(a) DNA encoding the amino acid sequence set forth in SEQ ID NOS:2, 4, 6, 8, 10, 12, 53, 56 or 58;
	(b) DNA that hybridizes to the DNA of (a) under moderately stringent conditions, wherein said DNA encodes biologically active DD, DED, or NB-ARC, or
	(c) DNA degenerate with (b), wherein said DNA encodes biologically active DD, DED, or NB-ARC domain.

Another exemplary nucleic acid encoding an invention DD, DED or NB-ARC domain can be selected from:
 

	(a) DNA encoding the amino acid sequence set forth in SEQ ID NOS:2, 4, 6, 8, 10, 12, 53, 56 or 58;
	(b) DNA that hybridizes to the DNA of (a) under moderately stringent conditions, wherein said DNA encodes biologically active DD, DED, or NB-ARC domain or
	(c) DNA degenerate with (b), wherein said DNA encodes biologically active DD, DED, or NB-ARC domain, wherein the nucleic acid sequence does not encode the amino acid sequence set forth in SEQ ID NOS:14, 24, 28, 55 or 57.

The invention additionally provides an isolated nucleic acid encoding a Death Domain (DD), Death Effector Domain (DED) or NB-ARC domain polypeptide, or functional fragments thereof, the nucleic acid encoding the amino acid sequence set forth in SEQ ID NOS:2, 4, 6, 8, 10, 12, 16, 18, 20, 22, 26, 53, 56 or 58. The invention also provides a nucleic acid having the same or substantially the same sequence as set forth in any of SEQ ID NOS:1, 3, 5, 7, 9, 11, 17, 21 or 52. The invention also provides a nucleic acid having the same sequence as that set forth in any of SEQ ID NOS:1, 3, 5, 7, 9, 11, 15, 17, 19, 21, 25 or 52.

Hybridization refers to the binding of complementary strands of nucleic acid (i.e., sense:antisense strands or probe:target-DNA) to each other through hydrogen bonds, similar to the bonds that naturally occur in chromosomal DNA. Stringency levels used to hybridize a given probe with target-DNA can be readily varied by those of skill in the art.

The phrase "stringent hybridization" is used herein to refer to conditions under which polynucleic acid hybrids are stable. As known to those of skill in the art, the stability of hybrids is reflected in the melting temperature (T_m) of the hybrids. In general, the stability of a hybrid is a function of sodium ion concentration and temperature. Typically, the hybridization reaction is performed under conditions of lower stringency, followed by washes of varying, but higher, stringency. Reference to hybridization stringency relates to such washing conditions.

As used herein, the phrase "moderately stringent hybridization" refers to conditions that permit target-DNA to bind a complementary nucleic acid that has about 60% identity, preferably about 75% identity, more preferably about 85% identity to the target DNA; with greater than about 90% identity to target-DNA being especially preferred. Preferably, moderately stringent conditions are conditions equivalent to hybridization in 50% formamide, 5× Denhart's solution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.2×SSPE, 0.2% SDS, at 42° C.

The phrase "high stringency hybridization" refers to conditions that permit hybridization of only those nucleic acid sequences that form stable hybrids in 0.018M NaCl at 65° C. (i.e., if a hybrid is not stable in 0.018M NaCl at 65° C., it will not be stable under high stringency conditions, as contemplated herein). High stringency conditions can be provided, for example, by hybridization in 50% formamide, 5× Denhart's solution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.1×SSPE, and 0.1% SDS at 65° C.

The phrase "low stringency hybridization" refers to conditions equivalent to hybridization in 10% formamide, 5× Denhart's solution, 6×SSPE, 0.2% SDS at 42° C., followed by washing in 1×SSPE, 0.2% SDS, at 50° C. Denhart's solution and SSPE (see, e.g., Sambrook et al., supra, 1989) are well known to those of skill in the art as are other suitable hybridization buffers.

As used herein, the term "degenerate" refers to codons that differ in at least one nucleotide from a reference nucleic acid, e.g., SEQ ID NOS:1, 3, 5, 7, 9, 11 or 52, but encode the same amino acids as the reference nucleic acid. For example, codons specified by the triplets "UCU", "UCC", "UCA", and "UCG" are degenerate with respect to each other since all four of these codons encode the amino acid serine.

Preferred nucleic acids encoding the invention polypeptide(s) hybridize under moderately stringent, preferably high stringency, conditions to substantially the entire sequence, or substantial portions (i.e., typically at least 15-30 nucleotides) of the nucleic acid sequence set forth in SEQ ID NOS:1, 3, 5, 7, 9, 11, 52, provided they do not comprise the sequence set forth in SEQ ID NOS:13, 23, 27 or 54, or a nucleic acid encoding SEQ ID NO:57.

The invention nucleic acids can be produced by a variety of methods well-known in the art, e.g., the methods described herein, employing PCR amplification using oligonucleotide primers from various regions of SEQ ID NOS:1, 3, 5, 7, 9, 11 or 52, and the like.

In accordance with a further embodiment of the present invention, optionally labeled DD, DED or NB-ARC encoding cDNAs, or fragments thereof, can be employed to probe library(ies) (e.g., cDNA, genomic, and the like) for additional nucleic acid sequences encoding novel bacterial or eukaryotic DD, DED or NB-ARC domains. Construction of suitable bacterial libraries or eukaryotic cDNA libraries is well-known in the art. Screening of such a cDNA library is initially carried out under low-stringency conditions, which comprise a temperature of less than about 42° C., a formamide concentration of less than about 50%, and a moderate to low salt concentration.

Presently preferred probe-based screening conditions comprise a temperature of about 37° C., a formamide concentration of about 20%, and a salt concentration of about 5× standard saline citrate (SSC; 20×SSC contains 3M sodium chloride, 0.3M sodium citrate, pH 7.0). Such conditions will allow the identification of sequences which have a substantial degree of similarity with the probe sequence, without requiring perfect homology. The phrase "substantial similarity" refers to sequences which share at least 50% homology. Preferably, hybridization conditions will be selected which allow the identification of sequences having at least 70% homology, at least 80%, at least 90%, at least 95%, or at least 98% with the probe, while discriminating against sequences which have a lower degree of homology with the probe. As a result, nucleic acids having the same or substantially the same nucleotide sequence as SEQ ID NOS:13, 15, 17, 19, 21, 25, 27 or 54 are obtained.

As used herein, a nucleic acid "probe" or "oligonucleotide" is single-stranded or double-stranded DNA or RNA, or analogs thereof, that has a sequence of nucleotides that includes at least 15, at least 20, at least 50, at least 100, at least 200, at least 300, at least 400, or at least 500 contiguous bases that are the same as (or the complement of) any contiguous bases set forth in any of SEQ ID NOS:1, 3, 5, 7, 9, 11, 15, 17, 19, 21 or 52. Oligonucleotides are useful, for example, as probes or as primers for amplification reactions such as the polymerase chain reaction (PCR). In addition, oligonucleotides can bind to the sense or anti-sense strands of other nucleic acids. Preferred regions from which to construct probes include 5′ and/or 3′ coding regions of SEQ ID NOS:1, 3, 5, 7, 9, 11, 15, 17, 19, 21 or 52. In addition, the entire cDNA encoding region of an invention DD, DED, or NB-ARC domain or the entire sequence corresponding to SEQ ID NOS:1, 3, 5, 7, 9, 11, 15, 17, 19, 21 or 52 can be used as a probe. Probes can be labeled by methods well-known in the art, as described hereinafter, and used in various diagnostic kits.

It is understood that an invention nucleic acid molecule, as used herein, specifically excludes previously known nucleic acid molecules consisting of nucleotide sequences having identity with the DD, DED and NB-ARC nucleotide sequence, such as Expressed Sequence Tags (ESTs), Sequence Tagged Sites (STSs) and genomic fragments, deposited in public databases such as the nr, dbest, dbsts, gss and htgs databases, which are available for searching at http://www.ncbi.nlm.nih.gov/blast/blast.cgi?Jform=0, using the program BLASTN 2.0.9 described by Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997).

In particular, a DD, DED or NB-ARC domain nucleic acid molecule specifically excludes nucleic acid molecules consisting of any of the nucleotide sequences having the Genbank (gb), EMBL (emb) or DDBJ (dbj) accession numbers described below. Similarly, a DD, DED or NB-ARC domain polypeptide fragment of DD, DED or NB-ARC domain containing-polypeptide specifically excludes the amino acid fragments encoded by the nucleotide sequences having the GenBank accession numbers described below. GenBank accession numbers specifically excluded include AW449244, AA218681, GI 4210498, GI 1832773, GI 6990020, GI 4758118 (accession No. NP_—004623), X83544, GI 7705841, GI 7705840, GI 5360131 (locus AF155118, accession No. AAD42884), AA114228, BE797255, BE242821, AW229739, AW227145, AV149215, GI 7190927, GI 7468151, GI 5353348, GI 4607778, and GI 6960635.

