WO2001029088A1

WO2001029088A1 - Novel genes encoding proteins having prognostic, diagnostic, preventive, therapeutic, and other uses

Info

Publication number: WO2001029088A1
Application number: PCT/US2000/017386
Authority: WO
Inventors: Charles Reay Mackay; Paul S. Myers; Susan J. Kirst; Christopher C. Fraser; Kevin R. Leiby
Original assignee: Millennium Pharmaceuticals, Inc.
Priority date: 1999-10-19
Filing date: 2000-06-23
Publication date: 2001-04-26
Also published as: AU5887700A; EP1240198A4; EP1240198A1

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 molecules 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

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.

The present invention provides sequence information for polynucleotides derived from human genes and for proteins encoded thereby, and thus enables the practitioner to assess, predict, and affect the physiological state of various human tissues.

Summary of the Invention The present invention is based, at least in part, on the discovery of a variety of human cDNA molecules which encode proteins which are herein designated TANGO 229,

INTERCEPT 289, INTERCEPT 309, MANGO 419, and INTERCEPT 429. These five 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, 11, 12, 41, 42, 51, 52, 71, 72, 81, and 82, the nucleotide sequence of a cDNA clone deposited with ATCC^® as either of Accession Nos. PTA-295 and PTA-455 ("a cDNA of a clone deposited as either of ATCC^® PTA-295 and PTA- 455"), 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, 550, 650, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, 2000, 2200, 2400, 2600, 2800, 3000, 3500, or 3743) consecutive nucleotide residues of any of SEQ ID NOs: 1, 2, 11, 12, 21, 22, 26, 27, 31, 32, 36, 37, 41, 42, 51, 52, 71, 72, 81, and 82, or a cDNA of a clone deposited as either of ATCC^® PTA-295 and PTA-455, 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, 13-15, 23-25, 28-30, 33-35, 38-40, 43-45, 53-61, 73-75, and 83-90, or the amino acid sequence encoded by a cDNA of a clone deposited as either of

ATCC^® PTA-295 and PTA-455, or a complement thereof.

In certain embodiments, the nucleic acid molecules have the nucleotide sequence of any of SEQ ID NOs: 1, 2, 11, 12, 21, 22, 26, 27, 31, 32, 36, 37, 41, 42, 51, 52, 71, 72, 81, and 82, or a cDNA of a clone deposited as either of ATCC^® PTA-295 and PTA-455.

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, 13-15, 23-25, 28-30, 33-35, 38-40, 43-45, 53-61, 73-75, and 83-90, the amino acid sequence encoded by a cDNA of a clone deposited as either of ATCC^® PTA-295 and PTA-455, the fragment including at least 8 (10, 15, 20, 25, 30, 40, 50, 75, 100, 125, 150, or 200) consecutive amino acids of any of SEQ ID NOs: 3-8, 13-15, 23-25, 28-30, 33-35, 38-40, 43-45, 53-61, 73-75, and 83-90, or the amino acid sequence encoded by a cDNA of a clone deposited as either of ATCC^® PTA-295 and PTA-455.

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, 13-15, 23-25, 28-30, 33-35, 38-40, 43-45, 53-61, 73-75, and 83-90, or the amino acid sequence encoded by a cDNA of a clone deposited as either of ATCC^® PTA-295 and PTA-455, 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, 11, 12, 21, 22, 26, 27, 31, 32, 36, 37, 41, 42, 51, 52, 71, 72, 81, and 82, or a cDN A of a clone deposited as either of ATCC^® PTA-295 and PTA-455 , 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, 13-15, 23-25, 28-30, 33-35, 38-40, 43-45, 53-61, 73-75, and 83-90.

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, 13-15, 23-25, 28-30, 33-35, 38-40, 43-45, 53-61, 73- 75, and 83-90, 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, 11, 12, 21, 22, 26, 27, 31, 32, 36, 37, 41, 42, 51, 52, 71, 72,

81, and 82. 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, 13-15, 23-25, 28-30, 33-35, 38-40, 43-45, 53-61, 73-75, and 83-90, or the amino acid sequence encoded by a cDNA of a clone deposited as either of ATCC^® PTA- 295 and PTA-455, 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, 11, 12, 21, 22, 26, 27, 31, 32, 36, 37, 41, 42, 51, 52, 71, 72, 81, and 82, 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, 11, 12, 21, 22, 26, 27, 31, 32, 36, 37, 41, 42, 51, 52, 71, 72, 81, and

82, a cDNA of a clone deposited as either of ATCC^® PTA-295 and PTA-455, 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, 550, 650, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, 2000, 2200, 2400, 2600, 2800, 3000, 3500, or 3743) 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, 11, 12, 21, 22, 26, 27, 31, 32, 36, 37,

41, 42, 51, 52, 71, 72, 81, and 82, a cDNA of a clone deposited as either of ATCC^® PTA-295 and PTA-455, 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 process mediated by interaction of the protein with a second protein. Such activities include, by way of example, formation of protein-protein interactions with proteins of one or more signaling pathways (e.g., with a protein with which the naturally-occurring polypeptide interacts); binding with a ligand of the naturally-occurring protein; and binding with an intracellular target of the naturally-occurring protein. Other activities include modulation of cellular proliferation, cellular differentiation, chemotaxis, cellular migration, cell death (e.g., apoptosis), or some combination of these.

By way of example, TANGO 229, compounds which modulate its activity, expression, or both, and compounds (e.g., antibodies) which interact with (e.g., bind with) TANGO 229 (collectively "TANGO 229-related molecules") can exhibit the ability to affect one or more of growth, proliferation, survival, differentiation, activity, morphology, and movement / migration of, for example, T cells and cells of heart, liver, pancreas, placenta, brain lung, skeletal muscle, kidney, spleen, lymph node, peripheral blood leukocyte, bone marrow, and thymus tissues. TANGO 229 protein can be involved in mediating cell binding and adhesion, including binding / adhesion of cells with other cells, with extracellular matrix, and with foreign materials. TANGO 229 protein can thus have a role in disorders associated with aberrant binding of these types. TANGO 229 protein can also be involved in mediating attraction and repulsion of cells and translocation of cells through, past, or along other cells or tissues. TANGO 229 protein can furthermore be involved in transducing signals across the cell membrane. Thus, TANGO 229-related molecules can be used to prognosticate, prevent, diagnose, or treat one or more disorders associated with these physiological processes.

Further by way of example, INTERCEPT 289, compounds which modulate its activity, expression, or both, and compounds (e.g., antibodies) which bind with INTERCEPT 289 (collectively "INTERCEPT 289-related molecules") can exhibit the ability to affect one or more of growth, proliferation, survival, differentiation, activity, morphology, and movement / migration of, for example, lymphocytes such as monocytes and macrophages. INTERCEPT 289 protein can be involved in activating one or more types of macrophages and monocytes, and thus can be involved in one or more immune disorders and other types of disorders mediated by monocytes and macrophages. Thus, INTERCEPT 289-related molecules can be used to prognosticate, prevent, diagnose, or treat one or more of these disorders.

Still further by way of example, INTERCEPT 309, compounds which modulate its activity, expression, or both, and compounds (e.g., antibodies) which bind with INTERCEPT 309 (collectively "INTERCEPT 309-related molecules") modulate one or more of growth, proliferation, survival, differentiation, activity, morphology, and movement / migration of cells of brain, liver, colon, prostate, kidneys, thyroid, and other epithelial and endothelial tissues. INTERCEPT 309 is a claudin-like protein, and can modulate tight-junction regulated intercellular and paracellular diffusion. INTERCEPT 309 also can participate in cell-to-cell adhesive mechanisms that do not necessarily involve tight junction formation. In addition, INTERCEPT 309 can mediate interaction of cells in which it is expressed with Clostridium perfringens enterotoxin, and can thus be involved in disorders mediated by C. perfringens and other pathogens. Furthermore, INTERCEPT 309 is associated with normal and aberrant apoptosis, and thus with disorders associated with aberrant apoptosis. Thus, INTERCEPT 309-related molecules can be used to prognosticate, prevent, diagnose, or treat one or more disorders associated with these physiological processes.

Yet further by way of example, MANGO 419, compounds which modulate its activity, expression, or both, and compounds (e.g., antibodies) which bind with MANGO 419 (collectively "MANGO 419-related molecules") can modulate one or more of growth, proliferation, survival, differentiation, activity, morphology, and movement / migration of, for example, cells of embryonic and mammary, prostate, and other epithelial and endothelial tissues. MANGO 419 protein can be involved in disorders which affect epithelial and endothelial tissues. Such disorders include cell proliferation disorders, disorders associated with aberrant epithelial / endothelial permeability, and disorders associated with aberrant binding or adhesion of cells with other cells, with extracellular matrix, or with foreign materials. Thus, MANGO 419- related molecules can be used to prognosticate, prevent, diagnose, or treat one or more such disorders. As an additional example, INTERCEPT 429, compounds which modulate its activity, expression, or both, and compounds (e.g., antibodies) which bind with INTERCEPT 429 (collectively "INTERCEPT 429-related molecules") can modulate one or more of growth, proliferation, survival, differentiation, activity, morphology, and movement / migration of, for example, cells of cardiac muscle, small intestine, and one or more of fetal lung, testis, and B cell tissues. INTERCEPT 429 can be involved in modulating growth, proliferation, survival, differentiation, and activity of cells of these tissues, in both normal and diseased tissues. Thus, INTERCEPT 429-related molecules can be used to prognosticate, prevent, diagnose, or treat one or more disorders which affect one or more of these tissues.

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 nucleotide residues to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences have a common structural 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 being 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. 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 (i.e., 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 (e.g., a small organic molecule). In yet another embodiment, the modulator is an antibody substance, as described herein.

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.

In yet a further aspect, the invention provides substantially purified antibodies or fragments thereof (i.e., antibody substances), including non-human antibodies or fragments thereof, which specifically bind with a polypeptide of the invention or with a portion thereof. In various embodiments, these substantially purified antibodies/fragments can be human, non-human, chimeric, and/or humanized antibodies. Non-human antibodies included in the invention include, by way of example, goat, mouse, sheep, horse, chicken, rabbit, and rat antibodies. In addition, the antibodies of the invention can be polyclonal antibodies or monoclonal antibodies. In a particularly preferred embodiment, the antibody substance of the invention specifically binds with an extracellular domain of one of TANGO 229, INTERCEPT 289, INTERCEPT 309, MANGO 419, and INTERCEPT 429. Preferably, the extracellular domain with which the antibody substance binds has an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 15, 25, 30, 35, 40, 45, 55, 59, and 88.

Any of the antibody substances of the invention can be conjugated with a therapeutic moiety or with a detectable substance. Non-limiting examples of detectable substances that can be conjugated with the antibody substances of the invention include an enzyme, a prosthetic group, a fluorescent material (i.e., a fluorophore), a luminescent material, a bioluminescent material, and a radioactive material (e.g., a radionuclide or a substituent comprising a radionuclide).

The invention also provides a kit containing an antibody substance of the invention conjugated with a detectable substance, and instructions for use. Still another aspect of the invention is a pharmaceutical composition comprising an antibody substance of the invention and a pharmaceutically acceptable carrier. In preferred embodiments, the pharmaceutical composition contains an antibody substance of the invention, a therapeutic moiety (preferably conjugated with the antibody substance), and a pharmaceutically acceptable carrier.

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 1A through IG. The nucleotide sequence (SEQ ID NO: 1) of a cDNA encoding the human TANGO 229 protein described herein is listed in Figures 1A through IF. The open reading frame (ORF; residues 72 to 2216; 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 229 is listed. Figure IG is a hydrophilicity plot of one embodiment of human TANGO 229 protein, 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 2 comprises Figures 2 A through 2Zvi.

The nucleotide sequence (SEQ ID NO: 11) of a cDNA encoding form la of the human INTERCEPT 289 protein described herein is listed in Figures 2 A through 2C. The ORF (residues 179 to 742; SEQ ID NO: 12) of the cDNA is indicated by nucleotide triplets, above which the amino acid sequence (SEQ ID NO: 13) of form la of human INTERCEPT 289 is listed.

The nucleotide sequence (SEQ ID NO: 21) of a cDNA encoding form lb of human INTERCEPT 289 protein described herein is listed in Figures 2D through 2G. The ORF (residues 179 to 712; SEQ ID NO: 22) of the cDNA is indicated by nucleotide triplets, above which the amino acid sequence (SEQ ID NO: 23) of form lb of human INTERCEPT 289 is listed.

The nucleotide sequence (SEQ ID NO: 26) of a cDNA encoding form 2a of human INTERCEPT 289 protein described herein is listed in Figures 2H through 2K. The ORF (residues 162 to 656; SEQ ID NO: 27) of the cDNA is indicated by nucleotide triplets, above which the amino acid sequence (SEQ ID NO: 28) of form 2a of human

INTERCEPT 289 is listed.

The nucleotide sequence (SEQ ID NO: 31) of a cDNA encoding form 2b of human INTERCEPT 289 protein described herein is listed in Figures 2L through 20. The ORF (residues 162 to 626; SEQ ID NO: 32) of the cDNA is indicated by nucleotide triplets, above which the amino acid sequence (SEQ ID NO: 33) of form 2b of human

INTERCEPT 289 is listed.

The nucleotide sequence (SEQ ID NO: 36) of a cDNA encoding form 3a of human INTERCEPT 289 protein described herein is listed in Figures 2P through 2S. The ORF (residues 162 to 596; SEQ ID NO: 37) of the cDNA is indicated by nucleotide triplets, above which the amino acid sequence (SEQ ID NO: 38) of form 3a of human

INTERCEPT 289 is listed.

The nucleotide sequence (SEQ ID NO: 41) of a cDNA encoding form 3b of human INTERCEPT 289 protein described herein is listed in Figures 2T through 2 V. The ORF (residues 162 to 566; SEQ ID NO: 42) of the cDNA is indicated by nucleotide triplets, above which the amino acid sequence (SEQ ID NO: 43) of form 3b of human

INTERCEPT 289 is listed.

Figure 2W is an alignment, made using the Wisconsin™ BestFit software

(Smith and Waterman, (1981) Adv. Appl. Math. 2:482-489; BLOSUM62 scoring matrix, gap opening penalty 10 / gap extension penalty 10) of the amino acid sequences of murine myeloid DNAX accessory protein associated lectin-1 ("M"; MDL-1; SEQ ID

NO: 18), murine INTERCEPT 289 ("R"; SEQ ID NO: 93), human MDL-1 ("H"; SEQ ID

NO: 16), form la of INTERCEPT 289 ("A"; SEQ ID NO: 13), form lb of INTERCEPT

289 ("B"; SEQ ID NO: 23), form 2a of INTERCEPT 289 ("C"; SEQ ID NO: 28), form 2b of INTERCEPT 289 ("D"; SEQ ID NO: 33), form 3a of INTERCEPT 289 ("E"; SEQ

ID NO: 38), and form 3b of INTERCEPT 289 ("F"; SEQ ID NO: 43).

Figures 2Xi through 2Xxiv is an alignment (made using the Wisconsin™

BestFit software; Smith and Waterman, (1981) Adv. Appl. Math. 2:482-489; gap opening penalty 10 / gap extension penalty 10), of the nucleotide sequences of cDNA molecules encoding form la of INTERCEPT 289 ("A"; SEQ ID NO: 11), form lb of INTERCEPT

289 ("B"; SEQ ID NO: 21), form 2a of INTERCEPT 289 ("C"; SEQ ID NO: 26), form 2b of INTERCEPT 289 ("D"; SEQ ID NO: 31), form 3a of INTERCEPT 289 ("E"; SEQ ID NO: 36), and form 3b of INTERCEPT 289 ("F"; SEQ ID NO: 41).

Figures 2Yi through 2Yvi is a series of hydrophilicity plots for individual forms of human INTERCEPT 289 protein. The plot corresponding to form la is shown in Figure 2Yi. The plot corresponding to form lb is shown in Figure 2Yii. The plot corresponding to form 2a is shown in Figure 2Yiii. The plot corresponding to form 2b is shown in Figure 2Yiv. The plot corresponding to form 3 a is shown in Figure 2Yv. The plot corresponding to form 3b is shown in Figure 2Yvi.

