WO2001051514A1

WO2001051514A1 - Novel genes encoding proteins having prognostic, diagnostic, preventive, therapeutic, and other uses

Info

Publication number: WO2001051514A1
Application number: PCT/US2001/000485
Authority: WO
Inventors: Christopher C. Fraser; Susan J. Kirst
Original assignee: Millennium Pharmaceuticals, Inc.
Priority date: 2000-01-07
Filing date: 2001-01-05
Publication date: 2001-07-19
Also published as: AU2769101A

Abstract

The invention provides isolated nucleic acids encoding a variety of proteins having diagnostic, preventive, therapeutic, and other uses. These nucleic and proteins are useful for diagnosis, prevention, and therapy of a number of human and other animal disorders. The invention also provides antisense nucleic acid molecules, expression vectors containing the nucleic acid molecules of the invention, host cells into which the expression vectors have been introduced, and non-human transgenic animals in which a nucleic acid molecule of the invention has been introduced or disrupted. The invention still further provides isolated polypeptides, fusion polypeptides, antigenic peptides and antibodies. Diagnostic, screening, and therapeutic methods using compositions of the invention are also provided. The nucleic acids and polypeptides of the present invention are useful as modulating agents in regulating a variety of cellular processes.

Description

TITLE

Novel Genes Encoding Proteins Having Prognostic,

Diagnostic, Preventive, Therapeutic, And Other Uses

BACKGROUND OF THE INVENTION

The molecular bases underlying many human and animal physiological states (e.g., diseased and homeostatic states of various tissues) remain unknown. Nonetheless, it is well understood that these states result from interactions among the proteins and nucleic acids present in the cells of the relevant tissues. In the past, the complexity of biological systems overwhelmed the ability of practitioners to understand the molecular interactions giving rise to normal and abnormal physiological states. More recently, though, the techniques of molecular biology, transgenic and null mutant animal production, computational biology, and pharmacogenomics have enabled practitioners to discern the role and importance of individual genes and proteins in particular physiological states. Knowledge of the sequences and other properties of genes (particularly including the portions of genes encoding proteins) and the proteins encoded thereby enables the practitioner to design and screen agents which will affect, prospectively or retrospectively, the physiological state of an animal tissue in a favorable way. Such knowledge also enables the practitioner, by detecting the levels of gene expression and protein production, to diagnose the current physiological state of a tissue or animal and to predict such physiological states in the future. This knowledge furthermore enables the practitioner to identify and design molecules which bind with the polynucleotides and proteins, in vitro, in vivo, or both.

Cadherins are a class of cell-surface adhesion molecules that mediate calciuro dependent cell-to-cell adhesion. Many cadherins exhibit homophilic adhesion; i.e. they bind with molecules of the same cadherin on a different cell. However, cadherins that bind specifically with other molecules have also been described (e.g. Telo et al., 1998, J. Biol. Chem. 273:17565-17572; Ludviksson et al., 1999 J. Immunol. 162:4975-4982). In addition to their binding capabilities, cadherins also exhibit transmembrane signaling and regulatable adhesion activity (e.g. Yap et al., 1997, 13:119-146; Gumbiner, 2000, J. Cell Biol. 148:399- 403). Despite the fact that numerous cadherins and cadherin-like proteins have been described, many others have not yet been characterized. A family of cadherin-like proteins which the inventor believes to be novel is described herein. 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 and transmembrane 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 in part, on the discovery of human cDNA molecules which encode proteins which are herein designated TANGO 416 and TANGO 457. These proteins, fragments thereof, derivatives thereof, and variants thereof are collectively referred to herein as the polypeptides of the invention or the 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 for regulating a variety of cellular processes. Accordingly, in one aspect, the present 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.

The invention also includes fragments of any of the nucleic acids described herein wherein the fragment retains a biological or structural function by which the full- length nucleic acid is characterized (e.g., an activity, an encoded protein, or a binding capacity). The invention furthermore includes fragments of any of the nucleic acids described herein wherein the fragment has a nucleotide sequence sufficiently (e.g., 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, or 99% or greater) identical to the nucleotide sequence of the corresponding full-length nucleic acid that it retains a biological or structural function by which the full-length nucleic acid is characterized (e.g., an activity, an encoded protein, or a binding capacity).

The invention also includes fragments of any of the polypeptides described herein wherein the fragment retains a biological or structural function by which the full- length polypeptide is characterized (e.g., an activity or a binding capacity). The invention furthermore includes fragments of any of the polypeptides described herein wherein the fragment has an amino acid sequence sufficiently (e.g., 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, or 99% or greater) identical to the amino acid sequence of the corresponding full- length polypeptide that it retains a biological or structural function by which the full-length polypeptide is characterized (e.g., an activity or a binding capacity). The invention also features nucleic acid molecules which are at least 40% (or

50%, 60%, 70%, 80%, 90%, 95%, or 98%) identical to the nucleotide sequence of any of SEQ ID NOs: 1, 2, 31, 32, 51, and 52, the TANGO 416 nucleotide sequence of the cDNA insert of a clone deposited on April 26, 1999 with the ATCC as accession no. PTA-1764, the TANGO 457 nucleotide sequence of the cDNA insert of a clone deposited on October 1, 1999 with the ATCC as PTA-817, or a complement thereof.

The invention features nucleic acid molecules which include a fragment of at least 15 (25, 40, 60, 80, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, 2000, 2200, 2400, 2600, 2800, 3000, 3500, 4000, 4500, 5000, or more) consecutive nucleotide residues of any of SEQ ID NOs: 1, 2, 31, 32, 51, and 52, the TANGO 416 nucleotide sequence of the cDNA insert of a clone deposited on April 26, 1999 with the ATCC as accession no. PTA-1764, the TANGO 457 nucleotide sequence of the cDNA insert of a clone deposited on October 1, 1999 with the ATCC as PTA-817, or a complement thereof.

The invention also features nucleic acid molecules which include a nucleotide sequence encoding a protein having an amino acid sequence that is at least 50% (or 60%,

70%, 80%, 90%, 95%, or 98%) identical to the amino acid sequence of any of SEQ ID NOs: 3-8, 33, 35, 38, and 53-60, or the amino acid sequence encoded by the TANGO 416 nucleotide sequence of the cDNA insert of a clone deposited on April 26, 1999 with the ATCC as accession no. PTA-1764, the TANGO 457 nucleotide sequence of the cDNA insert of a clone deposited on October 1 , 1999 with the ATCC as PTA-817, or a complement thereof.

In certain embodiments, the nucleic acid molecules have the nucleotide sequence of any of SEQ ID NOs: 1, 2, 31, 32, 51, and 52, the TANGO 416 nucleotide sequence of the cDNA insert of a clone deposited on April 26, 1999 with the ATCC as accession no. PTA-1764, or the TANGO 457 nucleotide sequence of the cDNA insert of a clone deposited on October 1, 1999 with the ATCC as PTA-817.

Also within the invention are nucleic acid molecules which encode a fragment of a polypeptide having the amino acid sequence of any of SEQ ID NOs: 3-8, 33, 35, 38, and 53-60, the fragment including at least 10 (12, 15, 20, 25, 30, 40, 50, 75, 100, 125, 150, 200, 250, 300, 400, 500, 750, 1000 or more) consecutive amino acid residues of any of SEQ ID NOs: 3-8, 33, 35, 38, and 53-60.

The invention includes nucleic acid molecules which encode a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of any of SEQ ID NOs: 3-8, 33, 35, 38, and 53-60, wherein the nucleic acid molecule hybridizes under stringent conditions to a nucleic acid molecule having a nucleic acid sequence of any of SEQ

ID NOs: 1, 2, 31, 32, 51, and 52, the TANGO 416 nucleotide sequence of the cDNA insert of a clone deposited on April 26, 1999 with the ATCC as accession no. PTA-1764, the TANGO 457 nucleotide sequence of the cDNA insert of a clone deposited on October 1, 1999 with the ATCC as PTA-817, or a complement thereof. Also within the invention are isolated polypeptides or proteins having an amino acid sequence that is at least about 50%, preferably 60%, 75%, 90%, 95%, or 98% identical to the amino acid sequence of any of SEQ ID NOs: 3-8, 33, 35, 38, and 53-60. Also within the invention are isolated polypeptides or proteins which are encoded by a nucleic acid molecule having a nucleotide sequence that is at least about 40%, preferably 50%, 60%, 75%, 85%, or 95% identical the nucleic acid sequence encoding any of SEQ ID NOs: 3-8, 33, 35, 38, and 53-60, and isolated polypeptides or proteins which are encoded by a nucleic acid molecule consisting of the nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule having the nucleotide sequence of any of SEQ ID NOs: 1, 2, 31, 32, 51, and 52, the TANGO 416 nucleotide sequence of the cDNA insert of a clone deposited on April 26, 1999 with the ATCC as accession no. PTA-1764, or the TANGO 457 nucleotide sequence of the cDNA insert of a clone deposited on October 1, 1999 with the ATCC as PTA-817.

Also within the invention are polypeptides which are naturally occurring allelic variants of a polypeptide that includes the amino acid sequence of any of SEQ ID

NOs: 3-8, 33, 35, 38, and 53-60, wherein the polypeptide is encoded by a nucleic acid molecule which hybridizes under stringent conditions to a nucleic acid molecule having the nucleotide sequence of any of SEQ ID NOs: 1, 2, 31, 32, 51, and 52, the TANGO 416 nucleotide sequence of the cDNA insert of a clone deposited on April 26, 1999 with the ATCC as accession no. PTA-1764, the TANGO 457 nucleotide sequence of the cDNA insert of a clone deposited on October 1, 1999 with the ATCC as PTA-817, or a complement thereof.

The invention also features nucleic acid molecules that hybridize under stringent conditions to a nucleic acid molecule having the nucleotide sequence of any of SEQ ID NOs: 1, 2, 31, 32, 51, and 52, the TANGO 416 nucleotide sequence of the cDNA insert of a clone deposited on April 26, 1999 with the ATCC as accession no. PTA-1764, the TANGO 457 nucleotide sequence of the cDNA insert of a clone deposited on October 1, 1999 with the ATCC as PTA-817, or a complement thereof. In other embodiments, the nucleic acid molecules are at least 15 (25, 40, 60, 80, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, 2000, 2200, 2400, 2600, 2800, 3000, 3500, 4000, 4500, 5000, or more) nucleotides in length and hybridize under stringent conditions to a nucleic acid molecule having the nucleotide sequence of any of SEQ ID NOs: 1, 2, 31, 32, 51, and 52, the TANGO 416 nucleotide sequence of the cDNA insert of a clone deposited on April 26, 1999 with the ATCC as accession no. PTA-1764, the TANGO 457 nucleotide sequence of the cDNA insert of a clone deposited on October 1 , 1999 with the ATCC as

PTA-817, or a complement thereof. In some embodiments, the isolated nucleic acid molecules encode a cytoplasmic, transmembrane, extracellular, or other domain of a polypeptide of the invention. In other embodiments, 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 invention provides isolated host cells, e.g., mammalian or non-mammalian cells, containing such a vector or a nucleic acid of the invention. The invention also provides methods for producing a polypeptide of the invention by culturing, in a suitable medium, a host cell of the invention containing a recombinant expression vector encoding a polypeptide of the invention such that the polypeptide of the invention 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 the invention refers to an activity exerted by a protein or polypeptide of the invention on a responsive cell as determined in vivo, or in vitro, according to standard techniques. Such activities can be a direct activity, such as an association with or an enzymatic activity exerted on a second protein or an indirect activity, such as a cellular processes mediated by interaction of the protein with a second protein.

The observations that expression of TANGO 416 protein is up-regulated in porcine endothelial cells, that TANGO 416 is a member of the cadherin family of proteins, and that at least one cadherin (designated E-cadherin), which is expressed in endothelial cells, binds to integrin αEβ7 indicate that TANGO 416 protein can also bind with integrin αEβ7. Thus, TANGO 416 nucleic acids, proteins, compounds which modulate their activity, expression, or both, and compounds (e.g., antibodies) which bind with TANGO 416 proteins (collectively "TANGO 416-related molecules") can modulate one or more of growth, proliferation, survival, differentiation, activity, morphology, and movement / migration of, for example, cells (e.g. endothelial cells) which normally express TANGO 416 and cells (e.g. certain T cells, eosinophils, mast cells, and other lymphocytes) which normally express integrin αEβ7.

Integrin αEβ7 (sometimes designated αE/HML-1, αE/human mucosal lymphocyte- 1 and CD 103, and described in international patent application publication number WO95/22610, published on August 24, 1995) is an integrin protein which is expressed by more than 90% of intestinal epithelial lymphocytes, on 40-50% of intestinal lamina propria T lymphocytes, and on about 2% of peripheral blood leukocyte. Integrin αEβ7 is also expressed by T lymphocytes at other mucosal epithelia and on about 40% of T cells obtained by bronchioalveolar lavage. Integrin αEβ7 is apparently not expressed on B cells. A putative endothelial ligand designated E-cadherin binds with integrin αEβ7.

Antibodies which bind specifically with the αE subunit of integrin αEβ7 have demonstrated efficacy for treatment of inflammatory bowel disease and for reducing pulmonary inflammation and airway hyper-responsiveness in murine models of these disorders. Integrin αEβ7 is believed to have a role in binding of lymphocytes with endothelial cells and with regulation of tissue levels of Tjjl and TJJ cytokines (e.g. IL-5, IL-13) and eotaxin. The ability of TANGO 416 to bind with integrin αEβ7 indicates that TANGO 416 protein and other TANGO 416-related molecules can be used to modulate the physiological activities associated with integrin αEβ7 function and to treat disorders to which such physiological activities contribute. In one embodiment of the invention, TANGO 416-related molecules are used to modulate interaction of cells which normally express integrin αEβ7 (e.g. binding with, movement over, among, or past, or activation of cellular function by) and cells which normally express TANGO 416. TANGO 416-related molecules can also be used to modulate production, release, or both, of cytokines and eotaxin by cells which normally express integrin αEβ7. TANGO 416 protein can thus be involved in disorders which affect epithelial and lymphocytic tissues. Such disorders include cell proliferation disorders, disorders associated with aberrant epithelial permeability, auto-, hypo-, and hyper-immune disorders, disorders associated with aberrant binding or adhesion of cells with other cells, and inflammatory disorders. TANGO 416-related molecules can be used to prognosticate, prevent, diagnose, or treat one or more such disorders. Examples of these disorders include acute and chronic inflammatory diseases of the bowel (e.g., Chrohn's disease, irritable bowel syndrome, and ulcerative colitis), colitis of various etiologies, gastrointestinal infections, gastritis, gastroesophageal reflux disorder, acute and chronic peritonitis, appendicitis, diarrhea, constipation, gastroenteritis, hemorrhoids, proctitis, chronic and acute bronchitis, asthma, pneumonia, hypersensitivity pneumonitis, allergic disorders, anemia, leukopenia, thrombocytopenia, lymphoproliferative diseases, transplant rejection, graft- ersus-host reactions, allergic reactions, hypersplenism, autoimmune disorders (e.g., multiple sclerosis, psoriasis, diabetes mellitus, insulin resistance, rheumatoid arthritis, scleroderma, glomerulonephritis, systemic lupus erythematosus, Goodpasture's syndrome, Graves' disease, myasthenia gravis), metastasis of tumor tissue, cystic fibrosis, various chronic obstructive pulmonary disorders, pericarditis, hypogonadism, and testosterone deficiency syndrome. Other disorders which can be treated using TANGO 416 proteins, nucleic acids encoding them, and agents that modulate activity or expression of either of these include disorders of bone and cartilage tissues (e.g., traumatic and degenerative injuries), disorders of the spleen (e.g., lymphoma and splenomegaly), disorders associated with aberrant processing of blood cells in splenic tissue (e.g., disorders involving aberrant macrophage activity), cardiovascular disorders (e.g., disorders of the cardiac muscle and disorders of blood vessels), disorders involving aberrant association (or non-association) of B and T lymphocytes with each other and with endothelial tissues (e.g., immune disorders and inflammatory disorders), pancreatic disorders (e.g., pancreatitis, pancreatic cysts, pancreatic tumors, diabetes mellitus, and islet cell tumors), and disorders of the prostate (e.g., inflammatory prostatic diseases, prostatic hyperplasia, and prostate tumors).

The present invention is based, at least in part, on the discovery of cDNA molecules which encode TANGO 457 proteins, which are transmembrane proteins with one or more immunoglobulin domains and which are encoded by sequences expressed in at least, uterus, fetal liver, fetal spleen, and placenta tissues.

The biological activities of TANGO 457 and modulators thereof include, e.g., (1) the ability to form, e.g., stabilize, promote, inhibit, or disrupt, protein-protein interactions (e.g., homophilic and/or heterophilic) with proteins in the signaling pathway of the naturally- occurring polypeptide; (2) the ability to bind a ligand of the naturally-occurring polypeptide; and (3) the ability to interact with a TANGO 457 receptor. Other activities include the ability to modulate function, survival, morphology, migration, proliferation and/or differentiation of cells of tissues (e.g., uterus, fetal liver, fetal spleen, and placenta) in which it is expressed. In one embodiment, a polypeptide of the invention has an amino acid sequence sufficiently identical to a polypeptide of the invention or to an identified domain thereof. As used herein, the term "sufficiently identical" refers to a first amino acid or nucleotide sequence which contains a sufficient or minimum number of identical or equivalent (e.g., with a similar side chain) amino acid residues or nucleotides to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences have a common domain and/or common functional activity. For example, amino acid or nucleotide sequences which contain a common domain having about 65% identity, preferably 75% identity, more preferably 85%, 95%, or 98% identity are defined herein as sufficiently identical. In one embodiment, the isolated polypeptide of the invention lacks both a transmembrane and a cytoplasmic domain. 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 with a heterologous amino acid sequence to form fusion proteins. The invention further features antibody substances that specifically bind a polypeptide of the invention, such as monoclonal or polyclonal antibodies, antibody fragments, and single-chain antibodies. The antibody substances of the invention can be conjugated antibody substances comprising, for example, an antibody conjugated with a therapeutic agent or a diagnostic agent. The therapeutic or diagnostic agent can be a moiety such as a cytotoxin, e.g., a cytostatic and/or cytocidal agent, a thrombotic or anti-angiogenic agent, a radioactive metal ion or a fluorescent label. In addition, the polypeptides of the invention or biologically active portions thereof can be incorporated into pharmaceutical compositions, which optionally include pharmaceutically acceptable carriers. These antibody substances can be made, for example, by providing the polypeptide of the invention to an immunocompetent vertebrate and thereafter harvesting blood or serum from the vertebrate.

In another aspect, the present invention provides methods for detecting the presence of the activity or expression of a polypeptide of the invention in 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 enhances) the activity or expression of a polypeptide of the invention such that activity or expression in the cell is modulated. In one embodiment, the agent is an antibody that specifically binds with 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 that is antisense with respect to the coding strand of an mRNA encoding a polypeptide of the invention.

