WO2000018800A1

WO2000018800A1 - Novel secreted immunomodulatory proteins and uses thereof

Info

Publication number: WO2000018800A1
Application number: PCT/US1999/022818
Authority: WO
Inventors: Samantha J. Busfield
Original assignee: Millennium Pharmaceuticals, Inc.
Priority date: 1998-09-30
Filing date: 1999-09-30
Publication date: 2000-04-06
Also published as: WO2000018800A9; AU6280999A

Abstract

The invention concerns cDNA molecules encoding TANGO 191 and TANGO 195, both of which are transmembrane proteins. The nucleic acids and polypeptides of the present invention are useful as modulating agents in regulating a variety of cellular processes. Accordingly, in one aspect, this invention provides isolated nucleic acid molecules encoding a polypeptide of the invention or biologically active portion thereof. The present invention also provides nucleic acid molecules which are suitable as primers or hybridization probes for the detection of nucleic acids encoding a polypeptide of the invention.

Description

NOVEL SECRETED I MuTSTOMODULATORY PROTEINS AND USES THEREOF

Background of the Invention

Many secreted proteins, for example, cytokines and cytokine receptors, play a vital role in the regulation of cell growth, cell differentiation, and a variety of specific cellular responses. A number of medically useful proteins, including erythropoietin, granulocyte- macrophage colony stimulating factor, human growth hormone, and various interleukins, are secreted proteins. Thus, an important goal in the design and development of new therapies is the identification and characterization of secreted proteins and the genes which encode them.

Many secreted proteins are receptors which bind a ligand and transduce an intracellular signal, leading to a variety of cellular responses. The identification and characterization of such a receptor enables one to identify both the ligands which bind to the receptor and the intracellular molecules and signal transduction pathways associated with the receptor, permitting one to identify or design modulators of receptor activity, e.g., receptor agonists or antagonists and modulators of signal transduction. Summary of the Invention The present invention is based, at least m part, on the discovery of cDNA molecules encoding TANGO 191 and TANGO 195, both of which are transmembrane proteins. These proteins, fragments, derivatives, and variants thereof are collectively referred to as "polypeptides of the invention" or "proteins of the invention." Nucleic acid molecules encoding polypeptides of the invention are collectively referred to as "nucleic acids of the invention. "

The nucleic acids and polypeptides of the present invention are useful as modulating agents m regulating a variety of cellular processes. Accordingly, m one aspect, this invention provides isolated nucleic acid molecules encoding a polypeptide of the invention or a biologically active portion thereof. The present invention also provides nucleic acid molecules which are suitable as primers or hybridization probes for the detection of nucleic acids encoding a polypeptide of the invention.

Tne invention features nucleic acid molecules which are at least about 45% (or 55%, 65%, 75%, 85%, 95%, or 98%) identical to the nucleotide sequence shown m SEQ ID NO: 1, 3, 4, 6 or , or the nucleotide sequence of the cDNA insert of either the clone deposited with the American Type Culture Collection, Manassas, VA (ATCC) as

Accession Number or the clone deposited with the

ATCC as Accession Number (the "cDNA of ATCC " or the "cDNA of ATCC "), or a complement thereof. Tne invention features nucleic acid molecules which include a fragment of at least 300 (325, 350, 375, 400, 425, 450, 500, 550, 600, 650, 700, 800, 900, 1000, or 1200 ^ nucleotides of the nucleotide sequence of SEQ ID NO:l, 3, 4, 6 or or the nucleotiαe sequence of the cDNA of ATCC or the cDNA of ATCC , or a complement thereof .

The invention also features nucleic acid molecules which include a nucleotide sequence encoding a protein having an ammo acid sequence that is at least about 45% (or 55%, 65%, 75%, 85%, 95%, or 98%) identical to the amino acid sequence of SEQ ID NO: 2, 5 or or the am o acid sequence encoded by the cDNA of ATCC or the cDNA of ATCC , or a complement thereof. In preferred embodiments, the nucleic acid molecules have the nucleotide sequence of SEQ ID NO : 1,

3, 4, 6 or or the nucleotide sequence of the cDNA of

ATCC , or the cDNA of ATCC .

Also withm the invention are nucleic acid molecules which encode a fragment of a polypeptide having the amino acid sequence of SEQ ID NO : 2 or 5 the fragment including at least 15 (25, 30, 50, 100, 150, 300, or 400) contiguous amino acids of SEQ ID NO : 2 or 5, the polypeptide encoded by the cDNA of ATCC , or the polypeptide encoded by the cDNA of ATCC .

The invention includes nucleic acid molecules which encode a naturally occurring allelic variant of a polypeptide comprising tne ammo acid sequence of SEQ ID NO: 2, 5 or the ammo acid sequence encoded by the cDNA of ATCC , or the ammo acid sequence encoded by the cDNA of ATCC , wherein the nucleic acid molecule hybridizes to a nucleic acid molecule having a nucleic acid sequence encoding SEQ ID NO: 2, 5 or or a complement thereof under stringent conditions. Also with the invention are isolated polypeptides or proteins navmg an ammo acid sequence that is at least about 65% ,_ preferably 75%, 85%, 95%, or 98% identical to the ammo acid sequence of SEQ ID NO: 2, 5 or Also withm the invention are isolated polypeptides or proteins which are encoded by a nucleic acid molecule having a nucleotide sequence that is at least about 65%, preferably 75%, 85%, or 95% identical to a nucleic acid sequence encoding SEQ ID NO : 2 , 5 or ; and isolated polypeptides or proteins which are encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic ac d molecule having the nucleotide sequence of SEQ ID NO:l, 3, 4, 6 or a complement thereof or the non-coding strand of the cDNA of ATCC or the cDNA of ATCC .

Also withm the invention are polypeptides which are a naturally occurring allelic variants of a polypeptide that includes the ammo acid sequence of SEQ

ID NO: 2, 5 or an ammo acid sequence encoded by the cDNA of ATCC , or an ammo acid sequence encoded by the cDNA of ATCC , wherein the polypeptide is encoded by a nucleic acid molecule which hybridizes to a nucleic acid molecule having the sequence of SEQ ID NO:

1, 3, 4, 6 or or a complement thereof under stringent; conditions .

Tne invention also features nucleic aciα moiecuiet that hybridize under stringent conditions to a nucleic acid molecule having the nucleotide sequence of SEQ ID

NO:l, 3, 4, or 6, the cDNA of ATCC or the cDNA of

ATCC , or a complement thereof. In other embodiments, the nucleic acid molecules are at least 300 (325 35C, 375, 400, 425, 450, 500, 550, 600, 650, 700, 800 90C 1000, or 1290) nucleotides in iengtn and hybridizes under stringent conditions to a nucleic acid molecule navmg the nucleotide sequence of SEQ ID NO : 1 ,

3, 4, 6, or tne cDNA ATCC , or tne cDNA of ATCC

, or a complement thereof . In preferred embodiments, the isolated nucleic acid molecules encode a cytoplasmic (SEQ ID NO: 11 or 16), transmembrane (SEQ ID NO:10 or 15), or extracellular (SEQ ID NO: 9 or 14) domain of a polypeptide of the invention or a complement thereof. In another embodiment , the invention provides an isolated nucleic acid molecule which is antisense to the coding strand of a nucleic acid of the invention.

Another aspect of the invention provides vectors, e.g., recombinant expression vectors, comprising a nucleic acid molecule of the invention. In another embodiment, the mvention provides host cells containing such a vector. The mvention also provides metnods for producing a polypeptide of the invention by culturmg, ir a suitable medium, a host cell of the invention containing a recombinant expression vector such that a the polypeptide is produced.

Another aspect of this invention features isolated or recombinant proteins and polypeptides of the invention. Preferred proteins and polypeptides possess at least one biological activity possessed by the corresponding naturally-occurring human polypeptide An activity, a biological activity, and a functional activity of a polypeptide of tne invention refers to an activity exerted by a protein, polypeptide or nucleic acid molecule of the invention on, for example, a responsive cell as determined m vivo, or in vi tro, according to standard techniques. Such activities can be a direct activity, such as an association with or an enzymatic activity on a second protein or an indirect such as a cellular signaling activity mediated by interaction of tne protein with a second protein. Thus sucn activities include, e.g., ( 1 ^ the ability to form prote rprotem interactions with proteins m the signaling pathway of the naturally-occurring polypeptide; (2) the ability to bind a ligand of the naturally-occurring polypeptide; (3) the ability to bind to an intracellular target of the naturally-occurring polypeptide. Other activities include: (1) the ability to modulate cellular proliferation; (2) the ability to modulate cellular differentiation; and (3) the ability to modulate cell death.

In one embodiment, a polypeptide of the invention has an ammo acid sequence sufficiently identical to at least one domain of a polypeptide of the invention. As used herein, the term "sufficiently identical" refers to a first ammo acid or nucleotide sequence whicn contains a sufficient or minimum number of identical or equivalent (e.g , with a similar side chain) ammo acid residues or nucleotides to a second ammo acid or nucleotide sequence such that the first and second ammo acid or nucleotide sequences have a common structural domain and/or common functional activity. For example, ammo acid or nucleotide sequences which contain a common structural domain having about 65% identity, preferably 75% identity, more preferably 85%, 95%, or 98% identity are defined herein as sufficiently identical.

Ii one embodiment, tne isolated polypeptide lacks both a transmembrane and a cytoplasmic αoraam In another embodiment the polypeptide lacks both a transmembrane domain and a cytoplasmic domain and is soluble under physiological conditions.

The polypeptides of the present invention, or biologically active portions thereof, can be operably linked tc a heterologous ammo acid sequence to form a fusion protein. The invention further features antibodies that specifically bind a polypeptide of the inventlor such as monoclonal or polyclonal antipodies. In addition, the polypeptides of the invention or biologically active portions thereof can be incorporated into pharmaceutical compositions, which optionally include pharmaceutically acceptable carriers.

In another aspect, the present invention provides methods for detecting the presence of the activity or expression of a polypeptide of the invention m a biological sample by contacting the biological sample with an agent capable of detecting an indicator of activity such that the presence of activity is detected in the biological sample. In another aspect, the invention provides methods for modulating activity of a polypeptide of the invention comprising contacting a cell with an agent that modulates (inhibits or stimulates) the activity or expression of a polypeptide of the invention such that activity or expression m the cell is modulated. In one embodiment, the agent is an antibody that specifically binds to a polypeptide of the invention.

In another embodiment, the agent modulates expression of a polypeptide of the invention by modulating transcription, splicing, or translation of an mRNA encoding a polypeptide of the invention. In yet another embodiment, the agent is a nucleic acid molecule having a nucleotide sequence tnat is antisense tc the coding strand of an mRNA encoding a polypeptide of the invention.

The present invention also provides methods for treating a subject having a disorder characterized by aberrant activity of a polypeptide of the invention or aberrant expression of a nucleic acid or polypeptide of the mvention by administering an agent which is a modulator of the activity of a polypeptide of tne invention or a modulator of tne expression of a nucleic acid or polypeptide of the invention to the subηect. In one embodiment, tne modulator is a protein of tne invention. In anotner embodiment, the modulator is a nucleic acid of the invention. In other embodiments, the modulator is a peptide, peptidomimetic, or other small molecule.

Tne present invention also provides diagnostic assays for identifying the presence or absence of a genetic lesion or mutation characterized by at least one of: d) aberrant modification or mutation of a gene encoding a polypeptide of the invention, (ii) mis- regulation of a gene encoding a polypeptide of the inventIOΓ, and (in) aberrant post-translational modification of a protein of the invention wherein a wild-type form of the gene encodes a protein having the activity of the protein of tne invention.

In another aspect, tne invention provides a metnod fcr identifying a compound that binds to or modulates the activity of a polypeptide of the invention In general, such methods entail measuring a biological activity of the polypeptide m the presence and absence of a test compound and identifying those compounds which alter the activity of the polypeptide.

Tne invention also features methods for identifying a compound which modulates the expression of a polypeptide or nucleic acid of tne invention b measuring the expression of the polypeptide or nucleic acid m tne presence and absence of the compound.

Other features and advantages of the invention will be apparent from the following detailed description and claims.

Brief Description of the Drawings Figure 1 depicts the cDNA sequence (SEQ ID NO:l> and predicted ammo acid sequence (SEQ ID NO: 21 of human TANGO 15.. The open reading frame of SEQ ID NO .1 extends from nuc±eotiαe 557 to 2353, inclusive (SEQ ID NO : 3 ) . Figure 2 is a hydropathy plot of TANGO 191. Relative hydrophobici y is shown above the line marked "0", and relative hydrophilicity is shown below the line marked " 0 " . Figure 3 depicts the cDNA sequence (SEQ ID NO: 4) and predicted ammo acid sequence (SEQ ID NO: 5) of a partial human TANGO 195 clone. The open reading frame or SEQ ID NO : 4 extends from nucleotide 166 to 1101, inclusive (SEQ ID NO: 6) . Figure 4 is a hydropathy plot of TANGO 195.

Relative hydrophobicity is shown above the line marked "0", and relative hydrophilicity is shown below the line marked " 0 " .

Figure 5 depicts an alignment of the ammo acid sequences of human TANGO 195 (SEQ ID NO: 5) and human SLA (SEQ ID NO: 20) . In this alignment the sequences are 22.8% identical overall.

Figure 6 depicts an alignment of portions of TANGO 191 with PF00047, an IG superfamily domain HMM (SEQ ID NOs:21, 22, and 23) .

Figure 7 depicts the cDNA sequence (SEQ ID NO: ) and predicted ammo acid sequence (SEQ ID NO: > of murine TANGO 195 Tne open reading frame of SEQ ID

NO: extends from nucleotide 42 to 876, inclusive (SEQ ID NO: ) .

Figure 8 depicts the cDNA sequence (SEQ ID NO: and predicted ammo acid sequence (SEQ ID NO: ) of a partial human TANGO 195 clone (T195Athpb93f1) . The open reading frame extends from nucleotide 159 to 1118, inclusive (SEQ ID NO: ) .

Figure 9 depicts the cDNA sequence (SEQ ID NO: and predicted ammo acid sequence (SEQ ID NO: ) of a full length TANGO 195 clone (T195AthLal70f10 ) The open reading frame extends from nucleotide 25 to nucleotide 879, inclusive (SEQ ID NO. ) . Detailed Description of the Invention The present invention is based on the discovery of cDNA molecules encoding TANGO 191 and TANGO 195, both of which are secreted proteins. The subsections and Tables summarize certain features of TANGO 191 and TANGO 195.

TANGO 191

The human TANGO 191 cDNA of SEQ ID NO : 1 has a 179"⁷ nucleotide open reading frame (SEQ ID NO : 3 ) encoding a 599 ammo acid protein (SEQ ID NO : 2 ) . The cDNA and protein sequences of human TANGO 191 are shown m Figure 1. This cDNA was isolated from a human mixed iympnocyte reaction library pased on its sequence similarity to genes encoding certain members of the mterleukm-1 (IL- 1) receptor superfamily. Human TANGO 191 is a transmembrane protein having a 19 ammo acid signal sequence (ammo acids 1 - 19 of SEQ ID NO: 2; SEQ ID NO: 7) followed by a 580 ammo acid mature protein (ammo acids 20 - 599 of SEQ ID NO : 2 ; SEQ ID NO: 8) . Mature TANGO 191 is predicted to have a transmembrane domain that extends from ammo acid 358 to ammo acid 382 of SEQ ID NO : 2 (SEQ ID NO:10), an extracellular domain that extends from ammo acid 20 to ammo acid 357 of SEQ ID NO : 2 (SEQ ID NO : 9 ) , and a cytoplamic domain extending from ammo acid 383 to ammo acid 599 of SEQ ID NO : 2 (SEQ ID NO:ll) .

TANGO 191 has a molecular weight of 68.3 kDa prior to cleavage of its signal peptide and a molecular weight of 66.1 kDa after cleavage of its signal peptide.

TANGO 191 has four potential N-glycosylation sites (ammo acids 21-24, 119-122, 152-155, and 345-248 of SEQ ID NO:2), 15 potential protein kinase C phosphorylation sites (ammo acids 26-28, 35-37, 63-65, 160-162, 203-205, 233-235, 272-275, 307- 309, 311-313, 327-329, 474-476, 506-508, 538-540, 575-577, and 590-592 of SEQ ID NO : 2 ) ; 12 potential casein kinase II phosphorylation sites (ammo acids 36-39, 89-92, 133-136, 224-227, 294-297, 301-304, 311-314, 327-330, 401-404, 427-430, 490-493, and 585-588 of SEQ ID NO : 2 ) ; one potential tyrosine kinase phosphorylation site (ammo acids 205-212 of SEQ ID NO: 2) ; and six potential N-myristoylation sites (ammo acids 117-122, 168-173, 217-222, 366-371, 460-465, and 583-588 of SEQ ID NO : 2 ) .