The nucleotide sequence (SEQ ID NO: 91) of a cDNA encoding murine INTERCEPT 289 protein described herein is listed in Figures 2Zi through 2Ziii. The

ORF (residues 198 to 767; SEQ ID NO: 92) of the cDNA is indicated by nucleotide triplets, beneath which the amino acid sequence (SEQ ID NO: 93) of murine INTERCEPT 289 is listed. Figures 2Ziv and 2Zv are a manual alignment of the nucleotide sequences of murine INTERCEPT 289 ORF ("MI289"; SEQ ID NO: 92) and the ORF of form la of human INTERCEPT 289 ("HI289"; SEQ ID NO: 12). Figure

2Zvi is a hydrophilicity plot for murine INTERCEPT 289 protein.

Figure 3 comprises Figures 3 A through 3T. The nucleotide sequence (SEQ ID NO: 51) of a cDNA encoding the human INTERCEPT 309 protein described herein is listed in Figures 3A through 3C. The ORF (residues 2 to 646; SEQ ID NO: 52) of the cDNA is indicated by nucleotide triplets, above which the amino acid sequence

(SEQ ID NO: 53) of human INTERCEPT 309 is listed. Figure 3D is a hydrophilicity plot of human INTERCEPT 309 protein. An alignment (made using the ALIGN software; paml20.mat scoring matrix, gap opening penalty = 12, gap extension penalty = 4) of the nucleotide sequences of a cDNA clone ("DKFZ"; SEQ ID NO: 64; GenBank accession no. AL049977) obtained from human fetal brain tissue and INTERCEPT 309 cDNA ("1309"; SEQ ID NO: 51) is shown in Figures 3E through 3K. An alignment (made using the ALIGN software; paml20.mat scoring matrix, gap opening penalty = 12, gap extension penalty = 4) of the nucleotide sequences of the cDNA encoding human INTERCEPT 309 ("1309"; SEQ ID NO: 51) and a portion of a cDNA encoding murine claudin-8 protein ("CLAUD8"; SEQ ID NO: 62) is shown in Figures 3L through 3R. An alignment (made using the ALIGN software; paml20.mat scoring matrix, gap opening penalty = 12, gap extension penalty = 4) of the amino acid sequences of human INTERCEPT 309 protein ("1309"; SEQ ID NO: 53) and murine claudin-8 protein ("CLAUD8"; SEQ ID NO: 63) is shown in Figure 3S. A manual alignment of individual alignments (made using the Wisconsin™ BestFit software; Smith and Waterman (1981) Adv. Appl. Math. 2:482-489; blosum62 scoring matrix, gap opening penalty 10 / gap extension penalty 10) of the amino acid sequences of human INTERCEPT 309 protein ("1309"; SEQ ID NO: 53) with each of human Clostridium perfringens enterotoxin receptor ("hCPE"; SEQ ID NO: 65), murine C. perfringens enterotoxin receptor ("mCPE"; SEQ ID NO: 66), and a protein encoded by a cDNA recovered from regressing rat ventral prostate tissue ("rRPV"; SEQ ID NO: 67) is shown in Figure 3T.

Figure 4 comprises Figures 4 A and 4B. The nucleotide sequence (SEQ ID NO: 71) of a cDNA encoding the human MANGO 419 protein described herein is listed in Figure 4A. The ORF (residues 84 to 323; SEQ ID NO: 72) of the cDNA is indicated by nucleotide triplets, above which the amino acid sequence (SEQ ID NO: 73) of human MANGO 419 is listed. Figure 4B is a hydrophilicity plot of human MANGO

419 protein.

Figure 5 comprises Figures 5A and 5B. The nucleotide sequence (SEQ ID NO: 81) of a cDNA encoding the human INTERCEPT 429 protein described herein is listed in Figure 5A. The ORF (residues 95 to 439; SEQ ID NO: 82) of the cDNA is indicated by nucleotide triplets, above which the amino acid sequence (SEQ ID NO: 83) of human INTERCEPT 429 is listed. Figure 5B is a hydrophilicity plot of human INTERCEPT 429 protein.

Detailed Description of the Invention The present invention is based, at least in part, on the discovery of a variety of human cDNA molecules which encode proteins which are herein designated TANGO 229, INTERCEPT 289, INTERCEPT 309, MANGO 419, and INTERCEPT 429. 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 229 A cDNA clone (designated jthtcOO 1 c06) encoding at least a portion of human TANGO 229 protein was isolated from a human T cell cDNA library. Human

TANGO 229 protein is a transmembrane protein.

The full length of the cDNA encoding human TANGO 229 protein

(Figure 1; SEQ ID NO: 1) is 3594 nucleotide residues. The open reading frame (ORF) of this cDNA, nucleotide residues 72 to 2216 of SEQ ID NO: 1 (i.e., SEQ ID NO: 2), encodes a 715-amino acid residue protein (Figure 1; SEQ ID NO: 3), corresponding to a

681 -residue transmembrane mature protein.

The invention thus includes purified human TANGO 229 protein, both in the form of the immature 715 amino acid residue protein (SEQ ID NO: 3) and in the form of the mature 681 amino acid residue protein (SEQ ID NO: 5). Mature human

TANGO 229 proteins can be synthesized without the signal sequence polypeptide at the amino terminus thereof, or they can be synthesized by generating immature TANGO 229 protein and cleaving the signal sequence therefrom.

In addition to full length mature and immature human TANGO 229 proteins, the invention includes fragments, derivatives, and variants of these TANGO

229 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 NO: 1, such as the portion which encodes mature TANGO 229 protein, immature TANGO 229 protein, or a domain of

TANGO 229 protein. These nucleic acids are collectively referred to as nucleic acids of the invention. TANGO 229 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 229 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 229 protein contains a signal sequence corresponding to amino acid residues 1 to 34 of SEQ ID NO: 3 (SEQ ID NO: 4). 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 32, 33, 34, 35, or 36 of SEQ ID NO: 3). The signal sequence is cleaved during processing of the mature protein.

TANGO 229 proteins include a transmembrane domain and two extra- membrane domains flanking the cell membrane. The transmembrane domain corresponds to about amino acid residues 456 to 480 of SEQ ID NO: 3 (i.e., the transmembrane domain having the sequence SEQ ID NO: 7). One of the extra- membrane domains corresponds to about amino acid residues 35 to 455 of SEQ ID NO: 3. This domain has the sequence SEQ ID NO: 6, and is most likely an extracellular domain. The other extra-membrane domain corresponds to about amino acid residues 481 to 715 of SEQ ID NO: 3. This domain has the sequence SEQ ID NO: 8, and is most likely a cytoplasmic domain. In one embodiment, the domain corresponding to about amino acid residues 35 to 455 of SEQ ID NO: 3 is a cytoplasmic domain, and the domain corresponding to about amino acid residues 481 to 715 is an extracellular 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 229 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 229 proteins using amino acid sequence comparison software (comparing the amino acid sequence of TANGO 229 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, myristoylases, and prenyl transferases. In various embodiments, the protein of the invention has at least 1, 2, 4, 6, 10, 15, or 20 or more of the post-translational modification sites described herein in Table I. Examples of additional domains present in human TANGO 229 protein include a CUB domain and a Factor V/VIII discoidin domain. 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 domains or signature sequences described herein in Table I. Preferably, the protein of the invention has at least one CUB domain and one Factor V/VIII discoidin domain.

CUB domains are extracellular domains of about 110 amino acid residues which occur in functionally diverse, mostly developmentally regulated proteins (Bork and Beckmann (1993) J Mol. Biol. 231:539-545; Bork (1991) FEBS Lett. 282:9-12). Many CUB domains contain four conserved cysteine residues, although some, like that of TANGO 202, contain only two of the conserved cysteine residues. The structure of the CUB domain has been predicted to assume a beta-barrel configuration, similar to that of immunoglobulins. Other proteins which comprise one or more CUB domains include, for example, mammalian complement sub-components Cls and Clr, hamster serine protease Casp, mammalian complement activating component of Ra-reactive factor, vertebrate enteropeptidase, vertebrate bone morphogenic protein 1 , sea urchin blastula proteins BP10 and SpAN, Caenorhabditis elegans hypothetical proteins F42A10.8 and R151.5, neuropilin (A5 antigen, in which a pair of Factor V/VHI discoidin domains also occur), sea urchin fibropellins I and III, mammalian hyaluronate-binding protein TSG-6 (PS4), mammalian spermadhesins, and Xenopus embryonic protein UVS.2. The presence of a CUB domain in TANGO 229 protein indicates that TANGO 229 is involved in one or more physiological processes in which these other CUB domain- containing proteins are involved, has a biological activity in common with one or more of these other CUB domain-containing proteins, or both. The presence of a CUB domain in TANGO 229 protein also indicates that TANGO 229 can be developmentally regulated.

Factor V/VIII discoidin domains are involved in binding with cell surface-attached carbohydrates. These domains occur in a variety of intracellular, extracellular, and transmembrane proteins, including human and murine coagulation factor V, human and murine coagulation factor VIII precursor, human and murine neuropilins, a variety of receptor-like tyrosine kinases (e.g., neurotrophic tyrosine kinases and cell adhesion tyrosine kinases), carboxypeptidases and carboxypeptidase-like proteins, milk fat globule glycoproteins, human breast epithelial antigen BA46, murine neurexin IV, human X-linked juvenile retinoschisis precursor protein, and human contactin associated protein. Presence of a Factor V/VIII discoidin domain in TANGO 229 indicates that this protein is involved in one or more physiological processes in which these other Factor V/VIII discoidin domain-containing proteins are involved, has biological activity in common with one or more of these other Factor V/VIII discoidin domain-containing proteins, or both. Presence of a Factor V/VIII discoidin domain in TANGO 229 protein is an indication that TANGO 229 is associated with binding of one or more glycosylated proteins at the surface of cells which express TANGO 229.

Binding of glycosylated proteins at the cell surface is associated with several physiologically relevant phenomena, including cell adhesion (including cell repulsion), transmembrane signal transduction, and nutrient binding and uptake by cells. The Factor V/VIII discoidin domain of human coagulation factor VIII protein is known to be involved in binding of factor VIII with von Willebrand factor and with membrane- associated lipids such as phosphatidylserine. Presence of a Factor V/VIII discoidin domain in TANGO 229 protein is thus an indication that the extracellular portion of TANGO 229 protein can interact with membrane lipids.

The signal peptide prediction program SIGNALP (Nielsen et al. (1997) Protein Engineering 10:1-6) predicted that human TANGO 229 protein includes a 34 amino acid residue signal peptide (amino acid residues 1 to 34 of SEQ ID NO: 3; SEQ ID NO: 4) preceding the mature TANGO 229 protein (amino acid residues 35 to 715 of SEQ ID NO: 3; SEQ ID NO: 5). Human TANGO 229 protein includes an extracellular domain (amino acid residues 35 to 455 of SEQ ID NO: 3; SEQ ID NO: 6), a transmembrane domain (amino acid residues 456 to 480 of SEQ ID NO: 3; SEQ ID NO:

7), and an intracellular domain (amino acid residues 481 to 715 of SEQ ID NO: 3; SEQ ID NO: 8). In an alternative embodiment, amino acid residues 35 to 455 of SEQ ID NO: 3 correspond to an intracellular domain of human TANGO 229 protein and residues 481 to 715 correspond to an extracellular domain. Figure IG depicts a hydrophilicity plot of human TANGO 229 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 34 of SEQ ID NO: 3 is the signal sequence of human TANGO 229 (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 229 protein from about amino acid residue 50 to about amino acid residue 70 appears to be located at or near the surface of the protein, while the region from about amino acid residue 195 to about amino acid residue 210 appears not to be located at or near the surface. The predicted molecular weight of human TANGO 229 protein without modification and prior to cleavage of the signal sequence is about 77.9 kilodaltons. The predicted molecular weight of the mature human TANGO 229 protein without modification and after cleavage of the signal sequence is about 72.3 kilodaltons.

Northern hybridization experiments using human tissue samples indicated that mRNA corresponding to cDNA encoding TANGO 229 is expressed in the tissues listed in Table IIA, wherein "++" indicates strongly detectable expression, "+" indicates a lesser degree of expression, and "+/-" indicates a still lesser degree of expression. In these tissues, two alternatively spliced forms of cDNA encoding TANGO 229 (having sizes of about 2.0 and 4.0 kilobases) were detected. Table IIA

Northern hybridization experiments using human immune system tissue samples indicated that mRNA corresponding to the cDNA encoding TANGO 229 is expressed in the tissues listed in Table IIB. In these tissues, two alternatively spliced forms of cDNA encoding TANGO 229 (having sizes of about 2.0 and 4.9 kilobases) were detected.

Table IIB

The nucleotide sequence (SEQ ID NO: 1) of TANGO 229 cDNA was aligned (using the LALIGN software {Huang and Miller (1991) Adv. Appl. Math. 12:373-381 }; paml20 scoring matrix, gap opening penalty = 12, gap extension penalty =

4) with the nucleotide sequence of the portion of human chromosome region 6q21 listed in GenBank Accession No. Z85999. This alignment indicated 45.8% identity between the two sequences in the 3826-residue overlapping portion. The nucleotide sequence (SEQ ID NO: 1) of TANGO 229 cDNA was also aligned (using the LALIGN software; paml20 scoring matrix, gap opening penalty = 12, gap extension penalty = 4) with an expressed sequence tag (EST) clone designated BP481 in P.C.T. Publication No. WO98/45435. This alignment indicated 72.9% identity between the two sequences in the 414-residue overlapping portion.

Biological function of TANGO 229 proteins, nucleic acids encoding them, and modulators of these molecules

TANGO 229 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 229 occurs in a human T cell cDNA library, and that RNA corresponding to TANGO 229 is detectable by Northern analysis of human heart, liver, pancreas, placenta, brain lung, skeletal muscle, kidney, spleen, lymph node, peripheral blood leukocyte, bone marrow, and thymus tissues, it is evident that TANGO 229 protein can be involved in one or more biological processes which occur in these tissues. In particular, TANGO 229 can be involved in modulating growth, proliferation, survival, differentiation, and activity of cells of these tissues (e.g., T cells and other cells of the immune system).

Expression of TANGO 229 in a variety of immune system tissues (e.g., T cells, peripheral blood leukocyte, and spleen, lymph node, bone marrow, and thymus tissues) is an indication that TANGO 229 can have a role in both normal immune processes and in a variety of disorders which affect or involve the immune system. Examples of such disorders include auto-immune diseases (e.g., rheumatoid and juvenile arthritis, rheumatism, systemic lupus erythamatosus, Grave's disease, and multiple sclerosis), bacterial, viral, and parasitic infections (e.g., sepsis, influenza, common colds, hepatitis, HIV infection, malaria, and gonorrhea), disorders associated with undesirable immune reactions with foreign material (e.g., transplant rejection, environmental {e.g., latex} hypersensitivity disorders, and allergic disorders), phagocytic dysfunction disorders (e.g., neutropenia and chronic granulomatous disease), anaphylaxis, urticaria, and immune deficiency disorders (e.g., T-cell and B-cell immunodeficiency disorders, AIDS). TANGO 229 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

The presence of a factor V/VIII discoidin domain in TANGO 229 protein is an indication that the protein can be involved in mediating cell binding and adhesion, including binding / adhesion of cells with other cells, with extracellular matrix, and with foreign materials (i.e., materials not originating in the body of the same individual). Cell binding and adhesion affected by TANGO 229 can encompass interactions between cells and between cells and extracellular components, which interactions lend structural and mechanical support to body tissues and containment of body fluids (e.g., by blood coagulation). However, TANGO 229 can also regulate cell-to-cell and cell-to- environment interactions which have little relevance to the structural integrity of the animal, but which permit information exchange between cells (e.g., cell-to-cell signaling such as that which occurs between helper T cells and antibody-producing B cells) or between cells and the environment (e.g., recognition by cells of the presence of a particular chemical entity, such as an antigen, in the environment). Certain cell-to- environment interactions mediated by TANGO 229 can also permit a cell which expresses it to exert an effect upon (e.g., degrade, absorb, or envelop) a component of the environment.

