the host cell is a eukaryotic host cell.
The polypeptide according to 0. 23. The polypeptide according to claim 17, which contains a heterologous polypeptide. 24. The polypeptide of claim 23, wherein said heterologous polypeptide contains an Fc portion of an antibody. 25. A composition comprising the polypeptide of claim 17 and a pharmaceutically acceptable carrier.