Figure 2 is a hydropathy plot of TANGO 191. Relative hydrophobicity is shown above the line marked "0", and relative hydrophilicity is shown below the line marked " 0 " .

Northern analysis of human TANGO 191 mRNA expression revealed that it is expressed m spleen, lympn node, peripheral blood lymphocytes, and bone marrow.

A clone (EPftX191a) containing a cDNA encoding TANGO 191 inserted into pZL-1 (GIBCO/BRL; Bethesda, MD) between the Notl and Sail sites was deposited with the American Type Culture Collection, Manassas, VA on September 9, 1998, and assigned Accession Number .

Human TANGO 191 appears to be a member of the IL-1 receptor superfamily. TANGO 191 includes three regions (ammo acids 71-128 of SEQ ID NO : 2 ; SEQ ID NO:!'⁷', ammo aicds 168-223 of SEQ ID NO : 2 ; SEQ ID NO:18j; ammo acids 266-339 of SEQ ID NO : 2 ; SEQ ID NO:19) which have homology to tne IG superfamily domain (PF00047) that is characteristic of members of the IL-1 superfamily (Figure 6) .

IL-1 receptor (IL-1R) plays a critical role the regulation of immune and inflammatory responses.

Signalling by IL-1R requires that IL-1R form a complex with IL-lAcP, a protein whicn may be required for ternalization of IL-1R It is thought that both IL-1R and IL-lAcP interact with IRAK-2. It has been proposed that this multiprotem complex interacts with TRAF6 , which engages a protein complex that acts to activate NK- yB . Members of the NK-yB family regulate many of immune and inflammatory genes that are induced by IL-1.

Since TANGO 191 has some similarity to IL-1 receptor, TANGO 191 nucleic acids and polypeptides as well as modulators of TANGO 191 expression or activity are useful m the treatment of a variety of immune and inflammatory disorders, e.g., asthma, graft vs-host disease, rheumatoid arthritis, psoriasis, inflammatory bowel disease, septic shock, ulcerative colitis, Crohn' s disease, chronic myelogenous leukemia, cancer, liver disease, Hodgkm' s disease osteoarthπtis , Lyme disease, cachexia, and autoimmune diseases, e.g., myasthenia gravis, autoimmune diabetes, and lupus.

TANGO 195

Tne human TANGO 195 partial cDNA of SEQ ID NO: 4 has a 936 nucleotide open reading frame (SEQ ID NO: 6) encoding a 312 ammo acid protein (SEQ ID NO: 5) The cDNA and protein sequences of human TANGO 195 clone are shown Figure 3. This partial TANGO 195 cDNA clone was isolated from a numan mixed lymphocyte reaction library based on its nomology to signalling lymphocyte activatior mar er (Cocks et al . (1995) Nature 376 :260-63) . Apparent full-length clones (3.0 kb and 1.3 kb) were isolated from the same library and a human mid-term placental library.

The portion of human TANGO 195 encoded by the cDNA of SEQ ID NO: is a transmembrane secreted protein having a 22 ammo acid signal sequence (ammo acids 1 - 22 of SEQ ID NO: 5; SEQ ID NO: 12" followed by a 291 ammo acic mature proteir (ammo aciαs 23 - 312 of SEQ ID NO : 5 ; SEQ ID NO: 13 , . The portion cf mature TANGO 195 encoded by tne cDNA of SEQ ID NO : 4 is predicted to have a transmembrane domain that extends from ammo acid 234 to ammo acid 254 of SEQ ID NO : 5 (SEQ ID NO:15), an extracellular domain that extends from ammo acid 23 to ammo acid 233 of SEQ ID NO : 5 (SEQ ID NO: 14), and a cytoplamic domain extending from ammo acid 255 to ammo acid 312 of SEQ ID NO : 5 (SEQ ID NO: 16) . TANGO 195 is a type I transmembrane protein belonging to the CD2 subgroup of the immunoglobulin superfamily.

Tne TANGO 195 of SEQ ID NO : 5 has three potential N-glycosylation sites (ammo acids 85-88, 100-103, and 156-159 of SEQ ID NO : 5 ) ; tnree potential protein kinase C phosphorylation sites (ammo acids 163-165, 230-232, and 308-310 of SEQ ID NO : 5 ) ; three potential casem kinase II phospftorylation sites (ammo acids 168-171, 215-218, and 230-233 cf SEQ ID NO : 5 ) ; one potential tyrosine kinase phosphorylation site (ammo acids 65-72 of SEQ ID NO:5); one potential cGMP-dependent protein kinase pnosphorylation site (ammo acids 165-168 of SEQ ID NO: 5, ; and three potential N-myristoylation sites (ammo acids 66-71, 110-115, and 183-188 of SEQ ID NO : 5 ) . Figure 4 is a hydropathy plot of the TANGO 195 of

SEQ ID NO : 5. Relative hydrophobicity is shown above the line marKed "0", and relative hydrophilicity is shown belov tne line mar ed "0¹.

Figure 7 depicts tne cDNA sequence (SEQ ID NO: and predicted ammo acid sequence (SEQ ID NO: ) of murine TANGO 195. The open reading frame of SEQ ID

NO: extends from nucleotide 42 to 876, inclusive (SEQ

ID NO: ) .

Figure 8 depicts the cDNA sequence (SEQ ID NO: ) and predicted ammo acid sequence (SEQ ID NO: , of a partial numan TANGO 195 clone (T195Athpb93f1) . The open reading frame extends from nucleotide 159 to 1116, inclusive (SEQ ID NO:

Figure 9 depicts tne cDNA sequence (SEQ ID NO: and predicted ammo acid sequence (SEQ ID NO: ) of a full length TANGO 195 clone (T195AthLal70f10) . The open reading frame extends from nucleotide 25 to nucleotide

879, inclusive (SEQ ID NO: ) .

The full-length TANGO 195 protein of Figure 9 is predicted to be a transmembrane protein having a 22 ammo acid signal sequence (ammo acids 1-22 of SEQ ID NO: ;

SEQ ID NO: ) followed by a 263 ammo acid mature protein (ammo acids 23-285 of SEQ ID NO: ) . This form of TANGO 195 is predicted to have a transmembrane domain extending from ammo acid 234 to ammo acid 254 of SEQ ID

NO: (SEQ ID NO: ) , an extracellular domain extending from ammo acid 23 to ammo acid 233 of SEQ ID NO:

(SEQ ID NO: ) and a cytoplasmic domain that extends from ammo acid 255 to ammo acid 285 of SEQ ID NO: (SEQ ID NO: ) .

In si tu expression analysis of TANGO 195 m adult mice revealed expression in the spleen (mutlifocal expression with expression highest m follicles) , thymus (multifocal expression) , and lymph node (multifocal expression) . No expression was detected m lung and stomach. In si tu expression analysis was also used to examine expression the spleens of adult mice 1, 3, 5, and 14 post -immunization with EFA/PBS In eacn case multifocal expression was observed witn expression Pemg highest m the follicles. The expression at 14 days pos -immunization was somewhat lower than m at other time points.

Northern analysis of TANGO 195 expression revealed the presence of a 1.8 kb transcript and a 3.4 kb transcript m spleen, lymph node and thymus and a 1.8 kb transcript was observed in bone marrow with expression being highest m lymph node. Additional Northern analysis revealed expression m the following tissues (m decreasing order of expression) : lympn node, stomach, small intestine, appendix, lung, spleen, and bone marrow However, a probe based on the open reading frame did not detect expression m lung or stomach.

Additional Northern analysis revealed that TANGO 195 is expressed m activated human monocytes/macrophages and, at lower level, m activated human lymphocytes. This analysis also revealed that cytok e induced differentiation of T cells appears to regulate TANGO 195 expression. To carry out this analysis, PBMCs were purified from human buffy coat by ficol gradient centrifugation as per manufacturers instructions (Sigma) . PBMCs were activated for 24 hours with lug/ml LPS . Using Miltenyi Biotech positive selection beads, CD4+, CD8+ anc CD19+ cells were isolated from resting PBMCs. The CD8+ cells were activated for 24 hours using plate bound anti- CD3. Resting monocytes were purified from PBMCs by gradient centrifugation and stimulated with 0, 0.01, 0.1 or lug/ml LPS either with or without 2ng/ml gIFN for 4 hours or with 0, 0.2 or 2 ng/ml gIFN for 4 hours. Activated T cells (predominantly CD4+ cells) were prepared from PBMCs by stimulation with plate bound anti- CD3 (TR66) for 3 days m RPMI 10% FBS . Cells were then expanded without antι-CD3 but m the presence of IL2 for 22 days. Cells were resuspended at 107/ml m fresn RPMI 10% FBS with no exogenous cytokmes, or IL10 (20ng/ml) plus IL4 (40ng/ml) or TNFa (5,000u/ml) plus gIFN

(15ng/ml) . (All cytokmes were purchased from Genzyme . ) Cells were harvested after 8 and 24 hours. RNA was prepared from all cell types using RNeasy mini kit (Qiagen) and expression was analyzed by standard Northern analysis using approx lOug total RNA. No expression was seen m resting leukocytes PBMCs, CD4-r, CD8+, CD19+ or monocytes A single band of approximately 3-3.5 Kb was seen m activated CD8-r cells and m T cells activated with no exogenous cytokmes or the combination of IL10 and IL4 , but not with TNFa and gIFN (after both 8 and 24 hours) . The strongest expression was seen m monocytes activated with gIFN (0.2 or 2ug/ml) giving 2 bands of approximately 1.5-2 kb and 3-3.5 kb (with the upper band stronger) . Addition of LPS did not increase expression m monocytes or PBMCs and may fact lead to a slight decrease m expression m gIFN stimulated monocytes .

TANGO 195 function was investigated by reconstituting irradiated mice with bone marrow cells infected with retrovirus expressing either full length murine TANG0195 ("T195fl") or the extracellular domain of murine TANGO 195 ("T195ex"). The donor and recipient mice were C57BL/6 and congenic for CD45 (CD45.1 for donor, CD45.2 for recipient) . Recipient mice were analyzed approximately 10 and 14 weeks after transplantation for blood chemistry, hematology and tissue histology Peripheral blood, spleen, lymph node and thymus cells were analyzed by FACs analysis and TANGO 195 RNA levels were analyzed m spleen. The percentage of infected cells, based on the percentage of G418-resιstant donor cells, was 54% (T195fl) and 69% (T195ex) . The level of TANGO 195 RNA expression m the spleen of recipient mice, based upon slot blot analysis (GAPDH control) , was 10 times tnat of control mice (328% of GAPDH for T195ex and 332% of GAPDH for T195fl compared to 33% of GAPDH m control mice) .

Expression of T195ex led to a statistically significant increase m triglyceride levels (ave = 97.6 mg/dl at 12 weeks; ave = 94.3 mg/dl at 16 weeks) m serum as compared to control mice (ave = 52.5 mg/dl at 12 week, ave = 63.1 mg/dl 16 weeks) or mice expressing T195fl (ave = 47 9 mg/dl at 12 weeks, ave = 67.5 mg/dl at 16 weeks) .

Expression of T195fl had a variety of effects on lymphocytes. In T195fl expressing mice, FACs analysis of peripheral blood showed an increase m total B220hι (CD45Rhι) , IgD+ B cells compared to control mice or mice expressing T195ex. The B220hι (CD45Rhι) , IgD+ B cells showed low levels of expression of Macl which is generally regarded as a marker for cells of the monocyte/macrophage lineage. FACs analysis of peripheral blood showed a slight decrease m CD4+ T cells m T195fl expressing mice compared to control mice and T195ex expressing mice. FACs analysis of spleen and lymph node cells showed a similar increase m B220+ IgD+ Macllo cells. These cells had similar levels of surface IgD and IgM compared to B220+ cells from control and T195ex mice CD45.1 staining confirmed that the B220+ IgD+ Macllo cells m spleen, lymph nodes and peripheral blood were derived from donor bone marrow. In T195fl expressing mice, FACs analysis of peritoneal lavage cells showed an increase m total B220- cells, an increase m B220+ CD23lo cells, and an increase B220+ Macllo cells compared to control mice of T195ex expressing mice B220+ cells showed slightly lower levels of IgD compared to B220+ cells from control mice or T195ex expressing mice. In addition, B220+ CD23lo cells in T195fl expressing mice showed significantly lower expression of B220 compared to control and T195ex mice Finally, it was observed that very few CD5- B220 were seen on peritoneal lavage from T195fl, T195ecd or control mice.

Taken together, these results suggest that increased expression of T195fl bone marrow derived cells leads to an increase m Bl like cells m the periphery Since Bl cells are not normally bone marrow derived the exact lineage of the cells is difficult to determine however they appear to oe Bib cells. These results also suggest that increased expression of T195fl on bone marrow derived cells may lead to a decrease m CD4+ T cells m tne peπpnery These results also suggest that TANGO 195 may play a role m B cell leukemia, immune response, and autoimmune disorders (e.g., arthritis) .

TANGO 195 maps to human chromosome locus hulq21. The flanking markers are AFMA323ZE5 and D1S2635. The among identified loci m close proximity to TANGO 195 are HYPLP1 (hyperlipidemial) and LPD1 (lipodystrophy) . Nearby known human genes include: SPTA1 (spectrm, alpha) , THBS3 (thrombospondm 3) , MTX (metaxm) , CTSS (cathepsm K,S), FLG (filaggrm) , PKLR (pyruvate kinase) HYPLIP1 (hyperlipidemia) .

The mouse chromosome corresponding to the human chromosomal locus is chromosome 3. Nearby mouse loci include: soc (soft coat), hyplipl (hyperlipidemia), ft (flaky tail) and ma (matted) . Nearby mapped mouse genes include. Imna (lamm A), fig (filaggrm) , bean (brevican) , gba (acid beta glucosidase) .

Rabbit polyclonal antibodies were raised against three peptides from murine TANGO 195 peptides to ammo acids 26-34, 102-117 and 161-176 Peptide purified sera from rabbits immunized with ammo acids 102-117 specifically recognizes mouse T195-hFc protein by stanαarad Western Blot. Additionally unpurified sera from rabbits immunized with ammo aciαs 102-117 recognize mouse T195-hFc by ELISA ELISA plates were coated with 5 Tg/ml mouse T195-hFc or human lg control m PBS overnight at 4°C. Plates were washed and blocked with PBS 1% PSA. Serial dilutions of serum were added and incubated for approximately 2 hours at room temperature . Plates were washed and rabbit immunoglobulin detected with anti- rabbit lg-HRP Serum from rabbits immunized with the peptide corresponding to ammo acids 102-117 showed greater than 20 fold higher titres against mouse T195-hFc compared to human Ig, and showed greater than 20 fold higher titres against mouse T195-hFc compared to control serum.

Several TANGO 195/immunoglobulin constant region fusion proteins were created. Using human TANGO 195 a fusion protein consisting of TANGO 195 (aal-233)- AAPGGASYKD-human IgGlfc was created. A second human TANGO 195 fusion substituted murine IgGlfc for human IgGlfc. Using murine TANGO 195 a fusion protein consisting of TANGO 195 (aal to 231) -AASGKASYKD-human IgGlfc was created. A second murine TANGO 195 fusion protein substituted murine IgGlfc for human IgGlfc.

A clone (Ep;j thPb093fol) containing a 1.3 kb cDNA encoding apparently full-length TANGO 195 m pMET7 between Notl and Sail was deposited with the American Type Culture Collection, Manassas, VA on September 9,

1998, and assigned Accession Number .

TANGO 195 has regions that are significantly similar to numan signalling lymphocyte activation molecule ("SLAM") (Accession Number U33017) . For example, the region of TANGO 195 from ammo acid 173 to ammo acid 250 has 32% identity (25/78 ammo aicds) and 50% identity (39/59 ammo acids) to the corresponding region of SLAM, tne region of TANGO 195 from ammo acid 134 to ammo acid 164 has 32% identity (10/31 ammo acids) and 41% identity (13/31 ammo acids) to the corresponding region of SLAM; and the region of TANGO 195 from ammo acid 117 to ammo acid 132 has 43% identity (7/16 ammo acids) and 75% identity (12/16 ammo acids) to the corresponding region of SLAM (Figure 5) . SLAM is tnought to enhance the expansion and differentiation of activated B cells (Punnonen et al .

(1997) J. Exp. Med 185:993-1004) and m the regulation of type 1 and type 2 cytokme production (Ferrante et al

(1998) J. Immunology 160.1514-21). TANGO 195 likely has a function similar to that of SLAM. Thus, modulators of TANGO 195 expression or activity may be useful in the treatment of diorders associated with abberrant B cell expansion or differentiation or abberrant cytokine production, e.g., allergic and autoimmune disorders.