Involvement of TANGO 229 protein in binding of cells is an indication that TANGO 229 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 extracellular matrix include those (e.g., metastasis of cancerous cells through or into extracellular matrix and away from the normal body location of the cells) in which the cells normally do, but aberrantly do not, bind with extracellular matrix as well as those (e.g., metastasis of cancers cells into extracellular matrix at body locations at which they do not normally occur, autoimmune disorders, liver fibrosis, abnormal blood coagulation, atherosclerosis, and arteriosclerosis) in which the cells normally do not bind with extracellular matrix, but aberrantly do. Examples of disorders involving aberrant binding or adhesion of cells with foreign materials include those (e.g., allergies and hypersensitivity disorders such as latex hypersensitivity) associated with aberrant binding with the foreign material and disorders in which the cells normally bind with the foreign material, but aberrantly do not. TANGO 229 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 certain known developmental proteins (e.g., human neuropilins; Kolodkin and Ginty (1997) Neuron 19:1159-1162), TANGO 229 protein contains both a CUB domain and a factor V/VIII discoidin domain. The presence of both of these types of domains is an indication that TANGO 229 protein is involved in mediating attraction and repulsion of cells and translocation of cells through, past, or along other cells or tissues. For example, TANGO 229 can, alone or in conjunction with one or more neuropilins, bind with a semaphorin protein to direct nerve growth. Apart from regulating the rate and direction of nerve growth, TANGO 229 can regulate the rate and direction of growth of other tissues, such as vascular tissues (e.g., during angiogenesis). TANGO 229 can also modulate the direction and rate of cell movement, relative to another cell or relative to a tissue, such as movement of leukocytes through vascular lumenal epithelium (e.g., during leukocytic extravasation) or movement of metastatic cells through a solid tissue. Another example of such modulation is the effect that TANGO 229 can have on the rate of cell growth, depending on contact between two cells or between two tissues. TANGO 229 can regulate cell growth such that the growth slows or substantially stops when two tissues contact one another (e.g., during wound healing). TANGO 229 is thus involved in disorders associated with aberrant growth or movement of cells through, past, or along other cells or tissues. Examples of disorders of these types include cancerous growth and proliferation of cells, metastasis of cancerous cells (i.e., including metastasis away from the normal body location of the cells, through tissues and extracellular matrix, and into body locations at which the cells do not normally occur), inflammation, atherosclerosis, arteriosclerosis, abnormal blood coagulation, asthma, and chronic obstructive pulmonary disorders. TANGO 229 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 229 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. For example, several tyrosine-protein kinases (e.g., human and murine cell adhesion kinase and neurotrophic receptor-related tyrosine kinase-3) comprise one or more factor V/VIII discoidin domains. The structure of TANGO 229 protein, which has several potential phosphorylation sites, is thus an indication that the protein can be involved in transducing signals across the cell membrane. Binding of a ligand of TANGO 229 protein 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 protein located on the other side. Thus, for example, a compound in the extracellular environment of a cell which expresses TANGO 229 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 229 protein can associate with other signal transduction proteins in the cell membrane, thereby modulating the intracellular activity of those other proteins. TANGO 229 protein 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, inflammation, aberrant or undesirable angiogenesis, and obesity. TANGO 229 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.

INTERCEPT 289 A cDNA clone (designated jthLal 86d06) encoding at least a portion of human INTERCEPT 289 protein was isolated from a human mixed lymphocyte reaction cDNA library. Human INTERCEPT 289 protein is a transmembrane protein which can occur in at least six alternative forms. These forms are herein designated "form la," "form lb," "form 2a," " form 2b," "form 3a," and "form 3b" for convenience. The properties of and variations among these forms are described herein. 1 a) The full length of the cDNA encoding INTERCEPT 289 protein form 1 a (Figures 2A-2C and SEQ ID NO: 11) is 4074 nucleotide residues. The ORF of this cDNA, nucleotide residues 179 to 742 of SEQ ID NO: 11 (i.e., SEQ ID NO: 12), encodes a 188-amino acid residue protein having the amino acid sequence SEQ ID NO: 13. lb) The full length of the cDNA encoding INTERCEPT 289 protein form lb (Figures 2D-2G and SEQ ID NO: 21) is 4018 nucleotide residues. The ORF of this cDNA, nucleotide residues 179 to 712 of SEQ ID NO: 21 (i.e., SEQ ID NO: 22), encodes a 178-amino acid residue protein having the amino acid sequence SEQ ID NO: 23.

2a) The full length of the cDNA encoding INTERCEPT 289 protein form 2a (Figures 2H-2K and SEQ ID NO: 26) is 3985 nucleotide residues. The ORF of this cDNA, nucleotide residues 162 to 656 of SEQ ID NO: 26 (i.e., SEQ ID NO: 27), encodes a 165-amino acid residue protein having the amino acid sequence SEQ ID NO: 28.

2b) The full length of the cDNA encoding INTERCEPT 289 protein form 2b (Figures 2L-2O and SEQ ID NO: 31) is 3958 nucleotide residues. The ORF of this cDNA, nucleotide residues 162 to 626 of SEQ ID NO: 31 (i.e., SEQ ID NO: 32), encodes a 155-amino acid residue protein having the amino acid sequence SEQ ID NO: 33.

3 a) The full length of the cDN A encoding INTERCEPT 289 protein form 3 a (Figures 2P-2S and SEQ ID NO: 36) is 3925 nucleotide residues. The ORF of this cDNA, nucleotide residues 162 to 596 of SEQ ID NO: 36 (i.e., SEQ ID NO: 37), encodes a 145-amino acid residue protein having the amino acid sequence SEQ ID NO: 38.

3b) The full length of the cDNA encoding INTERCEPT 289 protein form 3b (Figures 2T-2V and SEQ ID NO: 41) is 3898 nucleotide residues. The ORF of this cDNA, nucleotide residues 162 to 566 of SEQ ID NO: 41 (i.e., SEQ ID NO: 42), encodes a 135-amino acid residue protein having the amino acid sequence SEQ ID NO: 43. The mixed lymphocyte reaction library from which the cDNAs encoding INTERCEPT 289 were isolated was prepared as follows. Mononuclear cells were isolated from 50 milliliters of peripheral blood pooled from 22 human donors. Mononuclear cells were isolated using HISTOPAQUE™ 1077 (Sigma Chemical Co., St. Louis, MO) according to the manufacturer's instructions and collected in heparinized tubes. After pooling the mononuclear cells, CD19⁺ B cells were removed by positive selection using MACS™ beads and a VS+ separation column (Miltenyi Biotec, Germany) according to the manufacturer's instructions. CD 19^" cells were re-suspended at an approximate density of 10 x 10⁶ cells per milliliter in RPMI medium supplemented with 10% (v/v) fetal bovine serum, antibiotics, and L-glutamine. The cells were maintained at 37°C in a humidified incubator, and were harvested 4, 14, and 24 hours following re-suspension. Total RNA was isolated from the cells by guanidinium isothiocyanate / beta-mercaptoethanol lysis followed by cesium chloride gradient centrifugation. Isolated RNA was treated with DNase, and the poly-A-containing fraction of total RNA was further purified using OLIGOTEX™ beads (Qiagen, Inc.).

About 4.4 micrograms of poly-A-containing RNA was used to synthesize a cDNA library using the Superscript™ cDNA synthesis kit (Gibco BRL, Inc.; Gaithersburg, MD). cDNA was directionally cloned into expression plasmid pMET7 vectors using Sail and Nøtl polylinker restriction endonuclease sites in order to generate a plasmid library. Transformants were randomly selected and expanded in culture for single-pass nucleotide sequencing.

In addition to full length human INTERCEPT 289 proteins, the invention includes fragments, derivatives, and variants of these INTERCEPT 289 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 one of SEQ ID NOs: 11, 21, 26, 31, 36, and 41, such as the portion which encodes INTERCEPT 289 protein or a domain (e.g., the extracellular domain) of INTERCEPT 289 protein. These nucleic acids are collectively referred to as nucleic acids of the invention. In each form, INTERCEPT 289 protein includes a transmembrane domain and a portion corresponding to an extra-membrane (presumably extracellular) domain. In alternative embodiments, this extra-membrane domain is a cytoplasmic domain. The transmembrane domain corresponds to about amino acid residues 7 to 27 of SEQ ID NO: 13 (i.e., SEQ ID NO: 14 in form la), to about amino acid residues 7 to 27 of SEQ ID

NO: 23 (i.e., SEQ ID NO: 24 in form lb), to about amino acid residues 7 to 27 of SEQ ID NO: 28 (i.e., SEQ ID NO: 29 in form 2a), to about amino acid residues 7 to 27 of SEQ ID NO: 33 (i.e., SEQ ID NO: 34 in form 2b), to about amino acid residues 7 to 28 of SEQ ID NO: 38 (i.e., SEQ ID NO: 29 in form 3a), and to about amino acid residues 7 to 28 of SEQ ID NO: 43 (i.e., SEQ ID NO: 44 in form 3b).

Each form of INTERCEPT 289 protein also includes another extra- membrane portion. This portion corresponds to about amino acid residues 28 to 188 of SEQ ID NO: 13 (i.e., SEQ ID NO: 15 in form la), to about amino acid residues 28 to 178 of SEQ ID NO: 23 (i.e., SEQ ID NO: 25 in form lb), to about amino acid residues 28 to 165 of SEQ ID NO: 28 (i.e., SEQ ID NO: 30 in form 2a), to about amino acid residues 28 to 155 of SEQ ID NO: 33 (i.e., SEQ ID NO: 35 in form 2b), to about amino acid residues 29 to 145 of SEQ ID NO: 38 (i.e., SEQ ID NO: 40 in form 3a), and to about amino acid residues 29 to 135 of SEQ ID NO: 43 (i.e., SEQ ID NO: 45 in form 3b). INTERCEPT 289 proteins and nucleic acid molecules encoding them comprise a family of molecules having certain conserved structural and functional features, as illustrated in Figures 2W and 2Xi through 2Xxiv.

In Figure 2W, the amino acid sequences of various forms of INTERCEPT 289 ("A"-"F"; SEQ ID NOs: 13, 23, 28, 33, 38, and 43) are shown, as aligned using the Wisconsin™ BestFit software (Smith and Waterman, (1981) Adv. Appl. Math. 2:482-

489; blosum62 scoring matrix; gap opening penalty 10 / gap extension penalty 10). In Figures 2Xi through 2Xxiv, the nucleotide sequences (SEQ ID NOs: 11, 21, 26, 31, 36, and 41) of cDNA molecules encoding the six forms of INTERCEPT 289 protein described herein are aligned using the Wisconsin™ BestFit software (Smith and Waterman, (1981) Adv. Appl. Math. 2:482-489; gap opening penalty 10 / gap extension penalty 10). As indicated in these figures, the various forms of INTERCEPT 289 protein differ in the length of the polypeptide sequence between the transmembrane domain and the lectin C-type domain described below and in the amino acid sequence of the carboxyl-terminal portion of the protein.

INTERCEPT 289 proteins typically comprise a variety of potential post- translational modification sites and protein domains (often positioned within a domain located at or near the protein surface), such as those described herein in Table IIIA, as predicted by computerized sequence analysis of INTERCEPT 289 proteins using amino acid sequence comparison software (comparing the amino acid sequence of INTERCEPT 289 with the information in the PROSITE database {rel. 12.2; Feb, 1995} and the Hidden Markov Models database {Rel. PFAM 3.3}).

Table IIIA

In various embodiments, the protein of the invention has at least 1, 2, 4, 6, 8, 12, or more of the post-translational modification sites and domains described in Table IIIA.

An example of an additional domain present in INTERCEPT 289 proteins is a lectin C-type domain. 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 this domain. C-type lectin domains are conserved among proteins (e.g., animal lectins) which are involved in calcium-dependent binding of carbohydrates, although it has recently been recognized that these domains can also be involved in binding of proteins (Drickamer, (1988) J. Biol. Chem. 263:9557-9560;

Drickamer, (1993) Prog. Nucl. Acid Res. Mol. Biol. 45:207-232; Drickamer, (1993) Curr. Opin. Struct. Biol. 3:393-400). C-type lectins and their relevant properties are described in greater in P.C.T. Publication No. WO 98/28332, which, as with all references cited herein, is incorporated by reference. A cDNA clone (designated jtmMal 27f05) encoding at least a portion of murine INTERCEPT 289 protein was also isolated. Murine INTERCEPT 289 protein is a transmembrane protein. The properties of murine INTERCEPT 289 are described below.

In addition to full length murine INTERCEPT 289 proteins, the invention includes fragments, derivatives, and variants of murine INTERCEPT 289 proteins, as described herein. These proteins, fragments, derivatives, and variants are among those collectively referred to herein as polypeptides of the invention or proteins of the invention. Among the nucleic acid molecules of the invention are those which encode murine INTERCEPT 289 and portions thereof. Murine INTERCEPT 289 protein includes a transmembrane domain and a portion corresponding to an extra-membrane domain. In one embodiment, the domain is extracellular; in an alternative embodiments, this extra-membrane domain is a cytoplasmic domain. The transmembrane domain corresponds to about amino acid residues 7 to 27 of SEQ ID NO: 93 (i.e., SEQ ID NO: 94 in form la), and the extra- membrane portion corresponds to about amino acid residues 28 to 190 of SEQ ID NO:

93 (i.e., SEQ ID NO: 95). Murine INTERCEPT 289 proteins typically comprise a variety of potential post-translational modification sites and protein domains (often positioned within a domain located at or near the protein surface), such as those described herein in Table IIIB, as predicted by computerized sequence analysis of murine INTERCEPT 289 protein using amino acid sequence comparison software (comparing the amino acid sequence of murine INTERCEPT 289 with the information in the PROSITE database {rel. 12.2; Feb, 1995} and the Hidden Markov Models database {Rel. PFAM 3.3}).

Table IIIB

In various embodiments, the protein of the invention has at least 1, 2, 4, 6, 8, or more of the post-translational modification sites and domains described in Table IIIB. INTERCEPT 289 proteins and cDNAs exhibit homology with human myeloid DAP 12 (DNAX accessory protein, 12 kD) associated lectin- 1 (MDL-1), which is described in PCT Publication No. WO 99/06557, which is also incorporated herein by reference. In Figure 2W, the amino acid sequences of various forms of INTERCEPT 289 ("A"-"F" and "R"; SEQ ID NOs: 13, 23, 28, 33, 38, 43, and 93, respectively), human MDL-1 ("H"; SEQ ID NO: 16), and murine MDL-1 ("M"; SEQ ID NO: 18) proteins are shown, as aligned using the Wisconsin™ BestFit software (Smith and Waterman, (1981) Adv. Appl. Math. 2:482-489; BLOSUM62 scoring matrix; gap opening penalty 10 / gap extension penalty 10). Each of the seven forms of INTERCEPT 289 protein described herein has a lysine residue (i.e., at residue 116 of SEQ ID NOs: 13 and 23, at residue 93 of SEQ ID NOs: 28 and 33, at residue 73 of SEQ ID NOs: 38 and 43, and at residue 118 of SEQ ID NO: 93) that is not present in the described sequence (SEQ ID NO: 16) of human MDL-1 protein.

In the alignment shown in Figure 2W, the amino acid sequence (SEQ ID NO: 13) of form 1 a of INTERCEPT 289 protein is 100% identical to that of human

MDL-1 over the 187-amino acid residue overlapping region and about 72.7% identical to that of murine MDL-1 in the 165-amino acid residue overlapping region.

In the alignment shown in Figure 2W, the amino acid sequence (SEQ ID NO: 23) of form lb of INTERCEPT 289 protein is about 85.9% identical to that of human MDL-1 over the 177-amino acid residue overlapping region and about 60.0% identical to that of murine MDL-1 in the 155-amino acid residue overlapping region.

In the alignment shown in Figure 2W, the amino acid sequence (SEQ ID NO: 28) of form 2a of INTERCEPT 289 protein is 100% identical to that of human MDL-1 over the 164-amino acid residue overlapping region and about 71.5% identical to that of murine MDL-1 in the 165-amino acid residue overlapping region.

In the alignment shown in Figure 2W, the amino acid sequence (SEQ ID NO: 33) of form 2b of INTERCEPT 289 protein is about 83.8% identical to that of human MDL-1 over the 154-amino acid residue overlapping region and about 58.7% identical to that of murine MDL-1 in the 155-amino acid residue overlapping region. In the alignment shown in Figure 2W, the amino acid sequence (SEQ ID

NO: 38) of form 3a of INTERCEPT 289 protein is about 83.3% identical to that of human MDL-1 over the 144-amino acid residue overlapping region and about 74.5% identical to that of murine MDL-1 in the 145-amino acid residue overlapping region.

In the alignment shown in Figure 2W, the amino acid sequence (SEQ ID NO: 43) of form 3b of INTERCEPT 289 protein is about 63.4% identical to that of human MDL-1 over the 134-amino acid residue overlapping region and about 60.0% identical to that of murine MDL-1 in the 135-amino acid residue overlapping region.