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

The present invention also provides diagnostic assays for identifying the presence or absence of a genetic lesion or mutation characterized by at least one of: (i) aberrant modification or mutation of a gene encoding a polypeptide of the invention, (ii) mis- regulation of a gene encoding a polypeptide of the invention, and (iii) aberrant post- translational modification of a polypeptide of the invention wherein a wild-type form of the gene encodes a polypeptide having the activity of the polypeptide of the invention. In another aspect, the invention provides a method for identifying a compound that binds with or modulates the activity of a polypeptide of the invention. In general, such methods entail measuring a biological activity of the polypeptide in the presence and absence of a test compound and identifying those compounds which bind with or alter the activity of the polypeptide. The invention also features methods for identifying a compound which modulates the expression of a polypeptide or nucleic acid of the invention by measuring the expression of the polypeptide or nucleic acid in the 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 comprises Figures 1 A through II. The nucleotide sequence (SEQ ID NO: 1) of a cDNA encoding the human TANGO 416 protein described herein is listed in Figures 1 A through II. The open reading frame (ORF; residues 376 to 3780; SEQ ID NO: 2) of the cDNA is indicated by nucleotide triplets, above which the amino acid sequence (SEQ ID NO: 3) of human TANGO 416 is listed.

Figure 2 comprises Figures 2A through 21. The nucleotide sequence (SEQ ID NO: 31) of a cDNA encoding the human TANGO 416 protein described herein is listed in Figures 2A through 21. The open reading frame (ORF; residues 376 to 3777; SEQ ID NO: 32) of the cDNA is indicated by nucleotide triplets, above which the amino acid sequence (SEQ ID

NO: 33) of human TANGO 416 is listed.

Figure 3 is a hydrophobicity plot of the embodiment of human TANGO 416 protein listed in Figure 1, in which the locations of cysteine residues ("Cys") and potential N- glycosylation sites ("Ngly") are indicated by vertical bars and the predicted extracellular ("out"), intracellular ("ins"), or transmembrane ("TM") locations of the protein backbone is indicated by a horizontal bar.

Figure 4 is an alignment of a portion of the TANGO 416 cDNA sequence ("T416"; residues 1651-4000 of SEQ ID NO: 1) with a human testis cDNA clone having GenBank accession number AL137471 ("AL137471"; SEQ ID NO: 40). This alignment indicates that the two nucleotide sequences are about 98.6% identical over the overlapping region. The alignment was made using the ALIGN software (BLOSUM62 scoring matrix, gap opening penalty 12, gap extension penalty 4, frameshift gap penalty 5).

Figure 5 is an alignment of a portion of the TANGO 416 ORF nucleotide sequence ("T416"; residues 1-3405 of SEQ ID NO: 2) with the ORF nucleotide sequence ("m~

PC"; SEQ ID NO: 41) of murine protocadherin (sometimes designated vascular endothelial cadherin-2 or mVE-cad-2). This alignment indicates that the two nucleotide sequences are about 55.4% identical over the overlapping region. The alignment was made using the ALIGN software (BLOSUM62 scoring matrix, gap opening penalty 12, gap extension penalty 4, frameshift gap penalty 5).

Figure 6 is an alignment of a portion of the TANGO 416 protein amino acid sequence ("T416"; residues 1-1135 of SEQ ID NO: 3) with the amino acid sequence ("m-PC"; SEQ ID NO: 42) of murine protocadherin. This alignment indicates that the two amino acid sequences are about 32.8% identical over the overlapping region. The alignment was made using the ALIGN software (BLOSUM62 scoring matrix, gap opening penalty 12, gap extension penalty 4).

Figures 7A-7B depict a cDNA sequence of human TANGO 457 (SEQ ID NO: 51) and the predicted human TANGO 457 amino acid sequence encoded by the sequence (SEQ ID NO: 53). The open reading frame of TANGO 457, comprises nucleotide 149 to nucleotide 1243 of SEQ ID NO: 51 (SEQ ID NO: 52).

Figure 8 depicts a hydrophobicity plot of human TANGO 457. Relatively hydrophobic regions of the protein are above the dashed horizontal line, and relatively hydrophilic regions of the protein are below the dashed horizontal line. The cysteine residues (cys) and N- glycosylation site (Ngly) are indicated by short vertical lines just below the hydropathy trace. Figures 9A and 9B depict a local alignment of the nucleic acid of human TANGO 457 shown in SEQ ID NO: 51 and a portion of the nucleotide sequence of human chromosome llpl4.3 PAC clone pDJ239b22, from nucleic acids 121077 to 122478 (SEQ ID NO: 61; accession number AC003969). In the alignment, the TANGO 457 sequence is featured as the top strand, and the 1 lpl4.3 PAC clone pDJ239b22 sequences is on the bottom. The alignment shows that there is a 100% nucleotide sequence identity between the TANGO 457 sequence of SEQ ID NO: 51 and human chromosome 1 lpl4.3 PAC clone pDJ239b22, from nucleotides 908 to 2305 of TANGO 457. This alignment was performed using the ALIGN alignment program with a PAM120 scoring matrix, a gap length penalty of 12, and a gap penalty of 4.

DETAILED DESCRIPTION OF THE INVENTION The present invention is based, at least in part, on the discovery of a variety of cDNA molecules which encode proteins which are herein designated TANGO 416 and TANGO 457. These proteins exhibit a variety of physiological activities, and are included in a single application for the sake of convenience. It is understood that the allowability or non- allowability of claims directed to one of these proteins has no bearing on the allowability of claims directed to the others. The characteristics of each of these proteins and the cDNAs encoding them are now described separately.

TANGO 416

Expression of cDNA encoding a TANGO 416 protein was up-regulated in porcine endothelial cells that were activated using one or more of bacterial lipopolysaccharide, tumor necrosis factor alpha, and human serum. Up-regulation was detected by extracting total RNA from activated cells and subjecting the RNA to reverse transcriptase polymerase chain reaction for differential display. cDNA clones encoding at least a portion of human TANGO

416 protein and corresponding to the porcine TANGO 416 cDNA were isolated from human fetal spleen and osteoblast cDNA libraries (including a clone designated jthsa97d5 obtained from a fetal spleen library, a clone designated jthocl22e2 obtained from an osteoblast library, and a clone designated jthsal21fl0 obtained from a fetal spleen library). Human TANGO 416 protein is a transmembrane protein which can occur in at least two alternative forms, which vary in the presence or absence of a single amino acid residue (i.e. the glutamine residue at amino acid residue 830 of SEQ ID NO: 3 and corresponding nucleotide residues 2863-2865 in SEQ ID NO: 1). In this application, reference to amino acid and nucleotide residues is made to those residues in the longer form of TANGO 416 (i.e. the form having the amino acid sequence SEQ ID NO: 3 and the corresponding cDNA and ORF sequences SEQ ID NOs: 1 and 2, respectively). The longer form of TANGO 416 has a glutamine residue at amino acid residue 830 that is not present in the shorter form of TANGO 416. It is understood that both forms of TANGO 416 can exhibit the same biological properties, and that references to amino acid residues numbered 831 or higher in SEQ ID NO: 3 correspond to amino acid residues having the next lower number in SEQ ID NO: 33 (i.e. amino acid residue 901 in the longer form of TANGO 416 {SEQ ID NO: 3} corresponds to amino acid residue 900 in the shorter form of TANGO 416 {SEQ ID NO: 33}). Similarly, references to nucleotide residues numbered 2866 or higher in SEQ ID NO: 1 correspond to nucleotide residues having a number that is lower by 3 in SEQ ID NO: 31 (i.e. nucleotide residue 2903 in the longer form of TANGO 416 {SEQ ID NO: 1} corresponds to nucleotide residue 2900 in the shorter form of TANGO 416 {SEQ ID NO: 31}).

The full length of a cDNA which was isolated from a human fetal spleen cDNA library and which encodes human TANGO 416 protein (Figure 1; SEQ ID NO: 1; i.e. the longer form of TANGO 416) is 5121 nucleotide residues. The open reading frame (ORF) of this cDNA, nucleotide residues 376 to 3780 of SEQ ID NO: 1 (i.e., SEQ ID NO: 2), encodes a 1135-amino acid residue protein (Figure 1; SEQ ID NO: 3), corresponding to a 1108-residue transmembrane mature protein. The invention thus includes purified human TANGO 416 protein, both in the form of the immature 1135 amino acid residue protein (SEQ ID NO: 3, including the shorter 1134-residue protein {SEQ ID NO: 33}) and in the form of the mature 1108 amino acid residue protein (SEQ ID NO: 5, including the shorter 1107-residue protein {SEQ ID NO: 35}). Mature human TANGO 416 proteins can be synthesized without the signal sequence polypeptide at the amino terminus thereof, or they can be synthesized by generating immature TANGO 416 protein and cleaving the signal sequence therefrom.

In addition to full length mature and immature human TANGO 416 proteins, the invention includes fragments, derivatives, and variants of these TANGO 416 proteins, as described herein. These proteins, fragments, derivatives, and variants are collectively referred to herein as polypeptides of the invention or proteins of the invention.

The invention also includes nucleic acid molecules which encode a polypeptide of the invention. Such nucleic acids include, for example, a DNA molecule having the nucleotide sequence listed in SEQ ID NOs: 1, 2, 31, 32, 51, and 52, such as the portion which encodes a mature TANGO 416 protein, an immature TANGO 416 protein, or a domain of a

TANGO 416 protein. These nucleic acids are collectively referred to as nucleic acids of the invention.

TANGO 416 proteins and nucleic acid molecules encoding them comprise a family of molecules having certain conserved structural and functional features. As used herein, the term "family" is intended to mean two or more proteins or nucleic acid molecules having a common or similar domain structure and having sufficient amino acid or nucleotide sequence identity as defined herein. Family members can be from either the same or different species (e.g., human and mouse). For example, a family can comprise two or more proteins of human origin, or can comprise one or more proteins of human origin and one or more of non- human origin.

A common domain present in TANGO 416 proteins is a signal sequence. As used herein, a signal sequence includes a peptide of at least about 10 amino acid residues in length which occurs at the amino terminus of membrane-bound and secreted proteins and which contains at least about 45% hydrophobic amino acid residues such as alanine, leucine, isoleucine, phenylalanine, proline, tyrosine, tryptophan, or valine. In one embodiment, a signal sequence contains at least about 10 to 35 amino acid residues, preferably about 20 to 35 amino acid residues, and has at least about 35-60%, more preferably 40-50%, and more preferably at least about 45% hydrophobic residues. A signal sequence serves to direct a protein containing such a sequence to a lipid bi-layer. Thus, in one embodiment, a TANGO 416 protein contains a signal sequence corresponding to amino acid residues 1 to 27 of SEQ ID NO: 3 and 33 (i.e. SEQ ID NO: A). It is recognized that the carboxyl terminal boundary of the signal sequence can be located one or two residues from the residue identified above (i.e., following residues 25, 26, 27, 28, or 29 of SEQ ID NOs: 3 and 33). The signal sequence is cleaved during processing of the mature protein. TANGO 416 proteins include a transmembrane domain and two extra-membrane domains flanking the cell membrane. The transmembrane domain corresponds to about amino acid residues 701 to 721 of SEQ ID NOs: 3 and 33 (i.e., the transmembrane domain having the sequence SEQ ID NO: 7). One of the extra-membrane domains corresponds to about amino acid residues 28 to 700 of SEQ ID NOs: 3 and 33 (i.e. the domain having the sequence SEQ ID NO: 6). The other extra-membrane domain corresponds to about amino acid residues 722 to 1135 of SEQ ID NO: 3 (i.e. residues 722 to 1134 of SEQ ID NO: 33, this domain having the sequence SEQ ID NO: 8 in the longer form of TANGO 416 and SEQ ID NO: 38 in the shorter form). In one embodiment, the extra-membrane domain corresponding to SEQ ID NO: 6 is an extra-cellular domain and the other extra-membrane domain is an intracellular domain. In an alternative embodiment, the extra-membrane domain corresponding to SEQ ID NO: 6 is an intracellular domain and the other extra-membrane domain is an extra-cellular domain. As used herein, an "extracellular domain" refers to a portion of a protein which is localized to the non- cytoplasmic side of a lipid bi-layer of a cell when a nucleic acid encoding the protein is expressed in the cell. A "transmembrane domain" refers to an amino acid sequence which is at least about 20 to 25 amino acid residues in length and which contains at least about 65-70% hydrophobic amino acid residues such as alanine, leucine, phenylalanine, protein, tyrosine, tryptophan, or valine. As used herein, a "cytoplasmic domain" refers to a portion of a protein which is localized to the cytoplasmic side of a lipid bi-layer of a cell when a nucleic acid encoding the protein is expressed in the cell. TANGO 416 proteins typically comprise a variety of potential post-translational modification sites and protein domains (often positioned within an extracellular domain), such as those described herein in Table I, as predicted by computerized sequence analysis of TANGO 416 proteins using amino acid sequence comparison software (comparing the amino acid sequence of TANGO 416 with the information in the PROSITE database {rel. 12.2; Feb, 1995} and the Hidden Markov Models database {Rel. PFAM 3.3}). Table I

Table I (Continued)

Table I (Continued)

As used herein, the term "post-translational modification site or domain" refers to a protein region that includes about 3 to 10 amino acid residues, more preferably about 3 to 6 amino acid residues wherein the domain has an amino acid sequence which comprises a consensus sequence which is recognized and modified by a protein-modifying enzyme. The term also includes protein domains having greater lengths, as indicated herein. Examples of protein-modifying enzymes include amino acid glycosylases, cAMP- and cGMP-dependent protein kinases, protein kinase C, casein kinase II, tyrosine kinase, myristoylases, and prenyl transferases. In various embodiments, the protein of the invention has at least 1, 2, 4, 6, 10, 15, 20, 30, 40, or 50 or more of the post-translational modification sites described herein in Table I. In one embodiment, the protein of the invention has all 63 of the sites described in Table I

Examples of additional domains present in human TANGO 416 protein include cadherin extracellular repeated domains. In one embodiment, the protein of the invention has at least one domain or signature sequence that is at least 55%, preferably at least about 65%, 75%,

85%, or 95% identical to one of the cadherin extracellular domains or signature sequences described herein in Table I. Preferably, the protein of the invention has 2, 3, 4, or all 5, cadherin extracellular repeated domains.

Cadherin extracellular repeated domains have a conserved consensus sequence that occurs in numerous cadherins. The conserved extracellular cadherin repeated domain sequence, which is frequently repeated in cadherins is

{L or I or V}-X-{L or I or V}-X(i _or 2)-D-X-N-D-{N or H}-X-P, where X is any amino acid residue, and wherein the subscript '1 or 2' indicates that either one or two X residues can be present at that position. Folding of the extracellular repeated domain of cadherins is believed to roughly correspond to occurrence of extracellular cadherin repeated domains.

Cadherins are a family of cell-surface proteins which are involved in cell-to-cell binding, including specific cell adhesion processes which occur during development and adherens junction formation related to tissue organization in developing and adult organisms. Cadherins are also involved in intracellular signaling. Repeated cadherin extracellular domains occur in a variety of cadherins, including, for example, epithelial cadherin (sometimes designated E-cadherin, uvomorulin, L-CAM, or CDHl), neural cadherin (sometimes designated N-cadherin or CDH2), placental cadherin (sometimes designated P-cadherin or CDH3), retinal cadherin (sometimes designated R-cadherin or CDH4), vascular endothelial cadherin (sometimes designated VE-cadherin or CDH5), kidney cadherin (sometimes designated K- cadherin or CDH6), cadherin-8 (sometimes designated CDH8), osteoblast cadherin (sometimes designated OB-cadherin or CDHl 1), brain cadherin (sometimes designated BR-cadherin or CDH12), truncated cadherin (sometimes designated T-cadherin or CDH13), muscle cadherin (sometimes designated M-cadherin or CDH14), liver-intestine cadherin (sometimes designated Ll-cadherin), and EP-cadherin. Occurrence of repeated cadherin extracellular domains in TANGO 416 is an indication that TANGO 416 is a member of the cadherin family of proteins, and is thus involved in specific cell adhesion processes and regulation of intracellular signaling events in tissues in which it occurs.

The signal peptide prediction program SIGNALP (Nielsen et al. (1997) Protein Engineering 10:1-6) predicted that human TANGO 416 protein includes a 27 amino acid residue signal peptide (amino acid residues 1 to 27 of SEQ ID NOs: 3 and 33 {SEQ ID NO: 4}) preceding the mature TANGO 416 protein (about amino acid residues 28 to 1135 of SEQ ID NO: 3 {SEQ ID NO: 5} and about amino acid residues 28 to 1134 of SEQ ID NO: 33 {SEQ ID NO: 35}). Human TANGO 416 protein includes an extracellular domain (about amino acid residues 28 to 700 of SEQ ID NOs: 3 and 33 {SEQ ID NO: 6}), a transmembrane domain (about amino acid residues 701 to 721 of SEQ ID NOs: 3 and 33 {SEQ ID NO: 7}), and an intracellular domain (about amino acid residues 722 to 1135 of SEQ ID NO: 3 {SEQ ID NO: 8} and about amino acid residues 722 to 1134 of SEQ ID NO: 33 {SEQ ID NO: 38}).

Figure 3 depicts a hydrophilicity plot of human TANGO 416 protein. Relatively hydrophobic regions are above the dashed horizontal line, and relatively hydrophilic regions are below the dashed horizontal line. The hydrophobic region which corresponds to amino acid residues 1 to 27 of SEQ ID NOs: 3 and 33 is the signal sequence of human TANGO 416 (SEQ ID NO: 4). As described elsewhere herein, relatively hydrophilic regions are generally located at or near the surface of a protein, and are more frequently effective immunogenic epitopes than are relatively hydrophobic regions. For example, the region of human TANGO 416 protein from about amino acid residue 450 to about amino acid residue 470 appears to be located at or near the surface of the protein, while the region from about amino acid residue 335 to about amino acid residue 345 appears not to be located at or near the surface.

The predicted molecular weight of human TANGO 416 protein without modification and prior to cleavage of the signal sequence is about 126.0 kilodaltons. The predicted molecular weight of the mature human TANGO 416 protein without modification and after cleavage of the signal sequence is about 122.8 kilodaltons.

TANGO 416 DNA maps to chromosome 4, between chromosomal markers D4S422 and D4S1576, as assessed by comparing TANGO 416 sequence with an ESTs in a mapping database. Other genes which map to this chromosomal segment include those encoding endothelin-1 receptor precursor, surfactant protein A, transforming growth factor beta signaling protein-1 (Bsp-1), and a gene highly similar to Mus musculus hemoglobin zeta chain. Thus, disorders previously attributed to these loci by others can also be attributable to the chromosomal portion encoding TANGO 416. cDNA encoding TANGO 416 protein occurs in cDNA libraries generated from human fetal spleen tissue and from human osteoblasts. High homology of TANGO 416 cDNA was observed with expressed sequence tags (ESTs) obtained from EST libraries generated from human fetal heart, human fetal lung, human testis, human pancreas, human prostate, and human B cell tissues, indicating that TANGO 416 protein can be expressed in these tissues as well. Residues 1651-4000 of SEQ ID NO: 1 (the nucleotide sequence of TANGO 416 cDNA) were aligned (using the ALIGN software with gap length penalty of 12, and a gap penalty of 4) with the nucleotide sequence of the human testis cDNA clone DKFZp434B0923 listed in GenBank accession number AL137471. This alignment, shown in Figure 4, was generated using the ALIGN software (using the BLOSUM62 scoring matrix, a gap opening penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5), and indicated

98.6% identity between the two sequences in the 2350-residue overlapping portion. The nucleotide sequence (SEQ ID NO: 2) of the ORF encoding TANGO 416 was aligned using the ALIGN software (with gap length penalty of 12, and a gap penalty of 4)) with the nucleotide sequence of the ORF of a murine protocadherin (GenBank™ accession number Y08715; Telo et al., 1998, J. Biol. Chem. 273:17565-17572), as shown in Figure 5. This alignment was generated using the ALIGN software (using the BLOSUM62 scoring matrix, a gap opening penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5), and indicated 55.4% identity between the two sequences in the overlapping portion. Alignment of the amino acid sequence of TANGO 416 with the amino acid sequence of the murine protocadherin, as shown in Figure 6, indicated 32.8% sequence identity and 42.2 % sequence similarity. This alignment was generated using the ALIGN software (using the BLOSUM62 scoring matrix, a gap opening penalty of 12, a gap extension penalty of 4). Uses of TANGO 416 Nucleic acids, Polypeptides, and Modulators Thereof

TANGO 416 proteins are involved in disorders which affect both tissues in which they are normally expressed and tissues in which they are normally not expressed. Based on the observations that cDNA corresponding to TANGO 416 occurs in human fetal spleen and human osteoblast cDNA libraries, and that ESTs corresponding to portions of TANGO 416 can be detected in ESTs prepared from each of human fetal heart, human fetal lung, human testis, human pancreas, human prostate, and human B cell tissues, TANGO 416 protein can be involved in one or more biological processes which occur in these tissues. In particular, TANGO 416 can be involved in modulating growth, proliferation, survival, differentiation, adhesion, and activity of cells of these tissues. Furthermore, because TANGO 416 likely belongs to the cadherin family of proteins, it can also be involved in modulating movement of cells (e.g., T cells and other cells of the immune system) through tissues which express receptors for TANGO 416 (e.g. cells which express one or more cadherin receptors, e.g., cells which express integrin αEβ7).

