WO2001018016A1

WO2001018016A1 - Novel genes encoding proteins having prognostic, diagnostic, preventive, therapeutic, and other uses

Info

Publication number: WO2001018016A1
Application number: PCT/US2000/018174
Authority: WO
Inventors: Christopher C. Fraser; John D. Sharp; Nicholas Wrighton; Paul S. Myers; Andrew D. J. Goodearl
Original assignee: Millennium Pharmaceuticals, Inc.
Priority date: 1999-09-10
Filing date: 2000-06-30
Publication date: 2001-03-15
Also published as: EP1218392A4; AU6063900A; EP1218392A1

Abstract

The invention provides isolated nucleic acids encoding a variety of proteins having diagnostic, preventive, therapeutic, and other uses. These nucleic and proteins are useful for diagnosis, prevention, and therapy of a number of human and other animal disorders. The invention also provides antisense nucleic acid molecules, expression vectors containing the nucleic acid molecules of the invention, host cells into which the expression vectors have been introduced, and non-human transgenic animals in which a nucleic acid molecule of the invention has been introduced or disrupted. The invention still further provides isolated polypeptides, fusion polypeptides, antigenic peptides and antibodies. Diagnostic, screening, and therapeutic methods utilizing 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, pharmacogenomics, and the like 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 and murine genes and for proteins encoded thereby, and thus enables the practitioner to assess, predict, and affect the physiological state of various human and murine tissues.

Summary of the Invention The present invention is based, at least in part, on the discovery of a variety of human and murine cDNA molecules which encode proteins which are herein designated TANGO 210, TANGO 364, TANGO 366, INTERCEPT 394, INTERCEPT 400, and TANGO 405. These six 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 in 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 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) homologous with the amino acid sequence of the corresponding full-length polypeptide that is 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 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 an amino acid sequence sufficiently (e.g., 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, or 99% or greater) homologous with the nucleotide sequence of the corresponding full-length nucleic acid that is 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 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, 21, 22, 31, 32, 41, 42, 61, 62, 81, 82, 91, 92, 101, 102, 121, and 122, the nucleotide sequence of a cDNA clone deposited with ATCC^® as any of Accession Nos. PTA- 424, PTA-425, and PTA-438 ("a cDNA of a clone deposited as any of ATCC^® Accession Nos. PTA-424, PTA-425, and PTA-438"), 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, 31, 32, 41, 42, 61, 62, 81, 82,

91, 92, 101, 102, 121, and 122, or a cDNA of a clone deposited as any of ATCC^® Accession Nos. PTA-424, PTA-425, and PTA-438, 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: 3-5, 13-15, 23-28, 33-38, 43-54, 63-76, 83-92, 93, 103-105, and 123, or the amino acid sequence encoded by a cDNA of a clone deposited as any of ATCC^® Accession Nos. PTA-424, PTA- 425, and PTA-438, or a complement thereof.

In preferred embodiments, the nucleic acid molecules have the nucleotide sequence of any of SEQ ID NOs: 1, 2, 11, 12, 21, 22, 31, 32, 41, 42, 61, 62, 81, 82, 91, 92, 101, 102, 121, and 122, or a cDNA of a clone deposited as any of ATCC^® Accession Nos. PTA-424, PTA-425, and PTA-438.

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: 3-5, 13-15, 23-28, 33-38, 43-54, 63-76, 83-92, 93, 103-105, and 123, the amino acid sequence encoded by a cDNA of a clone deposited as any of ATCC^® Accession Nos. PTA-424, PTA-425, and PTA- 438, 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: 3-5, 13-15, 23-28, 33-38, 43-54, 63-76, 83-

92, 93, 103-105, and 123, or the amino acid sequence encoded by a cDNA of a clone deposited as any of ATCC^® Accession Nos. PTA-424, PTA-425, and PTA-438. 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: 3-5, 13-15, 23-28, 33-38, 43-54, 63-76, 83-92, 93, 103-105, and 123, or the amino acid sequence encoded by a cDNA of a clone deposited as any of ATCC^® Accession Nos. PTA-424, PTA-425, and PTA-438, 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, 31, 32, 41, 42, 61, 62, 81, 82, 91, 92, 101, 102, 121, and 122, or a cDNA of a clone deposited as any of ATCC^® Accession Nos. PTA-424, PTA-425, and PTA- 438, 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: 3-5, 13-15, 23-28, 33-38, 43-54, 63-76, 83- 92, 93, and 103-105.

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%, 75%, 85%, or 95% identical the nucleic acid sequence encoding any of SEQ ID NOs: 3-5, 13-15, 23-28, 33-38, 43-54, 63-76, 83-92, 93, 103-105, and 123, 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, 31 , 32, 41, 42, 61, 62, 81, 82, 91, 92, 101, 102, 121, and 122.

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: 3-5, 13-15, 23-28, 33-38, 43-54, 63-76, 83-92, 93, 103-105, and 123, or the amino acid sequence encoded by a cDNA of a clone deposited as any of ATCC^® Accession Nos. PTA-424, PTA- 425, and PTA-438, 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, 31, 32, 41, 42, 61, 62, 81, 82, 91, 92, 101, 102, 121, and 122, 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, 31, 32, 41, 42, 61, 62, 81, 82, 91, 92, 101, 102, 121, and 122, a cDNA of a clone deposited as any of ATCC^® Accession Nos. PTA-424, PTA-425, and PTA- 438, 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, 31, 32, 41, 42, 61, 62, 81, 82, 91 , 92, 101 , 102, 121 , and 122, a cDNA of a clone deposited as any of ATCC^® Accession Nos. PTA-424, PTA-425, and PTA-438, 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 and 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 on a second protein, or an indirect activity, such as a cellular processes 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 one or more of cellular proliferation, of cellular differentiation, of chemotaxis, of cellular migration, and of cell death (e.g., apoptosis). By way of example, TANGO 210, compounds which modulate its activity, expression, or both, and compounds (e.g., antibodies) which interact with (e.g., bind with) TANGO 210 (collectively "TANGO 210-related molecules") exhibit the ability to affect one or more of growth, proliferation, survival, differentiation, activity, morphology, and movement/migration of, for example, human adult kidney, fetal kidney, skin, and bone marrow cells and tissues. TANGO 210 modulates the structure of extracellular matrix within, or in fluid communication with, one or more of these tissues. For example, TANGO 210 exhibits proteinase activity that can enzymatically degrade one or more of the proteinaceous components of extracellular matrix. Thus, TANGO 210-related molecules can be used to prognosticate, prevent, diagnose, or treat disorders relating to aberrant formation or degradation of extracellular matrix. In various embodiments, for example, TANGO 210 is used to prognosticate, prevent, diagnose, or treat kidney, bone marrow, and skin disorders. Exemplary disorders for which TANGO 210-related molecules are useful include chronic and acute renal failure, immunologically mediated renal diseases, nephrotic syndromes, secondary glomerular diseases, chronic nephritic/proteinuric syndromes, chronic and acute tubulointerstitial nephritis, vascular hypertension, bone marrow failure, rejection of heterologous implanted bone marrow, ichthyosis, and the like. Aberrant degradation of extracellular matrix is also a characteristic of cancer cells, particularly including metastatic cancer cells. TANGO 210 can also be used to prognosticate, prevent, diagnose, or treat one or more cancers, including metastatic cancers. Further by way of example, TANGO 364, compounds which modulate its activity, expression, or both, and compounds (e.g., antibodies) which bind with TANGO 364 (collectively "TANGO 364-related molecules") exhibit the ability to affect one or more of growth, proliferation, survival, differentiation, activity, morphology, and movement/migration of, for example, human fetal and adult skin cells and tissue. Furthermore, TANGO 364 is involved in modulating cell-to-cell adhesion, tissue and extracellular matrix invasivity of cells, infectivity of cells by pathogens (e.g., bacteria and viruses), endocrine signaling processes, tissue developmental and organizational processes, and the like. Thus, TANGO 364-related molecules can be used to prognosticate, prevent, diagnose, or treat one or more disorders associated with these physiological processes. Such disorders include, for example, loss of control of cell growth, tumor metastasis, malformation of neurological connections, inflammation, immune and autoimmune responses, bacterial, fungal, and viral infections, and the like.

Still further by way of example, TANGO 366, compounds which modulate its activity, expression, or both, and compounds (e.g., antibodies) which bind with TANGO 366 (collectively "TANGO 366-related molecules") modulate one or more of growth, proliferation, survival, differentiation, activity, morphology, and movement/migration of human fibroblast cells and tissues in which fibroblasts normally or aberrantly occur. TANGO 366 is a cell surface protein-binding protein. TANGO 366 modulates binding of a cell which expresses it with one or more of an extracellular fluid protein, a protein component of the extracellular matrix, a surface protein another cell of the same animal, and a surface protein of a bacterium, fungus, or virus. TANGO 366 is therefore involved in cell-to-cell adhesion, tissue and extracellular matrix invasivity of cells, infectivity of cells by pathogens such as bacteria and viruses, endocrine signaling processes, tissue developmental and organizational processes, and the like. Thus, TANGO 366-related molecules can be used, for example, to prognosticate, prevent, diagnose, or treat disorders characterized by aberrant binding of cells with proteins, other cells of the same subject, or exogenous cells such as pathogens and disorders associated with aberrant motility of cells through extracellular matrix or another tissue. Exemplary disorders for which TANGO 366-related molecules have prognostic, preventive, diagnostic, and therapeutic uses include loss of control of cell growth, tumor metastasis, malformation of neurological connections, inflammation, immune and autoimmune responses, bacterial, fungal, and viral infections, and the like.

Yet further by way of example, INTERCEPT 394, compounds which modulate its activity, expression, or both, and compounds (e.g., antibodies) which bind with

INTERCEPT 394 (collectively "INTERCEPT 394-related molecules") modulate one or more of growth, proliferation, survival, differentiation, activity, morphology, and movement/migration of, for example, human adult and fetal kidney cells and tissues. INTERCEPT 394, a transmembrane protein, is involved in modulation of intracellular processes, including modulation that is effected upon binding of a ligand to an extracellular portion of INTERCEPT 394. INTERCEPT 394 protein is thus capable of transmitting signals across a membrane (e.g., from a signal source outside the cell to a molecule within the cell or from a signal source within the cell to a molecule outside the cell), along a membrane (i.e., between two or more molecules on a single side of a membrane), and combinations thereof. INTERCEPT 394 protein is also capable of interacting with other membrane-associated proteins to form complexes, the activity or specificity of which can be affected by association of INTERCEPT 394 therewith. INTERCEPT 394 protein is therefore involved in disorders associated with aberrant signal transmission and disorders associated with inappropriate association of membrane proteins. INTERCEPT 394-related molecules can be used to prognosticate, prevent, diagnose, and treat one or more of these disorders. Exemplary disorders for which INTERCEPT 394-related molecules have prognostic, preventive, diagnostic, and therapeutic use include, but are not limited to, carcinogenesis, tumor growth, tumor metastasis, angiogenesis, apoptosis, inappropriate blood coagulation, immune hypo- and hyper-stimulation, cell metabolism disorders, endocrine disorders, mineral import and export disorders, and the like.