In the alignment shown in Figure 2W, the amino acid sequence (SEQ ID NO: 93) of murine INTERCEPT 289 protein is 100% identical to that of murine MDL-1 over the 190-amino acid residue overlapping region and about 85.7% identical to that of human MDL-1 in the 188-amino acid residue overlapping region.

In the alignment shown in Figure 2Zv, the nucleotide sequence (SEQ ID NO: 92) of the ORF of murine INTERCEPT 289 is about 71.8% identical to that of the ORF of human INTERCEPT 289 form la. MDL-1 is a cell surface protein which is expressed by monocytes and macrophages and which binds with DAP 12. DAP 12 is a cell surface protein which is expressed by natural killer cells, peripheral blood granulocytes and monocytes, macrophages, and dendritic cells. DAP 12 is an immunoreceptor tyrosine-based activation motif-containing protein which associates non-covalently with activating isoforms of MHC class I receptors on natural killer cells (Bakker et al., 1999, Proc. Natl.

Acad. Sci. USA 96:9792-9796). Association of MDL-1 and DAP12 on the surface of monocytes and macrophages and binding of associated MDL-1 / DAP 12 with a ligand thereof (e.g., a surface protein, glycoprotein, or glycolipid on the surface of another cell of the same animal or on the surface of a foreign cell) causes activation of those cells. Upon activation, and depending on the type of the monocyte / macrophage, the monocyte / macrophage generates an oxidative burst, produces one or more cytokines, and other leukocyte-modulating molecules, releases one or more cytokines other leukocyte-modulating molecules, or some combination of these activities. MDL-1 and, by analogy, INTERCEPT 289 are therefore involved in modulation of immune function, including modulation of antibody and cytotoxic T cell responses, expansion of immune cell populations, inflammation, and generation of memory B cells.

The amino acid sequences (SEQ ID NOs: 13, 23, 28, 33, 38, and 43) of the six forms of INTERCEPT 289 protein described herein were aligned with the amino acid sequence of CD94 protein (GenBank Accession No. 5542082) using the Wisconsin™ BestFit software (Smith and Waterman, (1981) Adv. Appl. Math. 2:482-

489; BLOSUM62 scoring matrix; gap opening penalty 10 / gap extension penalty 10).

The amino acid sequence identity between CD94 protein and INTERCEPT 289 protein was 28.0% for form la in the 126-amino acid residue overlapping region, 25.2% for form lb in the 115-amino acid residue overlapping region, 28.0% for form 2a in the 125- amino acid residue overlapping region, 25.2% for form 2b in the 127-amino acid residue overlapping region, 27.2% for form 3a in the 125-amino acid residue overlapping region, and 24.3% for form 3b in the 115-amino acid residue overlapping region. CD94 protein is a cell-surface protein which has a C-type lectin domain in its carboxyl terminal portion and which acts as a receptor for natural killer (NK) cells. CD94 modulates the cytotoxic activity of NK cells, as well as production of cytokines by NK cells.

Figures 2Yi through 2Yvi depict hydrophilicity plots of the six forms of human INTERCEPT 289 protein described herein. Form la corresponds to Figure 2Yi, and has the amino acid sequence SEQ ID NO: 13. Form lb corresponds to Figure 2Yii, and has the amino acid sequence SEQ ID NO: 23. Form 2a corresponds to Figure 2Yiii, and has the amino acid sequence SEQ ID NO: 28. Form 2b corresponds to Figure 2Yiv, and has the amino acid sequence SEQ ID NO: 33. Form 3a corresponds to Figure 2Yv, and has the amino acid sequence SEQ ID NO: 38. Form 3b corresponds to Figure 2Yvi, and has the amino acid sequence SEQ ID NO: 43. Relatively hydrophobic regions are above the dashed horizontal line, and relatively hydrophilic regions are below the dashed horizontal line. 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. Figure 2Zvi depicts a hydrophilicity plot of the murine INTERCEPT 289 protein described herein.

The predicted molecular weights of the six forms of human INTERCEPT 289 protein described herein, without modification, is about 21.5 kilodaltons for form la, about 20.4 kilodaltons for form lb, about 19.1 kilodaltons for form 2a, about 18.0 kilodaltons for form 2b, about 16.9 kilodaltons for form 3a, and about 15.8 kilodaltons for form 3b. The predicted molecular weight of murine INTERCEPT 289, without modification is about 21.7 kilodaltons.

Expression of one or more forms of INTERCEPT 289 was detected in cDNA libraries prepared using human tissue and cell samples listed in Table IV, wherein "+" indicates detectable expression and "+/-" indicates weakly detectable expression. Table IV

Biological function of INTERCEPT 289 proteins, nucleic acids encoding them, and modulators of these molecules INTERCEPT 289 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 INTERCEPT 289 occurs in a human mixed lymphocyte reaction cDNA library, and that RNA corresponding to INTERCEPT 289 is detectable by PCR amplification, using primers which specifically amplify INTERCEPT 289 sequences, of nucleic acids (e.g., mRNA or cDNA) obtained from human leukemia, bone marrow, dendritic, ovarian ascitic, aortic endothelial, and cardiac (e.g., left ventricle cells obtained from a heart afflicted with congestive heart failure) cells, it is evident that INTERCEPT 289 protein can be involved in one or more biological processes which occur in these cells and in tissues which contain them. In particular, INTERCEPT 289 is involved in modulating growth, proliferation, survival, differentiation, and activity of cells of these cells and tissues (e.g., lymphocytes). Examples of disorders of such cells and tissues include various cancers (e.g., leukemias, lymphomas, and endothelial cancers such as ovarian cancers), atherosclerosis, arteriosclerosis, coronary artery disease, immune insufficiency disorders, immune hypersensitivity disorders, and congestive heart failure disorders (e.g., myocardial infarction, cardiomegaly, and cardiac valvular defects). INTERCEPT 289 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.

Presence of a C-type lectin domain in INTERCEPT 289 is an indication that this protein can specifically recognize particular surfaces, such as the surface of cells of a particular type. Further supportive of this observation is the fact that human

INTERCEPT 289 proteins exhibit significant sequence identity with MDL-1 which, in cooperation with DAP 12 protein associated therewith, is capable of binding one or more ligands and activating one or more types of macrophages and monocytes. Aberrant activation of macrophages and monocytes is associated with a variety of immunological disorders including, for example, inflammation, asthma, hypersensitivity disorders (e.g., allergies), atopic disorders (e.g., allergic rhinitis, allergic asthma, and atopic dermatitis), anaphylaxis, urticaria (i.e., hives), auto-immune disorders (e.g., rheumatoid and juvenile arthritis, rheumatism, systemic lupus erythamatosus, Grave's disease, and multiple sclerosis), graft and transplant rejection, leukemias (e.g., ALL, CML, CLL, and myelodysplastic syndrome), blood dyscrasias (e.g., multiple myeloma), polycythemia vera, myelofϊbrosis, leukopenias, lymphomas (e.g., Hodgkin's disease, non-Hodgkin's lymphoma, Burkitt's lymphoma, and mycosis fungoides), bacterial, viral, and parasitic infections (e.g., sepsis, influenza, common colds, hepatitis, HIV infection, malaria, and gonorrhea), immune insufficiency (e.g., AIDS), and immunodeficiency disorders. INTERCEPT 289 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.

INTERCEPT 309 A cDNA clone (designated jthYa038a01tl) encoding at least a portion of human INTERCEPT 309 protein was isolated from a human thyroid tissue cDNA library. Human INTERCEPT 309 protein is an integral membrane protein having three transmembrane regions and a fourth transmembrane region that can act as a signal sequence. Human INTERCEPT 309 protein is a claudin-like protein. The full length of the cDNA encoding human INTERCEPT 309 protein

(Figure 3; SEQ ID NO: 51) is 1909 nucleotide residues. The ORF of this cDNA, nucleotide residues 2 to 646 of SEQ ID NO: 51 (i.e., SEQ ID NO: 52), encodes an approximately 215-amino acid residue integral membrane protein (Figure 3; SEQ ID NO: 53) having three transmembrane regions in its mature (18 l-amino acid residue; SEQ ID NO: 68) form. In addition to full length human INTERCEPT 309 proteins, the invention includes fragments, derivatives, and variants of these INTERCEPT 309 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 NO: 51, such as the portion which encodes mature INTERCEPT 309 protein, immature INTERCEPT 309 protein, or a domain of INTERCEPT 309 protein. These nucleic acids are collectively referred to as nucleic acids of the invention. INTERCEPT 309 proteins and nucleic acid molecules encoding them comprise a family of molecules having certain conserved structural and functional features.

A common domain present in INTERCEPT 309 proteins is a signal sequence. In one embodiment, a INTERCEPT 309 protein contains a signal sequence corresponding to about amino acid residues 1 to 24 of SEQ ID NO: 53 (SEQ ID NO: 54).

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 22, 23, 24, 25, or 26 of SEQ ID NO: 53). The signal sequence is cleaved during processing of the mature protein. INTERCEPT 309 proteins include three transmembrane domains and two pairs of extra-membrane domains that flank the cell membrane. The three transmembrane domains correspond to about amino acid residues 72 to 92, 108 to 131, and 154 to 178 of SEQ ID NO: 53 (i.e., the transmembrane domains having the sequences SEQ ID NOs: 56, 58, and 60, respectively). One pair of extra-membrane domains corresponds to about amino acid residues 25 to 71 and 132 to 153 of SEQ ID

NO: 53 (these domains having the sequences SEQ ID NOs: 55 and 59). The other pair of extra-membrane domains corresponds to about amino acid residues 93 to 107 and 179 to 215 of SEQ ID NO: 53 (these domains having the sequences SEQ ID NOs: 57 and 61). In one embodiment, the first pair of extra-membrane domains (i.e., those having the sequences SEQ ID NOs: 55 and 59) are extracellular domains and the other pair of domains are cytoplasmic domains. However, in an alternative form, the first pair of extra-membrane domains are cytoplasmic and the other pair are extracellular domains.

It is recognized that, in certain forms, INTERCEPT 309 proteins can have an additional number of amino acid residues at their amino terminus. For example, the proteins can have from 1 to about 30 amino acid residues, more commonly 1 to about 12, 1 to about 10, or 1 to about 5 residues.

INTERCEPT 309 proteins typically comprise a variety of potential post- translational modification sites and protein domains (often positioned within an extracellular or protein surface domain), such as those described herein in Table V, as predicted by computerized sequence analysis of INTERCEPT 309 proteins using amino acid sequence comparison software (comparing the amino acid sequence of INTERCEPT 309 with the information in the PROSITE database {rel. 12.2; Feb, 1995} and the Hidden Markov Models database {Rel. PFAM 3.3}).

Table V

Table V (Continued)

In various embodiments, the protein of the invention has at least 1, 2, 4, 6, or all 11 of the post-translational modification sites and domains described herein in

Table V.

Figure 3D depicts a hydrophilicity plot of an embodiment of human INTERCEPT 309 protein. Relatively hydrophobic regions are above the dashed horizontal line, and relatively hydrophilic regions are below the dashed horizontal line. The hydrophobic regions which corresponds to about amino acid residues 72 to 92, 108 to 131, and 154 to 178 of SEQ ID NO: 53 are the transmembrane domains of human INTERCEPT 309 described above. 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 INTERCEPT 309 protein from about amino acid residue

90 to about amino acid residue 100 appears to be located at or near the surface of the protein, while the region from about amino acid residue 70 to about amino acid residue 85 appears not to be located at or near the surface.

The predicted molecular weight of human INTERCEPT 309 protein without modification and prior to cleavage of the signal sequence is about 23.8 kilodaltons. The predicted molecular weight of the mature human INTERCEPT 309 protein without modification and after cleavage of the signal sequence is about 21.4 kilodaltons.

INTERCEPT 309 protein exhibits amino acid sequence homology with murine claudin-8 protein, as indicated in the alignment (made using the ALIGN software {Myers and Miller (1989) CABIOS, ver. 2.0}; paml20.mat scoring matrix; gap opening penalty = 12, gap extension penalty = 4) of the amino acid sequences of INTERCEPT 309 (SEQ ID NO: 53) and murine claudin-8 (SEQ ID NO: 62) proteins shown in Figure 3S. In this alignment, the two amino acid sequences are about 80.0% identical. Furthermore, INTERCEPT 309 cDNA (SEQ ID NO: 51) is about 83.1% identical to the nucleotide sequence of cDNA encoding murine claudin-8 (SEQ ID NO: 63; GenBank accession no. AF087826) over the 639-residue overlapping region, as indicated in the alignment (made using the ALIGN software; paml20.mat scoring matrix; gap opening penalty = 12, gap extension penalty = 4) shown in Figures 3L through 3R. An alignment (made using the ALIGN software; paml20.mat scoring matrix; gap opening penalty = 12, gap extension penalty = 4) of the nucleotide sequences of a cDNA clone (SEQ ID NO: 64; GenBank accession no. AL049977) obtained from human fetal brain tissue and INTERCEPT 309 cDNA (SEQ ID NO: 51) is shown in Figures 3E through 3K and indicates 100% sequence identity between the sequences in the overlapping portion. The overlapping portion does not overlap the INTERCEPT 309

ORF, with the exception of nucleotide residues 1 and 28-32. It is recognized that 'overlap' of the human fetal brain cDNA clone sequence with these ORF residues is an artifact of the ALIGN software, and does not represent meaningful homology between residues 1 and 28-32 of the INTERCEPT 309 ORF and the corresponding residues of the human fetal brain cDNA clone. Nonetheless, isolation of this cDNA clone from fetal brain tissue is an indication that INTERCEPT 309 protein is expressed in fetal brain tissue.

An alignment (made using the LALIGN software {Huang and Miller, 1991, Adv. Appl. Math. 12:373-381}; paml20 scoring matrix, gap opening penalty = 12, gap extension penalty = 4) of the nucleotide sequence of INTERCEPT 309 cDNA (SEQ

ID NO: 51) with the nucleotide sequence encoding murine latent transforming growth factor-beta binding protein-3 (LTBP-3) indicated that the two sequences were 40.3% identical in a 1969-nucleotide residue overlapping portion. As disclosed in P.C.T. Publication No. WO 95/22611, latent transforming growth factor-beta binding protein 3 (LTBP-3) is a secreted protein that is expressed in murine epithelial, parenchymal, and stromal during embryonic development. LTBP-3 is thought to exhibit one or more of four activities i) modulating intracellular biosynthesis of latent transforming growth factor-beta; ii) binding latent transforming growth factor-beta with extracellular matrix; iii) modulating activation of latent transforming growth factor-beta complexes; and iv) targeting latent transforming growth factor-beta complexes to the cell surface. An alignment (made using the ALIGN software; paml 20.mat scoring matrix, gap opening penalty = 12, gap extension penalty = 4) of the amino acid sequence of INTERCEPT 309 cDNA (SEQ ID NO: 53) with the amino acid sequence of human peripheral myelin protein (PMP-22) indicated that the two protein sequences are 17.2% identical. PMP-22 is involved in myelination of peripheral nerves, particularly during development.

Individual alignments (made using the Wisconsin™ BestFit software; Smith and Waterman (1981) Adv. Appl. Math. 2:482-489; blosum62 scoring matrix, gap opening penalty 10 / gap extension penalty 10) of the amino acid sequence (SEQ ID NO:

53) of INTERCEPT 309 with the amino acid sequences of human (SEQ ID NO: 65; GenBank Accession No. 4502877) and murine (SEQ ID NO: 66; GenBank Accession No. BAA22985) receptors of Clostridium perfringens enterotoxin (CPE) and with the amino acid sequence (SEQ ID NO: 67) encoded by rat ventral prostate tissue during androgen withdrawal-induced tissue regression were manually aligned (by inserting a

'blank' at position 1 of the rRPV nucleotide sequence). The manually aligned alignments are shown in Figure 3T. The amino acid sequence of INTERCEPT 309 protein is about 43% identical to the human CPE receptor amino acid sequence, about 45% identical to the murine CPE receptor amino acid sequence, and about 43% identical to the amino acid sequence encoded by the transcript obtained from regressing rat ventral prostate tissue.