TABLE 1 Summary of Human TANGO 191 and TANGO 195 Sequence Information

TABLE 2: Summary of Domains of TANGO 191 and TANGO 195

Various aspects of the invention are described in further detail in the following subsections I . Isolated Nucleic Acid Molecules

One aspect of the invention pertains to isolated nucleic acid molecules that encode a polypeptide of the invention or a biologically active portion thereof, as well as nucleic acid molecules sufficient for use as hybridization probes to identify nucleic acid molecules encoding a polypeptide of the invention and fragments of such nucleic acid molecules suitable for use as PCR primers for the amplification or mutation of nucleic acid molecules. As used herein, the term "nucleic acid molecule" is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs. The nucleic acid molecule can be smgle- stranded or double-stranded, but preferably is double- stranded DNA.

An "isolated" nucleic acid molecule is one which is separated from other nucleic acid molecules which are present m the natural source of the nucleic acid molecule. Preferably, an "isolated" nucleic acid molecule is free of sequences (preferably protein encoding sequences) which naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) m the genomic DNA of the organism from which the nucleic acid is derived. For example, various embodiments, the isolated nucleic acid molecule can contain less than about 5 kB, 4 kB, 3 kB, 2 kB, 1 kB, 0.5 kB or 0.1 kB of nucleotide sequences which naturally flank the nucleic acid molecule m genomic DNA of the cell from which the nucleic acid is derived. Moreover, an "isolated" nucleic acid molecule, such as a cDNA molecule, can Pe substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when cnemically synthesized. A nucleic acid molecule of the present invention, e.g., a nucleic acid molecule having the nucleotide sequence of SEQ ID NO : 1 , 3, 4, or 6 , the cDNA of ATCC , or the cDNA of ATCC , or a complement thereof, can be isolated using standard molecular biology techniques and the sequence information provided herein. Using all or a portion of the nucleic acid sequences of

SEQ ID NO: 1, 3, 4, or 6 , the cDNA of ATCC , or the cDNA of ATCC as a hybridization probe, nucleic acid molecules of the invention can be isolated using standard hybridization and cloning techniques (e.g., as described m Sambrook et al . , eds., Molecular Cloning: A Labora tory Manual , 2nd ed . , Cold Spring Harbor Labora tory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) .

A nucleic acid molecule of the mvention can be amplified using cDNA, mRNA or genomic DNA as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to all or a portion of a nucleic acid molecule of the invention can be prepared b^Λ standard synthetic techniques, e.g., using an automated DNA synthesizer.

In another preferred embodiment, an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule which is a complement of the nucleotide sequence of SEQ ID NO:l, 3, 4, or 6 , the cDNA of ATCC , or the cDNA of ATCC , or a portion thereof. A nucleic acid molecule which is complementary to a given nucleotide sequence is one which is sufficiently complementary to the given nucleotide sequence that it can hybridize to the given nucleotide sequence thereby forming a stable duplex. Moreover, a nucleic acid molecule of the invention can comprise only a portion of a nucleic acid sequence encoding a full length polypeptide of the invention for example, a fragment which can be used as a probe or primer or a fragment encoding a biologically active portion of a polypeptide of the invention. The nucleotide sequence determined from the cloning one gene allows for the generation of probes and primers designed for use m identifying and/or cloning homologues m other cell types, e.g., from other tissues, as well as homologues from other mammals. The probe/primer typically comprises substantially purified oligonucleotide. The oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, preferably about 25, more preferably about 50, 75, 100, 125, 150, 175, 200, 250, 300, 350 or 400 consecutive nucleotides of the sense or anti- sense strand of SEQ ID NO : 1 , 3, 4, or 6 , the cDNA ATCC , or the cDNA of ATCC or of a naturally occurring mutant of SEQ NO : 1 , 3, 4, or 6, the cDNA of

ATCC , or the cDNA of ATCC .

Probes based on the sequence of a nucleic acid molecule of the invention can be used to detect transcripts or genomic sequences encoding the same protein molecule encoded by a selected nucleic acid molecule. The probe comprises a label group attached thereto, e.g., a radioisotope, a fluorescent compound, an enzyme, or an enzyme co- factor. Such probes can be used as part of a diagnostic test kit for identifying cells or tissues which mis-express the protein, such as by measuring levels of a nucleic acid molecule encoding the protein m a sample of cells from a subject, e.g., detecting mRNA levels or determining whether a gene encoding the protein has been mutated or deleted. A nucleic acid fragment encoding a "biologically active portion" of a polypeptide of the invention can be prepared by isolating a portion of any of SEQ ID NO : 3 or

6, the nucleotide sequence of the cDNA of ATCC , or the nucleotide sequence of the cDNA of ATCC which encodes a polypeptide having a biological activity, expressing the encoded portion of the polypeptide protein (e.g., by recombinant expression in vi tro) and assessing the activity of the encoded portion of the polypeptide. The invention further encompasses nucleic acid molecules that differ from the nucleotide sequence of SEQ

ID NO:l, 3, 4, or 6 , the cDNA of ATCC , or the cDNA of ATCC due to degeneracy of the genetic code and thus encode the same protein as that encoded by the nucleotide sequence of SEQ ID NO : 1 , 3, 4, or 6 , the cDNA of ATCC , or the cDNA of ATCC .

In addition to the nucleotide sequences shown SEQ

ID NO: 3 and 6 and present m the cDNA of ATCC and the cDNA of ATCC , it will be appreciated by those skilled m the art that DNA sequence polymorphisms that lead to changes m the ammo acid sequence may exist withm a population (e.g., the human population) . Such genetic polymorphisms may exist among individuals with a population due to natural allelic variation. An allele is one of a group of genes which occur alternatively at a given genetic locus. As used herein, the phrase "allelic variant" refers to a nucleotide sequence which occurs at a given locus or to a polypeptide encoded by the nucleotide sequence. As used herein, the terms "gene" and "recombinant gene" refer to nucleic acid molecules comprising an open reading frame encoding a polypeptide of the invention Such natural allelic variations can typically result m 1-5% variance m the nucleotide sequence of a given gene. Alternative alleles can be identified by sequencing the gene of interest m a number of different individuals. This can be readily carried out by using hybridization probes to identify the same genetic locus m a variety of individuals. Any and all such nucleotide variations and resulting amino acid polymorphisms or variations that are the result of natural allelic variation and that do not alter the functional activity are intended to be within the scope of the invention.

Moreover, nucleic acid molecules encoding proteins of the invention from other species (homologues) , which have a nucleotide sequence which differs from that of the protein described herein are intended to be withm the scope of the invention. Nucleic acid molecules corresponding to natural allelic variants and homologues of a cDNA of the invention can be isolated based on their identity to the nucleic acid molecule disclosed herein using a cDNA, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions. For example, a cDNA encoding a soluble form of a membrane-bound protein of the invention isolated based on its hybridization to a nucleic acid molecule encoding all or part of the membrane-bound form. Likewise, a cDNA encoding a membrane-bound form can be isolated based on its hybridization to a nucleic acid molecule encoding all or part of the soluble form.

Accordingly, m another embodiment, an isolated nucleic acid molecule of the invention is at least 300 (325, 350, 375, 400, 425, 450, 500, 550, 600, 650, 700, 800, 900, 1000, or 1290) nucleotides length and hybridizes under stringent conditions to the nucleic acid molecule comprising the nucleotide sequence, preferably the coding sequence, of SEQ ID NO:l, 3, 4, or 6 , the cDNA of ATCC , the cDNA of ATCC , or a complement thereof. As used herein, the term "hybridizes under stringent conditions" is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60% (65%, 70%, preferably 75%) identical to each otner typically remain hybridized to each other. Such stringent conditions are known to those skilled m the art and can be found m Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989) , 6.3.1-6.3.6. A preferred, non-limitmg example of stringent hybridization conditions are hybridization m 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes 0.2 X SSC, 0.1% SDS at 50-65°C. Preferably, an isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to the sequence of SEQ ID NO : 1 , 3, 4 , or 6, the cDNA of ATCC , or the cDNA of ATCC , or the complement thereof, corresponds to a naturally-occurring nucleic acid molecule. As used herein, a "naturally- occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs m nature (e.g., encodes a natural protein) .

In addition to naturally-occurring allelic variants of a nucleic acid molecule of the invention sequence that may exist m the population, the skilled artisan will further appreciate that changes can be introduced by mutation thereby leading to changes m the ammo acid sequence of the encoded protein, without altering the biological activity of the protein. For example, one can make nucleotide substitutions leading to ammo acid substitutions at "non-essential" ammo acid residues. A "non-essential¹ ammo acid residue is a residue that can be altered from the wild-type sequence without altering tne biological activity, whereas an "essential" ammo acid residue is required for biological activity. For example, ammo acid residues that are not conserved or only semi -conserved among homologues of various species may be non-essential for activity and thus would be likely targets for alteration. Alternatively, ammo acid residues that are conserved among the homologues of various species (e.g., murine and human) may be essential for activity and thus would not be likely targets for alteration.

Accordingly, another aspect of the invention pertains to nucleic acid molecules encoding a polypeptide of the invention that contain changes m ammo acid residues that are not essential for activity. Such polypeptides differ m ammo acid sequence from SEQ ID NO: 2, 5, 8, and 13 yet retain biological activity. In one embodiment, the isolated nucleic acid molecule includes a nucleotide sequence encoding a protein that includes an ammo acid sequence that is at least about 45% identical, 65%, 75%, 85%, 95%, or 98% identical to the ammo acid sequence of any of SEQ ID NO: 2, 5, 8, or 13. An isolated nucleic acid molecule encoding a variant protein can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotiαe sequence of SEQ ID NO:l, 3, 4, or 6 , the cDNA of ATCC , or the cDNA of ATCC such that one or more ammo acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced by standard techniques, such as site- directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative ammo acid substitutions are made at one or more predicted non-essential ammo acid residues. A "conservative ammo acid substitution" is one m which the ammo acid residue is replaced with an ammo acid residue having a similar side chain. Families of ammo acid residues having similar side chains have been defined m the art Tnese families include am o acids with basic side chains (e.g., lysine, argmme, histidme) , acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valme, leucme, isoleucine, prolme, phenylalanme, methionine, tryptophan), beta-branched side chains (e.g., threonine, valme, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanme, tryptophan, histidme). Alternatively, mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for biological activity to identify mutants that retain activity Following mutagenesis, the encoded protein can be expressed recombmantly and the activity of the protein can be determined.

In a preferred embodiment, a mutant polypeptide that is a variant of a polypeptide of the invention can be assayed for. (1) the ability to form protem:protem interactions with proteins m a signalling pathway of the polypeptide of the invention; (2) the ability to bind a ligand of the polypeptide of the invention; or (3) the ability to bind to an intracellular target protein of the polypeptide of the invention. In yet another preferred embodiment, the mutant polypeptide can be assayed for the ability to modulate cellular proliferation or cellular differentiation.

The present invention encompasses antisense nucleic acid molecules, i.e., molecules which are complementary to a sense nucleic acid encoding a polypeptide of the invention, e.g , complementary to the coding strand of a double- stranded cDNA molecule or complementary to an mRNA sequence. Accordingly, an antisense nucleic acid can hydrogen bond to a sense nucleic acid. The antisense nucleic acid can be complementary to an entire coding strand, or to only a portion thereof, e.g., all or part of the protein coding region (or open reading frame) . An antisense nucleic acid molecule can be antisense to all or part of a noncodmg region of the coding strand of a nucleotide sequence encoding a polypeptide of the invention. The noncodmg regions ("5' and 3' untranslated regions") are the 5' and 3' sequences which flank the coding region and are not translated into ammo acids . An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides m length An antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known m the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used. Examples of modified nucleotides which can be used to generate the antisense nucleic acid include 5- fluorouracil , 5-bromouracιl , 5-chlorouracιl , 5- lodouracil, hypoxanthme, xanthme, 4-acetylcytosme, 5- (carboxyhydroxylmethyl) uracil, 5- carboxymethylammomethyl-2 -thiouπdme, 5- carboxymethylammomethyluracil , dihydrouracil , beta-D- galactosylqueosme, mosme, N6 -1sopentenyladenine, 1- methylguanme, 1-methylmosιne, 2 , 2-dιmethylguanme, 2- methyladenme, 2 -methylguanme, 3-methylcytosme, 5- methylcytosme, N6-aαenme, 7 -methylguanme, 5^~- methylammomethyluracil , 5-methoxyammomethyl-2 - thiouracil, beta-D-mannosylqueosme, 5'- methoxycarboxymethyluracil , 5-methoxyuracil , 2- methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v) , wybutoxosine, pseudouracil , queosme, 2- thiocytosme, 5-methyl-2-thiouracil , 2-thiouracil, 4- thiouracil, 5-methyluracil , uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v) , 5 -methyl -2- thiouracil, 3- (3 -amino-3 -N-2 -carboxypropyl) uracil, (acp3)w, and 2 , 6-diaminopurine . Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned m an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection) . The antisense nucleic acid molecules of the invention are typically administered to a subject or generated in si tu such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a selected polypeptide of the invention to thereby inhibit expression, e.g., by inhibiting transcription and/or translation. The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, m the case of an antisense nucleic acid molecule which binds to DNA duplexes, through specific interactions in the major groove of the double helix. An example of a route of administration of antisense nucleic acid molecules of the invention includes direct injection at a tissue site. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically . For example, for systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antioodies which bind to cell surface receptors or antigens. The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of the antisense molecules, vector constructs m which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.

An antisense nucleic acid molecule of the invention can be an α-anomeric nucleic acid molecule. An a- anomeπc nucleic acid molecule forms specific double- stranded hybrids with complementary RNA m which, contrary to the usual -units, the strands run parallel to each other (Gaultier et al . (1987) Nucleic Acids Res . 15:6625-6641) . The antisense nucleic acid molecule can also comprise a 2 ' -o-methylπbonucleotide (Inoue et al . (1987) Nucleic Acids Res . 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al . (1987) FEBS Lett . 215:327 330)

The invention also encompasses ribozymes. Ribozymes are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region. Thus, ribozymes (e.g., hammerhead ribozymes (described m Haselhoff and Gerlach (1988) Nature 334 : 585-591) ^ can be used to catalytically cleave mRΝA transcripts to thereby inhibit translation of the protein encoded by the mRΝA. A ribozyme having specificity for a nucleic acid molecule encoding a polypeptide of the mvention can be designed based upon the nucleotide sequence of a cDΝA disclosed herein. For example, a derivative of a Tetrahymena L-19 IVS RΝA can be constructed m which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved m a Cech et al . U.S. Patent No. 4,987,071, and Cech et al . U.S. Patent No. 5,116,742. Alternatively, an mRNA encoding a polypeptide of the invention can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e . g. , Bartel and Szostak (1993) Science 261:1411-1418.

The invention also encompasses nucleic acid molecules which form triple helical structures. For example, expression of a polypeptide of the invention can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the gene encoding the polypeptide (e.g., the promoter and/or enhancer) to form triple helical structures that prevent transcription of the gene m target cells. See generally Helene (1991) Anti cancer Drug Des . 6(6) : 569-84; Helene (1992) Ann . N. Y. Acad . Sci . 660:27-36; and Maher (1992) Bioassays 14 (12 ): 807-15.

In preferred embodiments, the nucleic acid molecules of the mvention can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule. For example, the deoxyπbose phosphate backbone of the nucleic acids can be modified to generate peptide nucleic acids ( see Hyrup et al . (1996) Bioorganic & Medicinal Chemistry 4(1) 5-23) . As used herein, the terms "peptide nucleic acids" or "PNAs" refer to nucleic acid mimics, e.g. DNA mimics, m which the deoxyπbose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained. The neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described m Hyrup et al . (1996), supra ; Perry-O' Keefe et al . (1996) Proc . Natl . Acad. Sci . USA 93: 14670-675. PNAs can be used in therapeutic and diagnostic applications. For example, PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, e.g., inducing transcription or translation arrest or inhibiting replication. PNAs can also be used, e.g., in the analysis of single base pair mutations in a gene by, e.g., PNA directed PCR clamping; as artificial restriction enzymes when used in combination with other enzymes, e.g., SI nucleases (Hyrup (1996) , supra ; or as probes or primers for DNA sequence and hybridization (Hyrup (1996), supra ; Perry-O' Keefe et al. (1996) Proc . Natl . Acad . Sci . USA 93: 14670-675).

In another embodiment, PNAs can be modified, e.g., to enhance their stability or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known m the art. For example, PNA-DNA chimeras can be generated which may combine the advantageous properties of PNA and DNA. Such chimeras allow DNA recognition enzymes, e.g., RNAse H and DNA polymerases, to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity. PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (Hyrup (1996) , supra) . The synthesis of PNA-DNA chimeras can be performed as described Hyrup (1996), supra , and Finn et al . (1996) Nucleic Acids Res . 24(17) : 3357 -63. For example, a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry and modified nucleoside analogs. Compounds such as 5'-(4- methoxytπtyl) ammo-5 ' -deoxy-thymidine phosphoramidite can be used as a link between the PNA and the 5' end of DNA (Mag et al . (1989) Nucleic Acids Res . 17:5973-88). PNA monomers are then coupled m a stepwise manner to produce a chimeric molecule with a 5' PNA segment and a 3' DNA segment (Finn et al . (1996) Nuclei c Acids Res . 24 (17) : 3357-63) . Alternatively, chimeric molecules can be synthesized with a 5' DNA segment and a 3' PNA segment (Peterser et al . (1975) Bioorganic Med . Chem . Lett . 5:1119-11124) .