As used herein, the terms "label" and "indicating means" in their various grammatical forms refer to single atoms and molecules that are either directly or indirectly involved in the production of a detectable signal. Any label or indicating means can be linked to invention nucleic acid probes, expressed proteins, polypeptide fragments, or antibody molecules. These atoms or molecules can be used alone or in conjunction with additional reagents. Such labels are themselves well-known in clinical diagnostic chemistry.

The labeling means can be a fluorescent labeling agent that chemically binds to antibodies or antigens without denaturation to form a fluorochrome (dye) that is a useful immunofluorescent tracer. A description of immunofluorescent analytic techniques is found in DeLuca, "Immunofluorescence Analysis", in Antibody As a Tool, Marchalonis et al., eds., John Wiley & Sons, Ltd., pp. 189-231 (1982), which is incorporated herein by reference.

In one embodiment, the indicating group is an enzyme, such as horseradish peroxidase (HRP), glucose oxidase, and the like. In another embodiment, radioactive elements are employed as labeling agents. The linking of a label to a substrate, i.e., labeling of nucleic acid probes, antibodies, polypeptides, and proteins, is well known in the art. Detectable labels can be incorporated by chemical synthesis, chemical modification, in vitro enzymatic incorporation, or in vivo metabolic labeling. For instance, an invention antibody can be labeled by metabolic incorporation of radiolabeled amino acids provided in the culture medium. See, for example, Galfre et al., Meth. Enzymol., 73:3-46 (1981). Conventional means of protein conjugation or coupling by activated functional groups are particularly applicable. See, for example, Aurameas et al., Scand. J. Immunol., Vol. 8, Suppl. 7:7-23 (1978), Rodwell et al., Biotech., 3:889-894 (1984), and U.S. Pat. No. 4,493,795.

In another embodiment of the invention, nucleic acids are provided encoding chimeric proteins comprising an invention DD, DED, or NB-ARC domain or fragment thereof, having the sequence of SEQ ID NOS:2, 4, 6, 8, 10, 12, 53, 56 or 58, and further comprising one or more sequences from a heterologous protein. Functional fragments of DD, DED or NB-ARC include, for example, polypeptides having the sequence SEQ ID NO:2, 4, 6, 8, 10, 12, 53, 56 or 58. Nucleic acids encoding proteins with which the DD, DED or NB-ARC domain, or functional fragment thereof, are fused will also encode, for example, glutathione-S-transferase, an antibody, or other proteins or functional fragments thereof which facilitate recovery of the chimera. Nucleic acids of the invention can also encode proteins with which the DD, DED, or NB-ARC domain, or functional fragment thereof, are fused, for example, to luciferase, green fluorescent protein, an antibody, or other proteins or functional fragments thereof which facilitate identification of the chimera. Still further nucleic acids of the invention encode proteins with which the DD, DED or NB-ARC domain or functional fragment thereof are fused including, for example, the LexA DNA binding domain, ricin, α-sarcin, an antibody, or other proteins which have therapeutic properties or other biological activity.

The present invention also provides compositions containing an acceptable carrier and any of an isolated, purified DD-, DED- or NB-ARC-containing protein or functional polypeptide fragments thereof, alone or in combination with each other. These polypeptides or proteins can be recombinantly derived, chemically synthesized or purified from native sources. As used herein, the term "acceptable carrier" encompasses any of the standard pharmaceutical carriers, such as phosphate buffered saline solution, water and emulsions such as an oil/water or water/oil emulsion, and various types of wetting agents.

The DD, DED or NB-ARC compositions described herein can be used, for example, in methods for modulating the activity of members of the apoptotic pathway. Thus it is within the scope of the present invention that a protein comprising the sequence SEQ ID NOS:2, 4, 6, 8, 10, 12, 53, 56 or 58 or a nucleic acid encoding a protein comprising the sequence SEQ ID NOS:1, 3, 5, 7, 9, 11 or 52, modulates the activity of member of an apoptotic pathway.

In one embodiment, modulation of a member of FADD, caspases such as caspase-8 and caspase-10, DR4, DR5, Traf6, hToll, MyD88, Fas, Raidd, IRAK, IRAK-2, IRAK-M, p75NTR, Tradd, DAP kinase, RIP, NMP84, ankyrins, Flip, PEA15, Flash, BAP31, BAR, DEDT/DEDD, and DAP3 or a related polypeptide that binds an invention DD, DED or NB-ARC will comprise the step of contacting a member of FADD, caspases such as caspase-8 and caspase-10, DR4, DR5, Traf6, hToll, MyD88, Fas, Raidd, IRAK, IRAK-2, IRAK-M, p75NTR, Tradd, DAP kinase, RIP, NMP84, ankyrins, Flip, PEA15, Flash, BAP31, BAR, DEDT/DEDD, and DAP3 with a protein comprising the sequence SEQ ID NOS:2, 4, 6, 8, 10, 12, 16, 18, 20, 22, 26, 53, 56 or 58. Preferably, the method comprises contacting a cell with a protein comprising the sequence of SEQ ID NOS:2, 4, 6, 8, 10, 12, 53, 56 or 58.

In another embodiment, modulation of a member of FADD, caspases such as caspase-8 and caspase-10, DR4, DR5, Traf6, hToll, MyD88, Fas, Raidd, IRAK, IRAK-2, IRAK-M, p75NTR, Tradd, DAP kinase, RIP, NMP84, ankyrins, Flip, PEA15, Flash, BAP31, BAR, DEDT/DEDD, and DAP3, or a related polypeptide that binds an invention DD, DED, or NB-ARC will comprise the step of contacting a member of FADD, caspases such as caspase-8 and caspase-10, DR4, DR5, Traf6, hToll, MyD88, Fas, Raidd, IRAK, IRAK-2, IRAK-M, p75NTR, Tradd, DAP kinase, RIP, NMP84, ankyrins, Flip, PEA15, Flash, BAP31, BAR, DEDT/DEDD, and DAP3 or a related polypeptide that binds an invention DD, DED or NB-ARC domain, with a nucleic acid encoding a protein comprising the sequence SEQ ID NOS:2, 4, 6, 8, 10, 12, 16, 18, 20, 22, 26, 53, 56 or 58. Preferably, the method comprises contacting a cell with a nucleic acid encoding a protein comprising the sequence of SEQ ID NOS:2, 4, 6, 8, 10, 12, 16, 18, 20, 22, 26, 53, 56 or 58.

In another embodiment, the DD, DED or NB-ARC domain compositions described herein can be used, for example, in methods for modulating the activity of proteins containing domains that bind invention DDs, DEDs or NB-ARC domains. Thus, it is within the scope of the present invention that a protein comprising the sequence SEQ ID NOS:2, 4, 6, 8, 10, 12, 16, 18, 20, 22, 26, 53, 56 or 58, or a nucleic acid encoding a protein comprising the sequence SEQ ID NOS:2, 4, 6, 8, 10, 12, 16, 18, 20, 22, 26, 53, 56 or 58, modulates the activity of one or more proteins containing domains that bind invention DDs, DEDs or NB-ARC domains.

In one embodiment, modulation of a protein containing a domain that interacts with an invention DD, DED or NB-ARC domain will comprise the step of contacting a protein containing a domain that interacts with an invention DD, DED or NB-ARC domain with a protein comprising the sequence SEQ ID NOS:2, 4, 6, 8, 10, 12, 16, 18, 20, 22, 26, 53, 56 or 58. Preferably, the method comprises contacting a cell with a protein comprising the sequence of SEQ ID NOS:2, 4, 6, 8, 10, 12, 16, 18, 20, 22, 26, 53, 56 or 58.

In another embodiment, modulation of a protein containing a domain that interacts with an invention DD, DED or NB-ARC will comprise the step of contacting a protein containing a domain that interacts with an invention DD, DED or NB-ARC with a nucleic acid encoding a protein comprising the sequence SEQ ID NOS:2, 4, 6, 8, 10, 12, 16, 18, 20, 22, 26, 53, 56 or 58. Preferably, the method comprises contacting a cell with a nucleic acid encoding a protein comprising the sequence of SEQ ID NOS:2, 4, 6, 8, 10, 12, 16, 18, 20, 22, 26, 53, 56 or 58.