As an additional example, INTERCEPT 400, compounds which modulate its activity, expression, or both, and compounds (e.g., antibodies) which bind with INTERCEPT 400 (collectively "INTERCEPT 400-related molecules") modulate one or more of growth, proliferation, survival, differentiation, activity, morphology, and movement/migration of, for example, human adult and fetal keratinocytes and brain cells and tissues. INTERCEPT 400 is a transmembrane protein that is involved in modulating interactions between membrane components and cellular cytoskeletons, such as interactions involved in activation of leukocytes, interactions involved in affecting cellular metabolism, interactions involved in cellular growth, and interactions involved in cellular proliferation. INTERCEPT 400-related molecules can therefore be used to prognosticate, prevent, diagnose, and treat disorders associated with one or more of these physiological processes, and can be used to modulate the physiological processes even in the absence of a disorder. Disorders related to these processes include, by way of example and not limitation, obesity, unusual susceptibility or insensitivity to heat or cold, diabetes, arteriosclerosis, atherosclerosis, cancer, hypo- and hyper-immune disorders, aberrant immune proliferation, and the like.

Further by way of example, TANGO 405, compounds which modulate its activity, expression, or both, and compounds (e.g., antibodies) which bind with TANGO 405 (collectively "TANGO 405-related molecules") modulate one or more of growth, proliferation, survival, differentiation, activity, morphology, and movement/migration of human lymphocytes and bone marrow cells and tissues. As described herein, TANGO 405 is involved in activation of leukocytes, including modulating one or more of growth, proliferation, survival, differentiation, activity, morphology, movement/migration, and other cellular processes by which leukocytes are characterized. TANGO 405 is involved in disorders associated with aberrant activation of leukocytes, including both auto-immune disorders and disorders related to inappropriate activity or activation of leukocytes and disorders related to uncontrolled proliferation of leukocytes. Thus, TANGO 405-related molecules can be used to prognosticate, prevent, diagnose, and treat disorders such as leukemias, lymphomas, dyscrasias, auto-immune disorders, eosinophilic disorders, and the like.

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

In one embodiment, the isolated polypeptide of the invention lacks both a transmembrane and a cytoplasmic domain. In another embodiment, the polypeptide lacks both a transmembrane domain and a cytoplasmic domain and is soluble under physiological conditions. The polypeptides of the present invention, or biologically active portions thereof, can be operably linked to 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, single-chain antibodies, and the like. In addition, the polypeptides of the invention or biologically active portions thereof can be incorporated into pharmaceutical compositions, which optionally include pharmaceutically acceptable carriers. These antibody substances can be made, for example, by providing the polypeptide of the invention to an immunocompetent vertebrate and thereafter harvesting blood or serum from the vertebrate.

In another aspect, the present invention provides methods for detecting the presence of the activity or expression of a polypeptide of the invention in a biological sample by contacting the biological sample with an agent capable of detecting an indicator of activity such that the presence of activity is detected in the biological sample.

In another aspect, the invention provides methods for modulating activity of a polypeptide of the invention comprising contacting a cell with an agent that modulates (inhibits or enhances) the activity or expression of a polypeptide of the invention such that activity or expression in the cell is modulated. In one embodiment, the agent is an antibody that specifically binds to a polypeptide of the invention.

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

The present invention also provides methods to treat 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 polypeptide (e.g., an antibody or a fragment of a polypeptide of the invention), a peptidomimetic, or another small molecule (e.g., a small organic molecule).

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 to 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 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 compoimd. 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 210, TANGO 364, TANGO 366, INTERCEPT 394, INTERCEPT 400, and TANGO 405. Preferably, the extracellular domain with which the antibody substance binds has an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 26, 36, 48, 52, 66, 70, 74, and 105. 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 IA through 1 Y. The nucleotide sequence (SEQ ID NO: 1) of a cDNA encoding the human TANGO 210 protein described herein is listed in Figures IA, IB, 1C, and ID. The open reading frame (ORF; residues 45 to 1583; 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 210 is listed. Figure IE is a hydrophilicity plot of human TANGO 210 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 (the conformation of the alternative form of TANGO 210 protein, wherein the carboxyl terminal portion comprises a transmembrane domain, is shown here). The nucleotide sequence (SEQ ID NO: 11) of a cDNA encoding the murine TANGO 210 protein described herein is listed in Figures IF, 1G, IH, and II. The ORF (residues 22 to 927 and 1280 to 1906; collectively, SEQ ID NO: 12) of the cDNA is indicated by nucleotide triplets, above which the amino acid sequence (SEQ ID NO: 13) of murine TANGO 210 is listed. Figure 1 J is a hydrophilicity plot of murine TANGO 210 protein. An alignment of the amino acid sequences of human TANGO 210 protein (SEQ ID NO: 3) and murine TANGO 210 protein (SEQ ID NO: 13) amino acid sequences is shown in Figures IK and IL, wherein identical amino acid residues are indicated by ":" and similar amino acid residues are indicated by ".". An alignment of the nucleotide sequences of the human (SEQ ID NO: 1) and murine (SEQ ID NO: 11) cDNAs encoding TANGO 210 protein is shown in Figures IM through 1U, wherein identical nucleotide residues are indicated by ":" and similar nucleotide residues are indicated by ".". Figures IV and 1 W are an alignment of the amino acid sequences of human TANGO 210 protein ("210"; SEQ ID NO: 3) and human matrix metalloproteinase-8 (MMP-8; "MMP-8"; SEQ ID NO: 6). An alignment of the nucleotide sequences of the open reading frame (ORF) encoding human TANGO 210 ("210"; SEQ ID NO: 2) and the ORF encoding human MMP-8 (SEQ ID NO: 7) is shown in Figures lXi through lXvi. Figure 1 Y is a graph which depicts expression of TANGO 210 mRNA in selected human tissue and cell types, relative to TANGO 210 expression in the human fetal heart tissue.

Figure 2 comprises Figures 2A through 2K. The nucleotide sequence (SEQ ID NO: 21) of a cDNA encoding the human TANGO 364 protein described herein is listed in Figures 2A through 2E. The ORF (residues 235 to 1764; SEQ ID NO: 22) of the cDNA is indicated by nucleotide triplets, above which the amino acid sequence (SEQ ID NO: 23) of human TANGO 364 is listed. Figure 2F is a hydrophilicity plot of human TANGO 364 protein. The nucleotide sequence (SEQ ID NO: 121) of an alternatively-spliced form of the cDNA encoding the human TANGO 364 protein described herein is listed in Figures 2G through 21. The ORF (residues 2 to 898; SEQ ID NO: 122) of the cDNA is indicated by nucleotide triplets, above which the amino acid sequence (SEQ ID NO: 123) of the protein encoded by the splice variant is listed. Figures 2J and 2K are an alignment of the amino acid sequence of SEQ ID NOs: 23 and 123.

Figure 3 comprises Figures 3A through 3E. The nucleotide sequence (SEQ ID NO: 31) of a cDNA encoding the human TANGO 366 protein described herein is listed in Figures 3 A through 3D. The ORF (residues 86 to 1144; SEQ ID NO: 32) of the cDNA is indicated by nucleotide triplets, above which the amino acid sequence (SEQ ID NO: 33) of human TANGO 366 is listed. Figure 3E is a hydrophilicity plot of human TANGO 366 protein.

Figure 4 comprises Figures 4A through 4M. The nucleotide sequence (SEQ ID NO: 41) of a cDNA encoding the human INTERCEPT 394 protein described herein is listed in Figures 4A through 4F. The ORF (residues 303 to 2636; SEQ ID NO: 42) of the cDNA is indicated by nucleotide triplets, above which the amino acid sequence (SEQ ID NO: 43) of human INTERCEPT 394 is listed. Figure 4G is a hydrophilicity plot of human INTERCEPT 394 protein. The nucleotide sequence (SEQ ID NO: 41) of a cDNA encoding the human INTERCEPT 394 protein described herein is listed in Figures 4H through 4M. The alternative ORF (residues 120 to 2636; SEQ ID NO: 55) of the cDNA is indicated by nucleotide triplets, above which the amino acid sequence (SEQ ID NO: 56) of this alternative form of human INTERCEPT 394 protein is listed.

Figure 5 comprises Figures 5A through 5R. The nucleotide sequence (SEQ ID NO: 61 ) of a cDNA encoding the human INTERCEPT 400 protein described herein is listed in Figures 5A through 5C. The ORF (residues 206 to 1000; SEQ ID NO: 62) of the cDNA is indicated by nucleotide triplets, above which the amino acid sequence (SEQ ID NO: 63) of human INTERCEPT 400 is listed. Figure 5D is a hydrophilicity plot of human INTERCEPT 400 protein. The nucleotide sequence (SEQ ID NO: 81) of a cDNA encoding the murine INTERCEPT 400 protein described herein is listed in Figures 5E and 5F. The ORF (residues 3 to 524; SEQ ID NO: 82) of the cDNA is indicated by nucleotide triplets, above which the amino acid sequence (SEQ ID NO: 83) of murine INTERCEPT 400 is listed. Figure 5G is a hydrophilicity plot of murine INTERCEPT 400 protein. An alignment of the amino acid sequences of human INTERCEPT 400 protein (SEQ ID NO: 63) and murine INTERCEPT 400 protein (SEQ ID NO: 83) amino acid sequences is shown in Figure 5H. An alignment of the nucleotide sequences of the human (SEQ ID NO: 62) and murine (SEQ ID NO: 82) ORFs encoding INTERCEPT 400 protein is shown in Figures 51 through 5K. Figure 5L is an alignment of the amino acid sequences of human INTERCEPT 400 protein ("1400"; SEQ ID NO: 63) and murine Cig30 protein ("CIG30"; SEQ ID NO: 79). An alignment of the nucleotide sequences of the ORFs encoding human INTERCEPT 400 protein ("1400"; SEQ ID NO: 62) and the ORF encoding murine Cig30 ("CIG30"; SEQ ID NO: 78) is shown in Figures 5M through 5O. The nucleotide sequence (SEQ ID NO: 91) of a cDNA encoding the rat INTERCEPT 400 protein described herein is listed in Figures 5P and 5Q. The ORF (residues 1 to 432; SEQ ID NO: 92) of the cDNA is indicated by nucleotide triplets, above which the amino acid sequence (SEQ ID NO: 93) of rat INTERCEPT 400 is listed. Figure 5R is an alignment of the amino acid sequences of human (SEQ ID NO: 63), murine (SEQ ID NO: 83), and rat (SEQ ID NO: 93) INTERCEPT 400 proteins.

Figure 6 comprises Figures 6A through 6P. The nucleotide sequence (SEQ ID NO: 101) of a cDNA encoding the human TANGO 405 protein described herein is listed in Figures 6A through 6C. The ORF (residues 178 to 804; SEQ ID NO: 102) of the cDNA is indicated by nucleotide triplets, above which the amino acid sequence (SEQ ID NO: 103) of human TANGO 405 is listed. Figure 6D is a hydrophilicity plot of human TANGO 405 protein. The nucleotide sequence (SEQ ID NO: 111) of a cDNA encoding the murine TANGO 405 protein described herein is listed in Figures 6E and 6F. The ORF (residues 174 to 707; SEQ ID NO: 112) of the cDNA is indicated by nucleotide triplets, above which the amino acid sequence (SEQ ID NO: 113) of murine TANGO 405 is listed. Figure 6G is a hydrophilicity plot of murine TANGO 405 protein. An alignment of the amino acid sequences of human TANGO 405 protein (SEQ ID NO: 103) and murine TANGO 405 protein (SEQ ID NO: 113) amino acid sequences is shown in Figure 6H. An alignment of the nucleotide sequences of the human (SEQ ID NO: 102) and murine (SEQ ID NO: 112) ORFs encoding TANGO 405 protein is shown in Figures 61 through 6K. Figure 6L is an alignment of the amino acid sequences of murine TANGO 405 protein ("mT405"; SEQ ID NO: 113) and murine dectin-2 ("Dectin"; SEQ ID NO: 110). Figure 6M is an alignment of the amino acid sequences of human TANGO 405 protein ("hT405"; SEQ ID NO: 103) and murine dectin-2 ("Dectin"; SEQ ID NO: 110). The nucleotide sequence (SEQ ID NO: 124) of an alternative embodiment of a cDNA encoding the murine TANGO 405 protein described herein is listed in Figures 6N, 6O and 6P. The ORF (residues 179 to 805; SEQ ID NO: 125) of the cDNA is indicated by nucleotide triplets, above which the amino acid sequence (SEQ ID NO: 126) of the alternative embodiment of murine TANGO 405 is listed.