In other embodiments, the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors m vivo) , or agents facilitating transport across the cell membrane { see, e . g . , Letsmger et al . (1989) Proc . Natl . Acad . Sci . USA 86:6553-6556; Lemaitre et al . (1987) Proc . Natl . Acad . Sci . USA 84:648-652; PCT Publication No. W0 88/09810) or the blood-bram barrier ( see, e . g. , PCT Publication No. W0 89/10134) . In addition, oligonucleotides can be modified with hybridization-triggered cleavage agents ( see, e . g . , Krol et al . (1988) Bio /Techniques 6:958-976) or intercalating agents ( see, e . g. , Zon (1988) Pharm . Res . 5:539-549) . To this end, the oligonucleotide may be conjugated to another molecule, e.g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc.

II. Isolated Proteins and Antibodies One aspect of the invention pertains to isolated proteins and polypeptides of the invention, and biologically active portions thereof, as well as polypeptide fragments suitable for use as immunogens to raise antibodies directed against a polypeptide of the mvention. In one embodiment, the native polypeptide can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques. In another embodiment, polypeptides of the invention are produced Py recombinant DNA techniques. Alternative to recombinant expression, a polypeptide of the invention can be synthesized chemically using standard peptide synthesis techniques. An "isolated" or "purified" protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the protein is derived, or substantially free of chemical precursors or other chemicals when chemically synthesized. The language "substantially free of cellular material" includes preparations of protein m which the protein is separated from cellular components of the cells from which it is isolated or recombmantly produced. Thus, protein that is substantially free of cellular material includes preparations of protein having less than about 30%, 20%, 10%, or 5% (by dry weight) of heterologous protein (also referred to herein as a "contaminating protein") . When the protein or biologically active portion thereof is recombmantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, 10%, or 5% of the volume of the protein preparation. When the protein is produced by chemical synthesis, it is preferably substantially free of chemical precursors or other chemicals, i.e., it is separated from chemical precursors or other chemicals which are involved m the synthesis of the protein. Accordingly such preparations of the protein have less than about 30%, 20%, 10%, 5% (by dry weight) of chemical precursors or compounds other than the polypeptide of interest.

Biologically active portions of a polypeptide of the invention include polypeptides comprising ammo acid sequences sufficiently identical to or derived from the ammo acid sequence of the protein (e.g., the ammo acid sequence of SEQ ID NO : 2 , 5, 8, or 13), which include fewer amino acids than the full length protein, and exhibit at least one activity of the corresponding full- length protein. Typically, biologically active portions comprise a domain or motif with at least one activity of the corresponding protein. A biologically active portion of a protein of the invention can be a polypeptide which is, for example, 10, 25, 50, 100 or more amino acids in length. Moreover, other biologically active portions, in which other regions of the protein are deleted, can be prepared by recombinant techniques and evaluated for one or more of the functional activities of the native form of a polypeptide of the invention.

Preferred polypeptides have the ammo acid sequence of SEQ ID NO:2, 5, 7-11, and 12-16. Other useful proteins are substantially identical (e.g., at least about 45%, preferably 55%, 65%, 75%, 85%, 95%, or 99%) to SEQ ID NO: 2, 5, 7-11, and 12-16 and retain the functional activity of the protein of the corresponding naturally- occurring protein yet differ in amino acid sequence due to natural allelic variation or mutagenesis.

To determine the percent identity of two ammo acid sequences or of two nucleic acids, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced m the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence) . The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = # of identical positions/total # of positions (e.g., overlapping positions) x 100) . Preferably, the two sequences are the same length.

The determination of percent homology between two sequences can be accomplished using a mathematical algorithm A preferred, non- limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlm and Altschul (1990) Proc . Natl . Acad . Sci . USA 87:2264-2268, modified as m Karlm and Altschul (1993) Proc . Natl . Acad . Sci . USA 90:5873-5877 Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul, et al . (1990) J^". Mol . Biol 215:403-410. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to a nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain ammo acid sequences homologous to a protein molecules of the invention To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described m Altschul et al (1997) Nuclei c Acids Res . 25:3389-3402. Alternatively, PSI-Blast can be used to perform an iterated search which detects distant relationships between molecules Id . When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used See http ^• //www. ncbi .nlm.nih.gov. Another preferred, non-limitmg example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, (1988) CABIOS 4:11-17.

Such an algorithm is incorporated into the ALIGN program (version 2.0; which is part of the GCG sequence alignment software package When utilizing the ALIGN program for comparing ammo acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, only exact matches are counted.

The mvention also provides chimeric or fusion proteins. As used herein, a "chimeric protein" or "fusion protein" comprises all or part (preferably biologically active) of a polypeptide of the invention operably linked to a heterologous polypeptide (i.e., a polypeptide other than the same polypeptide of the invention) Withm the fusion protein, the term "operably linked" is intended to indicate that the polypeptide of the invention and the heterologous polypeptide are fused m-frame to each other. The heterologous polypeptide can be fused to the N-terminus or C-terminus of the polypeptide of the mvention.

One useful fusion protein is a GST fusion protein m which the polypeptide of the invention is fused to the C- termmus of GST sequences. Such fusion proteins can facilitate the purification of a recombinant polypeptide of the invention.

In another embodiment, the fusion protein contains a heterologous signal sequence at its N-terminus. For example, the native signal sequence of a polypeptide of the invention can be removed and replaced with a signal sequence from another protein. For example, the gp67 secretory sequence of the baculovirus envelope protein can be used as a heterologous signal sequence ( Current Protocols m Molecular Biology, Ausubel et al . , eds., John Wiley & Sons, 1992) . Other examples of eukaryotic heterologous signal sequences include the secretory sequences of melitt and human placental alkaline phosphatase (Stratagene; La Jolla, California) . In yet another example, useful prokaryotic heterologous signal sequences include the phoA secretory signal (Sambrook et al . , supra) and the protein A secretory signal (Pharmacia Biotech; Piscataway, New Jersey) . In yet another embodiment, the fusion protein is an immunoglobulin fusion protein m which all or part of a polypeptide of the invention is fused to sequences derived from a member of the immunoglobulin protein family. The immunoglobulin fusion proteins of the invention can be incorporated into pharmaceutical compositions and administered to a subject to inhibit an interaction between a ligand (soluble or membrane-bound) and a protein on the surface of a cell (receptor) , to thereby suppress signal transduction m vivo . The immunoglobulin fusion protein can be used to affect the bioavailability of a cognate ligand of a polypeptide of the invention. Inhibition of ligand/receptor interaction may be useful therapeutically, both for treating proliferative and differentiative disorders and for modulating (e.g promoting or inhibiting) cell survival.

Moreover, the immunoglobulin fusion proteins of the invention can be used as immunogens to produce antibodies directed against a polypeptide of the invention m a subject, to purify ligands and m screening assays to identify molecules which inhibit the interaction of receptors with ligands.

Chimeric and fusion protein of the invention can be produced by standard recombinant DNA techniques . In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence ( see, e . g. , Ausubel et al . , supra) . Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide) A nucleic acid encoding a polypeptide of the mvention can be cloned into such an expression vector such that the fusion moiety is linked m-frame to the polypeptide of the invention.

A signal sequence of a polypeptide of the invention (SEQ ID NO: 7 or 12) can be used to facilitate secretion and isolation of a secreted protein or other protein of interest. Signal sequences are typically characterized by a core of hydrophobic ammo acids which are generally cleaved from the mature protein during secretion m one or more cleavage events. Such signal peptides contain processing sites that allow cleavage of the signal sequence from the mature proteins as they pass through the secretory pathway. Thus, the invention pertains to the described polypeptides having a signal sequence, as well as to the signal sequence itself and to the polypeptide m the absence of the signal sequence (i.e., the cleavage products) . In one embodiment, a nucleic acid sequence encoding a signal sequence of the invention can be operably linked m an expression vector to a protein of interest, such as a protein which is ordinarily not secreted or is otherwise difficult to isolate. The signal sequence directs secretion of the protein, such as from a eukaryotic host into which the expression vector is transformed, and the signal sequence is subsequently or concurrently cleaved. The protein can then be readily purified from the extracellular medium by art recognized methods. Alternatively, the signal sequence can be linked to the protein of interest using a sequence which facilitates purification, such as with a GST domain. In another embodiment, the signal sequences of the present invention can be used to identify regulatory sequences, e.g , promoters, enhancers, repressors . Since signal sequences are the most ammo-termmal sequences of a peptide, it is expected that the nucleic acids which flank the signal sequence on its ammo-termmal side will be regulatory sequences which affect transcription. Thus, a nucleotide sequence which encodes all or a portion of a signal sequence can be used as a probe to identify and isolate signal sequences and their flanking regions, and these flanking regions can be studied to identify regulatory elements therein.

The present invention also pertains to variants of the polypeptides of the invention. Such variants have an altered ammo acid sequence which can function as either agonists (mimetics) or as antagonists. Variants can be generated by mutagenesis, e.g., discrete point mutation or truncation. An agonist can retain substantially the same, or a subset, of the biological activities of the naturally occurring form of the protein. An antagonist of a protein can inhibit one or more of the activities of the naturally occurring form of the protein by, for example, competitively binding to a downstream or upstream member of a cellular signaling cascade which includes the protein of interest. Thus, specific biological effects can be elicited by treatment with a variant of limited function. Treatment of a subject with a variant having a subset of the biological activities of the naturally occurring form of the protein can have fewer side effects m a subject relative to treatment with the naturally occurring form of the protein.

Variants of a protein of the invention which function as either agonists (mimetics) or as antagonists can be identified by screening combinatorial libraries of mutants, e g , truncation mutants, of the protein of the invention for agonist or antagonist activity. In one embodiment, a variegated library of variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library. A variegated library of variants can be produced by, for example, enzymatically ligatmg a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential protein sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) . There are a variety of methods which can be used to produce libraries of potential variants of the polypeptides of the invention from a degenerate oligonucleotide sequence. Methods for synthesizing degenerate oligonucleotides are known m the art ( see, e . g. , Narang (1983) Tetrahedron 39:3; Itakura et al . (1984) Annu . Rev. Biochem . 53:323; Itakura et al . (1984) Science 198:1056; Ike et al . (1983) Nucleic Acid Res . 11:477) . In addition, libraries of fragments of the coding sequence of a polypeptide of the invention can be used to generate a variegated population of polypeptides for screening and subsequent selection of variants. For example, a library of coding sequence fragments can be o generated by treating a double stranded PCR fragment of the coding sequence of interest with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturmg the DNA to form double stranded DNA which can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with SI nuclease, and ligatmg the resulting fragment library into an expression vector. By this method, an expression library can be derived which encodes N-terminal and internal fragments of various sizes of the protein of interest.

Several techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property. The most widely used techniques, which are amenable to high through-put analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates isolation of the vector encoding the gene whose product was detected. Recursive ensemble mutagenesis (REM) , a technique which enhances the frequency of functional mutants m the libraries, can be used m combination with the screening assays to identify variants of a protein of the invention (Arkm and Yourvan (1992) Proc . Natl . Acad Sci . USA 89:7811-7815; Delgrave et al . (1993) Protein Engineering 6(3) :327-331) .

An isolated polypeptide of the invention, or a fragment thereof, can be used as an immunogen to generate antibodies using standard techniques for polyclonal and monoclonal antibody preparation. The full-length polypeptide or protein can be used or, alternatively, the invention provides antigenic peptide fragments for use as immunogens The antigenic peptide of a protein of the invention comprises at least 8 (preferably 10, 15, 20, or 30) ammo acid residues of the ammo acid sequence of SEQ ID NO: 8 or 13 and encompasses an epitope of the protein such that an antibody raised against the peptide forms a specific immune complex with the protein.

Preferred epitopes encompassed by the antigenic peptide are regions that are located on the surface of the protein, e.g., hydrophilic regions. Figures 2 and 4 are hydrophobicity plots of the proteins of the invention. These plots or similar analyses can be used to identify hydrophilic regions. An immunogen typically is used to prepare antibodies by immunizing a suitable subject, (e.g., rabbit, goat, mouse or other mammal) . An appropriate immunogenic preparation can contain, for example, recombinantly expressed chemically synthesized polypeptide. The preparation can further include an adjuvant, such as Freund' s complete or incomplete adjuvant, or similar immunostimulatory agent. Accordingly, another aspect of the invention pertains to antibodies directed against a polypeptide of the invention. The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site which specifically binds an antigen, such as a polypeptide of the invention. A molecule which specifically binds to a given polypeptide of the invention is a molecule which binds the polypeptide, but does not substantially bind other molecules m a sample, e.g., a biological sample, which naturally contains the polypeptide. Examples of immunologically active portions of immunoglobulin molecules include F (ab) and F(ab')₂ fragments which can be generated by treating the antibody with an enzyme such as pepsin. The invention provides polyclonal and monoclonal antibodies. The term "monoclonal antibody" or "monoclonal antibody composition" , as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreactmg with a particular epitope.

Polyclonal antibodies can be prepared as described above by immunizing a suitable subject with a polypeptide of the invention as an immunogen. The antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized polypeptide. If desired, the antibody molecules can be isolated from the mammal (e.g., from the blood) and further purified by well-known techniques, such as protein A chromatography to obtain the IgG fraction. At an appropriate time after immunization, e.g., when the specific antibody titers are highest, antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler and Milstem (1975) Nature 256:495-497, the human B cell hybridoma technique (Kozbor et al . (1983) Immunol . Today 4:72), the EBV-hybridoma technique (Cole et al .

(1985) , Monoclonal Antibodi es and Cancer Therapy, Alan R Liss, Inc., pp. 77-96) or tπoma techniques. The technology for producing hybridomas is well known ( see generally Current Protocols m Immunology (1994) Coligan et al . (eds.) John Wiley & Sons, Inc., New York, NY) . Hybridoma cells producing a monoclonal antibody of the invention are detected by screening the hybridoma culture supernatants for antibodies that bind the polypeptide of interest, e.g., using a standard ELISA assay. Alternative to preparing monoclonal antibody-secreting hybridomas, a monoclonal antibody directed against a polypeptide of the invention can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g , an antibody phage display library) with the polypeptide of interest. Kits for generating and screening phage display libraries are commercially available (e.g., the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; and the Stratagene Surf ZAP™ Phage Display Ki t, Catalog No. 240612) Additionally, examples of methods and reagents particularly amenable for use in generating and screening antibody display library can be found in, for example, U.S. Patent No. 5,223,409; PCT Publication No. WO 92/18619; PCT Publication No. WO 91/17271; PCT Publication No. WO 92/20791; PCT Publication No. WO 92/15679; PCT Publication No. WO 93/01288; PCT Publication No. WO 92/01047; PCT Publication No. WO 92/09690; PCT Publication No. WO 90/02809; Fuchs et al . (1991) Bio/Technology 9:1370-1372; Hay et al . (1992) Hum . AntiJbod. Hybridomas 3:81-85; Huse et al . (1989) Science 246:1275-1281; Griffiths et al . (1993) EMBO J. 12:725- 734.

Additionally, recombinant antibodies, such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the invention. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in PCT Publication No. WO 87/02671; European Patent

Application 184,187; European Patent Application 171,496; European Patent Application 173,494; PCT Publication No. WO 86/01533; U.S. Patent No. 4,816,567; European Patent Application 125,023; Better et al . (1988) Science 240:1041-1043; Liu et al . (1987) Proc . Natl . Acad . Sci . USA 84:3439-3443; Liu et al . (1987) J. Immunol . 139:3521-3526; Sun et al . (1987) Proc . Natl . Acad . Sci . USA 84:214-218; Nishimura et al . (1987) Cane . Res . 47:999-1005; Wood et al . (1985) Nature 314:446-449; and Shaw et al . (1988) J^". Natl . Cancer Inst . 80:1553-1559); Morrison (1985) Sci ence 229:1202-1207; Oi et al . (1986) Bio/Techniques 4:214; U.S. Patent 5,225,539; Jones et al . (1986) Nature 321:552-525; Verhoeyan et al . (1988) Science 239:1534; and Beidler et al . (1988) J. Immunol . 141:4053-4060. Completely human antibodies are particularly desirable for therapeutic treatment of human patients. Such antibodies can be produced using transgenic mice which are incapable of expressing endogenous immunoglobulin heavy and light chains genes, but which can express human heavy and light chain genes. The transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of a polypeptide of the invention. Monoclonal antibodies directed against the antigen can be obtained using conventional hybridoma technology. The human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation. Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA 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 antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e . g. , U.S. Patent 5,625,126; U.S. Patent 5,633,425; U.S. Patent 5,569,825; U.S. Patent 5,661,016; and U.S. Patent 5,545,806. In addition, companies such as Abgenix, Inc. (Freemont, CA) , can be engaged to provide human antibodies directed against a selected antigen using technology similar to that described above .