In another embodiment, a DD, DED or NB-ARC domain comprising the sequence SEQ ID NOS:2, 4, 6, 8, 10, 12, 16, 18, 20, 22, 26, 53, 56 or 58, or a nucleic acid encoding a protein comprising the sequence SEQ ID NOS:2, 4, 6, 8, 10, 12, 16, 18, 20, 22, 26, 53, 56 or 58, modulates the activity of one or more associated proteins. Thus it is within the scope of the invention that an invention DD, DED or NB-ARC domain protein can modulate the activity of any protein with which the DD, DED or NB-ARC domain proteins are known to interact.

In one embodiment, modulation of a protein that binds an invention DD, DED or NB-ARC domain will comprise the step of contacting a with a protein comprising the sequence SEQ ID NOS:2, 4, 6, 8, 10, 12, 16, 18, 20, 22, 26, 53, 56 or 58. Preferably, the method comprises contacting a cell with a protein comprising the sequence of SEQ ID NOS:2, 4, 6, 8 10, 12, 16, 18, 20, 22, 26, 53, 56 or 58.

In another embodiment, modulation of a protein that interacts with an invention DD, DED or NB-ARC will comprise the step of contacting a protein that interacts with an invention DD, DED or NB-ARC with a nucleic acid encoding a protein comprising the sequence SEQ ID NOS:2, 4, 6, 8, 10, 12, 16, 18, 20, 22, 26, 53, 56 or 58. Preferably, the method comprises contacting a cell with a nucleic acid encoding a protein comprising the sequence of SEQ ID NOS:2, 4, 6, 8, 10, 12, 16, 18, 20, 22, 26, 53, 56 or 58.

DD or NB-ARC domain compositions can also be used, for example, in methods for modulating the activity of NF-κB or JNK. Proteins homologous to invention DD or NB-ARC domain, for example, the DD of IRAK4 (SEQ ID NO:6) is shown herein to modulate NF-κB activity. An invention NB-ARC domain, for example, the NB-ARC domain of DAP3, is expected to modulated NFκB activity based on previously known regulation of NFκB by the NB-ARC protein Nod1/CARD4. Thus, in accordance with another embodiment of the invention, a protein comprising the sequence SEQ ID NOS:2, 4, 6, 10, 12, 53, 56 or 58, or a nucleic acid encoding a protein comprising the sequence SEQ ID NOS:2, 4, 6, 10, 12, 53, 56 or 58, modulates the activity of NF-κB or JNK.

In one embodiment, modulation of NF-κB or JNK activity activity will comprise the step of contacting a cell containing NF-κB activity with a protein comprising the sequence SEQ ID NO:2, 4, 6, 10, 12, 53, 56 or 58. Preferably, the method comprises contacting a cell with a protein comprising the sequence of SEQ ID NO:2, 4, 6, 10, 12, 53, 56 or 58.

In another embodiment, modulation of NF-κB or JNK activity will comprise the step of contacting a cell containing NF-κB activity or JNK activity with a nucleic acid encoding a protein comprising the sequence SEQ ID NO:2, 4, 6, 10, 12, 53, 56 or 58. Preferably, the method comprises contacting a cell with a nucleic acid encoding a protein comprising the sequence of SEQ ID NO:2, 4, 6, 10, 12, 53, 56 or 58.

As disclosed herein, the N-terminal domain of DAP3 binds caspase-8, and DAP3 increases caspase-8 protease activity. Therefore, in another embodiment, modulation of caspase-8 activity comprises the step of contacting a cell containing caspase-8 activity with a nucleic acid encoding a protein comprising the NB-ARC domain (SEQ ID NO:4) of DAP3, or an invention DD- or DED-containing polypeptide.

The functions of the invention DDs, DEDs and NB-ARC domains support the role of DD, DED and NB-ARC domain containing polypeptides in modulating cellular pathways that effect apoptosis, cell proliferation, cell adhesion, cell stress responses, responses to microbial infection, and B cell immunoglobulin class switching. Thus, in accordance with another embodiment of the invention, a protein comprising the sequence SEQ ID NOS:2, 4, 6, 8, 10, 12, 53, 56 or 58, or SEQ ID NOS:16, 18, 20, 22 or 26, or a nucleic acid encoding a protein comprising the sequence SEQ ID NOS:2, 4, 6, 8, 10, 12, 53, 56 or 58, or SEQ ID NOS:16, 18, 20, 22 or 26, modulates apoptosis, cell proliferation, cell adhesion, cell stress responses, responses to microbial infection, or B cell immunoglobulin class switching.

In one embodiment, modulation of apoptosis, cell proliferation, cell adhesion, cell stress responses, responses to microbial infection, or B cell immunoglobulin class switching will comprise the step of contacting a cell with a protein comprising the sequence SEQ ID NOS:2, 4, 6, 8, 10, 12, 53, 56 or 58, or SEQ ID NOS:16, 18, 20, 22 or 26, whereby apoptosis, cell proliferation, cell adhesion, cell stress responses, responses to microbial infection, or B cell immunoglobulin class switching is modulated. Preferably, the method comprises contacting a cell with a protein comprising the sequence of SEQ ID NOS:2, 4, 6, 8, 10, 12, 53, 56 or 58, or SEQ ID NOS:16, 18, 20, 22 or 26.

In another embodiment, modulation of apoptosis, cell proliferation, cell adhesion, cell stress responses, responses to microbial infection, or B cell immunoglobulin class switching will comprise the step of contacting a cell with a nucleic acid encoding a protein comprising the sequence SEQ ID NOS:2, 4, 6, 8, 10, 12, 53, 56 or 58, or SEQ ID NOS:16, 18, 20, 22 or 26, whereby apoptosis, cell proliferation, cell adhesion, cell stress responses, responses to microbial infection, or B cell immunoglobulin class switching is modulated. Preferably, the method comprises contacting a cell with a nucleic acid encoding a protein comprising the sequence of SEQ ID NO:2, 4, 6, 8, 10, 12, 53, 56 or 58, or SEQ ID NOS:16, 18, 20, 22 or 26.

Also provided are antisense-nucleic acids having a sequence capable of binding specifically with full-length or any portion of an mRNA that encodes DD, DED or NB-ARC domain polypeptides so as to prevent translation of the mRNA. The antisense-nucleic acid can have a sequence capable of binding specifically with any portion of the sequence of the cDNA encoding DD, DED or NB-ARC domain polypeptides. As used herein, the phrase "binding specifically" encompasses the ability of a nucleic acid sequence to recognize a complementary nucleic acid sequence and to form double-helical segments therewith via the formation of hydrogen bonds between the complementary base pairs. An example of an antisense-nucleic acid is an antisense-nucleic acid comprising chemical analogs of nucleotides. Exemplary antisense molecules for the DED containing polypeptide DAP3 are described herein.

Compositions comprising an amount of the antisense-nucleic acid, described above, effective to reduce expression of DD, DED or NB-ARC domain polypeptides by passing through a cell membrane and binding specifically with mRNA encoding DD, DED or NB-ARC domain polypeptides so as to prevent translation and an acceptable hydrophobic carrier capable of passing through a cell membrane are also provided herein. Suitable hydrophobic carriers are described, for example, in U.S. Pat. Nos. 5,334,761; 4,889,953; 4,897,355, and the like. The acceptable hydrophobic carrier capable of passing through cell membranes may also comprise a structure which binds to a receptor specific for a selected cell type and is thereby taken up by cells of the selected cell type. The structure may be part of a protein known to bind to a cell-type specific receptor.

Antisense-nucleic acid compositions are useful to inhibit translation of mRNA encoding invention polypeptides. Synthetic oligonucleotides, or other antisense chemical structures are designed to bind to mRNA encoding DD, DED or NB-ARC domain polypeptides and inhibit translation of mRNA and are useful as compositions to inhibit expression of DD, DED or NB-ARC domain associated genes in a tissue sample or in a subject.

In accordance with another embodiment of the invention, kits for detecting mutations, duplications, deletions, rearrangements and aneuploidies in DD, DED or NB-ARC genes comprising at least one invention probe or antisense nucleotide.