Detailed Description of the Invention The present invention is based, at least in part, on the discovery of a variety of human, murine, and rat cDNA molecules which encode proteins which are herein designated TANGO 210, TANGO 364, TANGO 366, INTERCEPT 394, INTERCEPT 400, and TANGO 405. 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 210

A cDNA clone (designated jthke034a06) encoding at least a portion of human TANGO 210 protein was isolated from a human fetal skin cDNA library. A corresponding murine cDNA clone (designated jtmMa065g07) was isolated from a long term bone marrow cDNA library. The 'long term' bone marrow cDNA library was made by reverse transcription of mRNA obtained from bone marrow cells which were cultured for a period (generally two weeks) prior to stimulating the cells using yeast hyphae and thereafter obtaining mRNA from the cells. Human TANGO 210 protein is predicted by structural analysis to be a secreted protein although, in an alternative form, human TANGO 210 protein has a transmembrane region located near its carboxyl terminal end. Murine TANGO 210 protein is a secreted protein. The full length of the cDNA encoding human TANGO 210 protein (Figure 1;

SEQ ID NO: 1) is 1684 nucleotide residues. The open reading frame (ORF) of this cDNA, nucleotide residues 45 to 1583 of SEQ ID NO: 1 (i.e., SEQ ID NO: 2), encodes a 513-amino acid residue protein (Figure 1; SEQ ID NO: 3), corresponding to a 496-residue secreted protein. The invention thus includes purified human TANGO 210 protein, both in the form of the immature 513 amino acid residue protein (SEQ ID NO: 3) and in the form of the mature 496 amino acid residue protein (SEQ ID NO: 5). Mature human TANGO 210 protein can be in its secreted or membrane-bound form, as described below. The invention also includes purified murine TANGO 210 protein, both in the form of the immature 511 -amino acid residue protein (SEQ ID NO: 13) and in the form of the mature 494-amino acid residue protein (SEQ ID NO: 15). Mature human or murine TANGO 210 proteins can be synthesized without the signal sequence polypeptide at the amino terminus thereof, or they can be synthesized by generating immature TANGO 210 protein and cleaving the signal sequence therefrom. In addition to full length mature and immature human and murine TANGO 210 proteins, the invention includes fragments, derivatives, and variants of these TANGO 210 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 or some portion thereof or SEQ ID NO: 11 or some portion thereof, such as the portion which encodes mature human or murine TANGO 210 protein, immature human or murine TANGO 210 protein, or a domain of human or murine TANGO 210 protein. These nucleic acids are collectively referred to as nucleic acids of the invention.

TANGO 210 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, as described herein). 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 210 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 a preferred embodiment, a signal sequence contains at least about 10 to 35 amino acid residues, preferably about 10 to 20 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 bilayer. Thus, in one embodiment, a TANGO 210 protein contains a signal sequence corresponding to the portion of the protein from amino acid residue 1 to about amino acid residue 17 of SEQ ID NO: 3 (SEQ ID NO: 4) or to the portion of the protein from amino acid residue 1 to about amino acid residue 17 of SEQ ID NO: 13 (SEQ ID NO: 14). 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., at residue 15, 16, 17, 18, or 19 of SEQ ID NO: 3 or at residue 15, 16, 17, 18, or 19 of SEQ ID NO: 13). The signal sequence is cleaved during processing of the mature protein.

TANGO 210 proteins can also include 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 bilayer of a cell when a nucleic acid encoding the protein is expressed in the cell. Murine TANGO 210 protein is secreted. However, in one alternative form, the human TANGO 210 protein is a transmembrane protein having an extracellular domain located from about amino acid residue 25 to amino acid residue 488 of SEQ ID NO: 3 (i.e., SEQ ID NO: 8). In this alternative form, human TANGO 210 protein also has a transmembrane region (i.e., about amino acid residues 489 to 506 of SEQ ID NO: 3; SEQ ID NO: 9) and an intracellular domain (i.e., about amino acid residues 507 to 513 of SEQ ID NO: 3; SEQ ID NO: 10). In another alternative form, human TANGO 210 protein has an intracellular domain located from about amino acid residue 25 to amino acid residue 488 of SEQ ID NO: 3 (i.e., SEQ ID NO: 8), a transmembrane region (i.e., about amino acid residues 489 to 506 of SEQ ID NO: 3; SEQ ID NO: 9), and an extracellular domain (i.e., about amino acid residues 507 to 513 of SEQ ID NO: 3; SEQ ID NO: 10).

TANGO 210 proteins typically comprise a variety of potential post- translational modification sites (often within an extracellular domain), domains, or both, such as those described herein in Tables I (for human TANGO 210) and II (for murine TANGO 210), as predicted by computerized sequence analysis of TANGO 210 proteins using amino acid sequence comparison software (comparing the amino acid sequence of TANGO 210 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 II

Table II (Continued)

As used herein, the term "post-translational modification site" refers to a protein domain 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. Exemplary 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 Tables I and II.

Exemplary additional domains present in human and murine TANGO 210 protein include hemopexin domains and peptidase_M10 domains and signature sequences corresponding to hemopexin domains, zinc-binding domains, and matrix metalloproteinase (MMP) cysteine switches. 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%, more preferably at least about 75%, yet more preferably at least about 85%, and most preferably at least about 95% identical to one of the domains and signature sequences described herein in Tables I and II. Preferably, the protein of the invention has at least one hemopexin domain, one peptidase MlO domain, one hemopexin domain signature sequence, one zinc-binding domain signature sequence, and one MMP cysteine switch signature sequence. Hemopexin domains derive their name from a portion of a protein designated hemopexin. Hemopexin is a serum glycoprotein that binds with heme and transports it to the liver. Hemopexin domains facilitate binding of the protein comprising the domain with a variety of molecules and other proteins. Besides hemopexin, hemopexin domains occur in MMPs and in vitronectin, a cell adhesion and factor (Hunt et al. (1987) Prot. Seq. Data Anal. 1 :21-26; Stanley (1986) FEBS Lett. 199:249-253. A consensus hemopexin domain signature sequence has been identified (Pfam Accession PDOC00023), which has the structure

(L, I, A, or T)-X₃-W-X_{(2 or 3)}-(P or E)-X₂-(L, I, V, M, F, or Y)- (D, E, N, Q, or S)-(S, T, or A)-(A or V)-(L, I, V, M, F, or Y), wherein standard single-letter amino acid codes are used, X being any amino acid residue. Each of the human and murine TANGO 210 amino acid sequences include a single copy of this consensus sequence. This consensus sequence occurs in the amino acid sequences of many MMPs, including MMPs-1, -2, -3, -9, -10, -11, -12, -14, -15, and -16.

Peptidase_M10 domains are conserved amino acid sequences which occur in type 10 zinc-dependent metalloproteinases, according to the classification of Rawlings et al. (1995, Meth. Enzymol 248:183-228). Several mammalian MMPs are type 10 zinc-dependent metalloproteinases including, for example, MMP-1 (interstitial collagenase), MMP-2 (72 kD gelatinase), MMP-3 (stromelysin-1) MMP-7 (matrylisin), MMP-8 (neutrophil collagenase), MMP-9 (92 kD gelatinase), and MMP- 10 (stromelysin-2; Woessner, 1991, FASEB J. 5:2145- 2154). The peptidase_M10 domain includes a consensus zinc-binding domain signature sequence having the structure

(G, S, T, A, L, I, V, or N)-X₂-H-E-(L, I, V, M, F, Y, or W)- (D, E, G, H, R, K, or P)-H-X-(L, I, V, M, F, Y, W, G, S, P, or Q), wherein standard single-letter amino acid codes are used, X being any amino acid residue. The two histidine residues of the consensus sequence have been recognized as zinc ligands, and the glutamate residue is the (proteinase) active site residue. Each of the human and murine TANGO 210 amino acid sequences include this consensus sequence. Another distinguishing characteristic of mammalian extracellular MMPs is presence in the amino acid sequence of the MMP of an MMP cysteine switch signature. The consensus MMP cysteine switch signature sequence has the structure

P-R-C-(G or N)-X-P-(D or R)-(L, I, V, S, A, P, K, or Q) wherein standard single-letter amino acid codes are used, X being any amino acid residue. Each of the human and murine TANGO 210 amino acid sequences include a single copy of this consensus sequence. Human MMPs in which this consensus sequence occurs include MMPs-1, -2, -3, -7, -8, -9, -10, -11, -12, -13, -14, -15, and -16.

The signal peptide prediction program SIGN ALP (Nielsen et al. (1997) Protein Engineering 10:1-6) predicted that human TANGO 210 protein includes an approximately 17 amino acid signal peptide (amino acid residues 1 to 15, 16, 17, 18, or 19 of SEQ ID NO: 3; SEQ ID NO: 4) preceding the mature, secreted TANGO 210 protein (amino acid residues 18 to 513 of SEQ ID NO: 3; SEQ ID NO: 5). In one alternative form, human TANGO 210 protein includes an extracellular domain (amino acid residues 18 to 488 of SEQ ID NO: 3; SEQ ID NO: 8), a transmembrane domain (amino acid residues 489 to 506 of SEQ ID NO: 3; SEQ ID NO: 9), and a cytoplasmic domain (amino acid residues 507 to 513 of SEQ ID NO: 3 ; SEQ ID NO: 10). In another alternative form, human TANGO 210 protein includes a cytoplasmic domain (amino acid residues 18 to 488 of SEQ ID NO: 3; SEQ ID NO: 8), a transmembrane domain (amino acid residues 489 to 506 of SEQ ID NO: 3; SEQ ID NO: 9), and an extracellular domain (amino acid residues 507 to 513 of SEQ ID NO: 3; SEQ ID NO: 10).

Figure IE depicts a hydrophilicity plot of human TANGO 210 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 17 of SEQ ID NO: 3 is the signal sequence of human TANGO 210 (SEQ ID NO: 4). The hydrophobic region which corresponds to amino acid residues 489 to 506 of SEQ ID NO: 3 is the transmembrane portion in the alternative form of human TANGO 210 protein. 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 210 protein from about amino acid residue 190 to about amino acid residue 205 appears to be located at or near the surface of the protein, while the region from about amino acid residue 145 to about amino acid residue 155 appears not to be located at or near the surface.

The predicted molecular weight of human TANGO 210 protein without modification and prior to cleavage of the signal sequence is about 59.0 kilodaltons. The predicted molecular weight of the mature human TANGO 210 protein without modification and after cleavage of the signal sequence is about 57.0 kilodaltons.

Northern hybridization experiments using human tissue samples indicated that mRNA corresponding to the cDNA encoding TANGO 210 is expressed in the tissues listed in Table III, wherein "+" indicates detectable expression and "-" indicates failure to detect expression.