Expressed sequence tags (ESTs) which exhibit at least limited nucleotide sequence identity with SEQ ID NO: 51 have been isolated from human and murine liver, kidney, prostate, and colon tissues. Biological function of INTERCEPT 309 proteins, nucleic acids encoding them, and modulators of these molecules

INTERCEPT 309 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 INTERCEPT 309 occurs in human thyroid and fetal brain cDNA libraries, and that ESTs have been isolated from liver, kidney, prostate, and colon tissues, it is evident that INTERCEPT 309 protein is involved in one or more biological processes which occur in these tissues. In particular, INTERCEPT 309 is involved in modulating growth, proliferation, survival, differentiation, and activity (e.g., thyroid secretion activity) of cells of these tissues.

Thus, INTERCEPT 309 has a role in disorders which affect the brain, thyroid, and other tissues and one or more of growth, proliferation, survival, differentiation, activity, morphology, and movement / migration of cells in those tissues, as well as the biological function of organs (e.g., the brain, liver, colon, prostate, kidneys, and thyroid) comprising such tissues. Relevant disorders which involve these tissues are discussed separately below.

As indicated by its similarity to murine claudin-8 (e.g., as shown in Figure 3S), INTERCEPT 309 is a claudin-like protein, and can exhibit one or more of the activities exhibited by murine claudin-8 and other claudins. Claudins are proteins that are involved in formation, maintenance, and regulation of tight junctions, which are intercellular junctions that occur between cells of tissues (e.g., epithelia and endothelia) having selective permeability (Morita et al. (1999) Proc. Natl. Acad. Sci. USA 96:511- 516). Tight junctions can be associated with actin fibrils, and claudins can mediate interactions between actin fibrils and other components of the tight junction. Tissues in which tight junctions occur between adjacent cells can form sheets or other structures which exhibit selective trans-tissue permeability and in which the membrane and membrane-bound components of tissue-spanning cells can be selectively localized to one side (e.g., apical or basolateral side) of the tissue. By way of example, epithelial and endothelial tissues of kidney, liver, lung, and thyroid form barriers which permit transepithelial / transendothelial passage of certain compounds and cells (e.g., secreted / excreted products and immune system cells), but not others. Tight junction alterations have also been associated with tumor differentiation, particularly in thyroid tumors (Kerjaschki et al. (1979) Am. J. Pathol. 96:207-225; Cochand-Priollet et al. (1998) Ultrastruct. Pathol. 22:413-420). INTERCEPT 309 can have a role in each of these functions, both in normal tissue and in aberrant tissue (e.g., tissue of a patient afflicted with a disorder that affects the tissue).

An important feature of tight junctions is that the permeability of a tissue comprising such intercellular junctions can be regulated by cellular and other (e.g., endocrine) processes. Thus, depending on the cellular or other influences exerted on the components of the tight junction, the permeability of the tissue to water, solutes (e.g., urea), proteins (e.g., hormones), and immune cells (e.g., T cells and macrophages) can be regulated (Stevenson (1999) J. Clin. Invest. 104:3-4). Regulation of transmembrane permeability is critical to the function of many organs (e.g., kidney, colon, thyroid, liver, prostate, etc.). INTERCEPT 309, being a claudin-like protein can regulate transmembrane permeability in organs and tissues in which it is normally or aberrantly expressed.

One or more transmembrane proteins associated with tight junctions mediate transmembrane signal transduction which regulates, ter alia, the permeability of the junction (Fanning et al., (1999) J Am. Soc. Nephrol. 10:1337-1345). For example, inhibition of protein tyrosine phosphorylation (a common activity associated with transmembrane signaling) has been associated with aberrant thyroid epithelial cell junction formation (Yap et al., (1997) Endocrinology 138:2315-2324). INTERCEPT 309, being a transmembrane protein associated with tight junctions and having a potential tyrosine kinase phosphorylation site at residues 149-156 of SEQ ID NO: 53, can be involved in transmembrane transduction of signals between the cell interior and the extracellular milieu, including signal transduction associated with regulation of tight junction function.

Claudins can also participate in cell-to-cell adhesive processes that do not necessarily involve tight junction formation. Examples of such mechanisms include binding between cells forming the blood-brain barrier, adhesion of myelin to nerve fibers and to itself, and binding between skin cells to form a barrier to the passage of moisture and solutes to and from the environment. Similarity between the amino acid sequences of INTERCEPT 309 and PMP-22 is also indicative of a role of INTERCEPT 309 protein in mediating adhesion between myelin-producing cells and nerve cells (e.g., between Schwann cells and peripheral nerve cells). INTERCEPT 309 can therefore have a role in disorders (e.g., multiple sclerosis) involving aberrant (including insufficient) myelination or demyelination of nerve cells. INTERCEPT 309, being a cell surface claudin-like protein, can be a substrate for interaction of pathogens (e.g., bacteria, toxins, and viruses) with host cells, and can mediate interaction of pathogens with cells which express INTERCEPT 309. For example, Morita et al. (supra) determined that a murine claudin is a receptor for Clostridium perfringens enterotoxin (CPE). Similarity between the amino acid sequences of murine claudin-8 and INTERCEPT 309 indicates that INTERCEPT 309 can act as a receptor for CPE. Furthermore, amino acid sequence similarity between INTERCEPT 309 and other human and murine CPE receptors (e.g., GenBank Accession Nos. 4502877 and BAA22985, as indicated in Figure 3T) is a further indication that INTERCEPT 309 can mediate interaction of CPE with cells upon which CPE acts. INTERCEPT 309 proteins, nucleic acids encoding them, and agents that modulate activity or expression of either of these can be used to prognosticate, diagnose, and treat disorders mediated by C. perfringens. Such disorders include, by way of example, gastrointestinal disorders (e.g., diarrhea, gastroenteritis, and other disorders associated with food poisoning, and certain types of pseudomembranous colitis), disorders associated with wound healing (e.g., gangrene), and other pathogenic infections (e.g., sepsis with or without intravascular hemolysis). INTERCEPT 309 can, of course, also mediate interaction of other pathogens with cells which express it.

Being a claudin-like protein, INTERCEPT 309 can be involved in formation, maintenance, and regulation of structures (e.g., transmembrane protein complexes including INTERCEPT 309) that regulate the permeability of cell membranes with regard to various molecules and macromolecules. Regulation of trans-tissue (i.e., intercellular) diffusion of extracellular components (e.g., water, solutes, and immune cells) and diffusion of membrane-bound and integral membrane components from one side of a tissue (e.g., the apical face of an epithelium) to the other (e.g., the basolateral face of the same epithelium; i.e., paracellular diffusion) can be modulated by

INTERCEPT 309 proteins and nucleic acids and by small molecules which interact with INTERCEPT 309 proteins and nucleic acids encoding them. Actin is known to be associated with tight junction components, and modifications to the actin cytoskeleton of a cell can modulate tight junction-regulated intercellular and paracellular diffusion. Thus, compositions which affect the interaction between actin and INTERCEPT 309 protein can modulate tight junction regulation of intercellular and paracellular diffusion.

In addition, agents which act directly on an INTERCEPT 309 protein or nucleic acid, without affecting the interaction between the claudin and actin, can be used to modulate tight junction regulation of intercellular and paracellular diffusion. INTERCEPT 309 protein can also act as a receptor for C. perfringens enterotoxin and for other pathogens, and INTERCEPT 309 proteins, as described herein, can be used to modulate C. perfringens enterotoxin binding and toxicity, as well as binding of other pathogens with cells and tissues which express INTERCEPT 309.

The fact that cDNA encoding INTERCEPT 309 was isolated from human thyroid and fetal brain cDNA libraries and the fact that INTERCEPT 309 have been isolated from liver, kidney, prostate, and colon tissues indicates that INTERCEPT 309 can have a role in disorders of these tissues, particularly including those characterized above. Examples of disorders in which INTERCEPT 309 can have a role are described in the following paragraphs a)-f), which are organized, for convenience, by tissue type, a) Examples of brain disorders in which INTERCEPT 309 can have role include both CNS disorders, CNS-related disorders, focal brain disorders, global-diffuse cerebral disorders, and other neurological and cerebrovascular disorders. CNS disorders include, but are not limited to cognitive and neurodegenerative disorders such as Alzheimer's disease, senile dementia, Huntington's disease, amyotrophic lateral sclerosis, and Parkinson's disease, as well as Gilles de la Tourette's syndrome, autonomic function disorders such as hypertension and sleep disorders (e.g., insomnia, hypersomnia, parasomnia, and sleep apnea), neuropsychiatric disorders (e.g., schizophrenia, schizoaffective disorder, attention deficit disorder, dysthymic disorder, major depressive disorder, mania, and obsessive-compulsive disorder), psychoactive substance use disorders, anxiety, panic disorder, and bipolar affective disorder (e.g., severe bipolar affective disorder and bipolar affective disorder with hypomania and major depression).

CNS-related disorders include disorders associated with developmental, cognitive, and autonomic neural and neurological processes, such as pain, appetite, long term memory, and short term memory. Examples of focal brain disorders include aphasia, apraxia, agnosia, and amnesias (e.g., posttraumatic amnesia, transient global amnesia, and psychogenic amnesia). Global-diffuse cerebral disorders with which INTERCEPT 309 is associated include coma, stupor, obtundation, and disorders of the reticular formation. Cerebrovascular disorders include ischemic syndromes (e.g., stroke), hypertensive encephalopathy, hemorrhagic disorders, and disorders involving aberrant function of the blood-brain barrier (e.g., CNS infections such as meningitis and encephalitis, aseptic meningitis, metastasis of non-CNS tumor cells into the CNS, various pain disorders such as migraine, and CNS-related adverse drug reactions such as head pain, sleepiness, and confusion). INTERCEPT 309 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. b) Examples of thyroid disorders with which INTERCEPT 309 proteins and nucleic acids encoding them can be involved include hyper- and hypothyroidism, goiter, thyroiditis, thyroid cancers (e.g., adenomas and carcinomas), and autoimmune diseases involving thyroid autoantigens such as thyroglobulin and thyroperoxidase. INTERCEPT 309 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. c) Kidney disorders with which INTERCEPT 309 proteins and nucleic acids encoding them can be involved include acute and chronic renal failure, immunologically-mediated renal disorders (i.e., involving both renal antigens and extra- renal antigens that have become located within the kidneys), glomerular diseases such as acute and progressive nephritic syndromes and nephrotic syndromes, acute and chronic tubulointerstitial nephritis, infections of the kidney, nephrotoxic disorders (i.e., including those associated with antibiotics, analgesics, anti-cancer agents, anti-epileptic agents, etc.), nephrogenic diabetes insipidus, hereditary chronic nephropathies, urinary incontinence, urinary calculus formation, kidney infections, and kidney neoplasms. INTERCEPT 309 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. d) Examples of liver disorders in which INTERCEPT 309 can have a role include fibrosis, cirrhosis, hepatitis, hepatic adverse drug reactions such as hepatotoxicity, and hepatic neoplasms. INTERCEPT 309 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. e) Prostate disorders with which INTERCEPT 309 proteins and nucleic acids encoding them can be involved include prostate neoplasms, benign prostatic hyperplasia, and benign prostatic hypertrophy. INTERCEPT 309 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. f) Disorders of the colon in which INTERCEPT 309 can have a role include, for example, diarrhea, constipation, gastroenteritis, malabsorption syndromes such as celiac disease and tropical sprue, inflammatory bowel diseases such as Crohn's disease and ulcerative colitis, antibiotic-associated colitis, functional bowel disorders such as irritable bowel syndrome and functional diarrhea, diverticular diseases such as diverticulosis and diverticulitis, and benign and malignant neoplasms of the colon. INTERCEPT 309 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. INTERCEPT 309 (like claudins) regulates intercellular permeability in tissues through which one may wish to modulate the passage of drugs or other agents. Such tissues include, for example, the blood-brain barrier (e.g., at the choroid plexus), vascular endothelium, and liver epithelial tissues (i.e., other than fenestrated hepatic vascular epithelia). By way of example, one may wish either to enhance the permeability of a tissue with respect to a drug (e.g., a drug for which enhanced blood- brain barrier permeability is desired) or to reduce the permeability of a tissue with respect to a drug (e.g., a drug for which reduction of hepatic sequestration is desired). INTERCEPT 309 proteins and nucleic acids, and other compounds which modulate the structure or activity of INTERCEPT 309 proteins and nucleic acids, can be used to regulate the permeability of such tissues.

In addition to its structural and functional similarity with claudin proteins,

INTERCEPT 309 protein is also similar in sequence to at least one protein regulator of apoptosis. As shown in Figure 3T, the amino acid sequence of INTERCEPT 309 is similar to the amino acid sequence of a protein (rRPV) which is expressed specifically in regressing rat ventral prostate tissue and epididymis. As described by Briehl et al. (1991, Mol. Endocrinol. 5:1381-1388), expression of this rat protein is elevated 3- to 8-fold in ventral prostate tissue upon induction of tissue regression mediated by withdrawal of androgens. Androgen withdrawal induces apoptosis in rat ventral prostate tissue. Thus, the rat protein described by Briehl et al. (supra) is an apoptosis-associated protein. INTERCEPT 309, having a sequence similar to that of rRPV, can also modulate apoptosis in tissues in which it is expressed. Apoptosis is a process of controlled cell death that occurs normally in many tissues in which cell division occurs essentially continuously. Examples of such tissues include nearly all tissues other than adult brain and cardiac muscle tissues, and particularly include rapidly-growing and rapidly-replaced tissues such as epithelial and endothelial tissues. Elimination of abnormal or damaged cells from a tissue (other than adult brain or cardiac muscle tissues) frequently occurs by apoptosis of the abnormal or damaged cells, rather than by necrosis, which can lead to inflammation. Apoptosis thus represents an important homeostatic process in healthy individuals, and aberrance in normal apoptosis can lead to occurrence of one or more disorders. INTERCEPT 309 (which, as described above is similar to the rat protein of Briehl et al.) can also be associated with apoptosis. INTERCEPT 309 can modulate apoptosis in tissues in which it is expressed, both under normal (i.e., homeostatic, non-disorder-associated) conditions and in tissue affected by a disorder associated with aberrant apoptosis. Disorders associated with aberrant apoptosis include both disorders in which apoptosis occurs to a supra-normal degree (e.g., human immunodeficiency virus-mediated depletion of CD4+ T cells) and disorders in which apoptosis is inhibited, relative to normal levels (e.g., various cancers and viral infections characterized by survival of virus-infected cells). Examples of disorders associated with aberrant apoptosis include substantially all cancers and viral infections, obesity, diabetes, atherosclerosis, arteriosclerosis, coronary artery disease, and angiogenesis. INTERCEPT 309 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. MANGO 419

A cDNA clone (designated cohqf013f05) encoding at least a portion of human MANGO 419 protein was isolated from a human cDNA library prepared from prostate carcinoma tissue which had metastasized to liver. Human MANGO 419 protein is a secreted protein.

The full length of the cDNA encoding human MANGO 419 protein (Figure 4; SEQ ID NO: 71) is 323 nucleotide residues. The ORF of this cDNA, nucleotide residues 84 to 323 of SEQ ID NO: 71 (i.e., SEQ ID NO: 72), encodes an 80- amino acid residue (or longer) protein (Figure 4; SEQ ID NO: 73), corresponding to a

56-residue (or longer) secreted mature protein.

The invention thus includes purified human MANGO 419 protein, both in the form of the immature 80 amino acid residue protein (SEQ ID NO: 73) and in the form of the mature 56 amino acid residue protein (SEQ ID NO: 75). Mature human MANGO 419 proteins can be synthesized without the signal sequence polypeptide at the amino terminus thereof, or they can be synthesized by generating immature MANGO 419 protein and cleaving the signal sequence therefrom.

In addition to full length mature and immature human MANGO 419 proteins, the invention includes fragments, derivatives, and variants of these MANGO 419 proteins, as described herein. It is also recognized that MANGO 419 protein can have one or more amino acid residues attached at the carboxyl terminal end thereof. By way of example, there can be from 1 to about 500, 1 to 100, 1 to 50, 1 to 30, 1 to 20, or 1 to 10 additional amino acid residues. 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 NO: 71, such as the portion which encodes mature MANGO 419 protein, immature MANGO 419 protein, or a domain of MANGO 419 protein. These nucleic acids are collectively referred to as nucleic acids of the invention. MANGO 419 proteins and nucleic acid molecules encoding them comprise a family of molecules having certain conserved structural and functional features.