The present invention provides means to modulate levels of expression of DD, DED or NB-ARC polypeptides by employing synthetic antisense-nucleic acid compositions (hereinafter SANC) which inhibit translation of mRNA encoding these polypeptides. Synthetic oligonucleotides, or other antisense-nucleic acid chemical structures designed to recognize and selectively bind to mRNA, are constructed to be complementary to full-length or portions of a DD, DED or NB-ARC domain coding strand, including nucleotide sequences set forth in SEQ ID NOS:1, 3, 5, 7, 9, 11, 17, 21 or 52. The SANC is designed to be stable in the blood stream for administration to a subject by injection, or in laboratory cell culture conditions. The SANC is designed to be capable of passing through the cell membrane in order to enter the cytoplasm of the cell by virtue of physical and chemical properties of the SANC which render it capable of passing through cell membranes, for example, by designing small, hydrophobic SANC chemical structures, or by virtue of specific transport systems in the cell which recognize and transport the SANC into the cell. In addition, the SANC can be designed for administration only to certain selected cell populations by targeting the SANC to be recognized by specific cellular uptake mechanisms which bind and take up the SANC only within select cell populations. In a particular embodiment the SANC is an antisense oligonucleotide.

For example, the SANC may be designed to bind to a receptor found only in a certain cell type, as discussed supra. The SANC is also designed to recognize and selectively bind to target mRNA sequence, which may correspond to a sequence contained within the sequences shown in SEQ ID NOS:1, 3, 5, 7, 9, 11, 17, 21 or 52. The SANC is designed to inactivate target mRNA sequence by either binding thereto and inducing degradation of the mRNA by, for example, RNase I digestion, or inhibiting translation of mRNA target sequence by interfering with the binding of translation-regulating factors or ribosomes, or inclusion of other chemical structures, such as ribozyme sequences or reactive chemical groups which either degrade or chemically modify the target mRNA. SANCs have been shown to be capable of such properties when directed against mRNA targets (see Cohen et al., TIPS, 10:435 (1989) and Weintraub, Sci. American, January (1990), pp.40; both incorporated herein by reference).

In accordance with yet another embodiment of the present invention, there is provided a method for the recombinant production of invention DDs, DEDs or NB-ARC domains by expressing the above-described nucleic acid sequences in suitable host cells. Recombinant DNA expression systems that are suitable to produce DDs, DEDs or NB-ARC domains described herein are well-known in the art. For example, the above-described nucleotide sequences can be incorporated into vectors for further manipulation. As used herein, vector (or plasmid) refers to discrete elements that are used to introduce heterologous DNA into cells for either expression or replication thereof.

Suitable expression vectors are well-known in the art, and include vectors capable of expressing DNA operatively linked to a regulatory sequence, such as a promoter region that is capable of regulating expression of such DNA. Thus, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, a phage, recombinant virus or other vector that, upon introduction into an appropriate host cell, results in expression of the inserted DNA. Appropriate expression vectors are well known to those of skill in the art and include those that are replicable in eukaryotic cells and/or prokaryotic cells and those that remain episomal or those which integrate into the host cell genome.

As used herein, a promoter region refers to a segment of DNA that controls transcription of DNA to which it is operatively linked. The promoter region includes specific sequences that are sufficient for RNA polymerase recognition, binding and transcription initiation. In addition, the promoter region includes sequences that modulate this recognition, binding and transcription initiation activity of RNA polymerase. These sequences may be cis acting or may be responsive to trans acting factors. Promoters, depending upon the nature of the regulation, may be constitutive or regulated. Exemplary promoters contemplated for use in the practice of the present invention include the SV40 early promoter, the cytomegalovirus (CMV) promoter, the mouse mammary tumor virus (MMTV) steroid-inducible promoter, Moloney murine leukemia virus (MMLV) promoter, and the like.

As used herein, the term "operatively linked" refers to the functional relationship of DNA with regulatory and effector nucleotide sequences, such as promoters, enhancers, transcriptional and translational stop sites, and other signal sequences. For example, operative linkage of DNA to a promoter refers to the physical and functional relationship between the DNA and the promoter such that the transcription of such DNA is initiated from the promoter by an RNA polymerase that specifically recognizes, binds to and transcribes the DNA.

As used herein, expression refers to the process by which polynucleic acids are transcribed into mRNA and translated into peptides, polypeptides, or proteins. If the polynucleic acid is derived from genomic DNA, expression can, if an appropriate eukaryotic host cell or organism is selected, include splicing of the mRNA.

Prokaryotic transformation vectors are well-known in the art and include pBlueskript and phage Lambda ZAP vectors (Stratagene, La Jolla, Calif.), and the like. Other suitable vectors and promoters are disclosed in detail in U.S. Pat. No. 4,798,885, issued Jan. 17, 1989, the disclosure of which is incorporated herein by reference in its entirety.

Other suitable vectors for transformation of E. coli cells include the pET expression vectors (Novagen, see U.S Pat. No. 4,952,496), e.g., pET11a, which contains the T7 promoter, T7 terminator, the inducible E. coli lac operator, and the lac repressor gene; and pET 12a-c, which contain the T7 promoter, T7 terminator, and the E. coli ompT secretion signal. Another suitable vector is the pIN-IIIompA2 (see Duffaud et al., Meth. in Enzymology, 153:492-507, 1987), which contains the lpp promoter, the lacUV5 promoter operator, the ompA secretion signal, and the lac repressor gene.

Exemplary, eukaryotic transformation vectors, include the cloned bovine papilloma virus genome, the cloned genomes of the murine retroviruses, and eukaryotic cassettes, such as the pSV-2 gpt system (described by Mulligan and Berg, Nature Vol. 277:108-114 (1979)] the Okayama-Berg cloning system (Mol. Cell Biol. 2:161-170 (1982)), and the expression cloning vector described by Genetics Institute (Wong et al., Science 228:810-815 (1985)), are available which provide substantial assurance of at least some expression of the protein of interest in the transformed eukaryotic cell line.

Particularly preferred base vectors which contain regulatory elements that can be linked to the invention DD-, DED- or NB-ARC domain-encoding DNAs for transfection of mammalian cells are cytomegalovirus (CMV) promoter-based vectors such as pcDNA1 (Invitrogen, San Diego, Calif.), MMTV promoter-based vectors such as pMAMNeo (Clontech, Palo Alto, Calif.) and pMSG (Pharmacia, Piscataway, N.J.), and SV40 promoter-based vectors such as pSVβ (Clontech, Palo Alto, Calif.).

In accordance with another embodiment of the present invention, there are provided "recombinant cells" containing the nucleic acid molecules (i.e., DNA or mRNA) of the present invention. Methods of transforming suitable host cells, preferably bacterial cells, and more preferably E. coli cells, as well as methods applicable for culturing said cells containing a gene encoding a heterologous protein, are generally known in the art. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (2 ed.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (1989).

Exemplary methods of transformation include, e.g., transformation employing plasmids, viral, or bacterial phage vectors, transfection, electroporation, lipofection, and the like. The heterologous DNA can optionally include sequences which allow for its extrachromosomal maintenance, or said heterologous DNA can be caused to integrate into the genome of the host (as an alternative means to ensure stable maintenance in the host).

Host organisms contemplated for use in the practice of the present invention include those organisms in which recombinant production of heterologous proteins has been carried out. Examples of such host organisms include bacteria (e.g., E. coli), yeast (e.g., Saccharomyces cerevisiae, Candida tropicalis, Hansenula polymorpha and P. pastoris; see, e.g., U.S. Pat. Nos. 4,882,279, 4,837,148, 4,929,555 and 4,855,231), mammalian cells (e.g., HEK293, CHO and Ltk^- cells), insect cells, and the like. Presently preferred host organisms are bacteria. The most preferred bacteria is E. coli.

In one embodiment, nucleic acids encoding the invention DDs, DEDs or NB-ARC domains can be delivered into mammalian cells, either in vivo or in vitro using suitable viral vectors well-known in the art. Suitable retroviral vectors, designed specifically for "gene therapy" methods, are described, for example, in WIPO publications WO 9205266 and WO 9214829, which provide a description of methods for efficiently introducing nucleic acids into human cells. In addition, where it is desirable to limit or reduce the in vivo expression of the invention DD-, DED- or NB-ARC domain-containing, the introduction of the antisense strand of the invention nucleic acid is contemplated.

Viral based systems provide the advantage of being able to introduce relatively high levels of the heterologous nucleic acid into a variety of cells. Suitable viral vectors for introducing invention nucleic acid encoding an DD, DED or NB-ARC domain into mammalian cells (e.g., vascular tissue segments) are well known in the art. These viral vectors include, for example, Herpes simplex virus vectors (e.g., Geller et al., Science, 241:1667-1669 (1988)), Vaccinia virus vectors (e.g., Piccini et al., Meth. in Enzymology, 153:545-563 (1987); Cytomegalovirus vectors (Mocarski et al., in Viral Vectors, Y. Gluzman and S. H. Hughes, Eds., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1988, pp. 78-84), Moloney murine leukemia virus vectors (Danos et al., PNAS, USA, 85:6469 (1980)), adenovirus vectors (e.g., Logan et al., PNAS, USA, 81:3655-3659 (1984); Jones et al., Cell, 17:683-689 (1979); Berkner, Biotechniques, 6:616-626 (1988); Cotten et al., PNAS, USA, 89:6094-6098 (1992); Graham et al., Meth. Mol. Biol., 7:109-127 (1991)), adeno-associated virus vectors, retrovirus vectors (see, e.g., U.S. Pat. Nos. 4,405,712 and 4,650,764), and the like. Especially preferred viral vectors are the adenovirus and retroviral vectors.