Table III

Table III (Continued)

Human TANGO 210 exhibits sequence similarity to human MMP-8 (GENBANK™ Accession No. J05556), as indicated herein in Figures IV and 1 W, which list an alignment of the amino acid sequences of these proteins. Figures lXi through lXvi depict an alignment of the nucleotide sequences of the ORFs of human TANGO 210 (SEQ ID NO: 2) and MMP-8 (SEQ ID NO: 6). In these alignments (each made using the ALIGN software; paml20.mat scoring matrix; gap penalties -12/-4), the amino acid and ORF nucleotide sequences corresponding to these two proteins are 43.9% identical and 57.1% identical, respectively.

The full length of the cDNA encoding murine TANGO 210 protein (Figure 1; SEQ ID NO: 11) is 2467 nucleotide residues. The ORF of this cDNA, nucleotide residues 22 to 927 and about 1280 to 1906 of SEQ ID NO: 11 (i.e., collectively, SEQ ID NO: 12), encodes a 510-amino acid residue protein (Figure 1 ; SEQ ID NO: 13). It is recognized that the precise locations of the intron boundaries in SEQ ID NO: 11 have not been identified. Thus, murine TANGO 210 protein can comprise one or more additional or one or more fewer amino acid residues at the exon-exon boundary (i.e., between about residues 302 and 303 of SEQ ID NO: 13). The signal peptide prediction program SIGNALP (Nielsen et al. (1997) Protein

Engineering 10:1-6) predicted that murine TANGO 210 protein includes an approximately 17 amino acid signal peptide (amino acid residues 1 to about 17 of SEQ ID NO: 13; SEQ ID NO: 14) preceding the mature TANGO 210 protein (amino acid residues 18 to 511 of SEQ ID NO: 13; SEQ ID NO: 15). Murine TANGO 210 protein is a secreted protein. Figure 1 J depicts a hydrophilicity plot of murine TANGO 210 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 17 of SEQ ID NO: 13 is the signal sequence of murine TANGO 210 (SEQ ID NO: 14). 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 murine TANGO 210 protein from about amino acid residue 18 to about amino acid residue 28 appears to be located at or near the surface of the protein, while the region from about amino acid residue 148 to about amino acid residue 158 appears not to be located at or near the surface

The predicted molecular weight of murine TANGO 210 protein without modification and prior to cleavage of the signal sequence is about 58.7 kilodaltons. The predicted molecular weight of the mature murine TANGO 210 protein without modification and after cleavage of the signal sequence is about 56.2 kilodaltons.

Human and murine TANGO 210 proteins exhibit considerable sequence similarity, as indicated herein in Figures IK and IL. Figures IK and IL depict an alignment of human and murine TANGO 210 amino acid sequences (SEQ ID NOs: 3 and 13, respectively). In this alignment (made using the ALIGN software {Myers and Miller (1989) CABIOS, ver. 2.0}; paml20.mat scoring matrix; gap penalties -12/-4), the proteins are 77.2% identical in the overlapping region (i.e., 393 identical residues out of 509 residues in the overlapping region, which includes amino acid residues 1-509 of SEQ ID NO: 3 and amino acid residues 1-509 of SEQ ID NO: 13). The human and murine cDNAs encoding TANGO 210 are 76.2% identical in the overlapping portions (i.e., nucleotide residues 29-1601 of SEQ ID NO: 1 and nucleotide residues 8-927 and 1280-1935 of SEQ ID NO: 11), as assessed using the same software and parameters and as indicated in Figures IM through 1U. In the respective ORFs, SEQ ID NOs: 1 and 11 are 81.7% identical.

Human TANGO 210 Gene Expression Analysis

Expression of TANGO 210 in selected human tissues and cell types was analyzed as follows. Total RNA was prepared from selected human tissues using a single step extraction method using the RNA STAT-60™ kit according to the manufacturer's instructions (TelTest, Inc). Each RNA preparation was treated with DNase I (Ambion) at 37°C for 1 hour. DNase I treatment was considered to be complete if the sample required at least 38 PCR amplification cycles to reach a threshold level of fluorescence using β-2 microglobulin as an internal amplicon reference. The integrity of the RNA samples following DNase I treatment was confirmed by agarose gel electrophoresis and ethidium bromide staining. Following phenol extraction, cDNA was prepared from the sample using the SUPERSCRIPT™ Choice System following the manufacturer's instructions (Gibco BRL). A negative control of RNA without reverse transcriptase was mock reverse-transcribed for each RNA sample.

TANGO 210 expression was measured by TAQM AN^® quantitative PCR (Perkin Elmer Applied Biosystems) in cDNA prepared from the following normal human tissues: prostate, liver, breast, skeletal muscle, brain, colon, heart, ovary, kidney, lung, vein, aorta, testis, thyroid, placenta, fetal liver, fetal heart, osteoblasts (undifferentiated), small intestine, spleen, thymus, and lymph node. Probes were designed by PRIMEREXPRESS™ software (PE Biosystems) based on the sequence of each gene. Each gene probe was labeled using FAM (6-carboxy fluorescein), and the β2- microglobulin reference probe was labeled with a different fluorescent dye, VIC. The differential labeling of the target gene and internal reference gene thus enabled measurement in same well. Forward and reverse primers and the probes for both β2-microglobulin and target gene were added to the TAQMAN^® Universal PCR Master Mix (PE Applied Biosystems). Although the final concentration of primer and probe varied, each was internally consistent within a given experiment. A typical experiment contained 200 nanomolar forward and reverse primers and 100 nanomolar probe for β-2 microglobulin, and 600 nanomolar forward and reverse primers and 200 nanomolar probe for the target gene. TAQMAN^® matrix experiments were carried out using an ABI PRISM™ 7700 Sequence Detection System (PE Applied Biosystems). The thermal cycler conditions were as follows: hold for 2 minutes at 50°C and 10 minutes at 95°C, followed by two-step PCR for 40 cycles of 95°C for 15 seconds followed by 60°C for 1 minute.

The following method was used to quantitatively calculate TANGO 210 gene expression in the selected tissues relative to β-2 microglobulin expression in the same tissue. The threshold cycle (Ct) value is defined as the cycle at which a statistically significant increase in fluorescence is detected. A lower Ct value is indicative of a higher mRNA concentration. The Ct value of the kinase gene is normalized by subtracting the Ct value of the β-2 microglobulin gene to obtain a _ΔCt value using the following formula: _ΔCt=Ct_kinase - Ct p__{2 m}i_croglobuli_n- Expression is then calibrated against a cDNA sample showing a comparatively low level of expression of the kinase gene. The _ΔCt value for the calibrator sample is then subtracted from _ΔCt for each tissue sample according to the following formula: Ct=_ACt-_samp|e - _ΔCt-_calibrator. Relative expression is then calculated using the arithmetic formula given by 2^{" Q}. Expression of the target gene in each of the tissues tested is then graphically represented as discussed in more detail below.

Figure 1 Y depicts expression of TANGO 210 in various tissues and cell lines as described above, relative to expression in fetal heart tissue. The results indicate significant expression in breast, skeletal muscle, colon, vein, aorta, testis, thyroid, and small intestine tissues.

Biological function of TANGO 210 proteins, nucleic acids, and modulators thereof TANGO 210 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 210 occurs in a human fetal skin cDNA library and in a murine long term bone marrow cDNA library, and that RNA corresponding to TANGO 210 is detectable by Northern analysis of human adult and fetal kidney tissue, it is evident that TANGO 210 protein is involved in one or more biological processes which occur in these tissues. In particular, TANGO 210 is involved in modulating one or more of growth, proliferation, survival, differentiation, activity, morphology, and movement/migration of cells of these tissues. TANGO 210 is involved in modulating the structure of extracellular matrix which contacts or is in fluid communication with cells of these tissues. Thus, TANGO 210 has a role in disorders which affect these cells and one or more of their growth, proliferation, survival, differentiation, activity, morphology, and movement/migration, as well as the biological function of organs comprising one or more of these tissues.

The Northern analysis data described herein for human TANGO 210 indicate that nucleic acids corresponding to (i.e., homologous with or complementary to) all or part of human TANGO 210 cDNA or molecules (e.g., antibodies) which react specifically with human TANGO 210 protein or a portion thereof can be used to identify kidney tissue or to differentiate kidney tissue from other types of tissue, such as heart, brain, placenta, lung, liver, and pancreas tissues. Thus, human TANGO 210 proteins, nucleic acids, and compounds which interact specifically with either of these, can be used for one or more of tissue typing, identification, and separation.

TANGO 210 gene expression data described herein indicate that TANGO 210 can be expressed in at least breast, skeletal muscle, colon, vein, aorta, testis, thyroid, small intestine, and spleen tissues. Thus, TANGO 210 can have a role in disorders which affect cells of these tissues and one or more of their growth, proliferation, survival, differentiation, activity, morphology, and movement/migration, as well as the biological function of organs comprising one or more of these tissues.

The fact that TANGO 210 is expressed in breast tissue is an indication that TANGO 210 can be involved in both normal physiological function of breast tissue and in breast disorders. Examples of breast disorders include breast cancer, insufficient lactation, infant nutritional and growth disorders, mastalgia, fibroadenomas, breast infections, and gynecomastia.

In another example, TANGO 210 polypeptides, nucleic acids, and modulators thereof, can be involved in normal and aberrant functioning of skeletal muscle tissue, and can thus be involved in disorders of such tissue. Examples of skeletal muscle disorders include muscular dystrophy (e.g., Duchenne muscular dystrophy, Becker muscular dystrophy, Emery- Dreifuss muscular dystrophy, limb-girdle muscular dystrophy, facioscapulohumeral muscular dystrophy, myotonic dystrophy, oculopharyngeal muscular dystrophy, distal muscular dystrophy, and congenital muscular dystrophy), motor neuron diseases (e.g., amyotrophic lateral sclerosis, infantile progressive spinal muscular atrophy, intermediate spinal muscular atrophy, spinal bulbar muscular atrophy, and adult spinal muscular atrophy), myopathies (e.g., inflammatory myopathies (e.g., dermatomyositis and polymyositis), myotonia congenita, paramyotonia congenita, central core disease, nemaline myopathy, myotubular myopathy, and periodic paralysis), and metabolic diseases of muscle (e.g., phosphorylase deficiency, acid maltase deficiency, phosphofructokinase deficiency, de-brancher enzyme deficiency, mitochondrial myopathy, carnitine deficiency, carnitine palmityl transferase deficiency, phosphoglycerate kinase deficiency, phosphoglycerate mutase deficiency, lactate dehydrogenase deficiency, and myoadenylate deaminase deficiency). TANGO 210 polypeptides, nucleic acids, or modulators thereof can be used to prognosticate, diagnose, inhibit, prevent, or alleviate one or more of these disorders.

In another example, TANGO 210 polypeptides, nucleic acids, and modulators thereof can be used to treat colonic disorders, such as congenital anomalies (e.g., megacolon and imperforate anus), idiopathic disorders (e.g., diverticular disease and melanosis coli), vascular lesions (e.g., ischemic colitis, hemorrhoids, angiodysplasia), inflammatory diseases (e.g., idiopathic ulcerative colitis, pseudomembranous colitis, and lymphopathia venereum), and colon tumors (e.g., hypeφlastic polyps, adenomatous polyps, bronchogenic cancer, colonic carcinoma, squamous cell carcinoma, adenoacanthomas, sarcomas, lymphomas, argentaffinomas, carcinoids, and melanocarcinomas).