A common domain present in MANGO 419 proteins is a signal sequence. In one embodiment, a MANGO 419 protein contains a signal sequence corresponding to the portion of the protein from amino acid residue 1 to about amino acid residue 24 of SEQ ID NO: 73 (SEQ ID NO: 74). 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 22, 23, 24, 25, or 26 of SEQ ID NO: 73). The signal sequence is cleaved during processing of the mature protein.

MANGO 419 proteins typically comprise a variety of potential post- translational modification sites and protein domains (often positioned within an extracellular or protein surface domain), such as those described herein in Table VI, as predicted by computerized sequence analysis of MANGO 419 proteins using amino acid sequence comparison software (comparing the amino acid sequence of MANGO 419 with the information in the PROSITE database {rel. 12.2; Feb, 1995} and the Hidden Markov Models database {Rel. PFAM 3.3}).

Table VI

In various embodiments, the protein of the invention has at least 1 , 2, or all 3 of the post-translational modification sites and domains described herein in Table VI.

The signal peptide prediction program SIGN ALP (Nielsen et al. (1997) Protein Engineering 10:1-6) predicted that human MANGO 419 protein includes an approximately 24 amino acid residue signal peptide (amino acid residues 1 to about 24 of SEQ ID NO: 73; SEQ ID NO: 74) preceding the mature MANGO 419 protein (amino acid residues 25 to 80 of SEQ ID NO: 73; SEQ ID NO: 75).

Figure 4B depicts a hydrophilicity plot of human MANGO 419 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 about 24 of SEQ ID NO: 73 is the signal sequence of human MANGO 419 (SEQ ID NO: 74). 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 MANGO 419 protein from about amino acid residue 35 to about amino acid residue 55 appears to be located at or near the surface of the protein, while the region from about amino acid residue 60 to about amino acid residue 65 appears not to be located at or near the surface.

The predicted molecular weight of human MANGO 419 protein without modification and prior to cleavage of the signal sequence is about 8.8 kilodaltons. The predicted molecular weight of the mature human MANGO 419 protein without modification and after cleavage of the signal sequence is about 6.2 kilodaltons.

Expressed sequence tags (ESTs) which exhibit homology with SEQ ID NO: 71 have been isolated from murine mammary and embryonic tissues. Those ESTs have sequences that are similar to the sequence of a nucleic acid encoding an inner ear- specific collagen precursor.

Biological function of MANGO 419 proteins, nucleic acids encoding them, and modulators of these molecules MANGO 419 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 MANGO 419 occurs in a human metastatic prostate carcinoma cDNA library, and that ESTs obtained from mammary and embryonic tissues exhibit homology with MANGO 419 cDNA, it is evident that MANGO 419 protein can be involved in one or more biological processes which occur in these tissues. In particular, MANGO 419 can be involved in modulating growth, proliferation, survival, differentiation, and activity of cells of these tissues (e.g., mammary, prostate, and other epithelial and endothelial cells). MANGO 419 can have a role in disorders which affect epithelial and endothelial tissues including, for example, prostate, breast, and embryonic tissues. MANGO 419 proteins, nucleic acids encoding them, and small molecules which interact with either of these can be used to prognosticate, diagnose, and treat disorders of epithelial and endothelial tissues, particularly including carcinogenesis and metastasis of epithelial and endothelial neoplasms, such as prostate and mammary cancers.

Recovery of a cDNA encoding MANGO 419 from a library prepared using metastatic prostate carcinoma cells also indicates that MANGO 419 can affect the ability and propensity of a cell to adhere with other cells or with extracellular surfaces, and that MANGO 419 can affect the ability of cells which express it to move through other tissues and through extracellular matrix. Furthermore, the fact that MANGO 419 is a secreted protein indicates that it can be used (e.g., by detecting it in a body fluid) as a marker for the metastatic state of cancers, particularly including epithelial carcinomas. Expression of MANGO 419 protein in epithelial tissues such as prostate and mammary tissues is an indication that MANGO 419 protein and nucleic acids which encode them can be involved in disorders of epithelial and endothelial tissues. Examples of disorders of epithelial and endothelial tissues include cell binding, adhesion, and proliferation disorders and epithelial / endothelial permeability-related disorders. MANGO 419 protein is 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 a cancerous cells away from a solid tissue site at which they normally occur 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., 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 extracellular matrix include those (e.g., metastasis of a normally solid tumor tissue away from it site of origin) in which the cells normally do, but aberrantly do not, bind with extracellular matrix as well as those (e.g., metastasis of tumor cells into a tissue in which the cells do not normally occur, autoimmune disorders, liver fibrosis, abnormal blood coagulation, atherosclerosis, and arteriosclerosis) in which the cells normally do not bind with extracellular matrix, but aberrantly do. Examples of disorders involving aberrant binding or adhesion of cells with foreign materials include those (e.g., allergies and hypersensitivity disorders such as latex hypersensitivity) associated with aberrant binding with the foreign material and disorders in which the cells normally bind with the foreign material, but aberrantly do not. MANGO 419 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.

Expression of MANGO 419 protein in epithelial tissues such as prostate and mammary tissues is an indication that MANGO 419 proteins and nucleic acids can be involved in disorders associated with aberrant permeability of epithelial tissues (i.e., aberrant permeability with regard to water, solutes, proteins, immune cells, and pathogens). Such disorders include, by way of example, kidney disorders, liver disorders, gastrointestinal disorders, endocrine and exocrine disorders, prostate disorders, gynecological disorders, skin disorders, and brain disorders. Examples of disorders of these types are described separately, for convenience, in the following paragraphs a)-h). a) Kidney disorders with which MANGO 419 proteins and nucleic acids encoding them can be involved include acute and chronic renal failure, immunologically- mediated renal disorders (i.e., involving both renal antigens and extra-renal antigens that have located within the kidneys), acute and progressive nephritic syndromes, nephrotic syndromes, acute and chronic tubulointerstitial nephritis, infections of the kidney, nephrotoxic disorders (i.e., including those associated with antibiotics, analgesics, anti- cancer agents, anti-epileptic agents, etc.), nephrogenic diabetes insipidus, hereditary chronic nephropathies, urinary incontinence, urinary calculus formation, and kidney neoplasms. MANGO 419 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. b) Examples of liver disorders in which MANGO 419 can have a role include fibrosis, cirrhosis, hepatitis, hepatic adverse drug reactions such as hepatotoxicity, and hepatic neoplasms. MANGO 419 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. c) Disorders of the gastrointestinal tract in which MANGO 419 can have a role include, for example, gastroesophageal reflux disease, gastric ulcers, gastritis, appendicitis, peritonitis, diarrhea, constipation, gastroenteritis, malabsorption syndromes such as celiac disease and tropical sprue, inflammatory bowel diseases such as Crohn's disease and ulcerative colitis, antibiotic-associated colitis, functional bowel disorders such as irritable bowel syndrome and functional diarrhea, diverticular diseases such as diverticulosis and diverticulitis, and benign and malignant neoplasms of the colon. MANGO 419 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. d) Examples of endocrine and exocrine disorders with which MANGO 419 proteins and nucleic acids encoding them can be involved include diabetes mellitus, hypoglycemia, glucagon disorders, pituitary disorders such as diabetes insipidus, thyroid disorders such as hyper- and hypothyroidism, adrenal disorders such as Cushing's syndrome and hyperaldosteronism, multiple endocrine neoplasias, polyglandular deficiency syndromes, epithelial breast cancers, biliary calculi, cholecystitis, and neoplasms of the bile ducts, chronic and acute renal failure, immunologically mediated renal diseases, glomerular diseases such as acute neprhitic syndrome and nephrotic syndrome, tubulointerstitial diseases, nephrotoxic disorders, and infections of the kidney, goiter, thyroiditis, thyroid cancers, and autoimmune diseases involving endocrine (e.g., thyroid) autoantigens. MANGO 419 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. e) Prostate disorders with which MANGO 419 proteins and nucleic acids encoding them can be involved include prostate neoplasms, benign prostatic hyperplasia, and benign prostatic hypertrophy. MANGO 419 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. f) Gynecological disorders in which MANGO 419 can have a role include ovarian, cervical, vulvar, and vaginal cancers, infertility, and endometriosis. MANGO 419 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. g) Skin disorders with which MANGO 419 can be associated include psoriasis, infections, wounds (and healing of wounds), inflammation, dermatitis, acne, and benign and malignant dermatological tumors. MANGO 419 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. h) Examples of brain disorders in which MANGO 419 can have a role include both CNS disorders, CNS-related disorders, focal brain disorders, global-diffuse cerebral disorders, and other neurological and cerebrovascular disorders. CNS disorders include, but are not limited to cognitive and neurodegenerative disorders such as Alzheimer's disease, senile dementia, Huntington's disease, amyotrophic lateral sclerosis, and Parkinson's disease, as well as Gilles de la Tourette's syndrome, autonomic function disorders such as hypertension and sleep disorders (e.g., insomnia, hypersomnia, parasomnia, and sleep apnea), neuropsychiatric disorders (e.g., schizophrenia, schizoaffective disorder, attention deficit disorder, dysthymic disorder, major depressive disorder, mania, and obsessive-compulsive disorder), psychoactive substance use disorders, anxiety, panic disorder, and bipolar affective disorder (e.g., severe bipolar affective disorder and bipolar affective disorder with hypomania and major depression). CNS-related disorders include disorders associated with developmental, cognitive, and autonomic neural and neurological processes, such as pain, appetite, long term memory, and short term memory. Examples of focal brain disorders include aphasia, apraxia, agnosia, and amnesias (e.g., posttraumatic amnesia, transient global amnesia, and psychogenic amnesia). Global-diffuse cerebral disorders with which MANGO 419 can be associated include coma, stupor, obtundation, and disorders of the reticular formation. Cerebrovascular disorders can include ischemic syndromes (e.g., stroke), hypertensive encephalopathy, and hemorrhagic disorders. MANGO 419 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. INTERCEPT 429

A cDNA clone (designated jchrd012h06) encoding at least a portion of human INTERCEPT 429 protein was isolated from a human heart cDNA library. Human INTERCEPT 429 protein is a transmembrane protein.

The full length of the cDNA encoding human INTERCEPT 429 protein (Figure 5; SEQ ID NO: 81) is 546 nucleotide residues. The ORF of this cDNA, nucleotide residues 95 to 439 of SEQ ID NO: 81 (i.e., SEQ ID NO: 82), encodes a 115- amino acid residue protein (Figure 5; SEQ ID NO: 83), corresponding to a 93-residue transmembrane mature protein.

The invention includes purified human INTERCEPT 429 protein, both in the form of the immature 115 amino acid residue protein (SEQ ID NO: 83) and in the form of the mature 93 amino acid residue protein (SEQ ID NO: 85). Mature human INTERCEPT 429 proteins can be synthesized without the signal sequence polypeptide at the amino terminus thereof, or they can be synthesized by generating immature

INTERCEPT 429 protein and cleaving the signal sequence therefrom.

In addition to full length mature and immature human INTERCEPT 429 proteins, the invention includes fragments, derivatives, and variants of these INTERCEPT 429 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 NO: 81, such as the portion which encodes mature INTERCEPT 429 protein, immature INTERCEPT 429 protein, or a domain of INTERCEPT 429 protein. These nucleic acids are collectively referred to as nucleic acids of the invention.

INTERCEPT 429 proteins and nucleic acid molecules encoding them comprise a family of molecules having certain conserved structural and functional features. A common domain present in INTERCEPT 429 proteins is a signal sequence. In one embodiment, an INTERCEPT 429 protein contains a signal sequence corresponding to the portion of the protein from amino acid residue 1 to about amino acid residue 22 of SEQ ID NO: 83 (SEQ ID NO: 84). 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 20, 21, 22, 23, or 24 of SEQ ID NO: 83). The signal sequence is cleaved during processing of the mature protein.

INTERCEPT 429 proteins include two transmembrane domains, a pair of extra-membrane domains that flank the cell membrane on the same side of the membrane, and another extra-membrane domain that flanks the cell membrane on the opposite side of the membrane. The two transmembrane domains correspond to about amino acid residues 32 to 49 and 59 to 82 of SEQ ID NO: 83 (i.e., the transmembrane domains having the sequences SEQ ID NOs: 87 and 89). The pair of extra-membrane domains corresponds to about amino acid residues 23 to 31 and 83 to 115 of SEQ ID NO: 83 (these domains having the sequences SEQ ID NOs: 86 and 90). The other extra- membrane domain corresponds to about amino acid residues 50 to 58 of SEQ ID NO: 83 (this domain having the sequence SEQ ID NO: 88). In one embodiment, the pair of extra-membrane domains (i.e., those having the sequences SEQ ID NOs: 86 and 90) are intracellular domains and the other domain is an extracellular domain. However, in an alternative form, the pair of extra-membrane domains are extracellular and the other domain is cytoplasmic.

INTERCEPT 429 proteins typically comprise a variety of potential post- translational modification sites and protein domains (often positioned within an extracellular or protein surface domain), such as those described herein in Table VII, as predicted by computerized sequence analysis of INTERCEPT 429 proteins using amino acid sequence comparison software (comparing the amino acid sequence of INTERCEPT 429 with the information in the PROSITE database {rel. 12.2; Feb, 1995} and the Hidden Markov Models database {Rel. PFAM 3.3}). Table VII

In various embodiments, the protein of the invention has one or both of the post-translational modification sites and domains described herein in Table VII. Figure 5B depicts a hydrophilicity plot of human INTERCEPT 429 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 22 of SEQ ID NO: 83 is the signal sequence of human INTERCEPT 429 (SEQ ID NO: 84). 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 INTERCEPT 429 protein from about amino acid residue 85 to about amino acid residue 100 appears to be located at or near the surface of the protein. The predicted molecular weight of human INTERCEPT 429 protein without modification and prior to cleavage of the signal sequence is about 13.4 kilodaltons. The predicted molecular weight of the mature human INTERCEPT 429 protein without modification and after cleavage of the signal sequence is about 10.8 kilodaltons. Expressed sequence tags (ESTs) which exhibit homology with SEQ ID

NO: 81 have been isolated from murine small intestine tissue and from pooled human fetal lung, testis, and B cell tissues.

Biological function of INTERCEPT 429 proteins, nucleic acids encoding them. and modulators of these molecules

INTERCEPT 429 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 INTERCEPT 429 occurs in a human heart cDNA library, and that ESTs obtained from small intestine and one or more of fetal lung, testis, and B cell tissues exhibit homology with MANGO 419 cDNA, it is evident that INTERCEPT 429 protein can be involved in one or more biological processes which occur in these tissues. In particular, INTERCEPT 429 is involved in modulating growth, proliferation, survival, differentiation, and activity of cells of these tissues (e.g., cardiac muscle cells), both in normal (i.e., non-diseased) tissues and in tissues which are affected by one or more disorders. Examples of disorders with which INTERCEPT 429 protein can be associated are described in the following paragraphs.

Heart disorders with one or more of which INTERCEPT 429 proteins and nucleic acids can be involved include, by way of example, arteriosclerosis, hypertension, cardiac arrhythmias, cardiac insufficiency, myocardial ischemic disorders such as angina pectoris and coronary artery disease, cardiac arrest, various cardomyopathies (e.g., hypertrophic and restrictive cardiomyopathy), valvular heart diseases, endocarditis, pericardial disease, cardiac diseases, and muscular dystrophy. INTERCEPT 429 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. Muscular disorders in which INTERCEPT 429 proteins and nucleic acids can have a role include muscular dystrophies, myotonic myopathies, glycogen storage disorders and familial periodic paralysis. INTERCEPT 429 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. Lung disorders with which INTERCEPT 429 proteins and nucleic acids can be associated include, by way of example, asthma, chronic and acute bronchitis, chronic airway obstructive disorders, pulmonary embolism, pneumonia, and genesis and metastasis of lung tumors. INTERCEPT 429 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.

Testicular disorders which can involve INTERCEPT 429 proteins and nucleic acids include, for example, epididymo-orchitis, mumps orchitis, and genesis and metastasis of testicular cancers. INTERCEPT 429 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.

B cell disorders in which INTERCEPT 429 proteins and nucleic acids can be involved include leukemias, lymphomas, leukopenias, plasma cell dyscrasias, and splenomegaly. INTERCEPT 429 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.

Tables Al and Bl summarize sequence data corresponding to the human nucleic acids and proteins herein designated TANGO 229, INTERCEPT 289, INTERCEPT 309, and MANGO 419, INTERCEPT 429.

Table Al

Table Bl

Notes:

'it is recognized that the carboxyl terminal boundary of the signal sequence can be ± 1 or 2 residues from that indicated.