TANGO 416 can interact as a ligand with integrin αEβ7, as discussed above. Integrin αEβ7 (also designated human mucosal lymphocyte 1 antigen or CD 103) is expressed on more than 90% of intestinal epithelial lymphocytes (IEL), and about 40-50% of intestinal lamina propria T lymphocytes, but only on about 2% of peripheral blood leukocytes. Integrin αEβ7 is also expressed on T lymphocytes which are present at other mucosal epithelial (e.g. on about 40% of T cells recovered by bronchioalveolar lavage {BAL}). Integrin αEβ7 does not appear to be expressed on B lymphocytes. Transforming growth factor beta 1 (TGF-βl) induces expression of integrin αEβ7 on both T lymphocytes and cultured murine mast cells. Antibodies which bind specifically with integrin αEβ7 reduce morbidity and pathological effects associated with experimentally induced inflammatory bowel disease (IBD; e.g. using the CD45Rb SCID transfer model of IBD), transmural colitis, (e.g. using interleukin-2 {IL-2} knockout mice immunized using 2,4,6-trinitrophenol), and pulmonary inflammation (e.g. using mice sensitized intraperitoneally with and challenged with aerosol ovalbumin). In BAL fluids obtained from mice which were sensitized and challenged with ovalbumin and to which anti-integrin αEβ7 were administered, decreased numbers of eosinophils and leukocytes were detected, and levels of TJJ2 cytokines (e.g. IL-5 and IL-13) and eotaxin were decreased as well. Integrin αEβ7 knockout (i.e. nullizygous) mice exhibited decreased numbers of intestinal epithelial lymphocytes, decreased susceptibility to pulmonary inflammation, reduced airway hyper-responsiveness, and decreased levels of Tg cytokines in BAL fluids. These data indicate that integrin αEβ7 can function as a receptor for guiding lymphocytes (e.g. T cells, mast cells, and eosinophils) to mucosal epithelia and maintaining them at those locations. These data also indicate that integrin αEβ7 can modulate Tjjl and TJJ2 cytokine levels in tissues which contain integrin αEβ7-bearing cells (see international patent application publication number WO95/22610, published on August 24, 1995). TANGO 416 proteins can bind with integrin αEβ7 and thereby modulate the integrin's physiological effects. In particular, TANGO 416 proteins can modulate localization of integrin αEβ7-bearing lymphocytes at tissues which express TANGO 416 (e.g. at mucosal epithelia such as intestinal and pulmonary epithelia) and release and maintenance of Tjjl and Tpj2 cytokine levels in or near such tissues. Modulation of cytokine levels by TANGO 416 can, in turn, modulate proliferation, activity, and migration of cells of the immune system, such as are associated with a variety of inflammatory, auto-immune, hypo-immune, and hyper- immune disorders. TANGO 416 proteins, nucleic acids encoding them, and agents that modulate activity or expression of either of these can thus be used to modulate these processes. Disorders that involve proliferation, activity, and migration of immune cells in the vicinity of mucosal epithelia include, by way of example, acute and chronic inflammatory diseases of the bowel (e.g. inflammatory bowel disease and Crohn's disease), colitis (of various etiologies), gastrointestinal infections (e.g. formation and perseverance of peptic ulcers), gastritis, gastroesophageal reflux disorder, acute and chronic peritonitis, appendicitis, diarrhea, constipation, gastroenteritis, hemorrhoids, and proctitis. Such disorders also include disorders that involve proliferation, activity, and migration of immune cells in the vicinity of pulmonary mucosal epithelial, such as chronic and acute bronchitis, asthma, pneumonia (e.g. pneumococcal, staphylococcal, streptococcal, klebsiellal, hemophilal, viral, fungal, etc.), hyper- sensitivity pneumonitis, and allergic disorders (e.g. hay fever and the like). TANGO 416 proteins, nucleic acids encoding them, and agents that modulate activity or expression of either of these can thus be used to prognosticate, diagnose, and treat one or more of these disorders. Expression of TANGO 416 in osteoblasts is an indication that TANGO 416 can have a role in modulating bone formation, marrow cell differentiation and proliferation, and proliferation, differentiation, function, or some combination of these, of bone and cartilage cells. Examples of disorders which can be prognosticated, diagnosed, and treated using TANGO 416 proteins, nucleic acids encoding them, and agents that modulate activity or expression of either of these include disorders of bone and cartilage tissues including bone or cartilage injuries, such as those attributable to trauma (e.g., bone breakage and cartilage tearing), or to degeneration (e.g., osteoporosis and age-related degradation of cartilage). The compositions can also be used to treat disorders associated with degeneration of joints, such as arthritis (including rheumatoid arthritis), osteoarthritis, and bone wearing.

Occurrence of TANGO 416 cDNA in a fetal spleen library is an indication that TANGO 416 proteins, nucleic acids encoding them, and agents that modulate activity or expression of either of these can be used to modulate proliferation, differentiation, function, or some combination of these, of spleen cells (e.g., cells of the splenic connective tissue, splenic smooth muscle cells, and endothelial cells of splenic blood vessels). These compositions can thus be used to treat disorders of the spleen, (including both the fetal spleen and the adult spleen). Examples of splenic diseases and disorders include splenic lymphoma and splenomegaly. Occurrence of TANGO 416 in splenic tissue further indicates that TANGO 416 proteins, nucleic acids encoding them, and agents that modulate activity or expression of either of these can be used to modulate proliferation, differentiation, function, or some combination of these, of blood cells that are processed in splenic tissue. These cells include cells which are regenerated or phagocytized within the spleen, including, for example, erythrocytes, B and T lymphocytes, and macrophages. Examples of these disorders include phagocytotic disorders, such as disorders in which engulfment of bacteria and viruses in the bloodstream by macrophages in the spleen is inhibited.

Occurrence in a fetal heart library of an EST which exhibits homology with cDNA encoding TANGO 416 indicates that TANGO 416 proteins, nucleic acids encoding them, and agents that modulate activity or expression of either of these can be used to treat cardiovascular disorders, including disorders of the heart and disorders of the blood vessels. Examples of cardiac disorders which can be treated in this manner include ischemic heart diseases (e.g., angina pectoris, myocardial infarction and its aftermath, and chronic ischemic heart disease), hypertensive heart disease, pulmonary heart disease, valvular heart disease (e.g., rheumatic fever and rheumatic heart disease, endocarditis, mitral valve prolapse, and aortic valve stenosis), congenital heart disease (e.g., valvular and vascular obstructive lesions, atrial or ventricular septal defect, and patent ductus arteriosus), and myocardial disease (e.g., myocarditis, congestive cardiomyopathy, and hypertrophic cardiomyopathy). Examples of vascular disorders which can be treated in this manner include arteriosclerosis, atherosclerosis, aberrant or non-desired angiogenesis, stenosis and restenosis, and smooth muscle proliferation in response to traumatic injury. Involvement of TANGO 416 protein in binding of cells is an indication that

TANGO 416 can be involved in disorders associated with aberrant binding or adhesion of cells with other cells, with extracellular matrix, or with foreign materials. Disorders involving aberrant binding or adhesion of cells with other cells include both disorders in which cells normally bind with one another (e.g., metastasis of normally solid tumor tissue cells away from the tumor site of origin or immune hypersensitivity) and disorders in which the cells do not normally bind with one another, but do bind with one another in individuals afflicted with the disorder (e.g., metastasis of tumor cells into a tissue in which the cells do not normally occur, autoimmune disorders, infections, wherein cells with which T cells bind are not normally present in the animal, or disorders associated with abnormal blood coagulation). Disorders involving aberrant binding or adhesion of cells with tissue on which TANGO 416 is normally expressed, include those in which the cells normally do, but aberrantly do not, bind with TANGO 416-expressing tissue as well as those (e.g., metastasis of cancers cells into mucosal epithelium) in which the cells normally do not bind with TANGO 416-expressing tissue, but aberrantly do. TANGO 416 proteins, nucleic acids encoding them, and agents that modulate activity or expression of either of these can be used to prognosticate, diagnose, and treat one or more of these disorders.

Like many transmembrane signaling proteins, TANGO 416 protein comprises extracellular domains capable of interacting with environmental cues (e.g., the presence or absence of particular cells, proteins, or small molecules) and a cytoplasmic domain having a substantial size. Numerous cadherins interact with catenins, tyrosine kinases, and other proteins which can influence the structure of the intracellular matrix. TANGO 416 can also interact with such proteins, and the existence of numerous post-translationally modifiable sites (see Table I) on TANGO 416 is an indication that TANGO 416 can be involved in transducing signals across the cell membrane. Binding of a ligand of TANGO 416 protein (e.g. integrin αEβ7 on the surface of a different cell such as a leukocyte) with a portion of the protein located on one side of the membrane can affect one or more characteristics (e.g., conformation, phosphorylation state, or level or specificity of enzymatic activity) of a portion of the TANGO 416 protein located on the other side. Thus, for example, a compound in the extracellular environment of a cell which expresses TANGO 416 can bind with the extracellular domain of the protein, thereby effecting a change in a characteristic of the intracellular portion of the protein, leading to alteration of the physiology of the cell (e.g., effected by an activity exerted by the intracellular portion of the protein on another component of the cell). The compound in the extracellular environment can, for example, be a compound dissolved or suspended in a liquid, a compound attached to another cell of the same animal, or a compound attached to a foreign cell or virus particle.

TANGO 416 protein can associate with other signal transduction proteins in the cell membrane, thereby modulating the intracellular activity of those other proteins. TANGO 416 can also bind with a membrane-bound protein (e.g. integrin αEβ7) of another cell, thereby modulating physiological activities associated with signal transduction mediated by that membrane-bound protein. By way of example, signal transduction events associated with integrin αEβ7 include modulation of Tjjl and TJJ2 cytokine (and eotaxin) production and release by leukocytes and movement and adherence of leukocytes. TANGO 416 protein and fragments and variants thereof can modulate such activities. TANGO 416 proteins can thus have a role in disorders which involve aberrant transmembrane signal transduction. Examples of signal transduction-related disorders include cystic fibrosis, various chronic obstructive pulmonary disorders, lymphocyte localization and activation disorders (e.g. transmural colitis, airway hyper-responsiveness, and various allergic disorders), and inflammatory disorders such as inflammatory bowel disease. TANGO 416 proteins, nucleic acids encoding them, and agents that modulate activity or expression of either of these can be used to prognosticate, diagnose, and treat one or more of these disorders. Occurrence of ESTs which exhibit homology with TANGO 416 nucleic acids in EST libraries generated from tissues which comprise endothelial tissues (e.g. fetal heart tissue and testicular tissue) indicates that TANGO 416-related molecules can be used to prognosticate, diagnose, and treat one or more other disorders which afflict endothelial tissues and organs which contain them. By way of example, the heart is surrounded by an endothelial pericardium which can become inflamed, leading to pericarditis and other complications. Similarly, the endothelial lining of blood vessels is known to bind lymphocytes (e.g. during 'rolling' movement of lymphocytes through the vessel and during extravasation of lymphocytes). Disorders which can affect testicular endothelium include hypogonadism and testosterone deficiency syndrome. Interaction of one or more TANGO 416-related molecules with cells that modulate interaction of TANGO 416 with integrin αEβ7 cells can inhibit, prevent, or alleviate such disorders. Furthermore, interaction of TANGO 416-related molecules with sample material (e.g. blood or tissue) obtained by a patient can be used to diagnose such disorders.

Homology of an EST obtained from a human B cell EST library with a TANGO 416 nucleic acid is an indication that TANGO 416 can modulate disorders involving inappropriate interaction of B and T cells. Such disorders include hypo-, hyper-, and autoimmune disorders and also include inflammatory disorders. By modulating interactions of B and T lymphocytes with each other and with endothelial tissues (and other tissues which can express integrin αEβ7, TANGO 416 proteins, nucleic acids encoding them, and agents that modulate activity or expression of either of these can also be used to treat immune disorders and inflammatory disorders. By way of example, they can be used to treat autoimmune disorders (e.g., arthritis, graft rejection such as allograft rejection), T cell disorders (e.g., AIDS)), bacterial infection, psoriasis, bacteremia, septicemia, cerebral malaria, inflammatory bowel disease, arthritis (e.g., rheumatoid arthritis, osteoarthritis), and allergic inflammatory disorders (e.g., asthma or psoriasis).

Homology of an EST of a library made using cDNA obtained from pancreatic tissue with TANGO 416 cDNA sequence indicates that TANGO 416 proteins, nucleic acids encoding them, and agents that modulate activity or expression of either of these can be used to treat pancreatic disorders. Examples of pancreatic disorders which can be treated in this manner include pancreatitis (e.g., acute hemorrhagic pancreatitis and chronic pancreatitis), pancreatic cysts (e.g., congenital cysts, pseudocysts, and benign or malignant neoplastic cysts), pancreatic tumors (e.g., pancreatic carcinoma and adenoma), diabetes mellitus (e.g., insulin- and non-insulin-dependent types, impaired glucose tolerance, and gestational diabetes), and islet cell tumors (e.g., insulinomas, adenomas, ZoUinger-Ellison syndrome, glucagonomas, and somatostatinoma). cDNA encoding TANGO 416 also exhibits homology with an EST of a library made using cDNA obtained from prostate tissue. Thus, TANGO 416 proteins, nucleic acids encoding them, and agents that modulate activity or expression of either of these can be used to treat prostate disorders. Examples of prostate disorders which can be treated in this manner include inflammatory prostatic diseases (e.g., acute and chronic prostatitis and granulomatous prostatitis), prostatic hyperplasia (e.g., benign prostatic hypertrophy or hyperplasia), and prostate tumors (e.g., carcinomas).

Homology of TANGO 416 protein with murine vascular endothelial cadherin-2 (mVE-cad-2; Telo et al, 1998, J. Biol. Chem. 273:17565-17572; GenBank™ accession number Y08715; sometimes designated protocadherin; see Figures 5 and 6) is an indication that

TANGO 416 is a human orthologue of that mVE-cad-2, and exhibits one or more of the same activities. That is, TANGO 416 can be involved in adherens junction formation and maintenance, and can thereby modulate endothelial permeability to plasma proteins and circulating cells.

TANGO 457

The TANGO 457 proteins and nucleic acid molecules comprise families of molecules having certain conserved structural and functional features. As used herein, the terms "family" or "families" are intended to mean two or more proteins or nucleic acid molecules having a common structural domain and having sufficient amino acid or nucleotide sequence identity as defined herein. Family members can be from either the same or different species. For example, a family can comprise two or more proteins of human origin, or can comprise one or more proteins of human origin and one or more of non-human origin. Members of the same family may also have common structural domains. For example, the TANGO 457 proteins of the invention can have signal sequences. As used herein, a "signal sequence" includes a peptide of at least about 15 or 20 amino acid residues in length which occurs at the N-terminus of secretory and membrane-bound proteins and which contains at least about 70% hydrophobic amino acid residues such as alanine, leucine, isoleucine, phenylalanine, proline, tyrosine, tryptophan, or valine. In a preferred embodiment, a signal sequence contains at least about 10 to 40 amino acid residues, preferably about 19 to 34 amino acid residues, and has at least about 60-80%, more preferably at least about 65-75%, and more preferably at least about 70% hydrophobic residues. A signal sequence serves to direct a protein containing such a sequence to a lipid bilayer. A signal sequence is usually cleaved during processing of the mature protein.

In certain embodiments, a TANGO 457 protein has the amino acid sequence of SEQ ID NO: 55, and the signal sequence is located at amino acids 1 to 21, 1 to 22, 1 to 23, 1 to 24, 1 to 25, 1 to 26, or 1 to 27. In such embodiments of the invention, the domains and the mature protein resulting from cleavage of such signal peptides are also included herein. For example, the cleavage of a signal sequence consisting of amino acids 1 to 24 of SEQ ID NO: 53

(SEQ ID NO: 55) results in a cytoplasmic domain consisting of amino acids 25 to 264, a transmembrane domain consisting of amino acids 265 to 282, and an extracellular domain consisting of amino acids 283 to 365, of SEQ ID NO: 53 (SEQ ID NO: 56, 59, and 60, respectively) and the mature TANGO 457 protein corresponding to amino acids 25 to 365 of SEQ ID NO: 53 (SEQ ID NO: 54). The signal sequence is normally cleaved during processing of the mature protein.

A TANGO 457 family member can include one or more of the following domains: (1) an extracellular domain; (2) a transmembrane domain; and (3) a cytoplasmic domain. For example, in one embodiment, a TANGO 457 protein contains a cytoplasmic domain at about amino acid residues 1 to 264 of SEQ ID NO: 53 (SEQ ID NO: 56), a transmembrane domain at about amino acid residues 265 to 282 of SEQ ID NO: 53 (SEQ ID NO: 59), and an extracellular domain at about amino acid residues 283 to 365 of SEQ ID NO: 53 (SEQ ID NO: 60). In another embodiment, a human TANGO 457 protein contains an cytoplasmic domain at amino acid residues 283 to 365 of SEQ ID NO: 53 ( SEQ ID NO: 60), a transmembrane domain at amino acid residues 265 to 282 of SEQ ID NO: 53 ( SEQ ID NO: 59), and an extracellular domain at amino acid residues 1 to 264 of SEQ ID NO: 53 ( SEQ ID NO: 56).

A TANGO 457 family member can include one or more TANGO 457 Ig domains. A TANGO 457 Ig domain as described herein is about 68 to 84 amino acid residues in length and has the following consensus sequence, beginning about 1 to 15 amino acid residues, more preferably about 3 to 10 amino acid residues, and most preferably about 5 amino acid residues from the domain C-terminus: [FYL]-X-C-X-[VA] (SEQ ID NO: 70), wherein [FYL] is a phenylalanine, tyrosine or leucine residue (preferably tyrosine), where "X" is any amino acid, C is a cysteine residue, and [VA] is an alanine residue or a valine residue. In one embodiment, a TANGO 457 family member includes one or more Ig domains having an amino acid sequence that is at least about 55%, preferably at least about 65%, more preferably at least about 75%), yet more preferably at least about 85%, and most preferably at least about 95%> identical to amino acids 41 to 124 of SEQ ID NO: 53 (SEQ ID NO: 57). In another embodiment, a TANGO 457 family member includes one or more Ig domains having an amino acid sequence that is at least about 55%, preferably at least about 65%, more preferably at least about 75%, yet more preferably at least about 85%, and most preferably at least about 95% identical to amino acids 163 to 230 of SEQ ID NO: 53 (SEQ ID NO: 58).