In another example, TANGO 210 polypeptides, nucleic acids, and modulators thereof, can be used to treat cardiovascular disorders, such as ischemic heart disease (e.g., angina pectoris, myocardial infarction, and chronic ischemic heart disease), hypertensive heart disease, pulmonary heart disease, valvular heart disease (e.g., rheumatic fever and rheumatic heart disease, endocarditis, mitral valve prolapse, and aortic valve stenosis), congenital heart disease (e.g., valvular and vascular obstructive lesions, atrial or ventricular septal defect, and patent ductus arteriosus), and myocardial disease (e.g., myocarditis, congestive cardiomyopathy, and hypertrophic cardiomyopathy).

In another example, TANGO 210 polypeptides, nucleic acids, or modulators thereof, can be used to treat testicular disorders, such as unilateral testicular enlargement (e.g., non-tuberculous, granulomatous orchitis), inflammatory diseases resulting in testicular dysfunction (e.g., gonorrhea and mumps), and tumors (e.g., germ cell tumors, interstitial cell tumors, androblastoma, testicular lymphoma and adenomatoid tumors).

TANGO 210 polypeptides, nucleic acids, and modulators thereof, can be involved in disorders of the thyroid gland, such as hyperthyroidism (e.g., diffuse toxic hyperplasia, toxic multi-nodular goiter, toxic adenoma, and acute or sub-acute thyroiditis), hypothyroidism (e.g., cretinism and myxedema), thyroiditis (e.g., Hashimoto's thyroiditis, sub- acute granulomatous thyroiditis, sub-acute lymphocytic thyroiditis, Riedel's thryroiditis), Graves' disease, goiter (e.g., simple diffuse goiter and multi-nodular goiter), and tumors (e.g., adenoma, papillary carcinoma, follicular carcinoma, medullary carcinoma, undifferentiated malignant carcinoma, Hodgkin's disease, and non-Hodgkin's lymphoma). TANGO 210 polypeptides, nucleic acids, or modulators thereof can be used to prognosticate, diagnose, inhibit, prevent, or alleviate one or more of these disorders.

In another example, TANGO 210 polypeptides, nucleic acids, and modulators thereof can be used to treat intestinal disorders (e.g., disorders of the small intestine), such as ischemic bowel disease, infective enterocolitis, Crohn's disease, benign tumors, malignant tumors (e.g., argentaffinomas, lymphomas, adenocarcinomas, and sarcomas), malabsorption syndromes (e.g., celiac disease, tropical sprue, Whipple's disease, and abetalipoproteinemia), obstructive lesions, hernias, intestinal adhesions, intussusception, and volvulus.

TANGO 210 nucleic acids, proteins, and modulators thereof can be used to modulate the proliferation, differentiation, or function of cells that form the spleen (e.g., cells of the splenic connective tissue, splenic smooth muscle cells, and endothelial cells of the splenic blood vessels). TANGO 210 nucleic acids, proteins, and modulators thereof can also be used to modulate the proliferation, differentiation, and function of cells that are processed within the spleen (e.g., regenerated or phagocytized within the spleen, erythrocytes, B and T lymphocytes, and macrophages). Thus TANGO 210 nucleic acids, proteins, and modulators thereof can be used to treat disorders of the spleen (including disorders of the fetal spleen). Examples of splenic disorders include, splenic lymphoma, splenomegaly, and phagocytotic disorders (e.g., those in which macrophage engulfment of bacteria and viruses in the bloodstream is inhibited). There are several indications that TANGO 210 is an MMP. For instance, presence of each of a Peptidase_M10 domain, a zinc-binding domain signature, a hemopexin domain signature, and a cysteine switch in the amino acid sequences of both human and murine TANGO 210 indicates that TANGO 210 exhibits extracellular matrix proteinase activity (e.g., collagenase and basement membrane degradative activities). In addition, homology between the sequence of human TANGO 210 and MMPs (e.g., MMP-8, as described herein) is a further indication the TANGO 210 is an MMP.

MMPs degrade extracellular matrix (ECM), and are thus involved in maintenance, and in renewal and replacement of old ECM with new ECM. ECM serves numerous purposes in the body, including providing support, containment, or both, to specialized tissues (e.g., tissues of organs such as skin, kidney, bone marrow, etc.) and regulating fluid balance in tissues which line a void or fluid-filled compartment (e.g., skin, bladder, kidney, stomach, etc.). Demonstration, as described herein, that TANGO 210 is expressed in several of these tissues (fetal skin, bone marrow, kidney) indicates that TANGO 210 is involved in one or more of these processes.

An important function of kidney tissue is to regulate the volume and composition of body fluids. The kidneys regulate body fluids by selectively permitting water, electrolytes, metabolites, and the like to pass from the plasma into the bladder in a regulatable manner while retaining cells and proteins in the plasma. By regulating fluid balance, the kidneys also exert a significant effect on arterial blood pressure. The kidneys perform these functions in a manner analogous to filtration.

Fluid outflow from the plasma occurs through the membranes of kidney glomerular capillaries in structures designated Bowman's capsules. The membrane of glomerular capillaries has three layers (normal capillaries have only two). Glomerular capillaries have a highly fenestrated luminal endothelium which can serve to prevent passage of cells through the capillary membrane, but do not substantially inhibit passage of serum proteins. Surrounding the endothelium is an ECM basement membrane comprising collagen and peptidoglycan. An epithelial layer having gaps or channels through which glomerular filtrate is passed surrounds the basement membrane. The basement membrane is the layer of the glomerular capillary membrane which is principally responsible for retention of serum proteins. In order to maintain proper operation of the kidneys, it is critical that the relative porosity of the basement membrane be maintained. Mineral precipitates, circulating bacteria, and the like can clog the pores of the basement membrane. Turnover, renewal, or controlled degradation of the basement membrane ensures that the basement membrane remains functional. TANGO 210 is involved in regulating the thickness, porosity, and rate of degradation of the basement membrane of glomerular capillaries and other ECM components of kidney tissue. TANGO 210 is therefore involved in normal and abnormal formation and maintenance of functional kidney tissue. Thus, TANGO 210 is involved in a number of disorders which relate to aberrant kidney tissue formation and function. Such disorders include, for example, acute and chronic renal failure, immunologically mediated renal diseases (IMRD; e.g., IMRD associated with rejection of transplanted kidney tissue), nephrotic syndrome (e.g., nephrotic syndrome associated with one or more of minimal-change disease, focal glomerulosclerosis, membranous glomerulonephritis, membranoproliferative glomerulonephritis, etc.), secondary glomerular diseases (e.g., thin basement membrane disease, sickle cell disease, systemic lupus erythematosus, bacterial endocarditis, and the like), chronic nephritic/proteinuric syndromes (e.g., chronic glomerulonephritis and slowly progressive glomerular disease), chronic and acute tubulointerstitial nephritis, and vascular hypertension. TANGO 210 proteins, nucleic acids encoding them, and agents that modulate activity or expression of either of these can be used to prognosticate, diagnose, treat, and inhibit one or more of these disorders.

Recovery of a cDNA encoding TANGO 210 from a murine long term bone marrow cDNA library indicates that TANGO 210 is expressed in bone marrow, and is thus involved both in normal physiological processes which occur in bone marrow and in disorders which affect bone marrow. ECM is a significant component of bone marrow, and TANGO 210 is involved in degradation of ECM associated with turnover/renewal of bone marrow tissue, and with changes which occur in the bone marrow with age. As mammals age, the bone marrow becomes increasingly gelatinous and the ECM composition of the marrow changes. The cellular content of the bone marrow changes with time as well. In young mammals, most bones are filled with red marrow, which comprises large numbers of hematopoietic cells. As mammals age, red marrow is replaced by gelatinous, adipose cell- containing white and yellow marrows. TANGO 210 is involved in ECM changes which accompany age related changes in marrow composition. TANGO 210 is also involved in bone marrow-related disorders such as bone marrow failure (e.g., that associated with anemia) and rejection of heterologous implanted bone marrow. TANGO 210 proteins, nucleic acids encoding them, and agents that modulate activity or expression of either of these can be used to prognosticate, diagnose, treat, and inhibit one or more of these disorders. In addition, because TANGO 210 is associated with remodeling of bone marrow, TANGO 210 is also capable of modulating acceptance of donor bone marrow in a recipient.

Recovery of a cDNA encoding TANGO 210 from a human fetal skin cDNA library indicates that TANGO 210 is expressed in human skin, and is involved both in normal physiological processes which occur in skin and in skin disorders. Skin is a multi-layered tissue in which the various tissue layers can have different ECM compositions. Skin has a variety of roles in the normal mammal. Skin maintains the mechanical, osmotic, chemical, photic, and thermal integrity of the exterior surface of the mammal. TANGO 210, being expressed in the skin and able to modulate ECM composition, is therefore involved in regulating these characteristics in normal individuals and in individuals afflicted with disorders relating to aberrant regulation of these characteristics (e.g., ichthyosis). TANGO 210 is also involved in other disorders which occur in or affect ECM in skin. Such disorders include, by way of example, psoriasis, infections, wounds (and healing of wounds), inflammation, dermatitis, acne, benign and malignant dermatological tumors, and the like. TANGO 210 proteins, nucleic acids encoding them, and agents that modulate activity or expression of either of these can be used to prognosticate, diagnose, treat, and inhibit one or more of these disorders.

Numerous cancers are associated with aberrant MMP expression and activity. MMPs can aid cancer growth and metastasis by degrading ECM, thereby providing an avenue for angiogenesis, cell growth, or cell movement through a tissue. TANGO 210 is able to modulate the rate and extent of angiogenesis, and is therefore useful for prognosticating, diagnosing, treating, and inhibiting one or more disorders associated with aberrant angiogenesis, including, but not limited to cancers. Disorders associated with aberrant angiogenesis include both those associated with an abnormally high rate or extent of angiogenesis (e.g., cancerous growth and metastasis) and those associated with an abnormally or insufficiently low rate or extent of angiogenesis (e.g., impaired wound healing, transplanted tissue rejection, and acute and chronic ischemic disorders such as stroke). TANGO 210 proteins, nucleic acids encoding them, and agents that modulate activity or expression of either of these can be used to prognosticate, diagnose, treat, and inhibit one or more cancers or other disorders associated with aberrant angiogenesis.

TANGO 364 cDNA clones (designated jthke076a05 and jthkf069gl 1) encoding at least a portion of human TANGO 364 protein were isolated from a human fetal skin cDNA library by computerized sequence analysis of library ORFs which encode a signal sequence (SPOT analysis). Human TANGO 364 protein is predicted by structural analysis to be a transmembrane protein.

The full length of the cDNA encoding human TANGO 364 protein (Figure 2; SEQ ID NO: 21) is 3510 nucleotide residues. The ORF of this cDNA, nucleotide residues 235 to 1764 of SEQ ID NO: 21 (i.e., SEQ ID NO: 22), encodes a 510-amino acid residue protein (Figure 2; SEQ ID NO: 23), corresponding to a 479-residue transmembrane protein. TANGO 364 cDNA can exist in an alternatively-spliced form, as listed in Figures 2G through 21. In this alternative form, TANGO 364 cDNA is 2510 nucleotide residues in length (SEQ ID NO: 121). The ORF of this cDNA, nucleotide residues 2 to 898 of SEQ ID NO: 121 (i.e., SEQ ID NO: 122), encodes a 299-amino acid residue protein (Figure 2; SEQ ID NO: 123) which has the same sequence as the portions of full length TANGO 364 protein indicated in the alignment (made using the ALIGN software; paml20.mat scoring matrix; gap penalties -12/-4) listed in Figures 2 J and 2K. In the discussion which follows, the full length and alternatively- spliced forms of TANGO 364 molecules are referred to individually and collectively as TANGO 364 molecules of the corresponding type (e.g., cDNA and protein). The invention thus includes purified human TANGO 364 protein, both in the form of the immature 510 amino acid residue protein (SEQ ID NO: 23) and in the form of the mature 479 amino acid residue protein (SEQ ID NO: 25). Mature human TANGO 364 proteins can be synthesized without the signal sequence polypeptide at the amino terminus thereof, or it can be synthesized by generating immature TANGO 364 protein and cleaving the signal sequence therefrom.