²It is recognized that 'extracellular' and cytoplasmic' domains can have the opposite orientation in certain embodiments, as described herein.

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, 11, 12, 21, 22, 26, 27, 31, 32, 36, 37, 41, 42, 51, 52, 71, 72, 81, and 82, 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, 11, 12, 21, 22, 26, 27, 31, 32, 36, 37, 41, 42, 51, 52, 71, 72, 81, and 82 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, 11, 12, 21, 22, 26, 27, 31, 32, 36, 37, 41, 42, 51, 52, 71, 72, 81, and 82, 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 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, or 400 or more consecutive nucleotides of the sense or anti-sense sequence of any of SEQ ID NOs: 1, 2, 11, 12, 21, 22, 26, 27, 31, 32, 36, 37, 41, 42, 51, 52, 71, 72, 81, and 82, or of a naturally occurring mutant of any of SEQ ID NOs: 1, 2, 11, 12, 21, 22, 26, 27, 31, 32, 36, 37, 41, 42, 51, 52, 71, 72, 81, and 82. 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 one of SEQ ID

NOs: 2, 12, 22, 27, 32, 37, 42, 52, 72, and 82, 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, 11, 12, 21, 22, 26, 27, 31, 32,

36, 37, 41, 42, 51, 52, 71, 72, 81, and 82 due to degeneracy of the genetic code and thus encode the same protein as that encoded by the nucleotide sequence of any of SEQ ID NOs: 2, 12, 22, 27, 32, 37, 42, 52, 72, and 82.

In addition to the nucleotide sequences of any of SEQ ID NOs: 2, 12, 22, 27, 32, 37, 42, 52, 72, and 82, 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., % identity = # 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/bestfit.html#algorithm.

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. For example, TANGO 229 exhibits significant homology with an EST derived from chromosome 6q21, and allelic variants of TANGO 229 can be identified by sequencing the TANGO 229 gene at this locus 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 and murine 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 or murine 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, 550, 650, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, 2000, 2200, 2400, 2600, 2800, 3000, 3500, or 3743) 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, 11, 12, 21, 22, 26, 27, 31, 32, 36, 37, 41, 42, 51, 52, 71, 72, 81, and 82, 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, 11, 12, 21, 22, 26, 27, 31, 32, 36, 37, 41, 42, 51, 52, 71, 72, 81, and 82, 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, 13-15, 23-25, 28-30, 33-35, 38-40, 43-45, 53- 61, 73-75, and 83-90, 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, 13-15,

23-25, 28-30, 33-35, 38-40, 43-45, 53-61, 73-75, and 83-90.

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, 11, 12, 21, 22, 26, 27, 31, 32, 36, 37, 41 , 42, 51 , 52, 71 , 72, 81 , and 82, 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 protein:protein interactions with the polypeptide of the invention; (2) the ability to bind a ligand of the polypeptide of the invention (e.g., another protein identified herein); (3) the ability to bind with a modulator or substrate of the polypeptide of the invention; or (4) the ability to modulate a physiological activity of the protein, such as one of those disclosed herein. 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,

N₆-isopentenyladenine, 1 -methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2- methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7- methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, 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 α-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 Drug 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 Hyrup et al. (1996) Bioorganic & Medicinal Chemistry 4(1): 5-23). As used herein, the terms "peptide nucleic acids" or "PNAs" refer to nucleic acid mimics, e.g., DNA mimics, 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 (Hyrup (1996), supra; or as probes or primers for DNA sequence and hybridization (Hyrup (1996), supra; Perry-O'Keefe et al. (1996) Proc. Natl. Acad. Sci. USA 93: 14670-675). In another embodiment, PNAs can be modified, e.g., to enhance their stability or cellular uptake, by attaching lipophilic or other helper groups to PNA, by formation of PNA-DNA chimeras, or by use of liposomes or other techniques of drug 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 (Hyrup (1996), supra). The synthesis of PNA-DNA chimeras can be performed as described in Hyrup (1996), supra, and Finn et al. (1996) Nucleic Acids Res. 24(17):3357-63. For example, a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry and modified nucleoside analogs. Compounds such as 5'-(4- 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%, 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, 13-15, 23-25, 28-30, 33-35, 38-40, 43-45, 53-61, 73-75, and 83- 90), 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, 13-15, 23-25, 28-30, 33-35, 38-40, 43-45, 53-61, 73-75, and 83-90. 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, 13-15, 23-25, 28-30, 33-35, 38-40, 43-45, 53-61 , 73-75, and 83-90 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.

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 one of SEQ ID NOs: 3, 53, 73, and 83) 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 59:7811-7815; Delgrave et al. (1993) Protein Engineering 6(3):327-331). An isolated polypeptide of the invention, or a fragment thereof, can be used as an immunogen to generate antibodies using standard techniques for polyclonal and monoclonal antibody preparation. The full-length polypeptide or protein can be used or, alternatively, the invention provides antigenic peptide fragments for use as immunogens. The antigenic peptide of a protein of the invention comprises at least 8 (preferably 10, 15, 20, or 30 or more) amino acid residues of the amino acid sequence of any of SEQ ID NOs: 3-8, 13-15, 23-25, 28-30, 33-35, 38-40, 43-45, 53-61, 73-75, and 83-90, 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 IG, 2Yi through 2Yvi, 2Zvi, 3D, 4B, and 5B 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. Preferred polyclonal antibody compositions are ones that have been selected for antibodies directed against (i.e., which bind specifically with) one or more polypeptides of the invention. Particularly preferred polyclonal antibody preparations are ones that contain only antibodies directed against one or more polypeptides of the invention. Particularly preferred immunogen compositions are those that contain no other human proteins such as, for example, immunogen compositions made using a non-human host cell for recombinant expression of a polypeptide of the invention. In such a manner, the only human epitope or epitopes recognized by the resulting antibody compositions raised against this immunogen will be present as part of a polypeptide or polypeptides of the invention.

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. Alternatively, antibodies which bind specifically with a protein or polypeptide of the invention can be selected or purified (e.g., partially purified) using chromatographic methods, such as affinity chromatography. For example, a recombinantly expressed and purified (or partially purified) protein of the invention can be produced as described herein, and covalently or non-covalently coupled with a solid support such as, for example, a chromatography column. The column thus exhibits specific affinity for antibody substances which bind specifically with the protein of the invention, and these antibody substances can be purified from a sample containing antibody substances directed against a large number of different epitopes, thereby generating a substantially purified antibody substance composition, i.e., one that is substantially free of antibody substances which do not bind specifically with the protein. By a substantially purified antibody composition is meant, in this context, that the antibody sample contains at most only 30% (by dry weight) of contaminating antibodies directed against epitopes other than those on the desired protein or polypeptide of the invention, preferably at most 20%, more preferably at most 10%, most preferably at most 5% (by dry weight), of the sample is contaminating antibodies. A purified antibody composition means that at least 99% of the antibodies in the composition are directed against the desired protein or polypeptide of the invention. 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 supernatants for antibodies that bind the polypeptide of interest, e.g., using a standard ELISA assay. Alternative to preparing monoclonal antibody-secreting hybridomas, a monoclonal antibody directed against a polypeptide of the invention can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with the polypeptide of interest. Kits for generating and screening phage display libraries are commercially available (e.g., the Pharmacia

Recombinant Phage Antibody System, Catalog No. 27-9400-01 ; and the Stratagene 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 Nos. WO 92/18619, WO 91/17271, WO 92/20791, WO 92/15679, WO 93/01288, WO

92/01047, WO 92/09690, and 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) EMBOJ. 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 techniques, are within the scope of the invention. A chimeric antibody is a molecule in which different portions of the antibody amino acid sequence are derived from different animal species, such as those having a variable region derived from a murine monoclonal antibody and a constant region derived from a human immunoglobulin. (See, e.g., Cabilly et al., U.S. Patent No. 4,816,567; and Boss et al., U.S. Patent No. 4,816,397). Humanized antibodies are antibody molecules which are obtained from non-human species, which have one or more complementarity- determining regions (CDRs) derived from the non-human species, and which have a framework region derived from a human immunoglobulin molecule. (See, e.g., Queen, U.S. Patent No. 5,585,089). 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, for example, 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 radioactive material include ¹²⁵1, ¹³¹1, ³⁵S or Η.

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 drug moiety not being limited to classical chemical therapeutic agents. For example, the drug 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, interleukin-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 Drug Delivery", in Controlled Drug 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.

Accordingly, in one aspect, the invention provides substantially purified antibodies or fragment thereof, and non-human antibodies or fragments thereof, which antibodies or fragments specifically bind with a polypeptide having an amino acid sequence which comprises a sequence selected from the group consisting of

(i) SEQ ID NOs: 3-8, 13-15, 23-25, 28-30, 33-35, 38-40, 43-45, 53-61, 73-75, and 83-90; (ii) the amino acid sequences encoded by the cDNA of clones deposited as ATCC^®

PTA-295 and PTA-455; (iii) fragments of at least 15 amino acid residues of the amino acid sequence of at least one of SEQ ID NOs: 3-8, 13-15, 23-25, 28-30, 33-35, 38-40, 43-45, 53-61, 73-75, and 83-90;

(iv) amino acid sequences which are at least 95% identical to the amino acid sequence of at least one of SEQ ID NOs: 3-8, 13-15, 23-25, 28-30, 33-35, 38-40, 43-45,

53-61, 73-75, and 83-90, wherein the percent identity is determined using the ALIGN program of the GCG software package with a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4; and

(v) amino acid sequence which are encoded by nucleic acid molecules, the complement of which hybridizes with a nucleic acid molecule having the sequence of one of SEQ ID NOs: 1, 2, 11, 12, 21, 22, 26, 27, 31, 32, 36, 37, 41, 42, 51, 52, 71, 72, 81, and 82, or with a cDNA of a clone deposited as either of ATCC^® PTA-295 and PTA-455, under conditions of hybridization of 6x SSC (standard saline citrate buffer) at 45°C and washing in 0.2x SSC, 0.1% SDS at 65°C. In another aspect, the invention provides non-human antibodies or fragments thereof, which antibodies or fragments specifically bind with a polypeptide having an amino acid sequence which comprises a sequence selected from the group consisting of:

(i) SEQ ID NOs: 3-8, 13-15, 23-25, 28-30, 33-35, 38-40, 43-45, 53-61, 73-75, and 83-90;

(ii) the amino acid sequences encoded by the cDNA of clones deposited as ATCC^® PTA-295 and PTA-455;

(iii) fragments of at least 15 amino acid residues of the amino acid sequence of at least one of SEQ ID NOs: 3-8, 13-15, 23-25, 28-30, 33-35, 38-40, 43-45, 53-61, 73-75, and 83-90;

(iv) amino acid sequences which are at least 95% identical to the amino acid sequence of at least one of SEQ ID NOs: 3-8, 13-15, 23-25, 28-30, 33-35, 38-40, 43-45, 53-61, 73-75, and 83-90, wherein the percent identity is determined using the ALIGN program of the GCG software package with a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4; and (v) amino acid sequence which are encoded by nucleic acid molecules, the complement of which hybridizes with a nucleic acid molecule having the sequence of one of SEQ ID NOs: 1, 2, 11, 12, 21, 22, 26, 27, 31, 32, 36, 37, 41, 42, 51, 52, 71, 72, 81, and 82, or with a cDNA of a clone deposited as either of ATCC^® PTA-295 and PTA-455, under conditions of hybridization of 6x SSC (standard saline citrate buffer) at 45°C and washing in 0.2x SSC, 0.1% SDS at 65°C. Such non-human antibodies can be goat, mouse, sheep, horse, chicken, rabbit, or rat antibodies. Alternatively, the non-human antibodies of the invention can be chimeric and/or humanized antibodies. In addition, the non-human antibodies of the invention can be polyclonal antibodies or monoclonal antibodies. In still a further aspect, the invention provides monoclonal antibodies or fragments thereof, which antibodies or fragments specifically bind with a polypeptide having an amino acid sequence which comprises a sequence selected from the group consisting of:

(iv) amino acid sequences which are at least 95% identical to the amino acid sequence of at least one of SEQ ID NOs: 3-8, 13-15, 23-25, 28-30, 33-35, 38-40, 43-45, 53-61, 73-75, and 83-90, wherein the percent identity is determined using the ALIGN program of the GCG software package with a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4; and

(v) amino acid sequence which are encoded by nucleic acid molecules, the complement of which hybridizes with a nucleic acid molecule having the sequence of one of SEQ ID NOs: 1, 2, 11, 12, 21, 22, 26, 27, 31, 32, 36, 37, 41, 42, 51, 52, 71, 72, 81, and

82, or with a cDNA of a clone deposited as either of ATCC^® PTA-295 and PTA-455, under conditions of hybridization of 6x SSC (standard saline citrate buffer) at 45°C and washing in 0.2x SSC, 0.1% SDS at 65°C. The monoclonal antibodies can be human, humanized, chimeric and/or non-human antibodies. The substantially purified antibodies or fragments thereof can specifically bind with a signal peptide, a secreted sequence, an extracellular domain, a transmembrane or a cytoplasmic domain cytoplasmic membrane of a polypeptide of the invention. In a particularly preferred embodiment, the substantially purified antibodies or fragments thereof, the non-human antibodies or fragments thereof, and/or the monoclonal antibodies or fragments thereof, of the invention specifically bind with a secreted sequence or with an extracellular domain of one of TANGO 229, INTERCEPT 289, INTERCEPT 309, MANGO 419, and INTERCEPT 429. Preferably, the extracellular domain with which the antibody substance binds has an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 15, 25, 30, 35, 40, 45, 55, 59, and 88.

Any of the antibody substances of the invention can be conjugated with a therapeutic moiety or to a detectable substance. Non-limiting examples of detectable substances that can be conjugated with the antibody substances of the invention include an enzyme, a prosthetic group, a fluorescent material (i.e. a fluorophore), a luminescent material, a bioluminescent material, and a radioactive material (e.g. a radionuclide or a substituent comprising a radionuclide).

Still another aspect of the invention is a method of making an antibody that specifically recognizes one of TANGO 229, INTERCEPT 289, INTERCEPT 309,

MANGO 419, and INTERCEPT 429. This method comprises immunizing a vertebrate (e.g. a mammal such as a rabbit, goat, or pig) with a polypeptide. The polypeptide used as an immunogen has an amino acid sequence that comprises a sequence selected from the group consisting of: (i) SEQ ID NOs: 3-8, 13-15, 23-25, 28-30, 33-35, 38-40, 43-45, 53-61, 73-75, and (ii) the amino acid sequences encoded by the cDNA of clones deposited as ATCC^® PTA-295 and PTA-455;

(v) amino acid sequence which are encoded by nucleic acid molecules, the complement of which hybridizes with a nucleic acid molecule having the sequence of one of SEQ ID NOs: 1, 2, 11, 12, 21, 22, 26, 27, 31, 32, 36, 37, 41, 42, 51, 52, 71, 72, 81, and 82, or with a cDNA of a clone deposited as either of ATCC^® PTA-295 and PTA-455, under conditions of hybridization of 6x SSC (standard saline citrate buffer) at 45°C and washing in 0.2x SSC, 0.1% SDS at 65°C.

After immunization, a sample is collected from the vertebrate that contains an antibody that specifically recognizes the polypeptide with which the vertebrate was immunized. Preferably, the polypeptide is recombinantly produced using a non-human host cell. Optionally, an antibody substance can be further purified from the sample using techniques well known to those of skill in the art. The method can further comprise making a monoclonal antibody-producing cell from a cell of the vertebrate. Optionally, antibodies can be collected from the antibody-producing cell.

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 (Amann et al., (1988) Gene 69:301-315) and pET l id (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 R A polymerase transcription from a hybrid trp-lac fusion promoter. Target gene expression from the pET l id 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 HMS 174(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) EMBOJ. 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) Nαtwre 329:840) and pMT2PC (Kaufman et al. (1987) EMBOJ. 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. Νon-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 virus 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, DΕAΕ-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 drug 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, in Hogan, Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986), and in Wakayama et al, (1999) Proc. Natl. Acad. Sci. USA 96:14984-14989. 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 a 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 Nos. WO 90/11354, WO 91/01140, WO 92/0968, and WO 93/04169. In another embodiment, transgenic non-human animals can be produced which contain selected systems which allow for regulated expression of the transgene. One example of such a system is the cre/loxP recombinase system of 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 Nos. WO 97/07668 and WO 97/07669.