For example, in one embodiment of the present invention, adenovirus-transferrin/polylysine-DNA (TfAdpl-DNA) vector complexes (Wagner et al., PNAS, USA, 89:6099-6103 (1992); Curiel et al., Hum. Gene Ther., 3:147-154 (1992); Gao et al., Hum. Gene Ther., 4:14-24 (1993)) are employed to transduce mammalian cells with heterologous DD, DED or NB-ARC domain nucleic acid. Any of the plasmid expression vectors described herein may be employed in a TfAdpl-DNA complex.

As used herein, "retroviral vector" refers to the well-known gene transfer plasmids that have an expression cassette encoding an heterologous gene residing between two retroviral LTRs. Retroviral vectors typically contain appropriate packaging signals that enable the retroviral vector, or RNA transcribed using the retroviral vector as a template, to be packaged into a viral virion in an appropriate packaging cell line (see, e.g., U.S. Pat. No. 4,650,764).

Suitable retroviral vectors for use herein are described, for example, in U.S. Pat. No. 5,252,479, and in WIPO publications WO 92/07573, WO 90/06997, WO 89/05345, WO 92/05266 and WO 92/14829, incorporated herein by reference, which provide a description of methods for efficiently introducing nucleic acids into human cells using such retroviral vectors. Other retroviral vectors include, for example, the mouse mammary tumor virus vectors (e.g., Shackleford et al., PNAS, USA, 85:9655-9659 (1988)), and the like.

In accordance with yet another embodiment of the present invention, there are provided anti-DD, anti-DED or anti-NB-ARC domain antibodies having specific reactivity with one or more DD, DED or NB-ARC polypeptides of the present invention. Active fragments of antibodies are encompassed within the definition of "antibody". Invention antibodies can be produced by methods known in the art using invention polypeptides, proteins or portions thereof as antigens. For example, polyclonal and monoclonal antibodies can be produced by methods well known in the art, as described, for example, in Harlow and Lane, Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory (1988)), which is incorporated herein by reference. Invention polypeptides can be used as immunogens in generating such antibodies. Alternatively, synthetic peptides can be prepared (using commercially available synthesizers) and used as immunogens. Amino acid sequences can be analyzed by methods well known in the art to determine whether they encode hydrophobic or hydrophilic domains of the corresponding polypeptide. Altered antibodies such as chimeric, humanized, CDR-grafted or bifunctional antibodies can also be produced by methods well known in the art. Such antibodies can also be produced by hybridoma, chemical synthesis or recombinant methods described, for example, in Sambrook et al., supra., and Harlow and Lane, supra. Both anti-peptide and anti-fusion protein antibodies can be used. (see, for example, Bahouth et al., Trends Pharmacol. Sci. 12:338 (1991); Ausubel et al., Current Protocols in Molecular Biology (Supplement 47), John Wiley & Sons, New York (1999), which are incorporated herein by reference).

The invention provides isolated anti-DD, anti-DED, or anti-NB-ARC antibodies having specific reactivity with a polypeptide of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 53, 56, or 58. In addition, isolated anti-DD, anti-DED, or anti-NB-ARC antibodies are provided having specific reactivity with a polypeptide of SEQ ID NOS: 18 or 22. Furthermore, isolated anti-DD, anti-DED, or anti-NB-ARC antibodies are provided having specific reactivity with amino acids or peptides within the polypeptides of SEQ ID NOS: 16, 20, and 26 that differ from SEQ ID NOS: 24 and 28. Invention polypeptides, or fragments thereof, and synthetic peptides can be used as immunogens in generating the antibodies provided herein.

Antibody so produced can be used, inter alia, in diagnostic methods and systems to detect the level of DD, DED or NB-ARC polypeptides present in a mammalian, preferably human, body sample, such as tissue or vascular fluid. Such antibodies can also be used for the immunoaffinity or affinity chromatography purification of the invention DD, DED or NB-ARC domain. In addition, methods are contemplated herein for detecting the presence of an invention DD, DED or NB-ARC domain either within a cell, or on the surface of a cell, comprising contacting the cell with an antibody that specifically binds to DD, DED or NB-ARC domain polypeptides, under conditions permitting binding of the antibody to the DD, DED or NB-ARC domain polypeptides, detecting the presence of the antibody bound to the DD, DED or NB-ARC domain polypeptide, and thereby detecting the presence of invention polypeptides on the surface of the cell. With respect to the detection of such polypeptides, the antibodies can be used for in vitro diagnostic or in vivo imaging methods.

Immunological procedures useful for in vitro detection of target DD, DED or NB-ARC domain polypeptides in a sample include immunoassays that employ a detectable antibody. Such immunoassays include, for example, ELISA, Pandex microfluorimetric assay, agglutination assays, flow cytometry, serum diagnostic assays and immunohistochemical staining procedures which are well known in the art. An antibody can be made detectable by various means well known in the art. For example, a detectable marker can be directly or indirectly attached to the antibody. Useful markers include, for example, radionucleotides, enzymes, fluorogens, chromogens and chemiluminescent labels.

Invention anti-DD, anti-DED or anti-NB-ARC domain antibodies are contemplated for use herein to modulate the activity of the DD, DED or NB-ARC domain polypeptide in living animals, in humans, or in biological tissues or fluids isolated therefrom. The term "modulate" refers to a compound's ability to increase (e.g., via an agonist), decrease (e.g., via an antagonist), or otherwise modify (e.g., increasing a first DD, DED or NB-ARC domain activity while decreasing a second DD, DED or NB-ARC domain activity) the biological activity of an invention DD, DED or NB-ARC domain protein, such as binding to FADD, caspases such as caspase-8, DR4, DR5, Traf6, hToll, MyD88 and Fas, NF-κB or JNK modulating activity, or caspase such as caspase-8 modulating activity, apoptosis modulating activity, cell proliferation modulating activity, cell adhesion modulating activity, cell stress responses modulating activity, microbial infection response modulating activity, or B cell immunoglobulin class switching modulating activity, and the like. Accordingly, compositions comprising a carrier and an amount of an antibody having specificity for DD, DED or NB-ARC domain polypeptides effective to block naturally occurring ligands or other DD-, DED- or NB-ARC domain-associated proteins, and the like, from binding to invention DD, DED or NB-ARC domain polypeptides are contemplated herein. For example, a monoclonal antibody directed to an epitope of an invention DD, DED or NB-ARC domain polypeptide including an amino acid sequence set forth in SEQ ID NOS:2, 4, 6, 8, 10, 12, 53, 56 or 58, or SEQ ID NOS:16, 18, 20, 22 or 26, can be useful for this purpose.

The present invention further provides transgenic non-human mammals that are capable of expressing exogenous nucleic acids encoding DDs, DEDs or NB-ARC domains. As employed herein, the phrase "exogenous nucleic acid" refers to nucleic acid sequence which is not native to the host, or which is present in the host in other than its native environment (e.g., as part of a genetically engineered DNA construct). In addition to naturally occurring levels of DD-, DED- or NB-ARC domain-containing proteins, invention DDs, DEDs or NB-ARC domain can either be overexpressed or underexpressed (such as in the well-known knock-out transgenics) in transgenic mammals.

Also provided are transgenic non-human mammals capable of expressing nucleic acids encoding DD, DED or NB-ARC domain polypeptides so mutated as to be incapable of normal activity, i.e., do not express native DD, DED or NB-ARC domain polypeptides. The present invention also provides transgenic non-human mammals having a genome comprising antisense nucleic acids complementary to nucleic acids encoding DD, DED or NB-ARC domain polypeptides, placed so as to be transcribed into antisense mRNA complementary to mRNA encoding DD, DED or NB-ARC domain polypeptides, which hybridizes to the mRNA and, thereby, reduces the translation thereof. The nucleic acid can additionally comprise an inducible promoter and/or tissue specific regulatory elements, so that expression can be induced, or restricted to specific cell types. Examples of nucleic acids are DNA or cDNA having a coding sequence the same or substantially the same as the coding sequence of SEQ ID NOS:13, 15, 17, 19, 21 or 54, and preferably 1, 3, 5, 7, 9, 11 or 52. An example of a non-human transgenic mammal is a transgenic mouse. Examples of tissue specificity-determining elements are the metallothionein promoter and the L7 promoter.