In addition to full length mature and immature human TANGO 364 proteins, the invention includes fragments, derivatives, and variants of these TANGO 364 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: 21 or some portion thereof, such as the portion which encodes mature human TANGO 364 protein, immature human TANGO 364 protein, or a domain of human TANGO 364 protein. These nucleic acids are collectively referred to as nucleic acids of the invention.

TANGO 364 proteins and nucleic acid molecules encoding them comprise a family of molecules having certain conserved structural and functional features.

A common domain present in TANGO 364 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 a preferred embodiment, a signal sequence contains at least about 10 to 35 amino acid residues, preferably about 10 to 20 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 bilayer. Thus, in one embodiment, a TANGO 364 protein contains a signal sequence corresponding to the portion of the protein from amino acid residue 1 to about amino acid residue 31 of SEQ ID NO: 23 (SEQ ID NO: 24). 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., at residue 29, 30, 31, 32, or 33 of SEQ ID NO: 23). The signal sequence is cleaved during processing of the mature protein.

TANGO 364 proteins can include an extracellular domain. The human TANGO 364 protein extracellular domain is located from about amino acid residue 32 to amino acid residue 345 of SEQ ID NO: 23 (i.e., the extracellular domain has the sequence SEQ ID NO: 26).

In addition, TANGO 364 can include a transmembrane domain. As used herein, 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. In a preferred embodiment, a transmembrane domain contains at least about 15 to 30 amino acid residues, preferably about 20-25 amino acid residues, and has at least about 60- 80%, more preferably 65-75%, and more preferably at least about 70% hydrophobic residues. In one embodiment, a TANGO 364 protein of the invention contains a transmembrane domain corresponding to about amino acid residues 346 to 370 of SEQ ID NO: 23 (i.e., the transmembrane domain has the sequence SEQ ID NO: 27).

The present invention includes TANGO 364 proteins having a cytoplasmic domain, particularly including proteins having a carboxyl-terminal cytoplasmic domain. As used herein, a "cytoplasmic domain" refers to a portion of a protein which is localized to the cytoplasmic side of a lipid bilayer of a cell when a nucleic acid encoding the protein is expressed in the cell. The human TANGO 364 cytoplasmic domain is located from about amino acid residue 371 to amino acid residue 510 of SEQ ID NO: 23 (i.e., the cytoplasmic domain has the sequence SEQ ID NO: 28). In an alternative embodiment, TANGO 364 proteins have a cytoplasmic domain located from about amino acid residue 32 to amino acid residue 345 of SEQ ID NO: 23 (i.e., the extracellular domain has the sequence SEQ ID NO: 26); a transmembrane domain corresponding to about amino acid residues 346 to 370 of SEQ ID NO: 23 (i.e., the transmembrane domain has the sequence SEQ ID NO: 27); and an extracellular domain located from about amino acid residue 371 to amino acid residue 510 of SEQ ID NO: 23 (i.e., the extracellular domain has the sequence SEQ ID NO: 28).

TANGO 364 proteins typically comprise a variety of potential post- translational modification sites (often within an extracellular domain), such as those described herein in Table IV, as predicted by computerized sequence analysis of TANGO 364 proteins using amino acid sequence comparison software (comparing the amino acid sequence of TANGO 364 with the information in the PROSITE database {rel. 12.2; Feb, 1995} and the Hidden Markov Models database {Rel. PFAM 3.3}).

Table IV

Table IV (Continued)

As used herein, the term "post-translational modification site" refers to a protein domain 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. Exemplary 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 IV.

Exemplary additional domains present in human TANGO 364 protein include the RGD cell attachment sequence and Ig-/MHC-like domains. In one embodiment, the protein of the invention has at least one domain that is at least 55%, preferably at least about 65%, more preferably at least about 75%, yet more preferably at least about 85%, and most preferably at least about 95% identical to one of the Ig-/MHC-like domains described herein in Table IV. Preferably, the protein of the invention has at least one Ig-/MHC-like domain and one RGD cell attachment sequence.

Ig-/MHC-like domains are conserved among immunoglobulin (Ig) constant (CL) regions and one of the three extracellular domains of major histocompatibility proteins (MHC). Ig-/MHC-like domains are involved in protein-to-protein and protein-to-ligand binding. The signal peptide prediction program SIGNALP (Nielsen et al. (1997) Protein

Engineering 10:1-6) predicted that human TANGO 364 protein includes an approximately 31 amino acid signal peptide (amino acid residues 1 to about 31 of SEQ ID NO: 23; SEQ ID NO: 24) preceding the mature TANGO 364 protein (amino acid residues 32 to 510 of SEQ ID NO: 23; SEQ ID NO: 25). Human TANGO 364 protein includes an extracellular domain (amino acid residues 32 to 345 of SEQ ID NO: 23; SEQ ID NO: 26), a transmembrane domain (amino acid residues 346 to 370 of SEQ ID NO: 23; SEQ ID NO: 27), and a cytoplasmic domain (amino acid residues 371 to 510 of SEQ ID NO: 23; SEQ ID NO: 28).

Figure 2F depicts a hydrophilicity plot of human TANGO 364 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 31 of SEQ ID NO: 23 is the signal sequence of human TANGO 364 (SEQ ID NO: 24), and the hydrophobic region which corresponds to amino acid residues 346 to 370 of SEQ ID NO: 23 is the transmembrane region of TANGO 364 (SEQ ID NO: 27). 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 364 protein from about amino acid residue 371 to about amino acid residue 410 appears to be located at or near the surface of the protein, while the region from about amino acid residue 235 to about amino acid residue 245 appears not to be located at or near the surface.

The predicted molecular weight of human TANGO 364 protein without modification and prior to cleavage of the signal sequence is about 55.5 kilodaltons. The predicted molecular weight of the mature human TANGO 364 protein without modification and after cleavage of the signal sequence is about 52.1 kilodaltons.

TANGO 364 exhibits limited sequence similarity to numerous cell surface proteins, including proteins which serve as cell surface antigens, proteoglycans, and virus receptors.

Biological function of TANGO 364 proteins, nucleic acids, and modulators thereof

TANGO 364 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 364 occurs in a human fetal skin cDNA library, it is evident that TANGO 364 protein is involved in one or more biological processes which occur in skin tissues. In particular, TANGO 364 is involved in modulating one or more of growth, proliferation, survival, differentiation, activity, morphology, and movement/migration of skin cells. Thus, TANGO 364 has a role in disorders which affect skin cells and one or more of their growth, proliferation, survival, differentiation, activity, morphology, and movement/migration, as well as the biological function of skin. There are several indications that TANGO 364 is a cell surface protein which is involved in binding a ligand to the cell which expresses the protein. For instance, presence in TANGO 364 of an amino terminal extracellular domain that includes three Ig-/MHC-like domains exemplifies the cell-surface ligand-binding capability of TANGO 364. In addition, the amino acid sequence similarity which TANGO 364 exhibits with respect to several other cell surface ligand-binding proteins reinforces this view. Presence in TANGO 364 of an Ig- /MHC-like domain indicates that the corresponding region of TANGO 364 is structurally similar to this conserved extracellular region, and that TANGO 364 is involved in binding one or more of a ligand and a protein (including, for example, a serum protein and a cell-surface protein of another cell). Thus, molecules (e.g., antibodies and short peptides) which are able to interact specifically with an Ig-/MHC-like domain of a TANGO 364 protein can inhibit binding of TANGO 364 with its normal ligand, thereby disrupting one or more physiological processes associated with such binding. Furthermore, polypeptides (including, for example, full-length TANGO 364 protein and polypeptides of at least about 25 to 50 amino acid residues) which comprise all or part of an Ig-/MHC-like domain of TANGO 364 can bind with one or more of the normal ligands of TANGO 364, thereby replicating the normal physiological effect of binding between TANGO 364 and the ligand or inhibiting binding of endogenous TANGO 364 with the ligand. Therefore, TANGO 364 protein, polypeptides having at least one Ig-/MHC-like domain thereof, and molecules capable of interacting with such a domain are useful for prognosticating, diagnosing, treating, and inhibiting disorders associated with aberrant binding of TANGO 364 and its normal ligand.

TANGO 364 is involved in binding an animal cell which expresses it with one or more of a protein (e.g., an antibody, an major histocompatibility protein, a lectin, or another cell surface protein), a small molecule (e.g., a sugar, a hormone, or another molecule having a molecular weight less than about 5000, 1000, or 500 or less Daltons), a component of the extracellular matrix (e.g., a collagen protein), another cell of the same animal, a bacterial or fungal cell, and a virus. Thus, TANGO 364 is involved in modulating cell-to-cell adhesion, tissue and extracellular matrix invasivity of cells, infectivity of cells by pathogens (e.g., bacteria and viruses), endocrine signaling processes, tissue developmental and organizational processes, and the like. Thus, TANGO 364 is involved in disorders in which these physiological processes are relevant.

Disorders associated with aberrant cell-to-cell adhesion include tumor growth and metastasis, malformation or degradation of neurological connections, autoimmune disorders, immune insufficiency disorders, atherosclerosis, arteriosclerosis, abnormal blood coagulation, and the like. Disorders associated with tissue and extracellular matrix invasivity of cells include tumor metastasis, osteoporosis, inflammation, and the like. Disorders associated with pathogenic infections include infections associated with bacteria, fungi, mycoplasmas, viruses, eukaryotic parasites, and the like. Disorders associated with aberrant endocrine signaling processes include, for example, diabetes mellitus, hypoglycemia, glucagon disorders, pituitary disorders (e.g., diabetes insipidus), thyroid disorders (e.g., hyper- and hypothyroidism), adrenal disorders (e.g., Addison's disease, adrenal virilism, Cushing's syndrome, and hyperaldosteronism), multiple endocrine neoplasias, and polyglandular deficiency syndromes. Disorders associated with aberrant tissue developmental and organizational processes include, for example, birth defects, benign and malignant carcinogenesis, neurodegenerative disorders (e.g., Alzheimer's disease), and the like. TANGO 364 proteins, nucleic acids encoding them, and agents (e.g., antibodies, peptides, and small molecules) that modulate activity or expression of either of these can be used to prognosticate, diagnose, treat, and inhibit one or more of these disorders.

TANGO 366

A cDNA clone (designated jthqc016c02) encoding at least a portion of human TANGO 366 protein was isolated from a human normal prostate fibroblast cDNA library by SPOT analysis. Human TANGO 366 protein is predicted by structural analysis to be a transmembrane protein.

The full length of the cDNA encoding human TANGO 366 protein (Figure 3; SEQ ID NO: 31) is 2628 nucleotide residues. The ORF of this cDNA, nucleotide residues 86 to 1144 of SEQ ID NO: 31 (i.e., SEQ ID NO: 32), encodes a 353-amino acid residue protein (Figure 3; SEQ ID NO: 33), corresponding to a 337-residue transmembrane protein. The invention thus includes purified human TANGO 366 protein, both in the form of the immature 353 amino acid residue protein (SEQ ID NO: 33) and in the form of the mature 337 amino acid residue protein (SEQ ID NO: 35). Mature human TANGO 366 proteins can be synthesized without the signal sequence polypeptide at the amino terminus thereof, or it can be synthesized by generating immature TANGO 366 protein and cleaving the signal sequence therefrom.