In another embodiment, a TANGO 457 family member includes one or more TANGO 457 Ig domains having an amino acid sequence that is at least about 55%, preferably at least about 65%, more preferably at least about 75%, yet more preferably at least about 85%, and most preferably at least about 95% identical to amino acids 41 to 124 and/or 163 to 230 of SEQ ID NO: 53 (SEQ ID NO: 57 and 58), and has a conserved cysteine residue about 1 to 15, preferably 1 to 10, more preferably 1 to 8 residues downstream from the N-terminus of the Ig domain. Thus, in this embodiment, amino acids 48 and 163 of SEQ ID NO: 53 are cysteine residues.

In another embodiment, a TANGO 457 family member includes one or more TANGO 457 Ig domains having an amino acid sequence that is at least 55%, preferably at least about 65%, more preferably at least about 75%, yet more preferably at least about 85%, and most preferably at least about 95% identical to amino acids 41 to 124 and/or 163 to 230 of SEQ ID NO: 53 (SEQ ID NO: 57 and 58), and has a conserved cysteine residue about 1 to 8 residues downstream from the N-terminus of the TANGO 457 Ig domain, has a conserved cysteine within the consensus sequence that forms a disulfide with said first conserved cysteine, and has at least one TANGO 457 biological activity as described herein.

A cDNA encoding human TANGO 457 was identified by analyzing the sequences of clones present in a human uterine smooth muscle library for sequences that encode wholly secreted or transmembrane proteins. This analysis led to the identification of a clone, jthUa027hl2, encoding human TANGO 457. The human TANGO 457 cDNA of this clone is 2330 nucleotides long (Figure 7; SEQ ID NO: 51). The open reading frame of TANGO 457 comprises nucleotides 149 to 1243 of SEQ ID NO: 51 (SEQ ID NO: 52), and encodes a transmembrane protein comprising the 365 amino acid sequence depicted in Figure 7

(SEQ ID NO: 53).

The signal peptide prediction program SIGN ALP (Nielsen et al. (1997) Protein Engineering 10:1-6) predicted that human TANGO 457 includes a 24 amino acid signal peptide (amino acids 1 to about amino acid 24 of SEQ ID NO: 53; SEQ ID NO: 55) preceding the mature TANGO 457 protein (corresponding to about amino acid 25 to amino acid 365 of SEQ

ID NO: 53)(SEQ ID NO: 54). Human TANGO 213 is predicted to have a molecular weight of approximately 40.6 kDa prior to cleavage of its signal peptide and a molecular weight of approximately 38.0 kDa subsequent to cleavage of its signal peptide.

Secretion assays indicate that the polypeptide encoded by human TANGO 457 is not secreted and thus, likely a transmembrane protein. The secretion assays were performed essentially as follows: 8x10 293T cells were plated per well in a 6-well plate and the cells were incubated in growth medium (DMEM, 10% fetal bovine serum, penicillin/ streptomycin) at 37°C, 5% CO2 overnight. 293 T cells were transfected with 2 micrograms of full-length TANGO 457 inserted in the pMET7 vector/well and 10 micrograms LipofectAMINE (GIBCO/BRL Cat. # 18324-012) /well according to the protocol for GIBCO/BRL

LipofectAMINE. The transfectant was removed 5 hours later and fresh growth medium was added to allow the cells to recover overnight. The medium was removed and each well was gently washed twice with DMEM without methionine and cysteine (ICN Cat. # 16-424-54).

35 Next, 1 ml DMEM without methionine and cysteine with 50 microcuries Trans- S (ICN Cat. # 51006) was added to each well and the cells were incubated at 37°C, 5% CO2 for the appropriate time period. A 150 microliters aliquot of conditioned medium was obtained and 150 microliters of 2x SDS sample buffer was added to the aliquot. The sample was heat- inactivated and loaded on a 4-20% SDS-PAGE gel. The gel was fixed and the presence of secreted protein was detected by autoradiography. Clone 457, which encodes human TANGO 457, was deposited with the

American Type Culture Collection (10801 University Boulevard, Manassas, VA 20110-2209) on October 1, 1999 and assigned PTA-817. This deposit will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. This deposit was made merely as a convenience for those of skill in the art and is not an admission that a deposit is required under 35 U.S.C. §112.

Figure 8 depicts a hydropathy plot of the human TANGO 457 amino acid sequence shown in Figure 7. Relatively hydrophobic regions of the protein are shown above the horizontal line, and relatively hydrophilic regions of the protein are below the horizontal line. The cysteine residues (cys) and N-glycosylation site are indicated by short vertical lines just below the hydropathy trace.

In one embodiment, a TANGO 457 protein contains a cytoplasmic domain at about amino acid residues 1 to 264 of SEQ ID NO: 53 (SEQ ID NO: 56), a transmembrane domain at about amino acid residues 265 to 282 of SEQ ID NO: 53 (SEQ ID NO: 59), and an extracellular domain at about amino acid residues 283 to 365 of SEQ ID NO: 53 (SEQ ID NO: 60). In another embodiment, a human TANGO 457 protein contains an cytoplasmic domain at amino acid residues 283 to 365 of SEQ ID NO: 53 ( SEQ ID NO: 60), a transmembrane domain at amino acid residues 265 to 282 of SEQ ID NO: 53 (SEQ ID NO: 59), and an extracellular domain at amino acid residues 1 to 264 of SEQ ID NO: 53 ( SEQ ID NO: 56).

Human TANGO 457 includes an Ig domain at amino acids 41 to 124 and 163 to 230 of SEQ ID NO: 53, (SEQ ID NO: 57 and 8).

Seven N-glycosylation sites are present in TANGO 457. The first has the sequence NVTI (at amino acid residues 43 to 46 of SEQ ID NO: 53), the second has the sequence NITS (at amino acid residues 57 to 60 of SEQ ID NO: 53), the third has the sequence NITW (at amino acid residues 174 to 177 of SEQ ID NO: 53), the fourth has the sequence NVTS (at amino acid residues 208 to 211 of SEQ ID NO: 53), the fifth has the sequence NSSQ (at amino acid residues 216 to 219 of SEQ ID NO: 53), the sixth has the sequence NFTL (at amino acid residues 242 to 245 of SEQ ID NO: 53), and the seventh has the sequence NFSI (at amino acid residues 260 to 263 of SEQ ID NO: 53). TANGO 457 has one glycosaminoglycan attachment site with the sequence SGVG at amino acid residues 331 to 334 of SEQ ID NO: 53. Six protein kinase C phosphorylation sites are present in TANGO 457. The first has the sequence TWR (at amino acid residues 2 to 4 of SEQ ID NO: 53), the second has the sequence SLR (at amino acid residues 106 to 108 of SEQ ID NO: 53), the third has the sequence TQK (at amino acid residues 181 to 183 of SEQ ID NO: 53), the fourth has the sequence TIK (at amino acid residues 199 to 201 of SEQ ID NO: 53), the fifth has the sequence TEK (at amino acid residues 255 to 257 of SEQ ID NO: 53), and the sixth has the sequence SKK (at amino acid residues 301 to 303 of SEQ ID NO: 53). TANGO 457 has three casein kinase II phosphorylation sites. The first has the sequence TEGD (at amino acid residues 22 to 25 of SEQ ID NO: 53), the second has the sequence SSQE (at amino acid residues 217 to 220 of SEQ ID NO: 53), and the third has the sequence SLSE (at amino acid residues 251 to 254 of SEQ ID NO: 53). TANGO 457 has one tyrosine kinase phosphorylation site with the sequence

KENEDKY at amino acid residues 155 to 161 of SEQ ID NO: 53. Two N-myristoylation sites are present in TANGO 457. The first has the sequence GMKENE (at amino acid residues 153 to 158 of SEQ ID NO: 53) and the second has the sequence GNVGCV (at amino acid residues 334 to 339 of SEQ ID NO: 53). Lastly, TANGO 457 has an immunoglobulin and major histocompatibility complex protein site with the sequence YQCVVRH at amino acid residues 226 to 232 of SEQ ID NO: 53.

Figure 9 depicts a local alignment of the nucleic acid of human TANGO 457 shown in SEQ ID NO: 51 and a portion of the nucleotide sequence of human chromosome 1 lpl4.3 PAC clone pDJ239b22, from nucleic acids 121077 to 122478 (SEQ ID NO: 61; AC003969). The alignment shows that there is a 100% nucleotide sequence identity between the TANGO 457 sequence of SEQ ID NO: 51 and human chromosome 1 lpl4.3 PAC clone pDJ239b22, over the specified region. Genes known to map to the pl4 region of human chromosome 11 include those encoding fetal brain protein 239 and hepatitis B virus integration site-1 (see httpJ/www.ncbi.nlm.nih.gov/htbin-post/Omim/getmap?d3076). Expressed sequence tags (ESTs) which exhibit homology to TANGO 457 (SEQ ID NO: 51) have been isolated from a B-cell leukemia cell line and from fetal liver, fetal spleen, and placenta tissues. The dbEST accession numbers of these ESTs are AI361759, AA004711, AA004711, and AI189960, respectively. TANGO 457 (SEQ ID NO: 51) exhibits about 80% homology to AI361759 over about 445 base pairs, from nucleotides 1861 to 2306 of TANGO 457. TANGO 457 (SEQ ID NO: 51) exhibits about 77% homology to AA004711 over about 375 base pairs, from nucleotides 1830 to 2205 of TANGO 457. TANGO 457 (SEQ ID NO: 51) exhibits about 81% homology to AI189960 over about 415 base pairs, from nucleotides 1908 to 2320.

Uses of TANGO 457 Nucleic acids, Polypeptides, and Modulators Thereof

TANGO 457 proteins are involved in disorders which affect both tissues in which they are normally expressed and tissues in which they are normally not expressed. Based on the observations that cDNA corresponding to TANGO 457 occurs in a uterine smooth muscle cDNA library, bears homology to human chromosome 1 lpl4.3 PAC clone pDJ239b22, and bears homology to ESTs isolated from B-cell leukemia, liver, spleen, and placenta libraries, it is evident that TANGO 457 protein is involved in one or more biological processes which occur in these tissues. In particular, TANGO 457 is involved in modulating proliferation, migration, morphology, differentiation, and/or function of cells of these tissues. Relevant disorders which involve these tissues are discussed separately below.

As TANGO 457 was originally found in a uterine smooth muscle library, TANGO 457 polypeptides, nucleic acids, or modulators thereof, can be used to modulate the proliferation, migration, morphology, differentiation, and/or function of cells that form the uterus, e.g., endometrium endothelial cells and mesometrium smooth muscle cells, and thus to treat uterine disorders such as, e.g., hyperplasia of the endometrium, dysfunctional uterine bleeding (DUB), and uterine cancers (e.g., uterine leiomyomoma, uterine cellular leiomyoma, leiomyosarcoma of the uterus, malignant mixed mullerian tumor of uterus, uterine sarcoma). TANGO 457 polypeptides, nucleic acids, or modulators thereof can also be used to treat other reproductive disorders, including ovulation disorder, blockage of the fallopian tubes (e.g., due to pelvic inflammatory disease or endometriosis), disorders due to infections (e.g., toxic shock syndrome, chlamydia infection, Herpes infection, human papillomaviras infection), and ovarian disorders (e.g., ovarian endometriosis and ovarian cancers (e.g., ovarian fibroma, ovarian teratoma)). As TANGO 457 bears homology to regions of human chromosome 1 lpl4.3

PAC clone pDJ239b22, and since human chromosome 1 lpl4.3 is the location to which such genes as those encoding fetal brain protein 239 and hepatitis B virus integration site-1 are known to map, TANGO 457 nucleic acids, proteins, and modulators thereof can be used to modulate or treat disorders associated with hepatitis B infection (e.g., hepatitis, e.g., hepatitis B, and hepatocellular carcinomas) as well as CNS related disorders. Such CNS related disorders include but are not limited to bacterial and viral meningitis, Alzheimer's Disease, Huntington's disease, cerebral toxoplasmosis, Parkinson's disease, multiple sclerosis, brain cancers (e.g., cancers that have metastasized from other tissues (e.g., metastatic carcinoma of the brain), cancers of the supportive tissue of the brain (the glia; e.g., glioblastoma and astrocytoma), and cancers of other neural tissues (e.g., acoustic neuroma)), hydrocephalus, and encephalitis.

TANGO 457 is also expressed in the fetal liver, TANGO 457 nucleic acids, proteins, and modulators thereof can be used to modulate the proliferation, migration, morphology, differentiation, and/or function of cells that form the liver, e.g., hepatocytes, and thus to treat hepatic (liver) disorders, such as jaundice, hepatic failure, hereditary hyperbiliruinemias (e.g., Gilbert's syndrome, Crigler-Naijar syndromes and Dubin- Johnson and

Rotor's syndromes), hepatic circulatory disorders (e.g., hepatic vein thrombosis and portal vein obstruction and thrombosis), hepatitis (e.g., chronic active hepatitis, acute viral hepatitis, and toxic and drag-induced hepatitis), cirrhosis (e.g., alcoholic cirrhosis, biliary cirrhosis, and hemochromatosis), and malignant tumors (e.g., primary carcinoma, hepatoblastoma, and angiosarcoma).

In addition, TANGO 457 is expressed in the fetal spleen, TANGO 457 nucleic acids, proteins, and modulators thereof can be used to modulate the proliferation, migration, morphology, differentiation, and/or function of cells that form the spleen, e.g., cells of the splenic connective tissue, e.g., splenic smooth muscle cells and/or endothelial cells of the splenic blood vessels. TANGO 457 nucleic acids, proteins, and modulators thereof can also be used to modulate the proliferation, differentiation, and/or function of cells that are processed, e.g., regenerated or phagocytized within the spleen, e.g., erythrocytes and/or B and T lymphocytes and macrophages. Thus TANGO 457 nucleic acids, proteins, and modulators thereof can be used to treat spleen, e.g., the fetal spleen, associated diseases and disorders. Examples of splenic diseases and disorders include e.g., splenic lymphoma and/or splenomegaly, and/or phagocytotic disorders, e.g., those inhibiting macrophage engulfment of bacteria and viruses in the bloodstream.

As both fetal spleen and fetal liver are sites of hematopoiesis, TANGO 457 nucleic acids, proteins, and modulators thereof can also be used to modulate the proliferation, migration, morphology, differentiation, and/or function of hematopoietic cells, e.g., pluripotential stem cells (e.g., lymphoid cells and myeloid cells), and can be used to treat hematological disorders.

Hematological disorders include, but are not limited to, disorders associated with abnormal differentiation or hematopoiesis, morphology, migration, proliferation, or function of blood cells derived, for example, from myeloid multipotential cells in bone marrow, such as megakaryocytes (and ultimately platelets), monocytes, erythrocytes, and granulocytes (e.g., neutrophils, eosinophils, and basophils) and from lymphoid multipotential cells, such as T and B lymphocytes.

Platelet associated disorders include, but are not limited to, thrombocytopenia due to a reduced number of megakaryocytes in the bone marrow, for example, as a result of chemotherapy; invasive disorders, such as leukemia, idiopathic or drag- or toxin-induced aplasia of the marrow, or rare hereditary amegakaryocytic thrombocytopenias; ineffective thrombopoiesis, for example, as a result of megaloblastic anemia, alcohol toxicity, vitamin B12 or folate deficiency, myelodysplastic disorders, or rare hereditary disorders (e.g., Wiskott- Aldrich syndrome and May-hegglin anomaly); a reduction in platelet distribution, for example, as a result of cirrhosis, a splenic invasive disease (e.g., Gaucher's disease), or myelofibrosis with extramedullary myeloid metaplasia; increased platelet destruction, for example, as a result of removal of IgG-coated platelets by the mononuclear phagocytic system (e.g., idiopathic thrombocytopenic purpura (ITP), secondary immune thrombocytopenia (e.g., systemic lupus erythematosus, lymphoma, or chronic lymphocytic leukemia), drug-related immune thrombocytopenias (e.g., as with quinidine, aspirin, and heparin), post-transfusion purpura, and neonatal thrombocytopenia as a result of maternal platelet autoantibodies or maternal platelet alloantibodies). Also included are thrombocytopenia secondary to intravascular clotting and thrombin induced damage to platelets as a result of, for example, obstetric complications, metastatic tumors, severe gram-negative bacteremia, thrombotic thrombocytopenic purpura, or severe illness. Also included is dilutional thrombocytopenia, for example, due to massive hemorrhage.

Platelet associated disorders also include, but are not limited to, essential thrombocytosis and thrombocytosis associated with, for example, splenectomy, acute or chronic inflammatory diseases, hemolytic anemia, carcinoma, Hodgkin's disease, lymphoproliferative disorders, and malignant lymphomas.

Erythrocyte associated disorders include anemias such as, for example, hemolytic anemias due to hereditary cell membrane abnormalities, such as hereditary spherocytosis, hereditary elliptocytosis, and hereditary pyropoikilocytosis; hemolytic anemias due to acquired cell membrane defects, such as paroxysmal nocturnal hemoglobinuria and spur cell anemia; hemolytic anemias caused by antibody reactions, for example to the RBC antigens, or antigens of the ABO system, Lewis system, Ii system, Rh system, Kidd system, Duffy system, and Kell system; methemoglobinemia; a failure of erythropoiesis, for example, as a result of aplastic anemia, pure red cell aplasia, myelodysplastic syndromes, sideroblastic anemias, and congenital dyserythropoietic anemia; secondary anemia in non-hematolic disorders, for example, as a result of chemotherapy, alcoholism, or liver disease; anemia of chronic disease, such as chronic renal failure; and endocrine deficiency diseases.

Other erythrocyte associated disorders include polycythemias such as, for example, polycythemia vera, secondary polycythemia, and relative polycythemia. Neutrophil associated disorders include neutropenias that result from or accompany a number of conditions, including, but not limited to, chemotherapy; chronic idiopathic neutropenia; Felty's syndrome, acute infectious disease, lymphoma or aleukemic lymphocytic leukemia, myelodysplastic syndrome, and rheumatic diseases such as systemic lupus erythematosus, rheumatoid arthritis, and polymyositis. Also included is neutrophilia, for example, accompanying chronic myelogenous leukemia. Other hematological disorders include disorders associated with abnormal monocyte and/or macrophage function, such as impaired phagocytosis, chemotaxis, or secretion of cytokines, growth factors and acute-phase reactants, resulting from certain diseases, e.g., lysosomal storage diseases (e.g., Gaucher's disease); impaired monocyte cytokine production, for example, found in some patients with disseminated non-tuberculous mycobacterial infection who are not infected with HIV; leukocyte adhesion deficiency (LAD), hyperimmunoglobulin E- recurrent infection (HIE) or Job's syndrome, Chediak-Higashi syndrome (CHS), and chronic granulomatous diseases (CGD), certain autoimmune diseases, such as systemic lupus erythematosus and other autoimmune diseases characterized by tissue deposition of immune complexes, as seen in Sjogren's syndrome, mixed cryoglobulinemia, dermatitis herpetiformis, and chronic progressive multiple sclerosis. Also included are disorders or infections that impair mononuclear phagocyte function, for example, influenza virus infection and AIDS.