Animal model systems which elucidate the physiological and behavioral roles of DD, DED or NB-ARC domain polypeptides are also provided, and are produced by creating transgenic animals in which the expression of the DD, DED or NB-ARC domain polypeptide is altered using a variety of techniques. Examples of such techniques include the insertion of normal or mutant versions of nucleic acids encoding a DD, DED or NB-ARC domain polypeptide by microinjection, retroviral infection or other means well known to those skilled in the art, into appropriate fertilized embryos to produce a transgenic animal. (See, for example, Hogan et al., Manipulating the Mouse Embryo: A Laboratory Manual (Cold Spring Harbor Laboratory, (1986)).

Also contemplated herein, is the use of homologous recombination of mutant or normal versions of DD, DED or NB-ARC domain genes with the native gene locus in transgenic animals, to alter the regulation of expression or the structure of DD, DED or NB-ARC domain polypeptides (see, Capecchi et al., Science 244:1288 (1989); Zimmer et al., Nature 338:150 (1989); which are incorporated herein by reference). Homologous recombination techniques are well known in the art. Homologous recombination replaces the native (endogenous) gene with a recombinant or mutated gene to produce an animal that cannot express native (endogenous) protein but can express, for example, a mutated protein which results in altered expression of DD, DED or NB-ARC domain polypeptides.

In contrast to homologous recombination, microinjection adds genes to the host genome, without removing host genes. Microinjection can produce a transgenic animal that is capable of expressing both endogenous and exogenous DDs, DEDs or NB-ARC domains. Inducible promoters can be linked to the coding region of nucleic acids to provide a means to regulate expression of the transgene. Tissue specific regulatory elements can be linked to the coding region to permit tissue-specific expression of the transgene. Transgenic animal model systems are useful for in vivo screening of compounds for identification of specific ligands, i.e., agonists and antagonists, which activate or inhibit protein responses.

Invention nucleic acids, oligonucleotides (including antisense), vectors containing same, transformed host cells, polypeptides and combinations thereof, as well as antibodies of the present invention, can be used to screen compounds in vitro to determine whether a compound functions as a potential agonist or antagonist to invention DDs, DEDs or NB-ARC domains. These in vitro screening assays provide information regarding the function and activity of invention DDs, DEDs, or NB-ARC domains which can lead to the identification and design of compounds that are capable of specific interaction with one or more types of polypeptides, peptides or proteins.

By the known homology of invention DDs, DEDs and NB-ARC domains to known proteins containing these domains, it is within the scope of the invention that invention DD, DED or NB-ARC domain also have a role in cellular pathways that effect apoptosis, cell proliferation, cell adhesion, cell stress responses, responses to microbial infection, and B cell immunoglobulin class switching. Thus, invention DDs, DEDs or NB-ARC domains also provide drug discovery targets for a broad variety of pathologies including infection, autoimmunity, inflammation, allergy, allograph-rejection and sepsis, and for a broad variety of cancer pathologies, such as, gliomas, carcinomas, sarcomas, melanomas, hamartomas and the like. In certain aspects of the invention, invention DD, DED or NB-ARC domain proteins, agonist or antagonists thereto, are used to treat infection, autoimmunity, inflammation, allergy, allograph-rejection, sepsis, keratinocyte hyperplasia, neoplasia, keloid, benign prostatic hypertrophy, inflammatory hyperplasia, fibrosis, smooth muscle cell proliferation in arteries following balloon angioplasty (restenosis), and the like. Exemplary cancer pathologies contemplated herein for treatment include, gliomas, carcinomas, adenocarcinomas, sarcomas, melanomas, hamartomas, leukemias, lymphomas, and the like. Exemplary infections contemplated herein for treatment include bacterial infections such as infections caused by Chlamydia (Ojcius et al., J. Immunol. 161:4220-6 (1998)), Pseudomonas (Hauser and Engel, Infect. Immun. 67: 5530-7 (1999)), Salmonella (Hersh et al., Proc. Natl. Acad. Sci, USA, 96:2396-401 (1999)), Shigella (Zychlinsky, et al., Nature 358:167-9 (1992)), and Mycobacterium (Oddo, et al., J. Immunol. 160:5448-54 (1998)), which are incorporated herein by reference.

Chlamydia trachomatis is a eubacterial pathogen accounting for the major cause of blindness in Asia and Africa and is the most common sexually transmitted disease in the United States. Chlamydia infections have been linked to pelvic inflammatory disease, urethritis, and infertility. Different strains of Chlamydia have also been linked to arthritis, pneumonia, upper respiratory and ear infections, asthma, vasculitis, atherosclerosis, and other vascular diseases. In addition, chronic Chlamydia infections have also been linked to cancer. A recent longitudinal study provided evidence that patients infected with Chlamydia trachomatis serotype G carry a 6.6-fold increased risk of developing cervical cancer.

Also provided herein are methods of treating pathologies, said method comprising administering an effective amount of an invention therapeutic composition. Such compositions are typically administered in a physiologically compatible composition.

Methods of treating pathologies of abnormal cell proliferation include methods of modulating the activity of one or more oncogenic proteins, wherein the oncogenic proteins specifically interact with a DD, DED or NB-ARC domain. Methods of modulating the activity of such oncogenic proteins include contacting the oncogenic protein with a substantially pure DD, DED or NB-ARC domain or an active fragment (i.e., oncogenic protein-binding fragment) thereof. This contacting can modulate the activity of the oncogenic protein, thereby providing a method of treating a pathology caused by the oncogenic protein. Further methods of modulating the activity of oncogenic proteins include contacting the oncogenic protein with an agent, wherein the agent modulates the interactions between the DD, DED or NB-ARC domain and the oncogenic protein.

Methods of treating bacterial infections include methods of modulating the activity of one or more bacterial proteins that contain or specifically interact with a DD, DED or NB-ARC domain. Methods of modulating the activity of such a bacterial protein include contacting the bacterial protein with a substantially pure DD, DED or NB-ARC domain or an active fragment thereof. This contacting can modulate the activity of the bacterial protein, thereby providing a method of treating a pathology caused by the bacteria. Further methods of modulating the activity of bacterial proteins include contacting the bacterial protein with an agent, including, for example, a nucleic acid, a drug, a peptide, or a protein, including a secreted protein or an antibody, wherein the agent modulates a DD, DED or NB-ARC domain of a bacterial protein or the agent modulates the interactions between a DD, DED or NB-ARC domain and a bacterial protein.

Methods of treating bacterial infections can further include methods of modulating the activity of one or more host cell proteins that specifically interact with a bacterial protein that contains or specifically interacts with a DD, DED or NB-ARC domain. Methods of modulating the activity of such a host cell protein include contacting the host cell protein with a substantially pure DD, DED or NB-ARC domain or an active fragment thereof. This contacting can modulate the activity of the host cell protein, thereby providing a method of treating a pathology caused by the interaction of the host cell and bacterial proteins. Further methods of modulating the activity of host cell proteins include contacting the host cell protein with an agent, wherein the agent modulates the interactions between a host cell protein and a bacterial protein that contains or specifically interacts with a DD, DED or NB-ARC domain. All of the above methods for treating bacterial infections can be used alone or in combination with other methods of treating bacterial infections.

Methods of treating immune-based pathologies such as infection, autoimmunity, inflammation, allergy, allograft-rejection, and sepsis will include modulating the activity of one or more proteins that modulate immune response, wherein the protein that modulates immune response specifically interact with a DD, DED or NB-ARC domain. Methods of modulating the activity of such protein that modulates immune response will include contacting the protein that modulates immune response with a substantially pure DD, DED or NB-ARC domain or an active fragment (i.e., protein-binding fragment) thereof. This contacting will modulate the activity of the protein that modulates immune response, thereby providing a method of treating a pathology caused by the protein that modulates immune response. Further methods of modulating the activity of a protein that modulates immune response will include contacting the protein that modulates immune response with an agent, wherein the agent modulates the interactions between the DD, DED or NB-ARC domain and the protein that modulates immune response.

Also contemplated herein, are therapeutic methods using invention pharmaceutical compositions for the treatment of pathological disorders in which there is too little cell division, such as, for example, bone marrow aplasias, immunodeficiencies due to a decreased number of lymphocytes, and the like. Methods of treating a variety of inflammatory diseases with invention therapeutic compositions are also contemplated herein, such as treatment of sepsis, fibrosis (e.g., scarring), arthritis, graft versus host disease, and the like. Therapeutic methods using invention polypeptides or nucleic acids are also contemplated for treating infectious diseases.