In addition to full length mature and immature human TANGO 366 proteins, the invention includes fragments, derivatives, and variants of these TANGO 366 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: 31 or some portion thereof, such as the portion which encodes mature human TANGO 366 protein, immature human TANGO 366 protein, or a domain of human TANGO 366 protein. These nucleic acids are collectively referred to as nucleic acids of the invention.

TANGO 366 proteins and nucleic acid molecules encoding them comprise a family of molecules having certain conserved structural and functional features. A common domain present in TANGO 366 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 a preferred embodiment, a signal sequence contains at least about 10 to 35 amino acid residues, preferably about 10 to 20 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 bilayer. Thus, in one embodiment, a TANGO 366 protein contains a signal sequence corresponding to the portion of the protein from amino acid residue 1 to about amino acid residue 16 of SEQ ID NO: 33 (SEQ ID NO: 34). 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., at residue 14, 15, 16, 17, or 18 of SEQ ID NO: 33). The signal sequence is cleaved during processing of the mature protein. TANGO 366 proteins can include an extracellular domain. The human TANGO 366 protein extracellular domain is located from about amino acid residue 17 to amino acid residue 216 of SEQ ID NO: 33 (i.e., the extracellular domain has the sequence SEQ ID NO: 36). In addition, TANGO 366 can include a transmembrane domain. In one embodiment, a TANGO 366 protein of the invention contains a transmembrane domain corresponding to about amino acid residues 217 to 239 of SEQ ID NO: 33 (i.e., the transmembrane domain has the sequence SEQ ID NO: 37).

The present invention includes TANGO 366 proteins having a cytoplasmic domain, particularly including proteins having a carboxyl-terminal cytoplasmic domain. The human TANGO 366 cytoplasmic domain is located from about amino acid residue 240 to amino acid residue 353 of SEQ ID NO: 33 (i.e., the cytoplasmic domain has the sequence SEQ ID NO: 38).

In an alternative embodiment, TANGO 366 proteins can have a cytoplasmic domain located from about amino acid residue 17 to amino acid residue 216 of SEQ ID NO: 33 (i.e., the cytoplasmic domain has the sequence SEQ ID NO: 36); a transmembrane domain corresponding to about amino acid residues 217 to 239 of SEQ ID NO: 33 (i.e., the transmembrane domain has the sequence SEQ ID NO: 37); and an extracellular domain located from about amino acid residue 240 to amino acid residue 353 of SEQ ID NO: 33 (i.e., the extracellular domain has the sequence SEQ ID NO: 38)

TANGO 366 proteins typically comprise a variety of potential post- translational modification sites (often within an extracellular domain), such as those described herein in Table V, as predicted by computerized sequence analysis of TANGO 366 proteins using amino acid sequence comparison software (comparing the amino acid sequence of TANGO 366 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)

As used herein, the term "post-translational modification site" refers to a protein domain 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. Exemplary 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 V.

Exemplary additional domains present in human TANGO 366 protein include a glycosaminoglycan attachment site, several leucine rich repeat (LRR and LRRNT) domains, and a leucine zipper domain. In one embodiment, the protein of the invention has at least one domain that is at least 55%, preferably at least about 65%, more preferably at least about 75%, yet more preferably at least about 85%, and most preferably at least about 95% identical to one of the LRR or leucine zipper domains described herein in Table V. Preferably, the protein of the invention has at least one LRR domain, one leucine zipper domain, and one potential glycosaminoglycan attachment site sequence. One or more LRR domains are present in a variety of proteins involved in protein-protein interactions. Such proteins include, for example, proteins involved in signal transduction, cell-to-cell adhesion, cell-to-extracellular matrix adhesion, cell development, DNA repair, RNA processing, and cellular molecular recognition processes. Specialized LRR domains, designated LRR amino terminal (LRRNT) domains often occur near the amino ends of a series of LRR domains. TANGO 366 protein has nine LRR domains, arranged in two groups, the first group including (from the amino terminus toward the carboxyl terminus of TANGO 366) the LRRNT domain and four LRR domains, and the second group including four LRR domains. TANGO 366 is involved in one or more physiological processes in which these other LRR domain-containing proteins are involved, namely binding of cells with extracellular proteins such as soluble extracellular proteins and cell surface proteins of other cells.

TANGO 366 comprises a leucine zipper region at about amino acid residue 284 to about amino acid residue 305 (i.e., 284 LdlsgtnLvplpeaLllhlpaL 305). Leucine zipper regions are known to be involved in dimerization of proteins. Leucine zipper regions interact with one another, leading to formation of homo- or hetero-dimers between proteins, depending on their identity. Dimers of proteins having leucine zipper regions can also interact with DNA. The presence in TANGO 366 of a leucine zipper region is a further indication that this protein is involved in protein-protein interactions. The signal peptide prediction program SIGNALP (Nielsen et al. (1997) Protein

Engineering 10:1-6) predicted that human TANGO 366 protein includes an approximately 16 amino acid signal peptide (amino acid residues 1 to about 16 of SEQ ID NO: 33; SEQ ID NO: 34) preceding the mature TANGO 366 protein (amino acid residues 17 to 353 of SEQ ID NO: 33; SEQ ID NO: 35). Human TANGO 366 protein includes an extracellular domain (amino acid residues 17 to 216 of SEQ ID NO: 33; SEQ ID NO: 36), a transmembrane domain (amino acid residues 217 to 239 of SEQ ID NO: 33; SEQ ID NO: 37), and a cytoplasmic domain (amino acid residues 240 to 353 of SEQ ID NO: 33; SEQ ID NO: 38).

Figure 3E depicts a hydrophilicity plot of human TANGO 366 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 16 of SEQ ID NO: 33 is the signal sequence of human TANGO 366 (SEQ ID NO: 34), and the hydrophobic region which corresponds to amino acid residues 217 to 239 of SEQ ID NO: 33 is the transmembrane region of TANGO 366 (SEQ ID NO: 37). 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 366 protein from about amino acid residue 315 to about amino acid residue 330 appears to be located at or near the surface of the protein, while the region from about amino acid residue 290 to about amino acid residue 305 appears not to be located at or near the surface.

The predicted molecular weight of human TANGO 366 protein without modification and prior to cleavage of the signal sequence is about 37.8 kilodaltons. The predicted molecular weight of the mature human TANGO 366 protein without modification and after cleavage of the signal sequence is about 36.1 kilodaltons.

TANGO 366 exhibits limited sequence similarity to numerous cell surface proteins, including proteins which serve as cell surface antigens, proteoglycans, and protein receptors. TANGO 366 protein, cDNA, and ORF exhibit sequence homology to the sequences corresponding to a GENBANK™ record having Accession No. HSM800846. The nucleotide sequence of the DNA molecule described in GENBANK™ Accession No. HSM800846 is identical to nucleotide residues 418 to 2628 of SEQ ID NO: 31. The cDNA of GENBANK™ Accession No. HSM800846 was obtained from uterine tissue, indicating that TANGO 366 is expressed in uterine tissue and thus involved in normal and aberrant physiological processes in uterine tissue. In addition, nucleotide residues 36 to 319 of the reverse complement of SEQ ID NO: 31 exhibits significant homology with expressed sequence tag (EST) 01904, which is disclosed in an international patent application having PCT Publication No. WO93/16178. The ESTs described in that application were isolated from human brain tissue. This is an indication that TANGO 366 is expressed in brain tissue and thus is involved in normal and aberrant physiological processes in brain tissue. Biological function of TANGO 366 proteins, nucleic acids, and modulators thereof TANGO 366 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 366 occurs in a human normal prostate fibroblast, brain, and uterus cDNA libraries, it is evident that TANGO 366 protein is involved in one or more biological processes which occur in prostate, brain, uterus, and other solid tissues. In particular, TANGO 366 is involved in modulating one or more of growth, proliferation, survival, differentiation, activity, moφhology, and movement/migration of cells of prostate, brain, uterus, and other solid tissues. Thus, TANGO 366 has a role in disorders which affect the prostate, brain, uterus, and other solid tissues and one or more of growth, proliferation, survival, differentiation, activity, moφhology, and movement/migration of cells in those tissues, as well as the biological function of organs (e.g., the prostate) comprising such tissues. Disorders which affect the prostate include, by way of example, prostate cancer, benign prostatic hypeφlasia, benign prostatic hypertrophy, prostatitis, and the like. TANGO 366 proteins, nucleic acids encoding them, and agents that modulate activity or expression of either of these can be used to prognosticate, diagnose, treat, and inhibit one or more of these disorders. Exemplary brain disorders 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. Exemplary 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 TANGO 366 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 the like. TANGO 366 proteins, nucleic acids encoding them, and agents that modulate activity or expression of either of these can be used to prognosticate, diagnose, treat, and inhibit one or more of these disorders.

Disorders which involve uterus tissue include, by way of example, uterine cancers, endometriosis, female infertility, and primary and secondary dysmenorrheas. TANGO 366 proteins, nucleic acids encoding them, and agents that modulate activity or expression of either of these can be used to prognosticate, diagnose, treat, and inhibit one or more of these disorders.

There are several indications that TANGO 366 is a cell surface protein which is involved in binding a protein to the cell which expresses TANGO 366. For instance, presence in TANGO 366 of an amino terminal extracellular domain that includes an LRRNT and four LRR domains exemplifies the cell-surface protein interaction capability of TANGO 366. In addition, the amino acid sequence similarity which TANGO 366 exhibits with respect to several other cell surface protein-binding proteins reinforces this view. TANGO 366 is involved in binding an animal cell which expresses it with one or more of an extracellular fluid protein, a protein component of the extracellular matrix, a surface protein another cell of the same animal, and a surface protein of a bacterium, fungus, or virus. Thus, TANGO 366 is involved in modulating cell-to-cell adhesion, tissue and extracellular matrix invasivity of cells, infectivity of cells by pathogens (e.g., bacteria and viruses), endocrine signaling processes, tissue developmental and organizational processes, and the like. TANGO 366 is involved in disorders in which these physiological processes are relevant. Such disorders include, for example, loss of control of cell growth, tumor metastasis, malformation of neurological connections, inflammation, immune and autoimmune responses, bacterial, fungal, and viral infections, and the like. TANGO 366 proteins, nucleic acids encoding them, and agents that modulate activity or expression of either of these can be used to prognosticate, diagnose, treat, and inhibit one or more of these disorders.

INTERCEPT 394

A cDNA clone (designated jthKa041f02) encoding at least a portion of human INTERCEPT 394 protein was isolated from a human fetal kidney cDNA library. Human INTERCEPT 394 protein is predicted by structural analysis to be a transmembrane protein.