Monocyte associated disorders include monocytoses such as, for example, monocytoses associated with certain infections such as tuberculosis, brucellosis, subacute bacterial endocarditis, Rocky Mountain spotted fever, malaria, and visceral leishmaniasis (kala azar), in malignancies, leukemias, myeloproliferative syndromes, hemolytic anemias, chronic idiopathic neutropenias, and granulomatous diseases such as sarcoidosis, regional enteritis, and some collagen vascular diseases.

Other monocyte associated disorders include monocytopenias such as, for example, monocytopenias that can occur with acute infections, with stress, following administration of glucocorticoids, aplastic anemia, hairy cell leukemia, and acute myelogenous leukemia and as a direct result of administration of myelotoxic and immunosuppressive drugs.

Eosinophil associated disorders include eosinphilias such as, for example, eosinphilias that result from or accompany conditions such as allergic disorders, infections caused by parasites and other organisms, dermatologic diseases, pulmonary diseases, collagen vascular disease, neoplasms, immunodeficiency diseases, gastroenteritis, inflammatory bowel disease, chronic active hepatitis, pancreatitis, and hypopituitarism. Also included are hypereosinophilic syndrome (HES) and chronic and acute eosinophilic leukemias. Other eosinophil associated disorders include eosinopenias such as, for example, eosinopenias that occur with stress, such as acute bacterial infection, and following administration of glucocorticoids.

Basophil associated disorders include basophilias such as, for example, basophilias seen in myeloproliferative disorders (e.g., chronic myeloid leukemia, polycythemia vera, and myeloid metaplasia), following splenectomy, hemolytic anemia, ulcerative colitis, varicella infection, and Hodgkin's disease.

As TANGO 457 is expressed in the placenta, TANGO 457 nucleic acids, proteins, and modulators thereof can be used to modulate the proliferation, migration, morphology, differentiation, and/or function of cells that form the placenta, e.g., the decidual cells (which arise during pregnancy), and thus can be used to treat placental disorders, such as toxemia of pregnancy (e.g., preeclampsia and eclampsia), placentitis, or spontaneous abortion.

As TANGO 457 is expressed in B-cell, chronic lymphotic leukemia, TANGO 457 nucleic acids, proteins, and modulators thereof can be used to modulate the proliferation, migration, morphology, differentiation, and/or function of immune cells, e.g. B-cells, dendritic cells, natural killer cells and monocytes. TANGO 457 nucleic acids, proteins and modulators thereof can also be utilized to modulate immunoglobulins and formation of antibodies, and immune-related processes, e.g., the host immune response.

Such TANGO 457 compositions and modulators thereof can be utilized modulate or treat immune disorders that include, but are not limited to, immune proliferative disorders (e.g., carcinoma, lymphoma, e.g., follicular lymphoma), disorders associated with fighting pathogenic infections (e.g., bacterial (e.g., chlamydia) infection, parasitic infection, and viral infection (e.g., HSV or HIV infection)), pathogenic disorders associated with immune disorders (e.g., immunodeficiency disorders, such as HIV), autoimmune disorders (e.g., such as arthritis, graft rejection (e.g., allograft rejection), multiple sclerosis, Grave's disease, and

Hashimoto's disease), T cell disorders (e.g., AIDS), and inflammatory disorders (e.g., septicemia, cerebral malaria, inflammatory bowel disease, arthritis (e.g., rheumatoid arthritis, osteoarthritis), allergic inflammatory disorders (e.g., asthma, psoriasis), apoptotic disorders (e.g., rheumatoid arthritis, systemic lupus erythematosus, insulin-dependent diabetes mellitus), cytotoxic disorders, septic shock, and cachexia). As TANGO 457 contains one or more Ig domains, and as immunoglobulin superfamily proteins are cell surface molecules involved in signal transduction and cellular proliferation, TANGO 457 nucleic acids, proteins and modulators thereof can be utilized to modulate the development and progression of cancerous and non-cancerous cell proliferative disorders, such as deregulated proliferation (such as hyperdysplasia, hyper-IgM syndrome, or lymphoproliferative disorders), cirrhosis of the liver (a condition in which scarring has overtaken normal liver regeneration processes), treatment of keloid (hypertrophic scar) formation (disfiguring of the skin in which the scarring process interferes with normal renewal), psoriasis (a common skin condition characterized by excessive proliferation of the skin and delay in proper cell fate determination), benign tumors, fibrocystic conditions, and tissue hypertrophy (e.g., prostatic hyperplasia), cancers such as neoplasms or tumors (such as carcinomas, sarcomas, adenomas or myeloid lymphoma tumors, e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leimyosarcoma, rhabdotheliosarcoma, colon sarcoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hematoma, bile duct carcinoma, melanoma, choriocarcinoma, semicoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, small cell carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependynoma, pinealoma, hemangioblastoma, retinoblastoma), leukemias, (e.g. acute lymphocytic leukemia), acute myelocytic leukemia (myelolastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia), chronic leukemias (chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia), or polycythemia vera, or lymphomas (Hodgkin's disease and non- Hodgkin's diseases), multiple myelomas and Waldenstrδm's macroglobulinemia.

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 single-stranded or double-stranded.

An "isolated" nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in 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) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated nucleic acid molecule can contain less than about 5, 4, 3, 2, 1, 0.5, or 0.1 kilobases of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. Moreover, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.

A nucleic acid molecule of the present invention, e.g., a nucleic acid molecule having the nucleotide sequence of all or a portion of any of SEQ ID NOs: 1 , 2, 31 , 32, 51 , and 52, the TANGO 416 nucleotide sequence of the cDNA insert of a clone deposited on April 26, 1999 with the ATCC as accession no. PTA-1764, the TANGO 457 nucleotide sequence of the cDNA insert of a clone deposited on October 1, 1999 with the ATCC as PTA-817, or a complement thereof, or which has a nucleotide sequence comprising one of these sequences, 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 any of SEQ ID NOs: 1, 2, 31, 32, 51, and 52, the TANGO 416 nucleotide sequence of the cDNA insert of a clone deposited on April 26, 1999 with the ATCC as accession no. PTA- 1764, or the TANGO 457 nucleotide sequence of the cDNA insert of a clone deposited on October 1, 1999 with the ATCC as PTA-817 as a hybridization probe, nucleic acid molecules of the invention can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook et al., Eds., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). A nucleic acid molecule of the invention 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 by standard synthetic techniques, e.g., using an automated DNA synthesizer.

In another embodiment, an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule which is a complement of the nucleotide sequence of any of SEQ ID NOs: 1, 2, 31, 32, 51, and 52, the TANGO 416 nucleotide sequence of the cDNA insert of a clone deposited on April 26, 1999 with the ATCC as accession no. PTA-1764, the TANGO 457 nucleotide sequence of the cDNA insert of a clone deposited on October 1 , 1999 with the ATCC as PTA-817, 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 with the given nucleotide sequence thereby forming a stable duplex. Moreover, a nucleic acid molecule of the invention can comprise a portion of a nucleic acid sequence encoding a full length polypeptide of the invention, such as 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 cloning one gene allows generation of probes and primers designed for identifying and or cloning homologs in other cell types, e.g., from other tissues, as well as homologs 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 with at least about 15, preferably about 25, more preferably about 40, 60, 80, 100, 150, 200, 250, 300, 350, 400, 450, 550, 650, 700, 800, 900, 1000, 1200, 1400, 1410, 1600, 1800, 2000, 2200, 2400, 2600, 2800, 3000, 3500, 4000, 4500, 5000, or 5121 or more consecutive nucleotides of the sense or anti-sense sequence of any of SEQ ID NOs: 1, 2, 31, 32, 51, and 52, the TANGO 416 nucleotide sequence of the cDNA insert of a clone deposited on April 26, 1999 with the ATCC as accession no. PTA-1764, the TANGO 457 nucleotide sequence of the cDNA insert of a clone deposited on October 1, 1999 with the ATCC as PTA-817, or of a naturally occurring mutant of any of these sequences.

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 aberrantly express the protein, such as by measuring levels of a nucleic acid molecule encoding the protein in 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 either of SEQ ID NOs: 2, 32, and 52, expressing the encoded portion of the polypeptide protein (e.g., by recombinant expression in vitro), 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 any of SEQ ID NOs: 1 , 2, 31 , 32, 51 , and 52, the TANGO 416 nucleotide sequence of the cDNA insert of a clone deposited on April 26, 1999 with the

ATCC as accession no. PTA-1764, or the TANGO 457 nucleotide sequence of the cDNA insert of a clone deposited on October 1, 1999 with the ATCC as PTA-817, due to degeneracy of the genetic code and thus encode the same protein as that encoded by the nucleotide sequence of either of SEQ ID NOs: 2, 32, and 52. In addition to the nucleotide sequences of either of SEQ ID NOs: 2, 32, and 52, it will be appreciated by those skilled in the art that DNA sequence polymorphisms that lead to changes in the amino acid sequence can exist within a population (e.g., the human population). Such genetic polymorphisms can exist among individuals within a population due to natural allelic variation. An allele is one of a group of genes which occur alternatively at a given genetic locus.

To determine the percent identity of two amino acid sequences or of two nucleic acids, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in 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., % identify = # of identical positions / total # of positions

{e.g., overlapping positions} x 100). In one embodiment, the two sequences are the same length.

The determination of percent identity 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 Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin 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 amino acid sequences homologous to a protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic 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-limiting example of a mathematical algorithm utilized for the comparison of sequences is the local homology algorithm of Smith and Waterman

(Advances in Applied Mathematics 2: 482-489 (1981)). Such an algorithm is incorporated into the BestFit program, which is part of the Wisconsin™ package, and is used to find the best segment of similarity between two sequences. BestFit reads a scoring matrix that contains values for every possible GCG symbol match. The program uses these values to construct a path matrix that represents the entire surface of comparison with a score at every position for the best possible alignment to that point. The quality score for the best alignment to any point is equal to the sum of the scoring matrix values of the matches in that alignment, less the gap creation penalty multiplied by the number of gaps in that alignment, less the gap extension penalty multiplied by the total length of all gaps in that alignment. The gap creation and gap extension penalties are set by the user. If the best path to any point has a negative value, a zero is put in that position.

After the path matrix is complete, the highest value on the surface of comparison represents the end of the best region of similarity between the sequences. The best path from this highest value backwards to the point where the values revert to zero is the alignment shown by BestFit. This alignment is the best segment of similarity between the two sequences.

Further documentation can be found at http://ir.ucdavis.edu/GCGhelp/bestfιt.html#algorithm.

Additional algorithms for sequence analysis are known in the art and include ADVANCE and ADAM as described in Torellis and Robotti (1994) Comput. Appl. Biosci., 10:3-5; and FASTA described in Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 85:2444-8. Within FASTA, ktup is a control option that sets the sensitivity and speed of the search. If ktup=2, similar regions in the two sequences being compared are found by looking at pairs of aligned residues; if ktup=l, single aligned amino acids are examined, ktup can be set to 2 or 1 for protein sequences, or from 1 to 6 for DNA sequences. The default if ktup is not specified is 2 for proteins and 6 for DNA. For a further description of FASTA parameters, see http://bioweb.pasteur.fr/docs/man/man/fasta. I.html#sect2, the contents of which are incorporated herein by reference.

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, typically exact matches are counted.

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 phrase "allelic variant" refers to a nucleotide sequence which occurs at a given locus or to a polypeptide encoded by the nucleotide sequence. For example, the TANGO 416 gene maps to a region of chromosome 4 between chromosomal markers D4S422 and D4S 1576, and allelic variants of TANGO 416 can be identified by sequencing the TANGO 416 gene at this location in multiple individuals.

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 in 1-5% variance in the nucleotide sequence of a given gene. Alternative alleles can be identified by sequencing the gene of interest in a number of different individuals. This can be readily carried out by using hybridization probes to identify the same genetic locus in 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 (homologs), which have a nucleotide sequence which differs from that of the human proteins described herein are within the scope of the invention. Nucleic acid molecules corresponding to natural allelic variants and homologs of a cDNA of the invention can be isolated based on their identity to human nucleic acid molecules using the human cDNAs, 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 can be isolated based on its hybridization with 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 with a nucleic acid molecule encoding all or part of the soluble form.

Accordingly, in another embodiment, an isolated nucleic acid molecule of the invention is at least 15 (25, 40, 60, 80, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, 2000, 2200, 2400, 2600, 2800, 3000, 3500, 4000,

4500, 5000, or more) nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising the nucleotide sequence, preferably the coding sequence, of any of SEQ ID NOs: 1, 2, 31, 32, 51, and 52, the TANGO 416 nucleotide sequence of the cDNA insert of a clone deposited on April 26, 1999 with the ATCC as accession no. PTA- 1764, the TANGO 457 nucleotide sequence of the cDNA insert of a clone deposited on

October 1, 1999 with the ATCC as PTA-817, 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 other typically remain hybridized with each other. Such stringent conditions are known to those skilled in the art and can be found in Current

Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. A example of stringent hybridization conditions are hybridization in 6x sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2x 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 any of SEQ ID NOs: 1 , 2, 31 , 32, 51 , and 52, the TANGO 416 nucleotide sequence of the cDNA insert of a clone deposited on April 26, 1999 with the ATCC as accession no. PTA-1764, the TANGO 457 nucleotide sequence of the cDNA insert of a clone deposited on October 1, 1999 with the ATCC as PTA-817, or a 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 in nature (e.g., encodes a natural protein).

In addition to naturally-occurring allelic variants of a nucleic acid molecule of the invention sequence that can exist in the population, the skilled artisan will further appreciate that changes can be introduced by mutation thereby leading to changes in the amino acid sequence of the encoded protein, without altering the biological activity of the protein. For example, one can make nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues. A "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence without altering the biological activity, whereas an "essential" amino acid residue is required for biological activity. For example, amino acid residues that are not conserved or only semi-conserved among homologs of various species may be non-essential for activity and thus would be likely targets for alteration. Alternatively, amino acid residues that are conserved among the homologs 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 in amino acid residues that are not essential for activity. Such polypeptides differ in amino acid sequence from any of SEQ ID NOs: 3-8, 33, 35, 38, and 53-60, yet retain biological activity. In one embodiment, the isolated nucleic acid molecule includes a nucleotide sequence encoding a protein that includes an amino acid sequence that is at least about 40% identical, 50%, 60%,

70%, 80%, 90%, 95%, or 98% identical to the amino acid sequence of any of SEQ ID NOs: 3-8, 33, 35, 38, and 53-60, or the amino acid sequence encoded by the TANGO 416 nucleotide sequence of the cDNA insert of a clone deposited on April 26, 1999 with the ATCC as accession no. PTA-1764, or the TANGO 457 nucleotide sequence of the cDNA insert of a clone deposited on October 1 , 1999 with the ATCC as PTA-817.

An isolated nucleic acid molecule encoding a variant protein can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of any of SEQ ID NOs: 1, 2, 31, 32, 51, and 52, the TANGO 416 nucleotide sequence of the cDNA insert of a clone deposited on April 26, 1999 with the ATCC as accession no. PTA-1764, or the TANGO 457 nucleotide sequence of the cDNA insert of a clone deposited on October 1, 1999 with the ATCC as PTA-817, such that one or more amino acid residue 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 amino acid substitutions are made at one or more predicted non-essential amino acid residues. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). 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 recombinantly and the activity of the protein can be determined.

In one embodiment, a mutant polypeptide that is a variant of a polypeptide of the invention can be assayed for: (1) the ability to form proteimprotein interactions with the polypeptide of the invention; (2) the ability to bind a ligand of the polypeptide of the invention; (3) the ability to bind with a modulator or substrate of the polypeptide of the invention; (4) the ability to modulate a physiological activity of the protein, such as one of those disclosed herein; (5) ability to modulate cytokine production, release, or both, by cells comprising integrin αEβ7; (6) ability to modulate binding of cells comprising integrin αEβ7 to an endothelial tissue (e.g. intestinal, lung, or other mucosal endothelium); (7) ability to interact with a TANGO 457 receptor; (8) ability to modulate proliferation, migration, morphology, differentiation, or function of hematopoietic, immune, spleen or placental cells; (9) ability to modulate development or progression of a cell proliferative disorder.

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 non-coding region of the coding strand of a nucleotide sequence encoding a polypeptide of the invention. The non-coding regions ("5' and 3' non-translated regions") are the 5' and 3' sequences which flank the coding region and are not translated into amino acids.

An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 or more nucleotides in length. An antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in 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-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl- 2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, Ng-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2- methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7- methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D- mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6- isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2- thiocytosine, 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 sub-cloned in 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 situ such that they hybridize with or bind with 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, in the case of an antisense nucleic acid molecule which binds with 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 with receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies which bind with 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 in 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 -anomeric nucleic acid molecule. An α-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which 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-methylribonucleotide (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 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 as described in Haselhoff and Gerlach (1988) Nature 334:585-591) can be used to catalytically cleave mRNA transcripts to thereby inhibit translation of the protein encoded by the mRNA.

A ribozyme having specificity for a nucleic acid molecule encoding a polypeptide of the invention can be designed based upon the nucleotide sequence of a cDNA disclosed herein. For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the ribozyme active site is complementary to the nucleotide sequence to be cleaved, as described in 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 includes 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 in target cells. See generally Helene (1991) Anticancer Drag Des. 6(6):569-84; Helene (1992) Ann. N.Y. Acad. Sci. 660:27-36; and Maher (1992) Bioassays 14(12):807-15. "Expression" of a polypeptide, as used herein, refers individually and collectively to the processes of transcription of DNA to generate an RNA transcript and translation of an RNA to generate the polypeptide.

In various embodiments, the nucleic acid molecules of the invention 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 deoxyribose phosphate backbone of the nucleic acids can be modified to generate peptide nucleic acids (see Hyrap 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, in which the deoxyribose 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 specific hybridization with DNA and RNA under conditions of low ionic strength. Synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols such as those described in 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 anti-gene agents for sequence-specific modulation of gene expression by, e.g., inducing arrest of transcription or translation or by 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 (Hyrap (1996), supra; or as probes or primers for DNA sequence and hybridization (Hyrap (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 formation of PNA-DNA chimeras, or by use of liposomes or other techniques of drag delivery known in the art. For example, PNA-DNA chimeras can be generated which can 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 provides 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 (Hyrap (1996), supra). The synthesis of PNA-DNA chimeras can be performed as described in Hyrap (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-methoxytrityl)amino-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 in a step-wise manner to produce a chimeric molecule with a 5' PNA segment and a 3' DNA segment (Finn et al. (1996) Nucleic 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 can include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger 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. WO 88/09810) or the blood-brain barrier (see, e.g., PCT Publication No. WO 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 can be conjugated with 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 biologically active portions thereof, as well as polypeptide fragments suitable for use as immunogens to generate antibodies directed against a polypeptide of the invention. In one embodiment, the native polypeptide is 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 by recombinant DNA techniques. As an 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 in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly 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 recombinantly 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 in the synthesis of the protein. Accordingly such preparations of the protein have less than about 30%, 20%, 10%o, 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 polypeptide regions having an amino acid sequence sufficiently identical to or derived from the amino acid sequence of the protein (e.g., the amino acid sequence shown in any of SEQ ID NOs: 3-8, 33, 35, 38, and 53-60, or the amino acid sequence encoded by the TANGO 416 nucleotide sequence of the cDNA insert of a clone deposited on April 26, 1999 with the ATCC as accession no. PTA-1764, or the TANGO 457 nucleotide sequence of the cDNA insert of a clone deposited on October 1 , 1999 with the ATCC as PTA-817), 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.