Completely human antibodies which recognize a selected epitope can be generated using a technique referred to as "guided selection." In this approach a selected non-human monoclonal antibody, e.g., a murine antibody, is used to guide the selection of a completely human antibody recognizing the same epitope (Jespers et al . (1994) Bio/Technology 12:899-903). An antibody directed against a polypeptide of the invention (e.g., monoclonal antibody) can be used to isolate the polypeptide by standard techniques, such as affinity chromatography or immunoprecipitation. Moreover, such an antibody can be used to detect the protein (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the polypeptide. The antibodies can also be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen. 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, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, -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, dichlorotπazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include ¹⁵I, ¹³¹I, ³⁵S or ³H.

III. Recombinant Expression Vectors and Host Cells Another aspect of the invention pertains to vectors, preferably expression vectors, containing a nucleic acid encoding a polypeptide of the invention (or a portion thereof) . As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasuiid" , which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication m a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors) . Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors, expression vectors, are capable of directing the expression of genes to which they are operably linked. In general, expression vectors of utility m recombinant DNA techniques are often m the form of plasmids (vectors) . However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses) , which serve equivalent functions.

The recombinant expression vectors of the invention comprise a nucleic acid of the invention a form suitable for expression of the nucleic acid m a host cell . This means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operably linked to the nucleic acid sequence to be expressed. With a recombinant expression vector,

"operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence (s) m a manner which allows for expression of the nucleotide sequence (e.g., m an in vi tro transcription/translation system or m a host cell when the vector is introduced into the host cell) . The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals) . Such regulatory sequences are described, for example, in Goeddel, Gene Expression

Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990) . Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cell and those which direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences) . It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein. The recombinant expression vectors of the invention can be designed for expression of a polypeptide of the invention in prokaryotic or eukaryotic cells, e.g., bacterial cells such as E. coli , insect cells (using baculovirus expression vectors) , yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, supra . Alternatively, the recombinant expression vector can be transcribed and translated in vi tro, for example using T7 promoter regulatory sequences and T7 polymerase . Expression of proteins in prokaryotes is most often carried out m E . coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid m the purification of the recombinant protein by acting as a ligand m affinity purification Often, m fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokmase . Typical fusion expression vectors include pGEX (Pharmacia Biotech Ine; Smitn and Johnson (1988) Gene 67:31-40), pMAL (New England Biolabs, Beverly, MA) and pRIT5

(Pharmacia, Piscataway, NJ) which fuse glutathione S- transferase (GST) , maltose E binding protein, or protein A, respectively, to the target recombinant protein. Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al . , (1988) Gene 69:301-315) and pET lid (Studier et al . , Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 60-89) . Target gene expression from the pTrc vector relies on host RNA polymerase transcription from a hybrid trp-lac fusion promoter. Target gene expression from the pET lid vector relies on transcription from a T7 gnlO-lac fusion promoter mediated by a coexpressed viral RNA polymerase (T7 gnl) . This viral polymerase is supplied by host strains BL21'DE3) or HMS174(DE3) from a resident λ prophage harboring a T7 gnl gene under the transcriptional control of the lacUV 5 promoter.

One strategy to maximize recombinant protein expression E . coli is to express the protein m a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 119-128) . Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each ammo acid are those preferentially utilized m E . coli (Wada et al . (1992) Nuclei c Acids Res . 20:2111-2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.

In another embodiment, the expression vector is a yeast expression vector. Examples of vectors for expression m yeast S . cerivisae include pYepSecl (Baldaπ et al . (1987) EMBO J. 6:229-234), pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al . (1987) Gene 54:113-123), pYES2 (Invitrogen Corporation, San Diego, CA) , and pPicZ (Invitrogen Corp, San Diego, CA) .

Alternatively, the expression vector is a baculovirus expression vector Baculovirus vectors available for expression of proteins m cultured insect cells (e.g., Sf 9 cells) include the pAc series (Smith et al . (1983) Mol . Cell Biol . 3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170 : 31-39) .

In yet another embodiment, a nucleic acid of the invention is expressed m mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed (1987) Nature 329:840) and pMT2PC (Kaufman et al . (1987) EMBO J. 6:187- 195) . When used m mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook et al . , supra . In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially m a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid) . Tissue-specific regulatory elements are known m the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specifIC ; Pmkert et al . (1987) Genes Dev. 1:268-277) , lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol . 43:235-275), m particular promoters of T cell receptors (Wmoto and Baltimore (1989) EMBO J. 8.729-733) and lmmunoglobulms (Banerji et al . (1983) Cell 33:729-740 ; Queen and Baltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., the neurofIlament promoter; Byrne and Ruddle (1989) Proc . Natl . Acad . Sci . USA 86:5473-5477), pancreas-specific promoters (Edlund et al . (1985) Science 230:912-916), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Patent No. 4,873,316 and European Application Publication No. 264,166). Developmentally- regulated promoters are also encompassed, for example the murine hox promoters (Kessel and Gruss (1990) Sci ence 249:374-379) ana the a-fetoprotein promoter (Campes and Tilghman (1989) Genes ϋev. 3:537-546). The invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector m an antisense orientation. That is, the DNA molecule is operably linked to a regulatory sequence m a manner which allows for expression (by transcription of the DNA molecule) of an RNA molecule which is antisense to the mRNA encoding a polypeptide of the invention. Regulatory sequences operably linked to a nucleic acid cloned m the antisense orientation can De chosen which direct the continuous expression of the antisense RNA molecule m a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen which direct constitutive, tissue specific or cell type specific expression of antisense RNA. The antisense expression vector can be m the form of a recombinant plasmid, phagemid or attenuated virus m which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced. For a discussion of the regulation of gene expression using antisense genes see Wemtraub et al . (Reviews - Trends m Genetics, Vol. 1(1) 1986).

Another aspect of the invention pertains to host cells into which a recombinant expression vector of the invention has been introduced. The terms "host cell" and "recombinant host cell" are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur m succeeding generations due to either mutation or environmental influences, such progeny may not, m fact, be identical to the parent cell, but are still included withm the scope of the term as used herei . A host cell can be any prokaryotic (e.g., E. coli ) or eukaryotic cell (e.g , an insect cell, yeast, or a mammalian cell) .

Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms

"transformation" and "transfection" are intended to refer to a variety of ait-recognized techniques for introducing foreign nucleic acid into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE- dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfectmg host cells can be found m Sambrook, et al . ( supra) , and other laboratory manuals.

For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., for resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Preferred selectable markers include those which confer resistance to drugs, such as G418, hygromycm and methotrexate . Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while tne other cells die) .

A host cell of the invention, such as a prokaryotic or eukaryotic host cell culture, can be used to produce a polypeptide of the invention. Accordingly, the invention further provides methods for producing a polypeptide of the invention using the host cells of the invention. In one embodiment, tne method comprises culturmg the host cell of mvention (into which a recombinant expression vector encoding a polypeptide of the invention has been introduced) m a suitable medium such that the polypeptide is produced. In another embodiment, the method further comprises isolating the polypeptide from the medium or the host cell. The host cells of the invention can also be used to produce nonhuman transgenic animals. For example, m one embodiment, a host cell of the invention is a fertilized oocyte or an embryonic stem cell into which a sequences encoding a polypeptide of the invention have been introduced Such host cells can then be used to create non-human transgenic animals m which exogenous sequences encoding a polypeptide of the invention have been introduced into their genome or homologous recombinant animals in which endogenous encoding a polypeptide of the invention sequences have been altered. Such animals are useful for studying the function and/or activity of the polypeptide and for identifying and/or evaluating modulators of polypeptide activity. As used herein, a "transgenic animal" is a -non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more of the cells of the animal includes a transgene. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, etc. A transgene is exogenous DNA which is integrated into the genome of a cell from which a transgenic animal develops and which remains m the genome of the mature animal, thereby directing the expression of an encoded gene product m one or more cell types or tissues of the transgenic animal. As used herein, an "homologous recombinant animal" is a non-human animal, preferably a mammal, more preferably a mouse, in which an endogenous gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development of the animal .

A transgenic animal of the invention can be created by introducing nucleic acid encoding a polypeptide of the invention (or a homologue thereof) into the male pronuclei of a fertilized oocyte, e.g., by microinj ection, retroviral infection, and allowing the oocyte to develop m a pseudopregnant female foster animal. Intromc sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression of the transgene. A tissue- specific regulatory sequence (s) can be operably linked to the transgene to direct expression of the polypeptide of the invention to particular cells. Methods for generating transgenic animals via embryo manipulation and micromj ection, particularly animals such as mice, have become conventional m the art and are described, for example, m U.S. Patent NOS. 4,736,866 and 4,870,009, U.S. Patent No 4,873,191 and m Hogan, Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986) . Similar methods are used for production of othei transgenic animals. A transgenic founder animal can be identified based upon the presence of the transgene m its genome and/or expression of mRNA encoding the transgene m tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying the transgene can further be bred to other transgenic animals carrying other transgenes. To create an homologous recombinant animal, a vector is prepared which contains at least a portion of a gene encoding a polypeptide of the invention into which a deletion, addition or substitution has been introduced to thereby alter, e.g., functionally disrupt, the gene. In a preferred embodiment, the vector is designed such that, upon homologous recombination, the endogenous gene is functionally disrupted (i.e., no longer encodes a functional protein; also referred to as a "knock out" vector) . Alternatively, the vector can be designed such that, upon homologous recombination, the endogenous gene is mutated or otherwise altered but still encodes functional protein (e.g., the upstream regulatory region can de altered to thereby alter the expression of the endogenous protein) . In the homologous recombination vector the altered portion of the gene is flanked at its 5' and 3' ends by additional nucleic acid of the gene to allow for homologous recombination to occur between the exogenous gene carried by the vector and an endogenous gene in an embryonic stem cell. The additional flanking nucleic acid sequences are of sufficient length for successful homologous recombination with the endogenous gene. Typically, several kilobases of flanking DNA (both at the 5' and 3' ends) are included in the vector ( see, e . g . , Thomas and Capecchi (1987) Cell 51:503 for a description of homologous recombination vectors) . The vector is introduced into an embryonic stem cell line (e.g., by electroporation) and cells m which the introduced gene has homologously recombined with the endogenous gene are selected ( see, e . g . , Li et al . (1992) Cell 69:915). The selected cells are then injected into a blastocyst of an animal (e.g., a mouse) to form aggregation chimeras ( see, e . g. , Bradley m Teratocarcinomas and Embryonic Stem Cells : A Practical Approach, Robertson, ed. (IRL, Oxford, 1987) pp. 113- 152) . A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term. Progeny harboring the homologously recombined DNA in their germ cells can be used to breed animals in which all cells of the animal contain the homologously recombined DNA by germline transmission of the transgene. Methods for constructing homologous recombination vectors and homologous recombinant animals are described further in Bradley (1991) Current Opinion in Bio/Technology 2:823-829 and m PCT Publication NOS. WO 90/11354, WO 91/01140, WO 92/0968, and WO 93/04169.

In another embodiment, transgenic non-human animals can be produced which contain selected systems which allow for regulated expression of the transgene. One example of such a system is the cre/loxP recombinase system of bacteπophage PI. For a description of the cre/loxP recombinase system, see, e . g. , Lakso et al . (1992) Proc . Natl . Acad . Sci . USA 89:6232-6236. Another example of a recombinase system is the FLP recombinase system of Saccharomyces cerevisiae (0' Gorman et al . (1991) Science 251:1351-1355. If a cre/loxP recombinase system is used to regulate expression of the transgene, animals containing transgenes encoding both the Cre recombinase and a selected protein are required. Such animals can be provided through the construction of "double" transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase. Clones of the non-human transgenic animals described herein can also be produced according to the methods described m Wilmut et al . (1997) Nature 385:810-813 and PCT Publication NOS. WO 97/07668 and WO 97/07669.

IV. Pharmaceutical Compositions

The nucleic acid molecules, polypeptides, and antibodies (also referred to herein as "active compounds") of the invention can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier. As used herein the language

"pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art Except insofar as any conventional media or agent is incompatible with the active compound, use thereof m the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal , and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components : a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediammetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide . The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for mjectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteπostatic water, Cremophor EL™ (BASF; Parsippany, NJ) or phosphate buffered saline (PBS) . In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi . The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size m the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutano-, phenol, ascorbic acid, thimerosal, and the like In many cases, it will be preferable to include isotonic agents, for example, sugars,., polyalcohols such as mannitol, sorbitol, sodium chloride m the composition. Prolonged absorption of the mjectable compositions c_cn be brought about by including m the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

Sterile mjectable solutions can be prepared by incorporating the active compound (e.g., a polypeptide or antibody) m tne required amount m an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile mjectable solutions, the preferred methods of preparation are vacuum drying and freeze-drymg which yields a powde¹ of the active ingredient plus any additional des red ingredient from a previously sterile- filtered solution thereof. Oral compositions generally include an inert diluent or an edible carrier They can be enclosed m gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can o be incorporated with excipients and used the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound m the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as algmic acid, Pπmogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring .

For administration by inhalation, the compounds are delivered m tie form of an aerosol spray from a pressurized container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

Systemic administration can also be by transmucosal or transdermal means . For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used m the formulation. Such penetrants are generally known m the art, and include, for example, for transmucosal administration, detergents, bile salts, anc fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known m the art . The compounds can also be prepared m the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydπdes, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled m the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Ine Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled m the art, for example, as described m U.S. Patent No. 4, 522, 811.

It is especially advantageous to formulate oral or parenteral compositions m dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect m association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.

For antibodies, the preferred dosage is 0.1 mg/kg to 100 mg/kg of body weight (generally 10 mg/kg to 20 mg/kg) . If the antibody is to act in the brain, a dosage of 50 mg/kg to 100 mg/kg is usually appropriate. Generally, partially human antibodies and fully human antibodies have a longer half-life withm the human body than other antibodies. Accordingly, lower dosages and less frequent administration is often possible. Modifications such as lipidation can be used to stabilize antibodies and to enhance uptake and tissue penetration (e.g., into the brain) . A method for lipidation of antibodies is described by Cruikshank et al . ((1997) J. Acquired Immune Defi ciency Syndromes and Human Retrovirology 14:193) . The nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (U.S. Patent 5,328,470) or by stereotactic injection ( see, e . g. , Chen et al . (1994) Proc . Natl . Acad . Sci . USA 91:3054-3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g. retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system. The pharmaceutical compositions can be included a container, pack, or dispenser together with instructions for administration.

V. Uses and Methods of the Invention The nucleic acid molecules, proteins, protein homologues, and antibodies described herein can be used m one or more of the following methods: a) screening assays; b) detection assays (e.g., chromosomal mapping, tissue typing, forensic biology) ; c) predictive medicine (e.g., diagnostic assays, prognostic assays, monitoring clinical trials, and pharmacogenomics) ; and d) methods of treatment (e.g , therapeutic and prophylactic). For example, polypeptides of the invention can to used to (I) modulate cellular proliferation; (ii) modulate cellular differentiation; and (m) modulate cell survival. The isolated nucleic acid molecules of the invention can be used to express proteins (e.g., via a recombinant expression vector m a host cell m gene therapy applications), to detect mRNA (e.g., m a biological sample) or a genetic lesion, and to modulate activity of a polypeptide of the invention. In addition, the polypeptides of the invention can be used to screen drugs or compounds which modulate activity or expression of a polypeptide of the invention as well as to treat disorders characterized by insufficient or excessive production of a protein of the invention or production of a form of a protein of the invention which has decreased or aberrant activity compared to the wild type protein. In addition, the antibodies of the invention can be used to detect and isolate a protein of the and modulate activity of a protein of the invention.

This invention further pertains to novel agents identified by tne above-described screening assays and uses thereof fcr treatments as described herein. A. Screening Assays

The invention provides a method (also referred to herein as a "screening assay") for identifying modulators, i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) which bind to polypeptide of the invention or have a stimulatory or inhibitory effect on, for example, expression or activity of a polypeptide of the invention. In one embodiment, the invention provides assays for screening candidate or test compounds which bind to or modulate the activity of the membrane-bound form of a polypeptide of the invention or biologically active portion thereof . The test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the "one-bead one-compound" library method; and synthetic library methods using affinity chromatography selection. The biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam (1997) Anticancer Drug Des . 12:145) .