IV. Pharmaceutical Compositions

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

As used herein the language "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, 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 Retrovirology 14:193).

The nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (U.S. Patent 5,328,470), or by stereotactic injection (see, e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91 :3054- 3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.

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 (e.g., TANGO 229 proteins, INTERCEPT 289 proteins, INTERCEPT 309 proteins, MANGO 419 proteins, and INTERCEPT 429 proteins). 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) Nαtwre 354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (U.S. Patent No. 5,223,409), spores (U.S.

Patent Nos. 5,571,698; 5,403,484; and 5,223,409), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA 89:1865-1869) or phage (Scott and Smith (1990) Science 249:386- 390; Devlin (1990) Science 249:404-406; Cwiria et al. (1990) Proc. Natl. Acad. Sci. USA 87:6378-6382; and Felici (1991) J Mo/. 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. Determining 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 compound in a complex. For example, test compounds can be labeled with ¹²⁵1, ³⁵S, ¹ C, or ³H, 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 cellular second messenger of the target (e.g., an mRNA, intracellular Ca²⁺, 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 determining direct binding.

In an alternative embodiment, determining the ability of the test compound to modulate the activity of the polypeptide can be accomplished by determining the ability of the polypeptide of the invention to further modulate the target molecule. For example, the catalytic 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 determining 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 nonionic 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}-l -propane sulfonate (CHAPS), 3-{(3-cholamidopropyl) dimethylamminio } -2-hydroxy- 1 -propane sulfonate (CHAPSO), or N-dodecyl-N,N- dimethyl-3 -ammonio- 1 -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 Company; 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 non-bound 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.

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.

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.

Furthermore, the nucleic acid sequences disclosed herein can be used to perform searches against "mapping databases", e.g., BLAST-type search, such that the chromosome position of the gene is identified by sequence homology or identity with known sequence fragments which have been mapped to chromosomes. A polypeptide and fragments and sequences thereof and antibodies which bind specifically with such polypeptides/fragments can be used to map the location of the gene encoding the polypeptide on a chromosome. This mapping can be performed by specifically detecting the presence of the polypeptide/fragments in members of a panel of somatic cell hybrids between cells obtained from a first species of animal from which the protein originates and cells obtained from a second species of animal, determining which somatic cell hybrid(s) expresses the polypeptide, and noting the chromosome(s) of the first species of animal that it contains. For examples of this technique (see Pajunen et al., 1988, Cytogenet. Cell Genet. 47:37-41 and Van Keuren et al., 1986, Hum. Genet. 74:34-40). Alternatively, the presence of the polypeptide in the somatic cell hybrids can be determined by assaying an activity or property of the polypeptide (e.g., enzymatic activity, as described in Bordelon-Riser et al., 1979, Som. Cell Genet. 5:597-613 and Owerbach et al., 1978,

Proc. Natl. Acad. Sci. USA 75:5640-5644).

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, 11, 21, 26, 31, 36, 41, 51, 71, and 81 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, 12, 22, 27, 32, 37, 42, 52, 72, and 82 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 peφetrator 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, pharmacogenomics, and monitoring of clinical trials are used for prognostic (predictive) purposes to treat an individual prophylactically. Accordingly, one aspect of the present invention relates to diagnostic assays for determining expression of a polypeptide or nucleic acid of the invention, activity of a polypeptide of the invention, or some combination thereof, 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 or activity of a polypeptide of the invention. The invention also provides for prognostic (i.e., predictive) assays for determining whether an individual is at risk of developing a disorder associated with aberrant expression or activity of a polypeptide of the invention. For example, mutations in a gene of the invention can be assayed in a biological sample. Such assays can be used for prognostic or predictive purposes, or to prophylactically treat an individual prior to the onset of a disorder characterized by or associated with aberrant expression or activity of a polypeptide of the invention.

Another aspect of the invention provides methods for assessing expression of a nucleic acid or polypeptide of the invention or activity of a polypeptide of the invention in an individual to facilitate selection of appropriate therapeutic or prophylactic agents, and appropriate amounts (i.e., doses) of such agents), for that individual (referred to herein as "pharmacogenomics"). Pharmacogenomics allows selection of agents (e.g., drugs) and dose ranges for therapeutic or prophylactic treatment of an individual based on the genotype of the individual (e.g., the genotype of the individual examined to determine the ability of the individual to respond to a particular agent).

Alternatively, the expression level can be assessed as a relative expression level. To assess a relative expression level for a gene (e.g. an TANGO 229, INTERCEPT 289, INTERCEPT 309, MANGO 419, or INTERCEPT 429 gene, as described herein), the level of expression of the gene is determined for 10 or more samples (preferably 50 or more samples) of different isolates of cells in which the gene is believed to be expressed, prior to assessing the level of expression of the gene in the sample of interest. The mean expression level of the gene detected in the large number of samples is determined, and this value is used as a baseline expression level for the gene. The expression level of the gene assessed in the test sample (i.e. its absolute level of expression) is divided by the mean expression value to yield a relative expression level. Such a method can identify tissues or individuals which are afflicted with a disorder associated with aberrant expression of a gene of the invention. Preferably, the samples used in the baseline determination are generated either using cells obtained from a tissue or individual known to be afflicted with a disorder (e.g. a disorder associated with aberrant expression of one of the TANGO 229, INTERCEPT 289, INTERCEPT 309, MANGO 419, or INTERCEPT 429 genes) or using cells obtained from a tissue or individual known not to be afflicted with the disorder. Alternatively, levels of expression of these genes in tissues or individuals known to be or not to be afflicted with the disorder can be used to assess whether the aberrant expression of the gene is associated with the disorder (e.g., with onset of the disorder, or as a symptom of the disorder over time).

Another aspect of the invention provides methods for expression of a nucleic acid or polypeptide of the invention or activity of a polypeptide of the invention in an individual to thereby select appropriate therapeutic or prophylactic agents for that individual (referred to herein as "pharmacogenomics"). Pharmacogenomics allows for the selection of agents (e.g., drugs) for therapeutic or prophylactic treatment of an individual based on the genotype of the individual (e.g., the genotype of the individual examined to determine the ability of the individual to respond to a particular agent). Yet another aspect of the invention pertains to monitoring the influence of agents (e.g., drags 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 ofany of SEQ ID NOs: 1, 11, 21, 26, 31, 36, 41, 51, 71, and 81, 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 polyacrylamide 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 an example of an 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 stracture 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. Pharmaco genomics

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., drugs) for prophylactic or therapeutic treatments based on a consideration of the individual's genotype. Such pharmacogenomics can further be used to determine appropriate dosages and therapeutic regimens. Accordingly, the activity of a polypeptide of the invention, expression of a nucleic acid of the invention, or mutation content of a gene of the invention 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 drags 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 drugs act on the body are referred to as "altered drag action." Genetic conditions transmitted as single factors altering the way the body acts on drags 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 drug action. The discovery of genetic polymorphisms of drug metabolizing enzymes (e.g., N-acetyltransferase 2 {NAT 2 } and cytochrome P450 enzymes C YP2D6 and C YP2C 19) explains why some patients do not obtain the expected drag effects or exhibit exaggerated drag response and serious toxicity following administration of standard and safe doses of a drug. These polymoφhisms 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 polymoφhic, 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 drag 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 moφhine. 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 polymoφhic alleles encoding drag- 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., drag 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 drag, 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 drag 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.

D. 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 puφoses. 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 incoφorated by reference.

Deposits of Clones

Clones containing cDNA molecules encoding TANGO 229 and INTERCEPT 289 (clones EpT229 and EpI289, respectively), were deposited with the American Type Culture Collection (Manassas, VA) on October 1, 1999 as Accession No. PTA-295, 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 fragments as follows:

TANGO 229 (EpT229): 3.6 kilobases INTERCEPT 289 (EpI289): 1.9 kilobases

The identity of the strains can be inferred from the fragments liberated.

Clones containing cDNA molecules encoding INTERCEPT 429 (clone EpI429), were deposited with the American Type Culture Collection (Manassas, VA) on August 5, 1999 as Accession No. PTA-455, as part of a composite deposit representing a mixture of three 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: INTERCEPT 429 (EpI429): 0.5 kilobase

The identity of the strain containing INTERCEPT 429 can be inferred from the liberation of a fragment of the above identified size.

Clones containing cDNA molecules encoding INTERCEPT 309 and MANGO 419 (clones EpT309 and EpT419, respectively), were deposited with the American Type Culture Collection (Manassas, VA) on January 6, 2000 as Accession

Number PTA-1156, 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 fragments as follows:

TANGO 309 (EpT309): 1.9 kilobases MANGO 419 (EpT419): 0.3 kilobases

The identity of the strains can be inferred from the fragments liberated.

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

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 40% identical to the nucleotide sequence of any of SEQ ID NOs: 1, 2, 11, 12, 21, 22, 26, 27, 31, 32, 36, 37, 41, 42, 51, 52, 71, 72, 81, and 82, the nucleotide sequence of a cDNA clone deposited with ATCC^® as either of Accession Nos. PTA-295 and PTA-455, 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, 11, 12, 21, 22, 26, 27, 31, 32, 36, 37, 41, 42, 51, 52, 71, 72, 81, and 82, the nucleotide sequence of a cDNA clone deposited with ATCC^® as either of Accession Nos. PTA-295 and PTA-455, or a complement thereof; c) 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, 11, 12, 21, 22, 26, 27, 31, 32, 36, 37, 41, 42, 51, 52, 71, 72, 81, and 82, the nucleotide sequence of a cDNA clone deposited with ATCC^® as either of Accession Nos. PTA-295 and PTA-455, or a complement thereof; d) a nucleic acid molecule which encodes a polypeptide comprising the amino acid sequence of any of SEQ ID NOs: 3-8, 13-15, 23-25, 28-30, 33-35, 38-40, 43-45, 53- 61, 73-75, and 83-90, or the amino acid sequence encoded by a cDNA clone deposited with ATCC^® as either of Accession Nos. PTA-295 and PTA-455, or a complement thereof; e) a nucleic acid molecule which encodes a fragment of a polypeptide comprising the amino acid sequence of any of SEQ ID NOs: 3-8, 13-15, 23-25, 28-30, 33-35, 38-40, 43-45, 53-61, 73-75, and 83-90, or the amino acid sequence encoded by a cDNA clone deposited with ATCC^® as either of Accession Nos. PTA-295 and PTA- 455, wherein the fragment comprises at least 8 consecutive amino acid residues of any of SEQ ID NOs: 3-8, 13-15, 53-61, 73-75, and 83-90, or the amino acid sequence encoded by a cDNA clone deposited with ATCC^® as either of Accession Nos. PTA-295 and PTA-455; and f) 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, 13-15, 23-25, 28-30, 33-35, 38-40, 43-45, 53-61, 73-75, and 83-90, wherein the nucleic acid molecule hybridizes with a nucleic acid molecule consisting of the nucleotide sequence of any of SEQ ID NOs: 1, 2, 11, 12, 21, 22, 26, 27, 31, 32, 36, 37, 41, 42, 51, 52, 71, 72, 81, and 82, the nucleotide sequence of a cDNA clone deposited with ATCC^® as either of Accession Nos. PTA-295 and PTA-455, or a complement thereof under stringent conditions.

2. The isolated nucleic acid molecule of claim 1, which is selected from the group consisting of: a) a nucleic acid having the nucleotide sequence of any of SEQ ID NOs: 1, 2, 11, 12, 21, 22, 26, 27, 31, 32, 36, 37, 41, 42, 51, 52, 71, 72, 81, and 82, the nucleotide sequence of a cDNA clone deposited with ATCC^® as either of Accession Nos. PTA-295 and PTA-455, 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, 13-15, 23-25, 28-30, 33-35, 38-40, 43-45, 53-61, 73-75, and 83-90, or the amino acid sequence encoded by a cDNA clone deposited with ATCC^® as either of Accession Nos. PTA-295 and PTA-455, 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. 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, 13-15, 23-25, 28-30, 33-35, 38-40, 43-45, 53-61, 73-75, and 83-90, or the amino acid sequence encoded by a cDNA clone deposited with ATCC^® as either of Accession Nos. PTA-295 and PTA-455, wherein the fragment comprises at least 8 contiguous amino acids of any of SEQ ID NOs: 3-8, 13-15, 23-25, 28-30, 33-35, 38-40, 43-45, 53-61, 73-75, and 83-90, or the amino acid sequence encoded by a cDNA clone deposited with ATCC^® as either of Accession Nos. PTA-295 and PTA-455; b) a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of any of SEQ ID NOs: 3-8, 13-15, 23-25, 28-30, 33-35, 38-40, 43-45, 53- 61, 73-75, and 83-90, or the amino acid sequence encoded by a cDNA clone deposited with ATCC^® as either of Accession Nos. PTA-295 and PTA-455, 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, 11, 12, 21, 22, 26, 27, 31, 32, 36, 37, 41, 42, 51, 52, 71, 72, 81, and 82, the nucleotide sequence of a cDNA clone deposited with ATCC^® as either of Accession Nos. PTA-295 and PTA- 455, or a complement thereof under stringent conditions; and c) a polypeptide which is encoded by a nucleic acid molecule comprising a nucleotide sequence which is at least 40% identical to a nucleic acid consisting of the nucleotide sequence of any of SEQ ID NOs: 1, 2, 11, 12, 21, 22, 26, 27, 31, 32, 36, 37, 41, 42, 51, 52, 71, 72, 81, and 82, the nucleotide sequence of a cDNA clone deposited with ATCC^® as either of Accession Nos. PTA-295 and PTA-455, or a complement thereof.

9. The isolated polypeptide of claim 8 having the amino acid sequence of any of SEQ ID NOs: 3-8, 13-15, 23-25, 28-30, 33-35, 38-40, 43-45, 53-61, 73-75, and 83-90, or the amino acid sequence encoded by a cDNA clone deposited with ATCC^® as either of Accession Nos. PTA-295 and PTA-455, or a complement thereof.

10. The polypeptide of claim 8, wherein the amino acid sequence of the polypeptide further comprises heterologous amino acid residues.

11. An antibody which selectively binds with the polypeptide of claim 8.

12. A method for producing a polypeptide selected from the group consisting of: a) a polypeptide comprising the amino acid sequence of any of SEQ ID NOs: 3- 8, 13-15, 23-25, 28-30, 33-35, 38-40, 43-45, 53-61, 73-75, and 83-90, or the amino acid sequence encoded by a cDNA clone deposited with ATCC^® as either of Accession Nos. PTA-295 and PTA-455, or a complement thereof; b) a polypeptide comprising a fragment of the amino acid sequence of any of SEQ ID NOs: 3-8, 13-15, 23-25, 28-30, 33-35, 38-40, 43-45, 53-61, 73-75, and 83-90, or the amino acid sequence encoded by a cDNA clone deposited with ATCC^® as either of Accession Nos. PTA-295 and PTA-455, or a complement thereof, wherein the fragment comprises at least 8 contiguous amino acids of any of SEQ ID NOs: 3-8, 13-15, 23-25, 28-30, 33-35, 38-40, 43-45, 53-61, 73-75, and 83-90, or the amino acid sequence encoded by a cDNA clone deposited with ATCC^® as either of Accession Nos. PTA-295 and PTA-455, or a complement thereof; and c) a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of any of SEQ ID NOs: 3-8, 13-15, 23-25, 28-30, 33-35, 38-40, 43-45, 53- 61, 73-75, and 83-90, or the amino acid sequence encoded by a cDNA clone deposited with ATCC^® as either of Accession Nos. PTA-295 and PTA-455, 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, 11, 12, 21, 22, 26, 27, 31, 32, 36, 37, 41, 42, 51, 52, 71, 72, 81, and 82, the nucleotide sequence of a cDNA clone deposited with ATCC^® as either of Accession Nos. PTA-295 and PTA-455, 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.

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

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

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

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

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

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

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

20. The method of claim 19, wherein the binding of the test compound to the polypeptide is detected by a method selected from the group consisting of: a) detection of binding by direct detecting of test compound / polypeptide binding; b) detection of binding using a competition binding assay; c) detection of binding using an assay for an activity characteristic of the polypeptide.

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

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

23. An antibody substance which selectively binds with the polypeptide of claim 8, wherein the antibody substance is made by providing the polypeptide to an immunocompetent vertebrate and thereafter harvesting blood or serum from the vertebrate.