The present invention also provides therapeutic compositions useful for practicing the therapeutic methods described herein. Therapeutic compositions of the present invention, such as pharmaceutical compositions, contain a physiologically compatible carrier together with an invention DD, DED or NB-ARC domain (or functional fragment thereof), a DD, DED or NB-ARC domain modulating agent, such as a compound (agonist or antagonist) identified by the methods described herein, or an anti-DD, anti-DED or anti-NB-ARC domain antibody, as described herein, dissolved or dispersed therein as an active ingredient. In a preferred embodiment, the therapeutic composition is not immunogenic when administered to a mammal or human patient for therapeutic purposes.

As used herein, the terms "pharmaceutically acceptable", "physiologically compatible" and grammatical variations thereof, as they refer to compositions, carriers, diluents and reagents, are used interchangeably and represent that the materials are capable of administration to a mammal without the production of undesirable physiological effects such as nausea, dizziness, gastric upset, and the like.

The preparation of a pharmacological composition that contains active ingredients dissolved or dispersed therein is well known in the art. Typically such compositions are prepared as injectables either as liquid solutions or suspensions; however, solid forms suitable for solution, or suspension, in liquid prior to use can also be prepared. The preparation can also be emulsified.

The active ingredient can be mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient in amounts suitable for use in the therapeutic methods described herein. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like, as well as combinations of any two or more thereof. In addition, if desired, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and the like, which enhance the effectiveness of the active ingredient.

The therapeutic composition of the present invention can include pharmaceutically acceptable salts of the components therein. Pharmaceutically acceptable nontoxic salts include the acid addition salts (formed with the free amino groups of the polypeptide) that are formed with inorganic acids such as, for example, hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, phosphoric acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, anthranilic acid, cinnamic acid, naphthalene sulfonic acid, sulfanilic acid, and the like.

Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium hydroxide, ammonium hydroxide, potassium hydroxide, and the like; and organic bases such as mono-, di-, and tri-alkyl and -aryl amines (e.g., triethylamine, diisopropyl amine, methyl amine, dimethyl amine, and the like) and optionally substituted ethanolamines (e.g., ethanolamine, diethanolamine, and the like).

Physiologically tolerable carriers are well known in the art. Exemplary liquid carriers are sterile aqueous solutions that contain no materials in addition to the active ingredients and water, or contain a buffer such as sodium phosphate at physiological pH, physiological saline or both, such as phosphate-buffered saline. Still further, aqueous carriers can contain more than one buffer salt, as well as salts such as sodium and potassium chlorides, dextrose, polyethylene glycol and other solutes.

Liquid compositions can also contain liquid phases in addition to and to the exclusion of water. Exemplary additional liquid phases include glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions.

As described herein, an "effective amount" is a predetermined amount calculated to achieve the desired therapeutic effect, e.g., to modulate activity of an invention DD, DED or NB-ARC domain. The required dosage will vary with the particular treatment and with the duration of desired treatment; however, it is anticipated that dosages between about 10 micrograms and about 1 milligram per kilogram of body weight per day will be used for therapeutic treatment. It may be particularly advantageous to administer such compounds in depot or long-lasting form as discussed hereinafter. A therapeutically effective amount is typically an amount of an DD-, DED- or NB-ARC domain-modulating agent or compound identified herein that, when administered in a physiologically acceptable composition, is sufficient to achieve a plasma concentration of from about 0.1 μg/ml to about 100 μg/ml, preferably from about 1.0 μg/ml to about 50 μg/ml, more preferably at least about 2 μg/ml and usually 5 to 10 μg/ml. Therapeutic invention anti-DD, anti-DED or anti-NB-ARC domain antibodies can be administered in proportionately appropriate amounts in accordance with known practices in this art.

In accordance with still another embodiment of the present invention, there are provided methods for identifying compounds which bind to DD, DED or NB-ARC domain polypeptides. The invention proteins may be employed in a competitive binding assay. Such an assay can accommodate the rapid screening of a large number of compounds to determine which compounds, if any, are capable of binding to DDs, DEDs or NB-ARC domains. Subsequently, more detailed assays can be carried out with those compounds found to bind, to further determine whether such compounds act as modulators, agonists or antagonists of invention DDs, DEDs or NB-ARC domains. Compounds that bind to and/or modulate invention DDs, DEDs or NB-ARC domains can be used to treat a variety of pathologies mediated by invention DDs, DEDs or NB-ARC domains.

In another embodiment of the invention, there is provided a bioassay for identifying compounds which modulate the activity of invention DD, DED or NB-ARC domain polypeptides. Invention DD, DED or NB-ARC domain polypeptides are known to influence the activities of, for example, NF-κB, JNK, and caspase-8. Thus a reporter gene construct to assay for NF-κB activity can be used to test invention DED activity (see Examples). According to this method, invention DD, DED or NB-ARC domain polypeptides are contacted with an "unknown" or test substance, the activity of the invention DD, DED or NB-ARC domain polypeptide is monitored subsequent to the contact with the "unknown" or test substance, and those substances which effect a resultant modulation of, for example, NF-κB or JNK activity or caspase, such as caspase-8, activity are identified as functional ligands for DD, DED or NB-ARC domain polypeptides.

Alternative bioassays for identifying compounds which modulate the activity of invention DD, DED or NB-ARC domain polypeptides can be used which routinely are used to test for protein:protein interactions. Such bioassays include yeast two-hybrid assays, glutathione-S-transferase fusion protein binding assays, co-immmunoprecipitation assays, and the like. Such assays are well known in the art and can be found in standard reference texts such as Sambrook et al., supra, and Ausubel et al., supra, 1999.

In accordance with another embodiment of the present invention, transformed host cells that recombinantly express invention polypeptides can be contacted with a test compound, and the modulating effect(s) thereof can then be evaluated by comparing the DD-, DED-, or NB-ARC domain-medicated response (e.g., via reporter gene expression) in the presence and absence of test compound, or by comparing the response of test cells or control cells (i.e., cells that do not express DD, DED or NB-ARC domain polypeptides), to the presence of the compound.

As used herein, a compound or a signal that "modulates the activity" of invention DD, DED or NB-ARC domain polypeptides refers to a compound or a signal that alters the activity of DD, DED or NB-ARC domain polypeptides so that the activity of the invention polypeptide is different in the presence of the compound or signal than in the absence of the compound or signal. In particular, such compounds or signals include agonists and antagonists. An agonist encompasses a compound or a signal that activates DD, DED or NB-ARC domain protein expression. Alternatively, an antagonist includes a compound or signal that interferes with DD, DED or NB-ARC domain expression. Typically, the effect of an antagonist is observed as a blocking of agonist-induced protein activation. Antagonists include competitive and non-competitive antagonists. A competitive antagonist (or competitive blocker) interacts with or near the site specific for agonist binding. A non-competitive antagonist or blocker inactivates the function of the polypeptide by interacting with a site other than the agonist interaction site.

As understood by those of skill in the art, assay methods for identifying compounds that modulate DD, DED or NB-ARC domain activity generally require comparison to a control. One type of a "control" is a cell or culture that is treated substantially the same as the test cell or test culture exposed to the compound, with the distinction that the "control" cell or culture is not exposed to the compound. For example, in methods that use voltage clamp electrophysiological procedures, the same cell can be tested in the presence or absence of compound, by merely changing the external solution bathing the cell. Another type of "control" cell or culture may be a cell or culture that is identical to the transfected cells, with the exception that the "control" cell or culture do not express native proteins. Accordingly, the response of the transfected cell to compound is compared to the response (or lack thereof) of the "control" cell or culture to the same compound under the same reaction conditions.

In yet another embodiment of the present invention, the activation of DD, DED or NB-ARC domain polypeptides can be modulated by contacting the polypeptides with an effective amount of at least one compound identified by the above-described bioassays.