Examples of methods for the synthesis of molecular libraries can be found m the art, for example in: DeWitt et al . (1993) Proc . Natl . Acad . Sci . USA 90:6909; Erb et al . (1994) Proc . Natl . Acad . Sci . USA 91:11422; Zuckermann et al . (1994). J^". Med . Chem . 37:2678; Cho et al . (1993) Science 261:1303; Carrell et al . (1994) Angew. Chem . Int . Ed . Engl . 33:2059; Carell et al . (1994) Angew. Chem . Int . Ed . Engl . 33:2061; and Gallop et al . (1994) J. Med . Chem . 37:1233. Libraries of compounds may be presented in solution (e.g., Houghten (1992) Bio/Techniques 13:412-421), or on beads (Lam (1991) Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (U.S. Patent No. 5,223,409), spores (Patent NOS. 5,571,698; 5,403,484; and 5,223,409), plasmids (Cull et al . (1992) Proc . Natl . Acad . Sci . USA 89:1865-1869) or phage (Scott and Smith (1990) Science 249^386-390; Devlin (1990) Science 249:404-406; Cwirla et al . (1990) Proc . Natl . Acad . Sci . USA 87:6378-6382; and Felici (1991) J^". Mol . Biol . 222:301-310) .

In one embodiment, an assay is a cell-based assay in which a cell which expresses a membrane-bound form of a polypeptide of the invention, or a biologically active portion thereof, on the cell surface is contacted with a test compound and the ability of the test compound to bind to the polypeptide determined. The cell, for example, can be a yeast cell or a cell of mammalian origin. Determining the ability of the test compound to bind to the polypeptide can be accomplished, for example, by coupling the test compound with a radioisotope or enzymatic label such that binding of the test compound to the polypeptide or biologically active portion thereof can be determined by detecting the labeled compound in a complex. For example, test compounds can be labeled with ¹²⁵1 , ³⁵S, ¹⁴C, or ³H, either directly or indirectly, and the radioisotope detected by direct counting of radioemmission or by scintillation counting. Alternatively, test compounds can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product . In a preferred embodiment, the assay comprises contacting a cell which expresses a membrane-bound form of a polypeptide of the invention, or a biologically active portion thereof, on the cell surface with a known compound which binds the polypeptide to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with the polypeptide, wherein determining the ability of the test compound to interact with the polypeptide comprises determining the ability of the test compound to preferentially bind to the polypeptide or a biologically active portion thereof as compared to the known compound

In another embodiment, an assay is a cell-based assay comprising contacting a cell expressing a membrane-bound form of a polypeptide of the invention, or a biologically active portion thereof, on the cell surface with a test compound and determining the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of the polypeptide or biologically active portion thereof. Determining the ability of the test compound to modulate the activity of the polypeptide or a biologically active portion thereof can be accomplished, for example, by determining the ability of the polypeptide protein to bind to or interact with a target molecule.

Determining the ability of a polypeptide of the invention to bind to or interact with a target molecule can be accomplished by one of the methods described above for determining direct binding. As used herein, a "target molecule" is a molecule with which a selected polypeptide (e.g., a polypeptide of the invention binds or interacts with m nature, for example, a molecule on the surface of a cell which expresses the selected protein, a molecule on the surface of a second cell, a molecule m the extracellular milieu, a molecule associated with the internal surface of a cell membrane or a cytoplasmic molecule. A target molecule can be a polypeptide of the invention or some other polypeptide or protein. For example, a target molecule can be a component of a signal transduction pathway which facilitates transduction of an extracellular signal (e.g., a signal generated by binding of a compound to a polypeptide of the invention) through the cell membrane and into the cell or a second intercellular protein which has catalytic activity or a protein which facilitates the association of downstream signaling molecules with a polypeptide of the invention. Determining the ability of a polypeptide of the invention to bind to or interact with a target molecule can be accomplished by determining the activity of the target molecule. For example, the activity of the target molecule can be determined by detecting induction of a cellular second messenger of the target (e.g., intracellular Ca²⁺, diacylglycerol , IP3, etc.), detecting catalytic/enzymatic activity of the target on an appropriate substrate, detecting the induction of a reporter gene (e.g., a regulatory element that is responsive to a polypeptide of the invention operably linked to a nucleic acid encoding a detectable marker, e.g. luciferase), or detecting a cellular response, for example, cellular differentiation, or cell proliferation. In yet another embodiment, an assay of the present invention is a cell-free assay comprising contacting a polypeptide of the invention or biologically active portion thereof with a test compound and determining the ability of the test compound to bind to the polypeptide or biologically active portion thereof. Binding of the test compound to the polypeptide can be determined either directly or indirectly as described above. In a preferred embodiment, the assay includes contacting the polypeptide of the invention or biologically active portion thereof with a known compound which binds the polypeptide to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with the polypeptide, wherein determining the ability of the test compound to interact with the polypeptide comprises determining the ability of the test compound to preferentially bind to the polypeptide or biologically active portion thereof as compared to the known compound. In another embodiment, an assay is a cell -free assay comprising contacting a polypeptide of the invention or biologically active portion thereof with a test compound and determining the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of the polypeptide or biologically active portion thereof. Determining the ability of the test compound to modulate the activity of the polypeptide can be accomplished, for example, by determining the ability of the polypeptide to bind to a target molecule by one of the methods described above for determining direct binding. In an alternative embodiment, determining the ability of the test compound to modulate the activity of the polypeptide can be accomplished by determining the ability of the polypeptide of the invention to further modulate the target molecule. For example, the catalytic/enzymatic activity of the target molecule on an appropriate substrate can be determined as previously described.

In yet another embodiment, the cell -free assay comprises contacting a polypeptide of the invention or biologically active portion thereof with a known compound which binds the polypeptide to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with the polypeptide, wherein determining the ability of the test compound to interact with the polypeptide comprises determining the ability of the polypeptide to preferentially bind to or modulate the activity of a target molecule.

The cell -free assays of the present invention are amenable to use of both a soluble form or the membrane- bound form of a polypeptide of the invention. In the case of cell-free assays comprising the membrane-bound form of the polypeptide, it may be desirable to utilize a solubilizing agent such that the membrane-bound form of the polypeptide is maintained m solution. Examples of such solubilizing agents include non- ionic detergents such as n-octylglucoside, n-dodecylglucoside, n- dodecylmaltoside , octanoyl-N-methylglucamide, decanoyl -N- methylglucamide, Triton X-100, Triton X-114, Thesit, Isotπdecypoly (ethylene glycol ether) n, 3-[(3- cholamidopropyl) dimethylammmio] -1 -propane sulfonate (CHAPS) , 3- [ (3 -cholamidopropyl) dimethylammmio] -2 - hydroxy- 1 -propane sulfonate (CHAPSO) , or N-dodecyl=N,N- dimethyl- 3 -ammonιo-1 -propane sulfonate .

In more than one embodiment of the above assay methods of the present invention, it may be desirable to immobilize either the polypeptide of the invention or its target molecule to facilitate separation of complexed from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay. Binding of a test compound to the polypeptide, or interaction of the polypeptide with a target molecule m the presence and absence of a candidate compound, can be accomplished m any vessel suitable for containing the reactants. Examples of such vessels include microtitre plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix. For example, glutathione-S-transferase fusion proteins or glutathione-S-transferase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical; St. Louis, MO) or glutathione deπvatized microtitre plates, which are then combined with the test compound or the test compound and either the non-adsorbed target protein or A polypeptide of the invention, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH) . Following incubation, the beads or microtitre plate wells are washed to remove any unbound components and complex formation is measured either directly or indirectly, for example, as described above.

Alternatively, the complexes can be dissociated from the matrix, and the level of binding or activity of the polypeptide of the invention can be determined using standard techniques . Other techniques for immobilizing proteins on matrices can also be used m the screening assays of the invention. For example, either the polypeptide of the mvention or its target molecule can be immobilized utilizing conjugation of biotin and streptavidm. Biotinylated polypeptide of the invention or target molecules can be prepared from biotm-NHS (N-hydroxy- succmimide) using techniques well known m the art (e.g., biot ylation kit, Pierce Chemicals; Rockford, IL) , and immobilized m the wells of streptavidm-coated 96 well plates (Pierce Chemical) . Alternatively, antibodies reactive with the polypeptide of the invention or target molecules but which do not interfere with binding of the polypeptide of the invention to its target molecule can be derivatized to the wells of the plate, and unbound target or polypeptidede of the invention trapped m the wells by antibody conjugation. Methods for detecting such complexes, m addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the poiypeptide of the invention or target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the polypeptide of the invention or target molecule.

In another embodiment, modulators of expression of a polypeptide of the invention are identified a method m which a cell is contacted with a candidate compound and the expression of the selected mRNA or protein (i.e., the mRNA or protein corresponding to a polypeptide or nucleic acid of the invention) m the cell is determined. The level of expression of the selected mRNA or protein m the presence of the candidate compound is compared to the level of expression of the selected mRNA or protein the absence of the candidate compound. The candidate compound can then be identified as a modulator of expression of the polypeptide of the invention based on this comparison. For example, when expression of the selected mRNA or protein is greater (statistically significantly greater) m the presence of the candidate compound than m its absence, the candidate compound is identified as a stimulator of the selected mRNA or protein expression Alternatively, when expression of the selected mRNA or protein is less (statistically significantly less) m the presence of the candidate compound than m its absence, the candidate compound is identified as an inhibitor of the selected mRNA or protein expression The level of the selected mRNA or protein expression m the cells can be determined by methods described herein.

In yet another aspect of the invention, a polypeptide of the inventions can be used as "bait proteins" m a two-hybrid assay or three hybrid assay (see, e . g. , U.S. Patent No. 5,283,317; Zervos et al . (1993) Cell 72:223- 232; Madura et al (1993) J. Biol . Chem . 268:12046-12054; Bartel et al . (1993) Bio/Techniques 14:920-924; Iwabuchi et al . (1993) Oncogene 8:1693-1696; and PCT Publication No. WO 94/10300) , to identify other proteins, which bind to or interact with the polypeptide of the invention and modulate activity of the polypeptide of the invention. Such binding proteins are also likely to be involved m the propagation of signals by the polypeptide of the inventions as, for example, upstream or downstream elements of a signaling pathway involving the polypeptide of the invention.

This invention further pertains to novel agents identified by the above-described screening assays and uses thereof for treatments as described herein.

B . Detection Assays

Portions or fragments of the cDNA sequences identified herein (and the corresponding complete gene sequences) can be used m numerous ways as polynucleotide reagents. For example, these sequences can be used to: (I) map their respective genes on a chromosome and, thus, locate gene regions associated with genetic disease; (ii) identify an individual from a minute biological sample (tissue typing) ; and (m) aid m forensic identification of a biological sample. These applications are described in the subsections below.

1. Chromosome Mapping

Once the sequence (or a portion of the sequence) of a gene has been isolated, this sequence can be used to map the location of the gene on a chromosome. Accordingly, nucleic acid molecules described herein or fragments thereof, can be used to map the location of the corresponding genes on a chromosome. The mapping of the sequences to chromosomes is an important first step m correlating these sequences with genes associated with disease . Briefly, genes can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp m length) from the sequence of a gene of the invention. Computer analysis of the sequence of a gene of the invention can be used to rapidly select primers that do not span more than one exon m the genomic DNA, thus complicating the amplification process. These primers can then be used for PCR screening of somatic cell hybrids containing individual human chromosomes . Only those hybrids containing the human gene corresponding to the gene sequences will yield an amplified fragment. For a review of this technique, see D'Eustachio et al . ((1983) Science 220:919-924) .

PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular sequence to a particular chromosome. Three or more sequences can be assigned per day using a single thermal cycler. Using the nucleic acid sequences of the invention to design oligonucleotide primers, sublocalization can be achieved with panels of fragments from specific chromosomes.

Other mapping strategies which can similarly be used to map a gene to its chromosome include m si tu hybridization (described m Fan et al . (1990) Proc . Natl . Acad . Sci . USA 81 -. 6223 - 21 ) , pre-screenmg with labeled flow-sorted chromosomes (CITE) , and pre-selection by hybridization to chromosome specific cDNA libraries (CITE) . Fluorescence m si tu hybridization (FISH) of a DNA sequence to a metaphase chromosomal spread can further be used to provide a precise chromosomal location m one step. For a review of this technique, see Verma et al . , (Human Chromosomes: A Manual of Basic Techniques (Pergamon Press, New York, 1988)).

Reagents for chromosome mapping can be used individually to mark a single chromosome or a single site on that chromosome, or panels of reagents can be used for marking multiple sites and/or multiple chromosomes. Reagents corresponding to noncodmg regions of the genes actually are preferred for mapping purposes. Coding sequences are more likely to be conserved with gene families, thus increasing the chance of cross hybridizations during chromosomal mapping.

Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. (Such data are found, for example, m V.

McKusick, Mendelian Inheritance m Man, available on-line through Johns Hopkins University Welch Medical Library) . The relationship oetween genes and disease, mapped to the same chromosomal region, can then be identified through linkage analysis (co-inheritance of physically adjacent genes), described m, e.g., Egeland et al . (1987) Nature 325:783-787.

Moreover, differences m the DNA sequences between individuals affected and unaffected with a disease associated with a gene of the invention can be determined If a mutation is observed m some or all of the affected individuals but not m any unaffected individuals, then the mutation is likely to be the causative agent of the particular disease. Comparison of affected and unaffected individuals generally involves first looking for structural alterations the chromosomes such as deletions or translocations that are visible from chromosome spreads or detectable using PCR based on that DNA sequence. Ultimately, complete sequencing of genes from several individuals can be performed to confirm the presence of a mutation and to distinguish mutations from polymorphisms. 2. Tissue Typing

The nucleic acid sequences of the present invention can also be used to identify individuals from minute biological samples The United States military, for example, is considering the use of restriction fragment length polymorphism (RFLP) for identification of its personnel. In this technique, an individual's genomic DNA is digested with one or more restriction enzymes, and probed on a Southern blot to yield unique bands for identification. This method does not suffer from the current limitations of "Dog Tags" which can be lost, switched, or stolen, making positive identification difficult. The sequences of the present invention are useful as additional DNA markers for RFLP (described m U.S. Patent 5,272,057).

Furthermore, the sequences of the present invention can be used to provide an alternative technique which determines the actual base-by-base DNA sequence of selected portions of an individual's genome. Thus, the nucleic acid sequences described herein can be used to prepare two PCR primers from the 5' and 3' ends of the sequences. These primers can then be used to amplify an individual's DNA and subsequently sequence it.

Panels of corresponding DNA sequences from individuals, prepared m this manner, can provide unique individual identifications, as each individual will have a unique set of such DNA sequences due to allelic differences. The sequences of the present invention can be used to obtain such identification sequences from individuals and from tissue. The nucleic acid sequences of the invention uniquely represent portions of the human genome. Allelic variation occurs to some degree m the coding regions of these sequences, and to a greater degree m the noncodmg regions. It is estimated that allelic variation between individual humans occurs with a frequency of about once per each 500 bases. Each of the sequences described herein can, to some degree, be used as a standard against which DNA from an individual can be compared for identification purposes. Because greater numbers of polymorphisms occur m the noncodmg regions, fewer sequences are necessary to differentiate individuals. The noncodmg sequences of SEQ ID NO : 1 can comfortably provide positive individual identification with a panel of perhaps 10 to 1,000 primers which each yield a noncodmg amplified sequence of 100 bases. If predicted coding sequences, such as those m SEQ ID NO : 3 are used, a more appropriate number of primers for positive individual identification would be 500-2,000. If a panel of reagents from the nucleic acid sequences described herein is used to generate a unique identification database for an individual, those same reagents can later be used to identify tissue from that individual. Using the unique identification database, positive identification of the individual, living or dead, can be made from extremely small tissue samples.

3. Use of Partial Gene Sequences m Forensic Biology DNA-based identification techniques can also be used m forensic biology. Forensic biology is a scientific field employing genetic typing of biological evidence found at a crime scene as a means for positively identifying, for example, a perpetrator of a crime. To make such an identification, PCR ^rechnology can be used to amplify DNA sequences taken from very small biological samples such as tissues, e.g., hair or skm, or body fluids, e.g., blood, saliva, or semen found at a crime scene. The amplified sequence can then be compared to a standard, thereby allowing identification of the origin of the biological sample. The sequences of the present invention can be used to provide polynucleotide reagents, e.g., PCR primers, targeted to specific loci m the human genome, which can enhance the reliability of DNA-based forensic identifications by, for example, providing another

"identification marker" (i.e. another DNA sequence that is unique to a particular individual) . As mentioned above, actual base sequence information can be used for identification as an accurate alternative to patterns formed by restriction enzyme generated fragments.

Sequences targeted to noncodmg regions are particularly appropriate for this use as greater numbers of polymorphisms occur m the noncodmg regions, making it easier to differentiate individuals using this technique. Examples of polynucleotide reagents include the nucleic acid sequences of the invention or portions thereof, e.g., fragments derived from noncodmg regions having a length of at least 20 or 30 bases.

The nucleic acid sequences described herein can further be used to provide polynucleotide reagents, e.g., labeled or labelable probes which can be used m, for example, an in si tu hybridization technique, to identify a specific tissue, e.g., brain tissue. This can be very useful m cases where a forensic pathologist is presented with a tissue of unknown origin. Panels of such probes can be used to identify tissue by species and/or by organ type.