Examples of polypeptides have the amino acid sequence of any of SEQ ID NOs: 3-8, 33, 35, 38, and 53-60 or the amino acid sequence encoded by the TANGO 416 nucleotide sequence of the cDNA insert of a clone deposited on April 26, 1999 with the ATCC as accession no. PTA-1764, or the TANGO 457 nucleotide sequence of the cDNA insert of a clone deposited on October 1, 1999 with the ATCC as PTA-817. Other useful proteins are substantially identical (e.g., at least about 40%, preferably 50%, 60%, 70%, 80%, 90%, 95%, or 99%) to any of SEQ ID NOs: 3-8, 33, 35, 38, and 53-60 or the amino acid sequence encoded by the TANGO 416 nucleotide sequence of the cDNA insert of a clone deposited on April 26, 1999 with the ATCC as accession no. PTA-1764, or the TANGO 457 nucleotide sequence of the cDNA insert of a clone deposited on October 1, 1999 with the ATCC as PTA-817 and retain the functional activity of the protein of the corresponding naturally-occurring protein. Such proteins can differ in amino acid sequence owing, for example, to natural allelic variation or mutagenesis.

The invention 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 with a heterologous polypeptide (i.e., a polypeptide other than the same polypeptide of the invention). Within the fusion protein, the term "operably linked" is intended to indicate that the polypeptide of the invention and the heterologous polypeptide are fused in-frame with each other. The heterologous polypeptide can be fused with the amino-terminus or the carboxyl-terminus of the polypeptide of the invention. One useful fusion protein is a GST fusion protein in which the polypeptide of the invention is fused with the carboxyl terminus of GST sequences. Such fusion proteins can facilitate purification of a recombinant polypeptide of the invention.

In another embodiment, the fusion protein contains a heterologous signal sequence at its amino 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 in Molecular Biology, Ausubel et al., eds., John Wiley & Sons, 1992). Other examples of eukaryotic heterologous signal sequences include the secretory sequences of melittin 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 in which all or part of a polypeptide of the invention is fused with 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 in 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 can 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 in a subject, to purify ligands and in screening assays to identify molecules which inhibit the interaction of receptors with ligands. The immunoglobulin fusion protein can, for example, comprise a portion of a polypeptide of the invention fused with the amino-terminus or the carboxyl-terminus of an immunoglobulin constant region, as disclosed in U.S. Patent No. 5,714, 147, U.S. Patent No. 5,116,964, U.S. Patent No. 5,514,582, and U.S. Patent No. 5,455,165. Chimeric and fusion proteins 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 performed using anchor primers which give rise to complementary overhangs between two consecutive gene fragments and which can subsequently be annealed and re-amplified 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 invention can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the polypeptide of the invention. A signal sequence of a polypeptide of the invention (e.g., the signal sequence in either of SEQ ID NOs: 3 and 33) can be used to facilitate secretion and isolation of the secreted protein or another protein of interest. Signal sequences are typically characterized by a core of hydrophobic amino acids which are generally cleaved from the mature protein during secretion in 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 in 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 in an expression vector with 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 with 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 amino-terminal sequences of a peptide, the nucleic acids which flank the signal sequence on its amino-terminal side are likely 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 amino 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 with 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 in 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 (e.g., 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 ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential protein sequences can be expressed 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 in 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 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, re-naturing 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 ligating the resulting fragment library into an expression vector. By this method, an expression library can be derived which encodes amino 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 in the libraries, can be used in combination with the screening assays to identify variants of a protein of the invention (Arkin 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 10 (preferably 12, 15, 20, or 30 or more) amino acid residues of the amino acid sequence of any of SEQ ID NOs: 3-8, 33, 35, 38, and 53-60 or the amino acid sequence encoded by the TANGO 416 nucleotide sequence of the cDNA insert of a clone deposited on April 26, 1999 with the ATCC as accession no. PTA-1764, or the TANGO 457 nucleotide sequence of the cDNA insert of a clone deposited on October 1, 1999 with the ATCC as PTA-817, and encompasses an epitope of the protein such that an antibody raised against the peptide forms a specific immune complex with the protein.

Examples of epitopes encompassed by the antigenic peptide are regions that are located on the surface of the protein, e.g., hydrophilic regions. Figures 3 and 8 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 (i.e., immunocompetent) subject such as a rabbit, goat, mouse, or other mammal or vertebrate. An appropriate immunogenic preparation can contain, for example, recombinantly-expressed or chemically-synthesized polypeptide. The preparation can further include an adjuvant, such as Freund's complete or incomplete adjuvant, or a similar immunostimulatory agent.

Accordingly, another aspect of the invention pertains to antibodies directed against a polypeptide of the invention. The terms "antibody" and "antibody substance" as used interchangeably herein refer 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 with a given polypeptide of the invention is a molecule which binds the polypeptide, but does not substantially bind other molecules in 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 immunoreacting 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 harvested or isolated from the subject (e.g., from the blood or serum of the subject) 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 Milstein (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 Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) or trioma techniques. The technology for producing hybridomas is well known (see generally Current Protocols in 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 supematants 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. Eats 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 SurfZAP™ Phage Display Kit, 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. Antibod. Hybridomas 3:81-85; Huse et al. (1989)

Science 246:1275-1281; Griffiths et al. (1993) EMBO J. 12:725-734.

Recombinant antibodies, such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA tecliniques, 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) Cancer 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) Science 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, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable

125 131 35 3 radioactive material include I, I, S or H. An antibody (or fragment thereof) can be conjugated to a therapeutic moiety such as a cytotoxin, a therapeutic agent, or a radioactive agent (e.g., a radioactive metal ion). Cytotoxins and cytotoxic agents include any agent that is detrimental to cells. Examples of such agents include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1- dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, and 5- fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin {formerly designated daunomycin} and doxorubicin), antibiotics (e.g., dactinomycin {formerly designated actinomycin}, bleomycin, mithramycin, and anthramycin), and anti-mitotic agents (e.g., vincristine and vinblastine).

Conjugated antibodies of the invention can be used for modifying a given biological response, the drag moiety not being limited to classical chemical therapeutic agents. For example, the drag moiety can be a protein or polypeptide possessing a desired biological activity. Such proteins include, for example, toxins such as abrin, ricin A, Pseudomonas exotoxin, or diphtheria toxin; proteins such as tumor necrosis factor, alpha- interferon, beta-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator; and biological response modifiers such as lymphokines, interleukin-1, interIeukin-2, interleukin-6, granulocyte macrophage colony stimulating factor, granulocyte colony stimulating factor, or other growth factors. Techniques for conjugating a therapeutic moiety to an antibody are well known (see, e.g., Arnon et al., 1985, "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al., Eds., Alan R. Liss, Inc. pp. 243-256; Hellstrom et al., 1987, "Antibodies For Drag Delivery", in Controlled Drag Delivery, 2nd ed., Robinson et al., Eds., Marcel Dekker, Inc., pp. 623-653; Thorpe, 1985, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al., Eds., pp. 475-506; "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al., Eds., Academic Press, pp. 303-316, 1985; and Thorpe et al.,

1982, Immunol. Rev., 62:119-158). Alternatively, an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Patent No. 4,676,980.

III. Recombinant Expression Vectors and Host Cells

Another aspect of the invention pertains to vectors, including 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 "plasmid", 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 in 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, designated expression vectors, are capable of directing expression of genes with which they are operably linked. In general, expression vectors of utility in recombinant DNA techniques are often in 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 in a form suitable for expression of the nucleic acid in 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 with the nucleic acid sequence to be expressed. Within a recombinant expression vector, "operably linked" is intended to mean that the nucleotide sequence of interest is linked with the regulatory sequence(s) in a manner which allows expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system or in 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, and the level of expression of protein desired. 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 (e.g., E. coli) or eukaryotic cells (e.g., 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 vitro, for example using T7 promoter regulatory sequences and T7 polymerase.

Expression of proteins in prokaryotes is most often carried out in 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 in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in 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 enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith 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 (A ann 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 co- expressed 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 in E. coli is to express the protein in a host bacteria having 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 such that the individual codons for each amino acid are those preferentially used in E. coli (Wada et al. (1992) Nucleic Acids Res. 20:2111-2118). Such alteration of nucleic acid sequences of the invention can be performed by standard DNA synthesis techniques.

In another embodiment, the expression vector is a yeast expression vector. Examples of vectors for expression in yeast S. cerevisiae include pYepSecl (Baldari 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 in 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 in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed (1987) Nature 329:840) and ρMT2PC (Kaufman et al. (1987) EMBO J. 6:187-195). When used in 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 in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al. (1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol. 43:235- 275), in particular promoters of T cell receptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) and immunoglobulins (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 Grass (1990) Science 249:374-379) and the α-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3:537-546).

The invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operably linked with a regulatory sequence in a manner which allows for expression (by transcription of the DNA molecule) of an RNA molecule which is antisense, relative to the mRNA encoding a polypeptide of the invention. Regulatory sequences operably linked with a nucleic acid cloned in the antisense orientation can be selected which direct continuous expression of the antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be selected which direct constitutive, tissue specific, or cell type specific expression of antisense RNA. The antisense expression vector can be in the form of a recombinant plasmid, phagemid, or attenuated viras in 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 Weintraub et al. (Reviews - Trends in 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 can occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein. A host cell can be any prokaryotic (e.g., E. coli) or eukaryotic cell (e.g., insect cells, yeast or mammalian cells).

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 art-recognized techniques for introducing foreign nucleic acid into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, and electroporation. Suitable methods for transforming or transfecting host cells can be found in 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 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) can be introduced into the host cells along with the gene of interest. Examples of selectable markers include those which confer resistance to drags, such as G418, hygromycin and methotrexate. Cells stably transfected with the introduced nucleic acid can be identified by drag selection (e.g., cells that have incorporated the selectable marker gene survive, while other cells die).

A host cell of the invention, such as a prokaryotic or eukaryotic host cell in 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, the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding a polypeptide of the invention has been introduced) in 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 be used to produce non-human transgenic animals. For example, in 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 in 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 in the genome of the mature animal, thereby directing expression of an encoded gene product in 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 a nucleic acid encoding a polypeptide of the invention (or a homologue thereof) into the male pronuclei of a fertilized oocyte (e.g., by microinjection or retroviral infection) and allowing the oocyte to develop in a pseudopregnant female foster animal. Intronic 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 with the transgene to direct expression of a polypeptide of the invention to particular cells. Methods for generating transgenic animals via embryo manipulation and microinjection, particularly animals such as mice, have become conventional in the art and are described, for example, in U.S. Patent Nos. 4,736,866 and 4,870,009, U.S. Patent No. 4,873,191 and in Hogan, Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring

Harbor, N.Y., 1986). Similar methods are used for production of other transgenic animals. A transgenic founder animal can be identified based upon the presence of the transgene in its genome and/or expression of mRNA encoding the transgene in tissues or cells of the animals. A transgenic founder animal can be used to breed additional animals carrying the transgene. Moreover, transgenic animals harboring the transgene can further be bred to other transgenic animals harboring 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 one 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 be 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 in 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 in 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 in PCT Publication Numbers 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 bacteriophage 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 (O'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 be produced according to the methods described in Wilmut et al. (1997) Nature 385:810-813 and PCT Publication Numbers 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, anti-bacterial and anti-fungal 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 in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

The invention includes methods for preparing pharmaceutical compositions for modulating the expression or activity of a polypeptide or nucleic acid of the invention.

Such methods comprise formulating a pharmaceutically acceptable carrier with an agent which modulates expression or activity of a polypeptide or nucleic acid of the invention. Such compositions can further include additional active agents. Thus, the invention further includes methods for preparing a pharmaceutical composition by formulating a pharmaceutically acceptable carrier with an agent which modulates expression or activity of a polypeptide or nucleic acid of the invention and one or more additional active compounds.

The agent which modulates expression or activity can, for example, be a small molecule. For example, such small molecules include peptides, peptidomimetics, amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, organic or inorganic compounds (i.e., including heteroorganic and organometallic compounds) having a molecular weight less than about 10,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds.

It is understood that appropriate doses of small molecule agents and protein or polypeptide agents depends upon a number of factors within the ken of the ordinarily skilled physician, veterinarian, or researcher. The dose(s) of these agents will vary, for example, depending upon the identity, size, and condition of the subject or sample being treated, further depending upon the route by which the composition is to be administered, if applicable, and the effect which the practitioner desires the agent to have upon the nucleic acid or polypeptide of the invention. Examples of doses of a small molecule include milligram or microgram amounts per kilogram of subject or sample weight (e.g., about 1 microgram per kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 5 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram). Examples of doses of a protein or polypeptide include gram, milligram or microgram amounts per kilogram of subject or sample weight (e.g., about 1 microgram per kilogram to about 5 grams per kilogram, about 100 micrograms per kilogram to about 500 milligrams per kilogram, or about 1 milligram per kilogram to about 50 milligrams per kilogram). For antibodies, examples of dosages are from about 0.1 milligram per kilogram to 100 milligrams per kilogram of body weight (generally 10 milligrams per kilogram to 20 milligrams per kilogram). If the antibody is to act in the brain, a dosage of 50 milligrams per kilogram to 100 milligrams per kilogram is usually appropriate. It is furthermore understood that appropriate doses of one of these agents depend upon the potency of the agent with respect to the expression or activity to be modulated. Such appropriate doses can be determined using the assays described herein. When one or more of these agents is to be administered to an animal (e.g., a human) in order to modulate expression or activity of a polypeptide or nucleic acid of the invention, a physician, veterinarian, or researcher can, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained. In addition, it is understood that the specific dose level for any particular animal subject will depend upon a variety of factors including the activity of the specific agent employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drag combination, and the degree of expression or activity to be modulated.

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 ethylenediamine-tetraacetic 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 using acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable 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, bacteriostatic water, Cremophor EL (BASF; Parsippany, NJ) or phosphate buffered saline (PBS). The composition should be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and should 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 polyethylene 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 in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various anti-bacterial and anti-fungal agents, for example, parabens, chlorobutanol, 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, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound (e.g., a polypeptide or antibody) in the required amount in 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 then incorporating the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, examples of methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in 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 in the fluid carrier is applied orally and swished and expectorated or swallowed.

Pharmaceutically compatible binding agents, adjuvant materials, or both, 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 alginic acid, Primogel, 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 in the 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 in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and 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 in the art.

The compounds can also be prepared in 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, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes having monoclonal antibodies incorporated therein or thereon) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811. It is especially advantageous to formulate oral or parenteral compositions in 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 in 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.

Generally, partially human antibodies and fully human antibodies have a longer half-life within 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 Craikshank et al. ((1997) J. Acquired Immune Deficiency Syndromes and Human Retro virology 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.

It is recognized that the pharmaceutical compositions and methods described herein can be used independently or in combination with one another. That is, subjects can be administered one or more of the pharmaceutical compositions, e.g., pharmaceutical compositions comprising a nucleic acid molecule or protein of the invention or a modulator thereof, subjected to one or more of the therapeutic methods described herein, or both, in temporally overlapping or non-overlapping regimens. When therapies overlap temporally, the therapies may generally occur in any order and can be simultaneous (e.g., administered simultaneously together in a composite composition or simultaneously but as separate compositions) or interspersed. By way of example, a subject afflicted with a disorder described herein can be simultaneously or sequentially administered both a cytotoxic agent which selectively kills aberrant cells and an antibody (e.g., an antibody of the invention) which can, in one embodiment, be conjugated or linked with a therapeutic agent, a cytotoxic agent, an imaging agent, or the like.

The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

V. Uses and Methods of the Invention

The nucleic acid molecules, proteins, protein homologs, and antibodies described herein can be used in 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 for all of the purposes identified herein in portions of the disclosure relating to individual types of protein of the invention. The isolated nucleic acid molecules of the invention can be used to express proteins (e.g., via a recombinant expression vector in a host cell in gene therapy applications), to detect mRNA (e.g., in 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 drags 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 the above- described screening assays and uses thereof for 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 with a 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 with 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 useful for the synthesis of molecular libraries can be found in 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 can 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 numbers 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 with the polypeptide is determined. The cell, for example, can be a yeast cell or a cell of mammalian origin. Determimng the ability of the test compound to bind with 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

125 35 14 compound in a complex. For example, test compounds can be labeled with I, S, C,

3 or H, either directly or indirectly, and the radioisotope detected by direct counting of radio- emission 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 one 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 with the polypeptide or a biologically active portion thereof as compared to the known compound.

In another embodiment, the assay involves assessment of an activity characteristic of the polypeptide, wherein binding of the test compound with the polypeptide or a biologically active portion thereof alters (i.e., increases or decreases) the activity of the polypeptide.

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 to bind with or interact with a target molecule or to transport molecules across the cytoplasmic membrane.

Determining the ability of a polypeptide of the invention to bind with 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 in 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 in 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 association of downstream signaling molecules with a polypeptide of the invention. Determining the ability of a polypeptide of the invention to bind with 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

2+ cellular second messenger of the target (e.g., an mRNA, intracellular Ca , diacylglycerol,

IP3, and the like), detecting catalytic / enzymatic activity of the target on an appropriate substrate, detecting induction of a reporter gene (e.g., a regulatory element that is responsive to a polypeptide of the invention operably linked with 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 with the polypeptide or biologically active portion thereof. Binding of the test compound with the polypeptide can be determined either directly or indirectly as described above. In one 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 with 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 activity of the polypeptide can be accomplished, for example, by determining the ability of the polypeptide to bind with a target molecule by one of the methods described above for determimng 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 activity, the enzymatic activity, or both, 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 determimng the ability of the test compound to interact with the polypeptide. Ability of the test compound to interact with the polypeptide can be determined by assessing the ability of the polypeptide to preferentially bind with or modulate the activity of a target molecule, or by any other method.

The cell-free assays of the present invention are amenable to use of either soluble or membrane-bound forms (where applicable) of a polypeptide of the invention. In the case of cell-free assays comprising a membrane-bound form of the polypeptide, it can be desirable to use a solubilizing agent in order to maintain the membrane-bound form of the polypeptide in solution. Examples of such solubilizing agents include non-ionic detergents such as n-octylglucoside, n-dodecylglucoside, n-octylmaltoside, octanoyl-N- methylglucamide, decanoyl-N-methylglucamide, Triton X-100, Triton X- 114, Thesit, isotridecypoly(ethylene glycol ether)n, 3-{(3-cholamidopropyl) dimethylamminio}-!- propane sulfonate (CHAPS), 3-{(3-cholamidopropyl) dimethylamminio}-2-hydroxy-l- propane sulfonate (CHAPSO), or N-dodecyl-N,N-dimethyl-3-ammonio-l -propane sulfonate.

In one or more embodiments of the above assay methods of the present invention, it can be desirable to immobilize either the polypeptide of the invention or its target molecule in order to facilitate separation of complexed and non-complexed forms of one or both of the molecules, as well as to accommodate automation of the assay. Binding of a test compound with the polypeptide, or interaction of the polypeptide with a target molecule in the presence and absence of a candidate compound, can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter 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-derivatized microtiter plates, which are combined with the test compound and either the non-adsorbed target protein or a polypeptide of the invention. The combination is incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove unbound components, and complex formation is measured 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, such as those described herein.