In accordance with another embodiment of the present invention, there are provided methods for identifying a binding agent that binds a DD, DED or NB-ARC domain, where a DD, DED, or NB-ARC domain from DAP3, IRAK4, CTDD, DED4 or NIDD is contacted with a candidate binding agent and then the association of the domain and candidiate binding agent are detected. An association between the candidate binding agent and the domain identifies the candidate binding agent as a binding agent that binds a DD, DED, or NB-ARC domain from DAP3, IRAK4, CTDD, DED4 or NIDD. The association between the candidate binding agent and the domain can be detected using a variety of methods well known in the art, for example, co-immunoprecipitation assays and transcription based assays such as reporter assays and two-hybrid assays. Such assays are well known in the art and can be found in standard reference texts such as Sambrook et al., supra, and Ausubel et al., supra, 1999. Additional methods include, for example, scintillation proximity assay (SPA) (Alouani, Methods Mol. Biol. 138:135-41 (2000)), UV or chemical cross-linking (Fancy, Curr. Olpin. Chem. Biol. 4:28-33 (2000)), competition binding assays (Yamamura et al., Methods in Neurotransmitter Receptor Analysis, Raven Press, New York, 1990), biomolecular interaction analysis (BIA) (Weinberger et al., Pharmacogenomics 1:395-416 (2000)), mass spectrometry (MS) (McLafferty et al., Science 284:1289-1290 (1999) and Degterev, et al., Nature Cell Bioloqy 3:173-182 (2001)), nuclear magnetic resonance (NMR) (Shuker etal., Science 274:1531-1534 (1996), Hajduk et al., J. Med. Chem. 42:2315-2317 (1999), and Chen and Shapiro, Anal. Chem. 71:669A-675A (1999)), and fluorescence polarization assays (FPA) (Degterev et al., supra, 2001) which are incorporated herein by reference. The identified binding agent can be, for example, another protein, including an antibody or fragment thereof, or a drug or other agent.

In accordance with another embodiment of the present invention, there are provided methods for identifying an effective agent that modulates the association of a DD, DED or NB-ARC domain from DAP3, IRAK4, CTDD, DED4 or NIDD with a protein that binds the DD, DED or NB-ARC domain where the proteins are contacted under conditions that allow the domain and a protein that binds the domain to associate with an agent suspected of being able to modulate the association of the domain and protein that binds the domain. Detection of a modulated association of the domain and protein that binds the domain identifies the agent as an effective agent. An altered association can be detected, for example, by measuring the activity of NF-κB or caspases or by using other methods well known in the art and described herein. The effective agent can be, for example, another protein, including an antibody, or a drug.

In accordance with another embodiment of the present invention, there are provided methods of modulating a cell process such as apoptosis, cell proliferation, cell adhesion, cell stress responses, responses to microbial infection, and B cell immunoglobulin class switching, by contacting a cell with an effective agent that modulates the activity of a DD-, DED-, or NB-ARC domain. For example, a nucleic acid molecule encoding a DD, DED or NB-ARC domain from DAP3, IRAK4, CTDD, DED4 or NIDD, can be introduced into a cell and expression of the DD, DED or NB-ARC domain can modulate a cell process within the cell. In addition, an antisense nucleotide sequence that specifically hybridizes to a nucleic acid molecule encoding a DD, DED or NB-ARC domain from DAP3, IRAK4, CTDD, DED4 or NIDD, can be introduced into a cell where hybridization can reduce or inhibit the expression of the DD, DED or NB-ARC domain in the cell which modulates a cell process within the cell. Furthermore, a cell process can be modulated by contacting a cell with a DD, DED or NB-ARC domain or functional fragment thereof, an effective agent as described above, or an anti-DD, anti-DED or anti-NB-ARC domain antibody where the DD, DED, or NB-ARC domain is from DAP3, IRAK4, DED4 or NIDD.

Methods are also provided for modulating an activity mediated by a DD, DED or NB-ARC domain, by contacting the DD, DED or NB-ARC domain with an effective agent identified as described above. The modulated activity can be, for example, binding of a DD, DED or NB-ARC domain protein to a protein that binds a DD, DED or NB-ARC domain, NF-κB activity, caspase such as caspase-8 activity, apoptosis activity, cell proliferation activity, cell adhesion, cell stress response activity, responses to microbial infection activity, and B cell immunoglobulin class switching activity. For example, the activity of NF-κB or caspases can be modulated by a cell with an effective agent that modulates the activity of a DD-, DED-, or NB-ARC domain.

In accordance with another embodiment of the present invention, there are provided methods of diagnosing a pathology characterized by an increased or decreased level of a DD, DED or NB-ARC domain from DAP3, IRAK4, CTDD, DED4 or NIDD in a subject. For example, a test sample from a subject can be contacted with an agent that can bind the DD, DED or NB-ARC domain under suitable conditions, which allow specific binding of the agent to the DD, DED or NB-ARC domain, and then the amount of specific binding in the test sample can be compared with the amount of specific binding in a control sample, where an increased or decreased amount of specific binding in said test sample as compared to a control sample is diagnostic of a pathology. The agent that can bind the DD, DED or NB-ARC domain can be, for example, an anti-DD, anti-DED, or anti-NB-ARC domain antibody, FADD, caspases such as caspase-8 and caspase-10, DR4, DR5, Traf6, hToll, MyD88 Fas, Raidd, IRAK, IRAK-2, IRAK-M, p75NTR, Tradd, DAP kinase, RIP, NMP84, ankyrins, Flip, PEA15, Flash, BAP31, BAR, DEDT/DEDD, CTDD, or DAP3. In addition, a test sample containing nucleic acid molecules from a subject can be contacted under high stringency hybridization conditions with an oligonucleotide specific for one of the above DD, DED, or NB-ARC domain containing proteins, and the amount of specific binding in the test sample can be compared with the amount of specific binding in a control sample, where an increased or decreased amount of specific binding in the test sample as compared to said control sample is diagnostic of a pathology.

In accordance with another embodiment of the present invention, there are provided methods for diagnosing cancer, said method comprising detecting, in said subject, a defective sequence or mutant of SEQ ID NOS:1, 3, 5, 7, 9, 11, or 52.

In accordance with another embodiment of the present invention, there are provided methods for diagnosing a bacterial infection or monitoring the progression of a bacterial infection by detecting in a subject either nucleic acid molecules or proteins specific to a bacterial pathogen. For example, a Chlamydia infection can be detected by contacting a test sample from a subject with an antibody specifically reactive with a peptide or polypeptide consisting of any of SEQ ID NOS: 10, 20, 53, 56, or 58. In addition, a test sample from a subject can be contacted under high stringency conditions with a nucleic acid molecule encoding any of SEQ ID NOS: 10, 20, 53, 56, or 58.

In accordance with another embodiment of the present invention, there are provided diagnostic systems, preferably in kit form, comprising at least one invention nucleic acid in a suitable packaging material. The diagnostic nucleic acids are derived from the DD-, DED- or NB-ARC domain-encoding nucleic acids described herein. In one embodiment, for example, the diagnostic nucleic acids are derived from any of SEQ ID NOS:1, 3, 5, 7, 9, 11 or 52. Invention diagnostic systems are useful for assaying for the presence or absence of nucleic acid encoding DD, DED or NB-ARC domain polypeptides in either genomic DNA or in transcribed nucleic acid (such as mRNA or cDNA) encoding DD, DED or NB-ARC domain polypeptides.

A suitable diagnostic system includes at least one invention nucleic acid, preferably two or more invention nucleic acids, as a separately packaged chemical reagent(s) in an amount sufficient for at least one assay. Instructions for use of the packaged reagent are also typically included. Those of skill in the art can readily incorporate invention nucleic probes and/or primers into kit form in combination with appropriate buffers and solutions for the practice of the invention methods as described herein.

As employed herein, the phrase "packaging material" refers to one or more physical structures used to house the contents of the kit, such as invention nucleic acid probes or primers, and the like. The packaging material is constructed by well known methods, preferably to provide a sterile, contaminant-free environment. The packaging material has a label which indicates that the invention nucleic acids can be used for detecting a particular sequence encoding DD, DED or NB-ARC domain polypeptides including the nucleotide sequences set forth in SEQ ID NOS:1, 3, 5, 7, 9, 11 or 52 or mutations or deletions therein, thereby diagnosing the presence of, or a predisposition for, cancer. In addition, the packaging material contains instructions indicating how the materials within the kit are employed both to detect a particular sequence and diagnose the presence of, or a predisposition for, cancer.

The packaging materials employed herein in relation to diagnostic systems are those customarily utilized in nucleic acid-based diagnostic systems. As used herein, the term "package" refers to a solid matrix or material such as glass, plastic, paper, foil, and the like, capable of holding within fixed limits an isolated nucleic acid, oligonucleotide, or primer of the present invention. Thus, for example, a package can be a glass vial used to contain milligram quantities of a contemplated nucleic acid, oligonucleotide or primer, or it can be a microtiter plate well to which microgram quantities of a contemplated nucleic acid probe have been operatively affixed.
 

Claim 1 of 11 Claims

1. An isolated nucleic acid molecule consisting of

DNA encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:6.

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