C . Predictive Medicine

The present invention also pertains to the field of predictive medicine m which diagnostic assays, prognostic assays, pharmacogenomics , and monitoring clinical trails are used for prognostic (predictive) purposes to thereby treat an individual prophylactically Accordingly, one aspect of the present invention relates to diagnostic assays for determining expression of a polypeptide or nucleic acid of the invention and/or activity of a polypeptide of the invention, m the context of a biological sample (e.g., blood, serum, cells, tissue) to thereby determine whether an individual is afflicted with a disease or disorder, or is at risk of developing a disorder, associated with aberrant expression or activity of a polypeptide of the invention. The invention also provides for prognostic (or predictive) assays for determining whether an individual is at risk of developing a disorder associated with aberrant expression or activity of a polypeptide of the invention. For example, mutations m a gene of the invention can be assayed m a biological sample. Such assays can be used for prognostic or predictive purpose to thereby prophylactically treat an individual prior to the onset of a disorder characterized by or associated with aberrant expression or activity of a polypeptide of the invention. Another aspect of the invention provides methods for expression of a nucleic acid or polypeptide of the invention or activity of a polypeptide of the invention m an individual to thereby select appropriate therapeutic or prophylactic agents for that individual (referred to herein as "pharmacogenomics") .

Pharmacogenomics allows for the selection of agents (e.g., drugs) for therapeutic or prophylactic treatment of an individual based on the genotype of the individual (e.g., the genotype of the individual examined to determine the ability of the individual to respond to a particular agent)

Yet another aspect of the invention pertains to monitoring the influence of agents (e.g., drugs or other compounds) on the expression or activity of a polypeptide of the invention m clinical trials. These and other agents are described m further detail the following sections.

1. Diagnostic Assays

An exemplary method for detecting the presence or absence of a polypeptide or nucleic acid of the invention m a biological sample involves obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting a polypeptide or nucleic acid (e.g., mRNA, genomic DNA) of the invention such that the presence of a polypeptide or nucleic acid of the invention is detected m the biological sample A preferred agent for detecting mRNA or genomic DNA encoding a polypeptide of the invention is a labeled nucleic acid probe capable of hybridizing to mRNA or genomic DNA encoding a polypeptide of the invention. The nucleic acid probe can be, for example, a full-length cDNA, such as the nucleic acid of SEQ ID NO : 1 or 4, or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides m length and sufficient to specifically hybridize under stringent conditions to a mRNA or genomic DNA encoding a polypeptide of the invention. Other suitable probes for use m the diagnostic assays of the invention are described herein A preferred agent for detecting A polypeptide of the invention is an antibody capable of binding to A polypeptide of the invention, preferably an antibody with a detectable label Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab')_?) can be used. The term "labeled", with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotm such that it can be detected with fluorescently labeled streptavidm. The term "biological sample" is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present withm a subject That is, the detection method of the invention can be used to detect mRNA, protein, or genomic DNA m a biological sample m vi tro as well as in vivo . For example, m vi tro techniques for detection of mRNA include Northern hybridizations and in si tu hybridizations In vi tro techniques for detection of A polypeptide of the invention include enzyme linked immunosorbent assays (ELISAs) , Western blots, immunoprecipitations and immunofluorescence . In vi tro techniques for detection of genomic DNA include Southern hybridizations Furthermore, m vivo techniques for detection of a polypeptide of the invention include introducing into a subject a labeled antibody directed against the polypeptide. For example, the antibody can be labeled with a radioactive marker whose presence and location m a subject can be detected by standard imaging techniques .

In one embodiment, the biological sample contains protein molecules from the test subject Alternatively, the biological sample can contain mRNA molecules from the test subject oi genomic DNA molecules from the test subject A preferred biological sample is a peripheral blood leukocyte sample isolated by conventional means from a subject

In another embodiment, the methods further involve obtaining a control biological sample from a control subject, contacting the control sample with a compound or agent capable of detecting a polypeptide of the invention or mRNA or genomic DNA encoding a polypeptide of the invention, such that the presence of the polypeptide or mRNA or genomic DNA encoding the polypeptide is detected m the biological sample, and comparing the presence of the polypeptide or mRNA or genomic DNA encoding the polypeptide m the control sample with the presence of the polypeptide or mRNA or genomic DNA encoding the polypeptide m the test sample.

The invention also encompasses kits for detecting the presence of a polypeptide or nucleic acid of the invention m a biological sample (a test sample) . Such kits can be used to determine if a subject is suffering from or is at increased risk of developing a disorder associated with aberrant expression of a polypeptide of the invention ι,e.g., an immunological disorder) . For example, the kit can comprise a labeled compound or agent capable of detecting the polypeptide or mRNA encoding the polypeptide m a biological sample and means for determining the amount of the polypeptide or mRNA m the sample (e.g., an antibody which binds the polypeptide or an oligonucleotide probe which binds to DNA or mRNA encoding the polypeptide) . Kits may also include instruction fo observing that the tested subject is suffering from or is at risk of developing a disorder associated with aberrant expression of the polypeptide if the amount of the polypeptide or mRNA encoding the polypeptide is above or below a normal level. For antibody based kits, the kit may comprise, for example: (1) a first antibody (e.g., attached to a solid support) which binds to a polypeptide of the invention; and, optionally, (2) a second, different antibody which binds to either the polypeptide or the first antibody and is conjugated to a detectable agent. For oligonucleotide-based kits, the kit may comprise, for example: (1) an oligonucleotide, e.g., a detectably labeled oligonucleotide, which hybridizes to a nucleic acid sequence encoding a polypeptide of the invention or (2) a pair of primers useful for amplifying a nucleic acid molecule encoding a polypeptide of the invention.

The kit may also comprise, e.g., a buffering agent, a preservative, or a protein stabilizing agent. The kit may also comprise components necessary for detecting the detectable agent (e.g , an enzyme or a substrate) . The kit may also contain a control sample or a series of control samples which can be assayed and compared to the test sample contained Each component of the kit is usually enclosed withm an individual container and all of the various containers are withm a single package along with instructions for observing whether the tested subject is suffering from or is at risk of developing a disorder associated with aberrant expression of the polypeptide .

2. Prognostic Assays

The methods described herein can furthermore be utilized as diagnostic or prognostic assays to identify subjects having or at risk of developing a disease or disorder associated with aberrant expression or activity of a polypeptide of the invention. For example, the assays described herein, such as the preceding diagnostic assays or the following assays, can be utilized to identify a subπect having or at risk of developing a disorder associated with aberrant expression or activity of a polypeptide of the invention. Alternatively, the prognostic assays can be utilized to identify a subject having or at risk for developing such a disease or disorder Thus, the present invention provides a method m which a test sample is obtained from a subject and a polypeptide or nucleic acid (e.g., mRNA, genomic DNA) of the invention is detected, wherein the presence of the polypeptide or nucleic acid is diagnostic for a subject having or at risk of developing a disease or disorder associated with aberrant expression or activity of the polypeptide. As used herein, a "test sample" refers to a biological sample obtained from a subject of interest. For example, a test sample can be a biological fluid (e.g., serum), cell sample, or tissue. Furthermore, the prognostic assays described herein can be used to determine whether a subject can be administered an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate) to treat a disease or disorder associated with aberrant expression or activity of a polypeptide of the invention. For example, such methods can be used to determine whether a subject can be effectively treated with a specific agent or class of agents (e.g., agents of a type which decrease activity of the polypeptide) . Thus, the present invention provides methods for determining whether a subject can be effectively treated with an agent for a disorder associated with aberrant expression or activity of a polypeptide of the invention m which a test sample is obtained and the polypeptide or nucleic acid encoding the polypeptide is detected (e.g., wherein the presence of the polypeptide or nucleic acid is diagnostic for a subject that can be administered the agent to treat a disorder associated with aberrant expression or activity of the polypeptide) .

The methods of the invention can also be used to detect genetic lesions or mutations m a gene of the invention, thereby determining if a subject with the lesioned gene is at risk for a disorder characterized aberrant expression or activity of a polypeptide of the invention. In preferred embodiments, the methods include detecting, m a sample of cells from the subject, the presence or absence of a genetic lesion or mutation characterized by at least one of an alteration affecting the integrity of a gene encoding the polypeptide of the invention, or the mis-expression of the gene encoding the polypeptide of the invention. For example, such genetic lesions or mutations can be detected by ascertaining the existence of at least one of: 1) a deletion of one or more nucleotides from the gene; 2) an addition of one or more nucleotides to the gene; 3) a substitution of one or more nucleotides of the gene; 4) a chromosomal rearrangement of the gene; 5) an alteration m the level of a messenger RNA transcript of the gene; 6) an aberrant modification of the gene, such as of the methylation pattern of the genomic DNA; 7) the presence of a non-wild type splicing pattern of a messenger RNA transcript of the gene; 8) a non-wild type level of a the protein encoded by the gene; 9) an allelic loss of the gene; and 10) an inappropriate post-translational modification of the protein encoded by the gene. As described herein, there are a large number of assay techniques known m the art which can be used for detecting lesions m a gene.

In certain embodiments, detection of the lesion involves the use of a probe/primer m a polymerase chain reaction (PCR) (see, e . g. , U.S. Patent Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, m a ligation chain reaction (LCR) (see, e . g. , Landegran et al . (1988) Science 241:1077-1080; and Nakazawa et al . (1994) Proc . Natl . Acad . Sci . USA 91:360-

364) , the latter of which can be particularly useful for detecting point mutations m a gene ( see, e . g. , Abravaya et al . (1995) Nuclei c Acids Res . 23:675-682). This method can include the steps of collecting a sample of cells from a patient, isolating nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to the selected gene under conditions such that hybridization and amplification of the gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. It is anticipated that PCR and/or LCR may be desirable to use as a preliminary amplification step m conjunction with any of the techniques used for detecting mutations described herein.

Alternative amplification methods include: self sustained sequence replication (Guatelli et al . (1990) Proc . Natl . Acad . Sci . USA 87 :1874-1878) , transcriptional amplification system (Kwoh, et al . (1989) Proc . Natl . Acad . Sci . USA 86:1173-1177), Q-Beta Replicase (Lizardi et al . (1988) Bio/Technology 6:1197), or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill m the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present m very low numbers . In an alternative embodiment, mutations m a selected gene from a sample cell can be identified by alterations restriction enzyme cleavage patterns. For example, sample and control DNA is isolated, amplified (optionally) , digested with one or more restriction endonucleaseε, and fragment length sizes are determined by gel electrophoresis and compared. Differences m fragment length sizes between sample and control DNA indicates mutations the sample DNA Moreover, the use of sequence specific ribozymes (see, e . g. , U.S. Patent No. 5,498,531) can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site.

In other embodiments, genetic mutations can be identified by hybridizing a sample and control nucleic acids, e.g., DNA or RNA, to high density arrays containing hundreds or thousands of oligonucleotides probes (Cronm et al . (1996) Human Mutation 7:244-255; Kozal et al . (1996) Nature Medicine 2:753-759) . For example, genetic mutations can be identified m two- dimensional arrays containing light -generated DNA probes as described m Cron et al . , supra . Briefly, a first hybridization array of probes can be used to scan through long stretches of DNA m a sample and control to identify base changes between the sequences by making linear arrays of sequential overlapping probes. This step allows the identification of point mutations. This step is followed by a second hybridization array that allows the characterization of specific mutations by using smaller, specialized probe arrays complementary to all variants or mutations detected. Each mutation array is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene .

In yet another embodiment, any of a variety of sequencing reactions known m the art can be used to directly sequence the selected gene and detect mutations by comparing the sequence of the sample nucleic acids with the corresponding wild-type (control) sequence. Examples of sequencing reactions include those based on techniques developed by Maxim and Gilbert ((1977) Proc . Natl . Acad . Sci . USA 74:560) or Sanger ((1977) Proc . Natl . Acad . Sci . USA 74:5463). It is also contemplated that any of a variety of automated sequencing procedures can be utilized when performing the diagnostic assays ((1995) Bio /Techniques 19:448), including sequencing by mass spectrometry (see, e . g . , PCT Publication No. WO 94/16101; Cohen et al . (1996) Adv. Chromatogr . 36:127- 162; and Griffm et al . (1993) Appl . Biochem . Biotechnol . 38 :147-159) . Other methods for detecting mutations in a selected gene include methods m which protection from cleavage agents is used to detect mismatched bases m RNA/RNA or RNA/DNA heteroduplexes (Myers et al . (1985) Sci ence 230:1242) . In general, the technique of "mismatch cleavage" entails providing heteroduplexes formed by hybridizing (labeled) RNA or DNA containing the wild-type sequence with potentially mutant RNA or DNA obtained from a tissue sample. The double-stranded duplexes are treated with an agent which cleaves single-stranded regions of the duplex such as which will exist due to basepair mismatches between the control and sample strands. RNA/DNA duplexes can be treated with RNase to digest mismatched regions, and DNA/DNA hybrids can be treated with SI nuclease to digest mismatched regions. In other embodiments, either DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamme or osmium tetroxide and with pipeπdme m order to digest mismatched regions. After digestion of the mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine the site of mutation. See, e . g . , Cotton et al . (1988) Proc . Na tl . Acad . Sci . USA 85:4397; Saleeba et al . (1992) Methods Enzymol . 217:286-295. In a preferred embodiment, the control DNA or RNA can be labeled for detection. In still another embodiment, the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs m double-stranded DNA (so called "DNA mismatch repair" enzymes) m defined systems for detecting and mapping point mutations m cDNAs obtained from samples of cells For example, the mutY enzyme of E. coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T at G/T mismatches (Hsu et al . (1994) Carcinogenesis 15:1657-1662). According to an exemplary embodiment, a probe based on a selected sequence, e.g., a wild-type sequence, is hybridized to a cDNA or other DNA product from a test cell (s) . The duplex is treated with a DNA mismatch repair enzyme, and the cleavage products, if any, can be detected from electrophoresis protocols or the like. See, e . g . , U.S. Patent No. 5,459,039.

In other embodiments, alterations m electrophoretic mobility will be used to identify mutations m genes. For example, single strand conformation polymorphism (SSCP) may be used to detect differences m electrophoretic mobility between mutant and wild type nucleic acids (Orita et al . (1989) Proc . Natl . Acad . Sci . USA 86:2766; see also Cotton (1993) Mu tat . Res . 285:125- 144; Hayashi (1992) Genet. Anal . Tech . Appl . 9:73-79). Smgle-stranded DNA fragments of sample and control nucleic acids will be denatured and allowed to renature.

The secondary structure of smgle-stranded nucleic acids varies according to sequence, and the resulting alteration m electrophoretic mobility enables the detection of even a single base change. The DNA fragments may be labeled or detected with labeled probes The sensitivity of the assay may be enhanced by using RNA (rather than DNA) , m which the secondary structure is more sensitive to a change m sequence. In a preferred embodiment, the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes m electrophoretic mobility (Keen et al (1991) Trends Genet . 7:5).

In yet another embodiment, the movement of mutant or wild- type fragments m polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE) (Myers et al . (1985) Nature 313:495) . When DGGE is used as the method of analysis, DNA will be modified to insure that it does not completely denature, for example by adding a ^λGC clamp of approximately 40 bp of high-meltmg GC-πch DNA by PCR. In a further embodiment, a temperature gradient is used m place of a denaturing gradient to identify differences the mobility of control and sample DNA (Rosenbaum and Reissner (1987) Biophys . Chem . 265:12753). Examples of other techniques for detecting point mutations include, but are not limited to, selective oligonucleotide hybridization, selective amplification, or selective primer extension. For example, oligonucleotide primers may be prepared m which the known mutation is placed centrally and then hybridized to target DNA under conditions which permit hybridization only if a perfect match is found (Saiki et al . (1986) Nature 324:163); Saiki et al . (1989) Proc . Natl . Acad . Sci . USA 86:6230). Such allele specific oligonucleotides are hybridized to PCR amplified target DNA or a number of different mutations when the oligonucleotides are attached to the hybridizing membrane and hybridized with labeled target DNA.

Alternatively, allele specific amplification technology which depends on selective PCR amplification may be used conjunction with the instant invention. Oligonucleotides used as primers for specific amplification may carry the mutation of interest m the center of the molecule (so that amplification depends on differential hyoπdization) (Gibbs et al . (1989) Nucleic Acids Res . 17:2437-2448) or at the extreme 3' end of one primer where, under appropriate conditions, mismatch can prevent or reduce polymerase extension (Prossner (1993) Tibtech 11:238) In addition, it may be desirable to introduce a novel restriction site m the region of the mutation to create cleavage-based detection (Gasparmi et al . (1992) Mol . Cell Probes 6:1). It is anticipated that m certain embodiments amplification may also be performed using Taq ligase for amplification (Barany (1991) Proc . Na tl . Acad . Sci . USA 88:189) . In such cases, ligation will occur only if there is a perfect match at the 3' end of the 5' sequence making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification.