Other techniques for immobilizing proteins on matrices can also be used in the screening assays of the invention. For example, either the polypeptide of the invention or a target molecule thereof (e.g., a protein which binds therewith or a substrate or an analog of a substrate of the protein of the invention) can be immobilized using conjugation of biotin and streptavidin. Biotinylated polypeptide of the invention or target molecules can be prepared using biotin-NHS (biotin-N-hydroxy-succinimide) using techniques well known in the art (e.g., using a commercially available kit such as the biotinylation kit manufactured by Pierce Chemical Co.; Rockford, IL), and immobilized in the wells of streptavidin-coated 96-well plates (Pierce Chemical). Alternatively, antibodies which are reactive with the polypeptide of the invention or target molecules but which do not interfere with binding of the polypeptide of the invention with its target molecule can be derivatized to the wells of the plate, and unbound target or polypeptide of the invention can be trapped in the wells by antibody conjugation. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the polypeptide 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 in a method in which a cell is contacted with a candidate compound and expression of the selected mRNA or protein (i.e., mRNA or protein corresponding to a polypeptide or nucleic acid of the invention) in the cell is determined. The level of expression of the selected mRNA or protein in the presence of the candidate compound is compared with the level of expression of the selected mRNA or protein in 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, if expression of the selected mRNA or protein is greater (i.e., statistically significantly greater) in the presence of the candidate compound than in its absence, then the candidate compound is identified as a stimulator of expression of the selected mRNA or protein. Alternatively, if expression of the selected mRNA or protein is less (i.e., statistically significantly less) in the presence of the candidate compound than in its absence, then the candidate compound is identified as an inhibitor of expression of the selected mRNA or protein. The level of the selected mRNA or protein expression in the cells can be determined by methods described herein. In yet another aspect of the invention, a polypeptide of the invention can be used as a "bait protein" in 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 with 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 in 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 in 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 (iii) aid in 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. Mapping of sequences to chromosomes is an important first step in correlating these sequences with genes associated with occurrence of disease. For example, TANGO 416 DNA maps to chromosome 4, between chromosomal markers D4S422 and D4S1576.

Briefly, genes can be mapped to chromosomes by preparing PCR primers (preferably 15-25 nucleotide residues in 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 in the genomic DNA, which would complicate the amplification process. These primers can 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 one or more nucleic acid sequences of the invention to design oligonucleotide primers, sub-localization can be achieved using panels of fragments prepared from specific chromosomes. Other mapping strategies which can similarly be used to map a gene to its chromosomal location include in situ hybridization (described in Fan et al. (1990) Proc. Natl. Acad. Sci. USA 87:6223-27), pre-screening with labeled flow-sorted chromosomes, and pre-selection by hybridization with chromosome specific cDNA libraries. Fluorescence in situ hybridization (FISH) of a DNA sequence using a metaphase chromosomal spread can be used to provide a precise chromosomal location in 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 a chromosome. Alternatively, panels of reagents can be used for marking multiple sites, multiple chromosomes, or both. Reagents corresponding to non- coding regions of the genes actually are preferred for mapping purposes. Coding sequences are more likely to be conserved within gene families, thus increasing the chance of cross- hybridization 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, in V. McKusick, Mendelian Inheritance in Man, available on-line through Johns Hopkins University Welch Medical Library). The relationship between genes and disease, mapped to the same chromosomal region, can then be identified by linkage analysis (co-inheritance of physically adjacent genes), described in, e.g., Egeland et al. (1987) Nature 325:783-787.

Moreover, differences in the DNA sequences between individuals affected and non-affected with a disease associated with a gene of the invention can be determined. If a mutation is observed in some or all of the affected individuals, but not in any (or in very few) non-affected individuals, then the mutation is likely to be the causative agent of the particular disease. Comparison of affected and non-affected individuals generally involves first looking for structural alterations in 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 physical identification devices such as general issue "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 in 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. 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 to subsequently sequence it.

Panels of corresponding DNA sequences from individuals, prepared in this manner, can provide unique individual identifications, because (with the exception of identical twins) every individual has a unique set of such DNA sequences owing, at least in part, to allelic differences. 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 in the coding regions of these sequences, and to a greater degree in the non-coding regions. It is estimated that allelic variation between individual humans occurs with a frequency of about once per 500 nucleotide residues. 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 in the non-coding regions, fewer non-coding sequences are necessary to differentiate individuals. The non-coding sequences of any of SEQ ID NOs: 1, 31, and 51 can comfortably provide positive individual identification with a panel of perhaps 10 to 1,000 primers which each yield a non-coding amplified sequence of 100 bases. If predicted coding sequences, such as those in any of SEQ ID NOs: 2, 32, and 52 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 nucleic acids, cells, or tissue from that individual. Using the unique identification database, positive identification of the individual, living or dead, can be made from extremely small samples.

3. Use of Partial Gene Sequences in Forensic Biology

DNA-based identification techniques can be used in 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 technology can be used to amplify DNA sequences taken from very small biological samples such as tissues (e.g., hair or skin) or body fluids (e.g., blood, saliva, or semen) found at a crime scene. The amplified sequence can be compared with 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 in 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 nucleotide sequence information can be used for identification as an accurate alternative to patterns formed by restriction enzyme-generated fragments. Sequences of non-coding regions are particularly appropriate for this use, because greater numbers of polymorphisms occur in non-coding 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 non-coding regions having a length of at least 20 or 30 nucleotide residues.

The nucleic acid sequences described herein can further be used to provide polynucleotide reagents, e.g., labeled or labelable probes which can be used in, for example, an in situ hybridization technique, to identify a specific tissue, e.g., brain tissue. This can be very useful in 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 in which diagnostic assays, prognostic assays, and monitoring clinical trials 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 gene encoding a polypeptide of the invention as well as activity of a polypeptide of the invention, in 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 or unwanted expression of a gene encoding a polypeptide of the invention or aberrant or unwanted 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 a protein of the invention, with expression of a nucleic acid encoding a polypeptide of the invention, or with activity of a polypeptide of the invention. For example, mutations in a gene encoding a polypeptide of the invention can be assayed in 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 a polypeptide of the invention, expression of a nucleic acid encoding it, or its activity.

As an alternative to making determinations based on the absolute expression level of selected genes, determinations may be based on the normalized expression levels of these genes. Expression levels are normalized by correcting the absolute expression level of a gene encoding a polypeptide of the invention by comparing its expression to the expression of a different gene, e.g., a housekeeping gene that is constitutively expressed. Suitable genes for normalization include housekeeping genes such as the actin gene. This normalization allows the comparison of the expression level in one sample (e.g., a patient sample), to another sample, or between samples from different sources.

Alternatively, the expression level can be provided as a relative expression level. To determine a relative expression level of a gene, the level of expression of the gene is determined for 10 or more samples of different endothelial (e.g. intestinal endothelium, airway endothelium, or other mucosal epithelium) cell isolates, preferably 50 or more samples, prior to the determination of the expression level for the sample in question. The mean expression level of each of the genes assayed in the larger number of samples is determined and this is used as a baseline expression level for the gene(s) in question. The expression level of the gene determined for the test sample (absolute level of expression) is then divided by the mean expression value obtained for that gene. This provides a relative expression level and aids in identifying extreme cases of disorders associated with aberrant expression of a gene encoding a polypeptide of the invention protein or with aberrant expression of a ligand thereof.

Preferably, the samples used in the baseline determination will be from either or both of cells which aberrantly express a gene encoding a polypeptide of the invention or a ligand thereof (i.e. 'diseased cells') and cells which express a gene encoding a polypeptide of the invention at a normal level or a ligand thereof (i.e. 'normal' cells). The choice of the cell source is dependent on the use of the relative expression level. Using expression found in normal tissues as a mean expression score aids in validating whether aberrance in expression of a gene encoding a polypeptide of the invention occurs specifically in diseased cells. Such a use is particularly important in identifying whether a gene encoding a polypeptide of the invention can serve as a target gene. In addition, as more data is accumulated, the mean expression value can be revised, providing improved relative expression values based on accumulated data. Expression data from endothelial cells (e.g. mucosal endothelial cells) provides a means for grading the severity of the disorder. Another aspect of the invention pertains to monitoring the influence of agents (e.g., drugs, antibodies, antisense oligonucleotides, or other compounds) on the expression or activity of a polypeptide of the invention in clinical trials.

These and other agents are described in further detail in the following sections.

1. Diagnostic Assays

An example of a method for detecting the presence or absence of a polypeptide or nucleic acid of the invention in 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. An example of an agent for detecting mRNA or genomic DNA encoding a polypeptide of the invention is a labeled nucleic acid probe capable of hybridizing with 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 either of SEQ ID NOs: 1, 31, and 51 , or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or

500 nucleotides in length and sufficient to specifically hybridize under stringent conditions with a mRNA or genomic DNA encoding a polypeptide of the invention. Other suitable probes for use in the diagnostic assays of the invention are described herein.

An example of an agent for detecting a polypeptide of the invention is an antibody capable of binding with a polypeptide of the invention, such as an antibody having a detectable label. Antibodies can be polyclonal or, preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab')2) can be used. The term "labeled," with regard to the probe or antibody, includes 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 coupling it 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 biotin such that it can be detected with fluorescently labeled streptavidin. 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 within a subject. That is, the detection method of the invention can be used to detect mRNA, protein, or genomic DNA in a biological sample in vitro as well as in vivo. For example, in vitro techniques for detection of mRNA include Northern hybridization methods and in situ hybridization methods. In vitro techniques for detection of a polypeptide of the invention include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitation, and immunofluorescence. In vitro techniques for detection of genomic

DNA include Southern hybridizations. Furthermore, in 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, the presence and location of which in a subject can be detected using standard imaging techniques.

In one embodiment, the biological sample contains protein molecules obtained from the test subject. Alternatively, the biological sample can contain mRNA molecules obtained from the test subject or genomic DNA molecules obtained from the test subject. An example of a biological sample is a peripheral blood leukocyte-containing sample obtained by conventional means from a subject (e.g., isolated peripheral blood leukocytes).

In another embodiment, the methods further involve obtaining a control biological sample from a control (i.e., non-afflicted) 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. The presence or amount of the polypeptide, mRNA, or genomic DNA encoding the polypeptide in the control and test samples can be compared to assess the degree, if any, to which the presence or amount in the test sample differs from that in the control sample.

The invention also encompasses kits for detecting the presence of a polypeptide or nucleic acid of the invention in a biological sample obtained from a subject. 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 (e.g., one of the disorders described in the section of this disclosure wherein the individual polypeptide of the invention is discussed). For example, the kit can comprise a labeled compound or agent capable of detecting the polypeptide or mRNA encoding the polypeptide in a biological sample. The kit can also, or alternatively, contain means for determining the amount of the polypeptide or mRNA in the sample (e.g., an antibody which specifically binds with the polypeptide or an oligonucleotide probe which binds with a nucleic acid encoding the polypeptide). Kits can include instructions for assessing whether 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 can comprise, for example: (1) a first antibody (e.g., attached to a solid support) which specifically binds with a polypeptide of the invention; and, optionally, (2) a second, different antibody which specifically binds with either the polypeptide or the first antibody and is conjugated with a detectable agent.

For oligonucleotide-based kits, the kit can comprise, for example: (1) an oligonucleotide (e.g., a detectably labeled oligonucleotide) which hybridizes with a nucleic acid encoding a polypeptide of the invention or (2) a pair of primers useful for amplifying a nucleic acid encoding a polypeptide of the invention. The kit can comprise, for example, a buffering agent, a preservative, or a protein stabilizing agent. The kit can also comprise components necessary for detecting the detectable agent (e.g., an enzyme or a substrate). The kit can contain a control sample or a series of control samples which can be assayed and compared with the test sample assay results. Each component of the kit can be enclosed within an individual container and all of the various containers can furthermore be within a single package, optionally with instructions for assessing 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 used 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 (e.g., one of the disorders described in the section of this disclosure wherein the individual polypeptide of the invention is discussed). Thus, the present invention provides a method in 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, level, or activity of the polypeptide or nucleic acid in the sample is associated with an enhanced or diminished risk of developing a disease or disorder associated with aberrant expression or activity of the polypeptide.

Furthermore, the prognostic assays described herein can be used to determine whether an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drag candidate) can be administered to a subject in order 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 using 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 an agent can be administered to a subject in order to effectively treat a disorder associated with aberrant expression or activity of a polypeptide of the invention. When efficacious agents are known or found, such assays can also be used to estimate tan efficacious dose of the agent. The methods of the invention can be used to detect genetic lesions or mutations in a gene of the invention in order to assess if a subject having the lesioned or mutated gene is at risk for a disorder characterized aberrant expression or activity of a polypeptide of the invention. In certain embodiments, the methods include detecting, in a sample of cells obtained 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 in 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) a non-wild type splicing pattern of a messenger RNA transcript of the gene; 8) a non-wild type level of 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 in the art which can be used for detecting such lesions and mutations in a gene.

In certain embodiments, detection of the lesion involves the use of an oligonucleotide primer in 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, in 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 in a gene (see, e.g., Abravaya et al. (1995) Nucleic 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 with 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. The method can also include detecting the size of the amplification product and comparing the length to the length of a corresponding product obtained in the same manner from a control sample. PCR, LCR, or both can be used as a preliminary amplification step in 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 any of a variety of techniques well known to those of skill in the art. These detection schemes are especially useful for detection of nucleic acid molecules if such molecules are present in very low numbers.

In an alternative embodiment, mutations in a selected gene can be identified in a sample by detecting alterations in restriction enzyme cleavage patterns. For example, sample and control DNA is isolated, (optionally) amplified, digested with one or more restriction endonucleases, and fragment length sizes are determined by gel electrophoresis and compared. Differences in fragment length sizes between sample and control DNA indicates occurrence of mutations or other sequence differences in the sample DNA. Moreover, 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 are identified by hybridizing a sample and control nucleic acids, e.g., DNA or RNA, with high density arrays containing hundreds or thousands of oligonucleotides probes (Cronin et al. (1996) Human Mutation 7:244-255; Kozal et al. (1996) Nature Medicine 2:753-759). For example, genetic mutations can be identified using two-dimensional arrays of light-generated DNA probes fixed to a surface, as described in Cronin et al., supra. Briefly, a first hybridization array of probes can be used to scan through long stretches of DNA in 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 hybridization of the nucleic acid sample with a second hybridization array in order to characterize specific mutations using smaller, specialized probe arrays complementary to many or all potential variants or mutations. 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 methods known in 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 used 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 Griffin et al. (1993) Appl. Biochem. Biotechnol. 38:147-159).

Other methods for detecting mutations in a selected gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA / RNA or RNA / DNA heteroduplexes (Myers et al. (1985) Science 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 that cleaves single-stranded regions of the duplex such as those which exist due to base pair 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, DNA / DNA or RNA / DNA duplexes can be treated with hydroxylamine or osmium tetroxide and with piperidine in order to digest mismatched regions. After digestion of the mismatched regions, the resulting material is separated by size on denaturing poly aery lamide gels to determine the site of the mutated or mismatched region. See, e.g., Cotton et al. (1988) Proc. Natl. Acad. Sci. USA 85:4397; Saleeba et al. (1992) Methods Enzymol. 217:286-295. In one embodiment, the control DNA or RNA is labeled for detection. In still another embodiment, the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called DNA mismatch repair enzymes) in defined systems for detecting and mapping point mutations in cDNAs obtained from samples of cells. For example, the mutY enzyme of E. coli cleaves following A residues at G / A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves following T residues at G / T mismatches (Hsu et al. (1994) Carcinogenesis

15:1657-1662). According to one embodiment, a probe based on a selected sequence, e.g., a wild-type sequence, is hybridized with a cDNA or other DNA product obtained from a test cell(s). The duplex is treated with a DNA mismatch repair enzyme, and the cleavage products, if any, are detected using an electrophoresis protocol or another polynucleotide- separating method. See, e.g., U.S. Patent No. 5,459,039.

In other embodiments, alterations in electrophoretic mobility are used to identify mutations in genes. For example, single strand conformation polymorphism (SSCP) analysis can be used to detect differences in electrophoretic mobility between mutant and wild type nucleic acids (Orita et al. (1989) Proc. Natl. Acad. Sci. USA 86:2766; see also Cotton (1993) Mutat. Res. 285:125-144; Hayashi (1992) Genet. Anal. Tech. Appl. 9:73-79). Single-stranded DNA fragments of sample and control nucleic acids are denatured and allowed to re-nature. The secondary structure of single-stranded nucleic acids varies according to their nucleotide sequence, and the resulting alteration in electrophoretic mobility enables detection of even a single base change. The DNA fragments can be labeled or detected using labeled probes. The sensitivity of the assay can be enhanced by using RNA

(rather than DNA), because the secondary structure of RNA is more sensitive to sequence changes. In one embodiment, the method uses heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility (Keen et al. (1991) Trends Genet. 7:5). In yet another embodiment, the movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE), as described (Myers et al. (1985) Nature 313:495). When DGGE is used as the method of analysis, DNA is modified to ensure that it does not completely denature, for example by adding a 'GC clamp' of approximately 40 nucleotide residues of high-melting GC-rich DNA to one or both ends of the DNA strands, for example using a PCR method. In a further embodiment, a temperature gradient is used in place of a denaturing gradient to identify differences in 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, and selective primer extension. For example, oligonucleotide primers can be prepared in which the known mutation is located centrally. The primers are hybridized with target DNA under conditions which permit hybridization only if a perfect complementary nucleotide sequence match occurs (Saiki et al. (1986) Nature 324:163); Saiki et al. (1989) Proc. Natl. Acad. Sci. USA 86:6230). Such allele specific oligonucleotides are hybridized with PCR-amplified target

DNA or attached to a surface for hybridization.

Alternatively, allele specific amplification technology can be used in conjunction with the methods of the invention. Oligonucleotides used as primers for specific amplification have a sequence complementary to the nucleotide sequence of a mutation of interest in the center of the molecule, so that occurrence of amplification depends on occurrence of the mutation in the sample nucleic acid (Gibbs et al. (1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3' end of one primer where, under appropriate conditions, mismatching can prevent or inhibit polymerase extension (Prossner (1993) Tibtech 11 :238). In addition, it can be desirable to introduce a novel restriction site in the region of the mutation in order to facilitate cleavage-based detection (Gasparini et al. (1992) Mol. Cell

Probes 6:1). Amplification can be performed using Taq ligase (Barany (1991) Proc. Natl. 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, thereby making it possible to assess the presence of a known mutation at a specific site by looking for the presence or absence of amplification. The methods described herein can be performed, for example, using prepackaged diagnostic kits comprising at least one probe nucleic acid or antibody reagent described herein. Such kits can be used, for example, in clinical settings to diagnose patients exhibiting symptoms or a family history of a disorder involving a gene encoding a polypeptide of the invention. Furthermore, any cell type or tissue in which the polypeptide of the invention is expressed (e.g., a blood sample containing peripheral blood leukocytes for proteins which are secreted or which occur on or in peripheral blood leukocytes) can be used in the prognostic assays described herein.

3. Pharmacogenomics Agents 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 for example, 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 drag) of the individual can be considered. Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration of the pharmacologically active drag. Thus, the pharmacogenomics of the individual permits selection of effective agents (e.g., drags) 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 in an individual can be determined to facilitate selection of one or more appropriate agents for therapeutic or prophylactic treatment of the individual. Pharmacogenomics deals with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See, e.g., Linder (1997) Clin. 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 drags act on the body are referred to as "altered drug action." Genetic conditions transmitted as single factors altering the way 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 (G6PD) deficiency is a common inherited enzymopathy in which the main clinical complication is hemolysis after ingestion of oxidant drugs (anti-malarials, sulfonamides, analgesics, nitrofurans) and consumption of fava beans.