The methods described herein may be performed, for example, by utilizing pre-packaged diagnostic kits comprising at least one probe nucleic acid or antibody reagent described herein, which may be conveniently used, e.g., m clinical settings to diagnose patients exhibiting symptoms or family history of a disease or illness involving a gene encoding a polypeptide of the invention.

Furthermore, any cell type or tissue, preferably peripheral blood leukocytes, m which the polypeptide of the invention is expressed may be utilized the prognostic assays described herein.

3. Pharmacogenomics Agents, or modulators which have a stimulatory or inhibitory effect on activity or expression of a polypeptide of the invention as identified by a screening assay described herein can be administered to individuals to treat (prophylactically or therapeutically) disorders associated with aberrant activity of the polypeptide. In conjunction with such treatment, the pharmacogenomics (i.e., the study of the relationship between an individual's genotype and that individual's response to a foreign compound or drug) of the individual may be considered. Differences m metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation oetween dose and blood concentration of the pharmacologically active drug. Thus, the pharmacogenomics of the individual permits the selection of effective agents (e.g., drugs) for prophylactic or therapeutic treatments based on a consideration of the individual's genotype. Such pharmacogenomics can further be used to determine appropriate dosages and therapeutic regimens. Accordingly, the activity of a polypeptide of the invention, expression of a nucleic acid of the invention, or mutation content of a gene of the invention m an individual can be determined to thereby select appropriate agent (s) for therapeutic or prophylactic treatment of the individual .

Pharmacogenomics deals with clinically significant hereditary variations m the response to drugs due to altered drug disposition and abnormal action m affected persons. See, e . g. , Lmder (1997) Clm . Chem . 43(2):254- 266. In general, two types of pharmacogenetic conditions can be differentiated. Genetic conditions transmitted as a single factor altering the way drugs act on the body are referred to as "altered drug action." Genetic conditions transmitted as single factors altering the wav the body acts on drugs are referred to as "altered drug metabolism" . These pharmacogenetic conditions can occur either as rare defects or as polymorphisms. For example, glucose-6-phosphate dehydrogenase deficiency (G6PD) is a common inherited enzymopathy m which the mam clinical complication is haemolysis after mgestion of oxidant drugs (anti-malaπals , sulfonamides, analgesics, nitrofurans) and consumption of fava beans.

As an illustrative embodiment, the activity of drug metabolizing enzymes is a major determinant of both the intensity and duration of drug action. The discovery of genetic polymorphisms of drug metabolizing enzymes (e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymes CYP2D6 and CYP2C19) has provided an explanation as to why some patients do not obtain the expected drug effects or show exaggerated drug response and serious toxicity after taking the standard and safe dose of a drug. These polymorphisms are expressed m two phenotypes m the population, the extensive metabolizer (EM) and poor metabolizer (PM; . The prevalence of PM is different among different populations. For example, the gene coding for CYP2D6 is highly polymorphic and several mutations have been identified m PM, which all lead to the absence of functional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quite frequently experience exaggerated drug response and side effects when they receive standard doses If a metabolite is the active therapeutic moiety, a PM will show no therapeutic response, as demonstrated for the analgesic effect of codeine mediated by its CYP2D6-formed metabolite morphine The other extreme are the so called ultra- rapid metabolizers who do not respond to standard doses. Recently, the molecular basis of ultra-rapid metabolism has been identified to be due to CYP2D6 gene amplification. Thus, the activity of a polypeptide of the invention, expression of a nucleic acid encoding the polypeptide, or mutation content of a gene encoding the polypeptide m an individual can be determined to thereby select appropriate agent (s) for therapeutic or prophylactic treatment of the individual. In addition, pharmacogenetic studies can be used to apply genotypmg of polymorphic alleles encoding drug-metabolizmg enzymes to the identification of an individual's drug responsiveness phenoty e This knowledge, when applied to dosmg or drug selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic or prophylactic efficienc/ when treating a subject with a modulator of activity or expression of the polypeptide, such as a modulator identified by one of the exemplary screening assays described herein.

4. Monitoring of Effects During Clinical Trials Monitoring the influence of agents (e.g., drugs, compounds) on the expression or activity of a polypeptide of the invention (e g the ability to modulate aberrant cell proliferation and/or differentiation) can be applied not only m basic drug screening, but also m clinical trials. For example, the effectiveness of an agent, as determined by a screening assay as described herein, to increase gene expression, protein levels or protein activity, can be monitored m clinical trials of subjects exhibiting decreased gene expression, protein levels, or protein activity. Alternatively, the effectiveness of an agent, as determined by a screening assay, to decrease gene expression, protein levels or protein activity, can be monitored clinical trials of subjects exhibiting increased gene expression, protein levels, or protein activity In such clinical trials, expression or activity of a polypeptide of the invention and preferably, that of otner polypeptide that have been implicated m for example, a cellular proliferation disorder, can be used as a marker of the immune responsiveness of a particular cell

For example, and not by way of limitation, genes, including those of the invention, that are modulated m cells by treatment with an agent (e.g., compound, drug or small molecule) which modulates activity or expression of a polypeptide of the invention (e.g , as identified m a screening assay descπoed herein) can be identified. Thus, to study the effect of agents on cellular proliferation disorders, for example, m a clinical trial, cells can be isolated and RNA prepared and analyzed for the levels of expression of a gene of the invention and other genes implicated m the disorder. The levels of gene expression (i.e., a gene expression pattern) can be quantified by Northern blot analysis or RT-PCR, as described herein, or alternatively by measuring the amount of protein produced, by one of the methods as described herein, or by measuring the levels of activity of a gene of the invention or other genes. In this way, the gene expression pattern can serve as a marker, indicative of the physiological response of the cells to the agent. Accordingly, this response state may be determined before, and at various points during, treatment of the individual with the agent.

In a preferred embodiment, the present invention provides a method for monitoring the effectiveness of treatment of a subject with an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate identified by the screening assays described herein) comprising the steps of (I) obtaining a pre-admmistration sample from a subject prior to administration of the agent; (ii) detecting the level of the polypeptide or nucleic acid of the invention m the preadmmistration sample; (m) obtaining one or more post -administration samples from the subject; (iv) detecting the level the of the polypeptide or nucleic acid of the invention m the post- admmistration samples, (v) comparing the level of the polypeptide or nucleic acid of the invention m the preadmmistration sample with the level of the polypeptide or nucleic acid of the invention m the post- administration sample or samples; and (vi) altering the administration of the agent to the subject accordingly. For example, increased administration of the agent may be desirable to increase the expression or activity of the polypeptide to highei levels than detected, i.e., to increase the effectiveness of the agent. Alternatively, decreased administration of the agent may be desirable to decrease expression or activity of the polypeptide to lower levels than detected, i.e., to decrease the effectiveness of the agent.

C. Methods of Treatment

The present invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with aberrant expression or activity of a polypeptide of the mvention.

1. Prophylactic Methods In one aspect, the invention provides a method for preventing m a subject, a disease or condition associated with an aberrant expression or activity of a polypeptide of the invention, by administering to the subject an agent which modulates expression or at least one activity of the polypeptide. Subjects at risk for a disease which is caused or contributed to by aberrant expression or activity of a polypeptide of the invention can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein. Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the aberrancy, such that a disease or disorder is prevented or, alternatively, delayed m its progression. Depending on the type of aberrancy, for example, an agonist or antagonist agent can be used for treating the sub_ject. The appropriate agent can be determined based on screening assays described herein. 2. Therapeutic Methods

Another aspect of the invention pertains to methods of modulating expression or activity of a polypeptide of the invention for therapeutic purposes. The modulatory method of the invention involves contacting a cell with an agent that modulates one or more of the activities of the polypeptide An agent that modulates activity can be an agent as described herein, such as a nucleic acid or a protein, a naturally-occurring cognate ligand of the polypeptide, a peptide, a peptidomimetic, or other small molecule In one embodiment, the agent stimulates one or more of the biological activities of the polypeptide. Examples of such stimulatory agents include the active polypeptide of the invention and a nucleic acid molecule encoding the polypeptide of the invention that has been introduced into the cell. In another embodiment, the agent inhibits one or more of the biological activities of the polypeptide of the invention. Examples of such inhibitory agents include antisense nucleic acid molecules and antibodies. These modulatory methods can be performed m vi tro (e.g., by culturmg the cell with the agent) or, alternatively, in vivo (e.g, by administering the agent to a subject) As such, the present invention provides methods of treating an individual afflicted with a disease or disorder characterized by aberrant expression or activity a polypeptide of the invention. In one embodiment, the method involves administering an agent (e.g., an agent identified by a screening assay described herein; , or combination of agents that modulates (e.g., upregulates or downregulates) expression or activity. In another embodiment, the method involves administering a polypeptide of the invention or a nucleic acid molecule of the invention as therapy to compensate for reduced or aberrant expression or activity of the polypeptide. Stimulation of activity is desirable m situations m which activity or expression is abnormally low downregulated and/or m which increased activity is likely to have a beneficial effect. Conversely, inhibition of activity is desirable m situations m which activity or expression is abnormally high or upregulated and/or m which decreased activity is likely to have a beneficial effect.

This invention is further illustrated by the following examples which should not be construed as limiting. The contents of all references, patents and published patent applications cited throughout this application are hereby incorporated by reference .

Equivalents Those skilled m the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

What is claimed is :

Claims

1. An isolated nucleic acid molecule selected from the group consisting of: a) a nucleic acid molecule comprising a nucleotide sequence which is at least 55% identical to the nucleotide sequence of SEQ ID NO : 1 , 3, 4, or 6 , the cDNA insert of the plasmid deposited with ATCC as Accession

Number , the cDNA insert of the plasmid deposited with ATCC as Accession Number , or a complement thereof; b) a nucleic acid molecule comprising a fragment of at least 300 nucleotides of the nucleotide sequence of SEQ ID NO:l, 3, 4, or 6 , the cDNA insert of the plasmid deposited with ATCC as Accession Number , the cDNA insert of the plasmid deposited with ATCC as Accession Number , or a complement thereof; c) a nucleic acid molecule which encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or 5, amino acid sequence encoded by the cDNA insert of the plasmid deposited with ATCC as Accession Number , or the amino acid sequence of the cDNA insert of the plasmid deposited with ATCC as Accession Number

d) a nucleic acid molecule which encodes a fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or 5, the polypeptide encoded by the cDNA insert of the plasmid deposited with ATCC as Accession

Number , or the polypeptide encoded by the cDNA insert of the plasmid deposited with ATCC as Accession Number , wherein the fragment comprises at least 15 contiguous amino acids of SEQ ID NO : 2 or 5, the polypeptide encoded by the cDNA insert of the plasmid deposited with ATCC as Accession Number , or the polypeptide encoded by the cDNA insert of the plasmid deposited with ATCC as Accession Number ; and e) a nucleic acid molecule which encodes a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO : 2 or 5, the amino acid sequence encoded by the cDNA insert of the plasmid deposited with ATCC as Accession Number , or the amino acid sequence encoded by the cDNA insert of the plasmid deposited with ATCC as Accession Number , wherein the nucleic acid molecule hybridizes to a nucleic acid molecule comprising SEQ ID NO: 3 or 6 or a complement thereof under stringent conditions.

2. The isolated nucleic acid molecule of claim 1, which is selected from the group consisting of: a) a nucleic acid comprising the nucleotide sequence of SEQ ID NO:l, 3, 4, or 5 , the cDNA insert of the plasmid deposited with ATCC as Accession Number , the cDNA insert of the plasmid deposited with ATCC as Accession Number , or a complement thereof; and b) a nucleic acid molecule which encodes a polypeptide comprising the amino acid sequence of SEQ ID NO : 2 or 5, the amino acid sequence encoded by the cDNA insert of the plasmid deposited with ATCC as Accession

Number , or the amino acid sequence encoded by the cDNA insert of the plasmid deposited with ATCC as Accession Number .

3. The nucleic acid molecule of claim 1 further comprising vector nucleic acid sequences.

4. The nucleic acid molecule of claim 1 further comprising nucleic acid sequences encoding a heterologous polypeptide .

5. A host cell which contains the nucleic acid molecule of claim 1.

6. The host cell of claim 5 which is a mammalian host cell .

7. A non-human mammalian host cell containing the nucleic acid molecule of claim 1.

8. An isolated polypeptide selected from the group consisting of: a) a fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO : 2 or 5, wherein the fragment comprises at least 15 contiguous amino acids of SEQ ID NO: 2 or 5; b) a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO : 2 or 5, the amino acid sequence encoded by the cDNA insert of the plasmid deposited with ATCC as Accession Number , or the amino acid sequence encoded by the cDNA insert of the plasmid deposited with ATCC as

Accession Number , wherein the polypeptide is encoded by a nucleic acid molecule which hybridizes to a nucleic acid molecule comprising SEQ ID NO: 1 or 4 or a complement thereof under stringent conditions; and c) a polypeptide which is encoded by a nucleic acid molecule comprising a nucleotide sequence which is at least 55% identical to a nucleic acid comprising the nucleotide sequence of SEQ ID NO : 1 or 4 or a complement thereof.

9. The isolated polypeptide of claim 8 comprising the amino acid sequence of SEQ ID NO : 2 or 5, the amino acid sequence encoded by the cDNA insert of the plasmid deposited with ATCC as Accession Number , or the amino acid sequence encoded by the cDNA insert of the plasmid deposited with ATCC as Accession Number .

10. The polypeptide of claim 8 further comprising heterologous ammo acid sequences.

11. An antibody which selectively binds to a polypeptide of claim 8.

12. A method for producing a polypeptide selected from the group consisting of: a) a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or 5, the amino acid sequence encoded by the cDNA insert of the plasmid deposited with ATCC as Accession Number , or the amino acid sequence encoded by the cDNA insert of the plasmid deposited with ATCC as Accession Number ; b) a polypeptide comprising a fragment of the amino acid sequence of SEQ ID NO : 2 or 5, the amino acid sequence encoded by the cDNA insert of the plasmid deposited with ATCC as Accession Number , or the amino acid sequence encoded by the cDNA insert of the o plasmid deposited with ATCC as Accession Number , wherein the fragment comprises at least 15 contiguous amino acids of SEQ ID NO : 2 or 5, the amino acid sequence encoded by the cDNA insert of the plasmid deposited with

ATCC as Accession Number or the amino acid sequence encoded by the cDNA insert of the plasmid deposited with ATCC as Accession Number ; and c) a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or 5, the ammo acid sequence encoded by the cDNA insert of the plasmid deposited with ATCC as Accession Number or the amino acid sequence encoded by the cDNA insert of the plasmid deposited with ATCC as

Accession Number , wherein the polypeptide is encoded by a nucleic acid molecule which hybridizes to a nucleic acid molecule comprising SEQ ID NO : 1 or 4 or a complement thereof under stringent conditions; comprising culturing the host cell of claim 5 under conditions in which the nucleic acid molecule is expressed.

13. A method for detecting the presence of a polypeptide of claim 8 in a sample, comprising: a) contacting the sample with a compound which selectively binds to a polypeptide of claim 8; and b) determining whether the compound binds to the polypeptide in the sample.

14. The method of claim 13, wherein the compound which binds to the polypeptide is an antibody.

15. A kit comprising a compound which selectively binds to a polypeptide of claim 8 and instructions for use .

16. A method for detecting the presence of a nucleic acid molecule of claim 1 in a sample, comprising the steps of : a) contacting the sample with a nucleic acid probe or primer which selectively hybridizes to the nucleic acid molecule; and b) determining whether the nucleic acid probe or primer binds to a nucleic acid molecule in the sample.

17. The method of claim 16, wherein the sample comprises mRNA molecules and is contacted with a nucleic acid probe .

18. A kit comprising a compound which selectively hybridizes to a nucleic acid molecule of claim 1 and instructions for use.

19. A method for identifying a compound which binds to a polypeptide of claim 8 comprising the steps of: a) contacting a polypeptide, or a cell expressing a polypeptide of claim 8 with a test compound; and b) determining whether the polypeptide binds to the test compound.

20. The method of claim 19, wherein the binding of the test compound to the polypeptide is detected by a method selected from the group consisting of: a) detection of binding by direct detecting of test compound/polypeptide binding; b) detection of binding using a competition binding assay; c) detection of binding using an assay for TANGO 191 or TANGO 195 -mediated signal transduction.

21. A method for modulating the activity of a polypeptide of claim 8 comprising contacting a polypeptide or a cell expressing a polypeptide of claim 8 with a compound which binds to the polypeptide in a sufficient concentration to modulate the activity of the polypeptide .

22. A method for identifying a ^compound which modulates the activity of a polypeptide of claim 8, comprising : a) contacting a polypeptide of claim 8 with a test compound; and b) determining the effect of the test compound on the activity of the polypeptide to thereby identify a compound which modulates the activity of the polypeptide.