As an illustrative embodiment, the activity of drag metabolizing enzymes is a major determinant of both the intensity and duration of drag action. The discovery of genetic polymorphisms of drug metabolizing enzymes (e.g., N-acetyltransferase 2 {NAT 2} and cytochrome P450 enzymes CYP2D6 and CYP2C19) explains why some patients do not obtain the expected drug effects or exhibit exaggerated drag response and serious toxicity following administration of standard and safe doses of a drag. These polymorphisms are expressed in two phenotypes in the population, the extensive metabolizer (EM) and poor metabolizer (PM). The prevalence of PM is different among different populations. For example, the gene encoding CYP2D6 is highly polymorphic, and several mutations have been identified in PM. Each of these mutations results in absence of functional CYP2D6.

Poor metabolizers of CYP2D6 and CYP2C19 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. At 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, activity of a polypeptide of the invention, expression of a nucleic acid encoding the polypeptide, or mutation content of a gene encoding the polypeptide in an individual can be determined to facilitate selection of appropriate agents for therapeutic or prophylactic treatment of the individual. In addition, pharmacogenetic studies can be used to ' apply genotyping of polymorphic alleles encoding drug-metabolizing enzymes to identification of an individual's drag responsiveness phenotype. This knowledge, when applied to dosing or drag selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic or prophylactic efficiency when treating a subject with a modulator of activity or expression of the polypeptide, such as a modulator identified by one of the examples of screening assays described herein.

4. Monitoring of Effects During Clinical Trials Monitoring the influence of agents (e.g., drug compounds) on expression or activity of a polypeptide of the invention (e.g., ability to modulate aberrant cell proliferation chemotaxis, differentiation, or both) can be applied not only in basic drug screening, but also in 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 in 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 in 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, optionally, that of other polypeptide that have been implicated in similar disorders, can be used as a marker of the immune responsiveness of a particular cell.

For example, genes (including those of the invention) that are modulated in cells by treatment with an agent (e.g., a peptide, a drug, or another small molecule) which modulates activity or expression of a polypeptide of the invention (e.g., as identified in a screening assay described herein) can be identified. Thus, to study the effect of agents on cellular proliferation disorders, for example, in a clinical trial, cells can be isolated and their RNA can be prepared and analyzed to determine the level of expression of one or more genes of the invention and, optionally, other genes implicated in the disorder. The levels of gene expression (i.e., a gene expression pattern) can be quantified by Northern blot analysis or by RT-PCR, as described herein, or by assessing the amount of protein produced, by one of the methods as described herein, or by measuring the level of activity of a gene of the invention or other gene(s). In this way, the gene expression pattern can serve as an indicator of the physiological response of the cells to the agent. Accordingly, this response state can be determined before, and at various points during, or after treatment of the individual with the agent (or, of course, at more than one of these stages).

In one 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 (i) obtaining a pre- administration sample from a subject prior to administration of the agent; (ii) detecting the level of the polypeptide or nucleic acid of the invention in the pre-administration sample; (iii) obtaining one or more post-administration samples from the subject; (iv) detecting the level the of the polypeptide or nucleic acid of the invention in the post-administration sample(s); (v) comparing the level of the polypeptide or nucleic acid of the invention in the pre- administration sample with the level of the polypeptide or nucleic acid of the invention in the post-administration sample(s); and (vi) altering the administration of the agent to the subject accordingly. For example, increased administration of the agent can be desirable to increase the expression or activity of the polypeptide to levels higher than those detected, i.e., to increase the effectiveness of the agent. Alternatively, decreased administration of the agent can be desirable to decrease expression or activity of the polypeptide to levels lower than those detected, i.e., to decrease the effectiveness of the agent.

C. Methods of Treatment

The present invention provides both prophylactic and therapeutic methods of treating a subject afflicted with, at risk for developing, or susceptible to a disorder associated with aberrant expression or activity of a polypeptide of the invention. Such disorders are described elsewhere in this disclosure.

1. Prophylactic Methods In one aspect, the invention provides a method for preventing in a subject, a disorder associated with aberrant expression or activity of a polypeptide of the invention, by administering to the subject an agent which modulates expression of the polypeptide 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 one or combination of the diagnostic and prognostic assays described herein. Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the aberrance, so that the disease or disorder is prevented or, alternatively, delayed in its onset or progression. Depending on the type of aberrance, for example, an agonist or antagonist agent can be used for treating the subject. 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 a small molecule. In one embodiment, the agent stimulates one or more of the biological activities of the polypeptide. Examples of such stimulatory agents include a polypeptide of the invention, a biologically active portion of such a polypeptide, a portion of such a polypeptide which comprises an epitope of the native polypeptide, and a nucleic acid molecule encoding the polypeptide of the invention that has been introduced into the cell. In another embodiment, the agent inhibits a biological activity of the polypeptide of the invention or expression of a protein or nucleic acid of the invention. Examples of such inhibitory agents include antisense nucleic acid molecules and antibodies. These modulatory methods can be performed in vitro (e.g., by culturing 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 of 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., up-regulates or down-regulates) 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 or substitute for reduced or aberrant expression or activity of the polypeptide.

Stimulation of activity is desirable in situations in which activity or expression is abnormally low or in which increased activity is likely to have a beneficial effect. Conversely, inhibition of activity is desirable in situations in which activity or expression is abnormally high or in which decreased activity is likely to have a beneficial effect. The contents of all references, patents, and published patent applications cited in this disclosure are hereby incorporated by reference.

Deposits of Clones

A clone containing a cDNA molecule encoding TANGO 416 (clone EpT416), was deposited with the American Type Culture Collection (Manassas, VA) on April 26, 1999 as Accession Number accession no. PTA-1764, as an Escherichia coli strain carrying a recombinant plasmid harboring the clone.

To isolate the cDNA clone, an aliquot of the strain can be streaked out to yield single colonies on nutrient medium (e.g., Luria broth plates) supplemented with 100 micrograms per milliliter ampicillin. Single colonies are grown, and plasmid DNA is extracted from single colonies using a standard mini-preparation procedure. Next, a sample of the DNA mini-preparation is digested using a combination of the restriction enzymes Sal I and Not I, and the resulting products are resolved on a 0.8% (w/v) agarose gel using standard DNA electrophoresis conditions. The digest liberates a fragment as follows: TANGO 416 (EpT416): 5.1 kilobases. The identity of the strain containing TANGO 416 can be inferred from the liberation of a fragment of the above identified size.

A clone containing a cDNA molecule encoding TANGO 457 (clone 457), was deposited with the American Type Culture Collection (Manassas, VA) on October 1, 1999 as Accession Number PTA-817, as part of a composite deposit representing a mixture of four strains, each carrying one recombinant plasmid harboring a particular cDNA clone.

To distinguish the strains and isolate a strain harboring a particular cDNA clone, an aliquot of the mixture can be streaked out to yield single colonies on nutrient medium (e.g., Luria broth plates) supplemented with 100 micrograms per milliliter ampicillin. Single colonies are grown, and plasmid DNA is extracted from single colonies using a standard mini- preparation procedure. Next, a sample of the DNA mini-preparation is digested using a combination of the restriction enzymes Sal I and Not I, and the resulting products are resolved on a 0.8% (w/v) agarose gel using standard DNA electrophoresis conditions. The digest liberates a fragment as follows: TANGO 457 (457): 2.3 kilobases

The identity of the strain containing TANGO 457 can be inferred from the liberation of a fragment of the above identified size.

Equivalents Those skilled in 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 encompassed by the following claims.

Claims

CLAIMS What is claimed is:

1. An isolated nucleic acid molecule selected from the group consisting of: a) a nucleic acid molecule having a nucleotide sequence which is at least 90% identical to the nucleotide sequence of any of SEQ ID NOs: 1 , 2, 31 , 32, 51 , and 52, the

TANGO 416 nucleotide sequence of the cDNA insert of a clone deposited on April 26, 1999 with the ATCC as accession no. PTA-1764, the TANGO 457 nucleotide sequence of the cDNA insert of a clone deposited on October 1, 1999 with the ATCC as PTA-817, or a complement thereof; b) a nucleic acid molecule comprising at least 15 nucleotide residues and having a nucleotide sequence identical to at least 15 consecutive nucleotide residues of any of SEQ ID NOs: 1, 2, 31, 32, 51, and 52, the TANGO 416 nucleotide sequence of the cDNA insert of a clone deposited on April 26, 1999 with the ATCC as accession no. PTA-1764, the TANGO 457 nucleotide sequence of the cDNA insert of a clone deposited on October 1, 1999 with the ATCC as PTA-817, or a complement thereof; c) a nucleic acid molecule which encodes a polypeptide comprising the amino acid sequence of any of SEQ ID NOs: 3-8, 33, 35, 38, and 53-60, and the amino acid sequence encoded by the TANGO 416 nucleotide sequence of the cDNA insert of a clone deposited on April 26, 1999 with the ATCC as accession no. PTA-1764, or the TANGO 457 nucleotide sequence of the cDNA insert of a clone deposited on October 1, 1999 with the ATCC as PTA-817; d) a nucleic acid molecule which encodes a fragment of a polypeptide comprising the amino acid sequence of any of SEQ ID NOs: 3-8, 33, 35, 38, and 53-60 and the amino acid sequence encoded by the TANGO 416 nucleotide sequence of the cDNA insert of a clone deposited on April 26, 1999 with the ATCC as accession no. PTA- 1764, or the TANGO 457 nucleotide sequence of the cDNA insert of a clone deposited on October 1, 1999 with the ATCC as PTA-817, wherein the fragment comprises at least 10 consecutive amino acid residues of any of SEQ ID NOs: 3-8, 33, 35, 38, and 53-60 and the amino acid sequence encoded by the TANGO 416 nucleotide sequence of the cDNA insert of a clone deposited on April 26, 1999 with the ATCC as accession no. PTA-1764, or the TANGO 457 nucleotide sequence of the cDNA insert of a clone deposited on October 1, 1999 with the ATCC as PTA-817; e) a nucleic acid molecule which encodes a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of any of SEQ ID NOs: 3-8, 33, 35, 38, and 53-60, wherein the nucleic acid molecule hybridizes with a nucleic acid molecule consisting of the nucleotide sequence of any of SEQ ID NOs: 1, 2, 31, 32, 51, and 52, the TANGO 416 nucleotide sequence of the cDNA insert of a clone deposited on April 26, 1999 with the ATCC as accession no. PTA-1764, the TANGO 457 nucleotide sequence of the cDNA insert of a clone deposited on October 1, 1999 with the ATCC as PTA-817, or a complement thereof under stringent conditions; and f) a nucleic acid molecule which encodes at least 10 consecutive nucleotide residues of an extracellular domain of one of TANGO 416 and TANGO 457, the extracellular domain having the amino acid sequence of one of SEQ ID NO: 6 and 60.

2. The isolated nucleic acid molecule of claim 1 , which is selected from the group consisting of: a) a nucleic acid having the nucleotide sequence of any of SEQ ID NOs: 1, 2, 31, 32, 51, and 52, the TANGO 416 nucleotide sequence of the cDNA insert of a clone deposited on April 26, 1999 with the ATCC as accession no. PTA-1764, the TANGO 457 nucleotide sequence of the cDNA insert of a clone deposited on October 1, 1999 with the ATCC as PTA-817, or a complement thereof; and b) a nucleic acid molecule which encodes a polypeptide having the amino acid sequence of any of SEQ ID NOs: 3-8, 33, 35, 38, and 53-60 and the amino acid sequence encoded by the TANGO 416 nucleotide sequence of the cDNA insert of a clone deposited on April 26, 1999 with the ATCC as accession no. PTA- 1764, the TANGO 457 nucleotide sequence of the cDNA insert of a clone deposited on October 1, 1999 with the ATCC as PTA-817, or a complement thereof.

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. The nucleic acid molecule of claim 1, having the nucleotide sequence SEQ ID NO: 1.

9. The nucleic acid molecule of claim 1 , having the nucleotide sequence SEQ

ID NO: 2.

10. The nucleic acid molecule of claim 1, having the nucleotide sequence SEQ ID NO: 31.

11. The nucleic acid molecule of claim 1, having the nucleotide sequence SEQ ID NO: 32.

12. The nucleic acid molecule of claim 1, having the nucleotide sequence SEQ ID NO: 51.

13. The nucleic acid molecule of claim 1, having the nucleotide sequence SEQ ID NO: 52.

14. An isolated polypeptide selected from the group consisting of: a) a fragment of a polypeptide comprising the amino acid sequence of any of SEQ ID NOs: 3-8, 33, 35, 38, and 53-60 and the amino acid sequence encoded by the TANGO 416 nucleotide sequence of the cDNA insert of a clone deposited on April 26, 1999 with the ATCC as accession no. PTA-1764, or the TANGO 457 nucleotide sequence of the cDNA insert of a clone deposited on October 1 , 1999 with the ATCC as PTA-817, wherein the fragment comprises at least 10 contiguous amino acids of any of SEQ ID NOs: 3-8, 33, 35, 38, and 53-60 and the amino acid sequence encoded by the TANGO 416 nucleotide sequence of the cDNA insert of a clone deposited on April 26, 1999 with the ATCC as accession no. PTA-1764, or the TANGO 457 nucleotide sequence of the cDNA insert of a clone deposited on October 1 , 1999 with the ATCC as PTA-817; b) a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of any of SEQ ID NOs: 3-8, 33, 35, 38, and 53-60, wherein the polypeptide is encoded by a nucleic acid molecule which hybridizes with a nucleic acid molecule consisting of the nucleotide sequence of any of SEQ ID NOs: 1, 2, 31, 32, 51, and 52, the TANGO 416 nucleotide sequence of the cDNA insert of a clone deposited on April 26, 1999 with the

ATCC as accession no. PTA-1764, the TANGO 457 nucleotide sequence of the cDNA insert of a clone deposited on October 1, 1999 with the ATCC as PTA-817, or a complement thereof under stringent conditions; c) a polypeptide which is encoded by a nucleic acid molecule comprising a nucleotide sequence which is at least 90% identical to a nucleic acid consisting of the nucleotide sequence of any of SEQ ID NOs: 1, 2, 31, 32, 51, and 52, the TANGO 416 nucleotide sequence of the cDNA insert of a clone deposited on April 26, 1999 with the ATCC as accession no. PTA-1764, the TANGO 457 nucleotide sequence of the cDNA insert of a clone deposited on October 1, 1999 with the ATCC as PTA-817, or a complement thereof; and d) a polypeptide comprising at least 10 consecutive amino acid residues of an extracellular domain of one of TANGO 416 and TANGO 457, the extracellular domain having the amino acid sequence of one of SEQ ID NO: 6 and 60.

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15. The isolated polypeptide of claim 14, having the amino acid sequence of any of SEQ ID NOs: 3-8, 33, 35, 38, and 53-60 and the amino acid sequence encoded by the TANGO 416 nucleotide sequence of the cDNA insert of a clone deposited on April 26, 1999 with the ATCC as accession no. PTA-1764, or the TANGO 457 nucleotide sequence of the cDNA insert of a clone deposited on October 1 , 1999 with the ATCC as PTA-817.

16. The isolated polypeptide of claim 14, having the amino acid sequence SEQ ID NO: 3.

17. The isolated polypeptide of claim 14, having the amino acid sequence

SEQ ID NO: 33.

18. The isolated polypeptide of claim 14, having the amino acid sequence SEQ ID NO: 53.

19. The polypeptide of claim 14, wherein the amino acid sequence of the polypeptide further comprises heterologous amino acid residues.

20. An antibody which selectively binds with the polypeptide of claim 14.

21. A method for producing a polypeptide selected from the group consisting of: a) a polypeptide comprising the amino acid sequence of any of SEQ ID NOs: 3-8, 33, 35, 38, and 53-60 and the amino acid sequence encoded by the TANGO 416 nucleotide sequence of the cDNA insert of a clone deposited on April 26, 1999 with the ATCC as accession no. PTA-1764, or the TANGO 457 nucleotide sequence of the cDNA insert of a clone deposited on October 1, 1999 with the ATCC as PTA-817; b) a polypeptide comprising a fragment of the amino acid sequence of any of SEQ ID NOs: 3-8, 33, 35, 38, and 53-60 and the amino acid sequence encoded by the TANGO 416 nucleotide sequence of the cDNA insert of a clone deposited on April 26, 1999 with the ATCC as accession no. PTA-1764, or the TANGO 457 nucleotide sequence of the cDNA insert of a clone deposited on October 1, 1999 with the ATCC as PTA-817, wherein the fragment comprises at least 10 contiguous amino acids of any of SEQ ID NOs: 3-8, 33, 35, 38, and 53-60 and the amino acid sequence encoded by the TANGO 416 nucleotide sequence of the cDNA insert of a clone deposited on April 26, 1999 with the ATCC as accession no. PTA-1764, or the TANGO 457 nucleotide sequence of the cDNA insert of a clone deposited on October 1, 1999 with the ATCC as PTA-817; and c) a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of any of SEQ ID NOs: 3-8, 33, 35, 38, and 53-60, or a complement thereof, wherein the polypeptide is encoded by a nucleic acid molecule which hybridizes with a nucleic acid molecule consisting of the nucleotide sequence of any of SEQ ID NOs: 1, 2, 31, 32, 51, and 52, the TANGO 416 nucleotide sequence of the cDNA insert of a clone deposited on April 26, 1999 with the ATCC as accession no. PTA-1764, the TANGO 457 nucleotide sequence of the cDNA insert of a clone deposited on October 1, 1999 with the ATCC as PTA-817, or a complement thereof under stringent conditions; the method comprising culturing the host cell of claim 5 under conditions in which the nucleic acid molecule is expressed.

22. A method for detecting the presence of a polypeptide of claim 14 in a sample, comprising: a) contacting the sample with a compound which selectively binds with a polypeptide of claim 14; and b) determining whether the compound binds with the polypeptide in the sample.

23. The method of claim 22, wherein the compound which binds with the polypeptide is an antibody.

24. A kit comprising a compound which selectively binds with a polypeptide of claim 14 and instructions for use.

25. 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 with the nucleic acid molecule; and b) determining whether the nucleic acid probe or primer binds with a nucleic acid molecule in the sample.

26. The method of claim 25, wherein the sample comprises mRNA molecules and is contacted with a nucleic acid probe.

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

28. A method for identifying a compound which binds with a polypeptide of claim 14 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 with the test compound.

29. The method of claim 28, 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 an activity characteristic of the polypeptide.

30. A method for modulating the activity of a polypeptide of claim 14 comprising contacting a polypeptide or a cell expressing a polypeptide of claim 14 with a compound which binds with the polypeptide in a sufficient concentration to modulate the activity of the polypeptide.

31. A method for identifying a compound which modulates the activity of a polypeptide of claim 14, comprising: a) contacting a polypeptide of claim 14 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.

32. An antibody substance which selectively binds with the polypeptide of claim 14.

33. A method of making an antibody substance which selectively binds with the polypeptide of claim 14, the method comprising providing the polypeptide to an immunocompetent vertebrate and thereafter harvesting blood or serum from the vertebrate.

34. A method of making an antibody substance which selectively binds with the polypeptide of claim 14, the method comprising contacting a polypeptide of claim 14 with a plurality of particles which individually comprise an antibody substance and a nucleic acid encoding the antibody substance, segregating a particle which binds with the polypeptide, and expressing the antibody substance from the nucleic acid of the segregated particle.