The full length of the cDNA encoding human INTERCEPT 394 protein (Figure 4; SEQ ID NO: 41) is 3743 nucleotide residues. The ORF of this cDNA, nucleotide residues 303 to 2636 of SEQ ID NO: 41 (i.e., SEQ ID NO: 42), encodes a 778-amino acid residue protein (Figure 4; SEQ ID NO: 43), corresponding to a 778-residue transmembrane protein. It is recognized that, in an alternative form, transcription of INTERCEPT 394 protein can be initiated at the ATG codon located at nucleotide residues 120-122 of SEQ ID NO 41. In this alternative form, INTERCEPT 394 protein has, at the amino-terminal end of SEQ ID NO: 43, an additional 61 amino acid residues, this additional portion having the amino acid sequence encoded by nucleotide residues 120-319 of SEQ ID NO: 41. The sequences corresponding to the cDNA (SEQ ID NO: 41), ORF (SEQ ID NO: 55), and protein (SEQ ID NO: 56) of this alternate form are listed in Figures 4H through 4M. In the following discussion, molecules of the two forms of INTERCEPT 394 are referred to individually and collectively as molecules of the corresponding type (e.g., cDNA or protein).

The invention thus includes purified human INTERCEPT 394 protein, both in the form of the immature 778 amino acid residue protein (SEQ ID NO: 43) and in the form of the mature 753 amino acid residue protein (SEQ ID NO: 45). Mature human INTERCEPT 394 proteins can be synthesized without the signal sequence polypeptide at the amino terminus thereof, or it can be synthesized by generating immature INTERCEPT 394 protein and cleaving the signal sequence therefrom. In addition to full length mature and immature human INTERCEPT 394 proteins, the invention includes fragments, derivatives, and variants of these INTERCEPT 394 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: 41 or some portion thereof, such as the portion which encodes mature human INTERCEPT 394 protein, immature human INTERCEPT 394 protein, or a domain of human INTERCEPT 394 protein. These nucleic acids are collectively referred to as nucleic acids of the invention.

INTERCEPT 394 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 394 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 a preferred embodiment, a signal sequence contains at least about 10 to 35 amino acid residues, preferably about 10 to 20 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 bilayer. Thus, in one embodiment, a INTERCEPT 394 protein contains a signal sequence corresponding to the portion of the protein from amino acid residue 1 to about amino acid residue 25 of SEQ ID NO: 43 (SEQ ID NO: 44). 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., at residue 23, 24, 25, 26, or 27 of SEQ ID NO: 43). The signal sequence is cleaved during processing of the mature protein.

INTERCEPT 394 proteins can include an extracellular domain. Human INTERCEPT 394 protein extracellular domains are located at about amino acid residues 88 to 228 and 337 to 345 of SEQ ID NO: 43 (i.e., the extracellular domains having the sequences SEQ ID NOs: 48 and 52, respectively).

In addition, INTERCEPT 394 can include a transmembrane domain. In one embodiment, a INTERCEPT 394 protein of the invention contains transmembrane domains corresponding to about amino acid residues 71 to 87, 229 to 253, 320 to 336, and 346 to 364 of SEQ ID NO: 43 (i.e., the transmembrane domains having the sequences SEQ ID NOs: 47, 49, 51, and 53, respectively).

The present invention includes INTERCEPT 394 proteins having a cytoplasmic domain. The INTERCEPT 394 cytoplasmic domains are located from about amino acid residue 26 to 70, 254 to 319, and 365 to 778 of SEQ ID NO: 43 (i.e., the cytoplasmic domains having the sequences SEQ ID NOs: 46, 50, and 54, respectively).

In an alternative form, INTERCEPT 394 proteins have cytoplasmic domains located at about amino acid residues 88 to 228 and 337 to 345 of SEQ ID NO: 43 (i.e., the cytoplasmic domains having the sequences SEQ ID NOs: 48 and 52, respectively); transmembrane domains corresponding to about amino acid residues 71 to 87, 229 to 253, 320 to 336, and 346 to 364 of SEQ ID NO: 43 (i.e., the transmembrane domains having the sequences SEQ ID NOs: 47, 49, 51, and 53, respectively); and extracellular domains located from about amino acid residue 26 to 70, 254 to 319, and 365 to 778 of SEQ ID NO: 43 (i.e., the extracellular domains having the sequences SEQ ID NOs: 46, 50, and 54, respectively). INTERCEPT 394 proteins typically comprise a variety of potential post- translational modification sites (often within an extracellular domain), such as those described herein in Table VI, as predicted by computerized sequence analysis of INTERCEPT 394 proteins using amino acid sequence comparison software (comparing the amino acid sequence of INTERCEPT 394 with the information in the PROSITE database {rel. 12.2; Feb, 1995} and the Hidden Markov Models database {Rel. PFAM 3.3 }). Table VI

Table VI (Continued)

As used herein, the term "post-translational modification site" refers to a protein domain 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. Exemplary 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 VI.

The signal peptide prediction program SIGNALP (Nielsen et al. (1997) Protein Engineering 10:1-6) predicted that human INTERCEPT 394 protein includes an approximately 25 amino acid signal peptide (amino acid residues 1 to about 25 of SEQ ID NO: 43; SEQ ID NO: 44) preceding the mature INTERCEPT 394 protein (amino acid residues 26 to 778 of SEQ ID NO: 43; SEQ ID NO: 45). Human INTERCEPT 394 protein includes two extracellular domains (amino acid residues 88 to 228 and 337 to 345 of SEQ ID NO: 43; SEQ ID NOs: 48 and 52, respectively), four transmembrane domains (amino acid residues 71 to 87, 229 to 253, 320 to 336, and 346 to 364 of SEQ ID NO: 43; SEQ ID NOs: 47, 49, 51, and 53, respectively), and three cytoplasmic domains (amino acid residues 26 to 70, 254 to 319, and 365 to 778 of SEQ ID NO: 43; SEQ ID NOs: 46, 50, and 54, respectively).

Figure 4G depicts a hydrophilicity plot of human INTERCEPT 394 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 25 of SEQ ID NO: 43 is the signal sequence of human INTERCEPT 394 (SEQ ID NO: 44). Hydrophobic regions which corresponding to amino acid residues 71 to 87, 229 to 253, 320 to 336, and 346 to 364 of SEQ ID NO: 43 are the transmembrane regions of INTERCEPT 394 (SEQ ID NOs: 47, 49, 51, and 53, respectively). 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 394 protein from about amino acid residue 205 to about amino acid residue 225 appears to be located at or near the surface of the protein, while the region from about amino acid residue 410 to about amino acid residue 340 appears not to be located at or near the surface.

The predicted molecular weight of human INTERCEPT 394 protein without modification and prior to cleavage of the signal sequence is about 87.4 kilodaltons. The predicted molecular weight of the mature human INTERCEPT 394 protein without modification and after cleavage of the signal sequence is about 84.5 kilodaltons. Nucleotide residues 2944 to 3482 of the reverse complement of SEQ ID NO: 41 exhibits significant homology with EST clone BJ38, which is disclosed in an international patent application having PCT Publication No. WO98/45435. The ESTs described in that application were isolated from human tissues. This is an indication that INTERCEPT 394 is expressed in the same tissues as this EST clone and thus is involved in normal and aberrant physiological processes in these tissues.

Biological function of INTERCEPT 394 proteins, nucleic acids, and modulators thereof

INTERCEPT 394 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 394 occurs in a human fetal kidney cDNA library, it is evident that INTERCEPT 394 protein is involved in one or more biological processes which occur in kidney and other fetal and adult human tissues. In particular, INTERCEPT 394 is involved in modulating one or more of growth, proliferation, survival, differentiation, activity, moφhology, and movement/migration of cells of kidney and other tissues. Thus, INTERCEPT 394 has a role in disorders which affect kidney and other tissues and one or more of growth, proliferation, survival, differentiation, activity, moφhology, and movement/migration of cells in those tissues, as well as the biological function of organs (e.g., the kidneys) comprising such tissues. Exemplary kidney disorders 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, kidney neoplasms, and the like.

The relatively large size of the carboxyl-terminal cytoplasmic domain of INTERCEPT 394 is an indication that INTERCEPT 394 protein is involved in modulation of one or more intracellular processes. The presence of extracellular domains indicates that the activity of INTERCEPT 394 can be modulated by binding thereto of ligands (i.e., either naturally-occurring ligands or non-naturally-occurring ligands such as pharmaceutical agents). Because INTERCEPT 394 protein is an integral membrane protein, it is capable of exerting its physiological effect either by itself or in combination with one or more other membrane proteins. INTERCEPT 394 is thus involved in either or both of generation of signals which can be transmitted either to another protein (or other molecule) on the same side of the membrane or to a protein (or other molecule) on the opposite side of the membrane the membrane. INTERCEPT 394 can transmit such signals by binding a ligand, whereby its conformation is altered such that the ability of INTERCEPT 394 to interact with another molecule (e.g., to catalyze a reaction involving the molecule or by binding with the molecule) is altered upon binding the ligand. Alternatively, INTERCEPT 394 can be altered by being post-translationally modified (e.g., phosphorylated, glycosylated, or myristoylated) such that the ability of INTERCEPT 394 to interact with another molecule is altered upon post- translational modification.

Involvement of INTERCEPT 394 in one or more signal transmission pathways is an indication that INTERCEPT 394 is involved in physiological pathways involving such transmission. Thus, INTERCEPT 394 is also involved in disorders which involve these signal transmission pathways. Exemplary physiological pathways that involve signal transmission include cell nutrition and metabolism, cell proliferation, cell differentiation, apoptosis, chemotactic and chemokinetic activities, cell aggregation and attachment, cell movement, immune stimulation, hematopoiesis, metastasis, and the like. INTERCEPT 394 is thus involved in disorders relating to aberrant activity of one or more of these signal transmission pathways. Such disorders include, for example, carcinogenesis, tumor growth, tumor metastasis, angiogenesis, apoptosis, inappropriate blood coagulation (e.g., that involved in atherosclerosis, arteriosclerosis, and stroke), immune hypo- and hyper-stimulation, cell metabolism disorders (e.g., diabetes), endocrine disorders (e.g., hypo- and hyper-thyroidism), mineral import and export disorders (e.g., osteoporosis, kidney stone formation, and hemochromatosis), and the like.

Presence of INTERCEPT 394 in the membrane of cells in which it is expressed indicates that INTERCEPT 394 can be used as a diagnostic target for detection or imaging of such cells. Furthermore, a portion of INTERCEPT 394 (e.g., an extracellular domain) can be used to interfere with binding of a virus which normally binds with INTERCEPT 394, thereby inhibiting, reducing, or eliminating pathological effects associated with infection of a human by the virus.

INTERCEPT 400 A cDNA clone (designated jthkfO 14a09) encoding at least a portion of human

INTERCEPT 400 protein was isolated from a human normal embryonic keratinocyte cDNA library. A corresponding murine cDNA clone (designated jtmba232b 12) was isolated from a brain polysome cDNA library. Human and murine INTERCEPT 400 proteins are predicted by structural analysis to be transmembrane proteins. The full length of the cDNA encoding human INTERCEPT 400 protein (Figure

5; SEQ ID NO: 61) is 2989 nucleotide residues. The open reading frame (ORF) of this cDNA, nucleotide residues 206 to 1000 of SEQ ID NO: 61 (i.e., SEQ ID NO: 62), encodes a 265- amino acid residue immature protein (Figure 5; SEQ ID NO: 63), corresponding to a 219- residue transmembrane protein. The invention thus includes purified human INTERCEPT 400 protein, both in the form of the immature 265 amino acid residue protein (SEQ ID NO: 63) and in the form of the mature 219 amino acid residue protein (SEQ ID NO: 65). The invention also includes purified murine INTERCEPT 400 protein, which is a 180-amino acid residue transmembrane protein (SEQ ID NO: 83). Mature human INTERCEPT 400 proteins can be synthesized without the signal sequence polypeptide at the amino terminus thereof, or it can be synthesized by generating immature INTERCEPT 400 protein and cleaving the signal sequence therefrom. In addition to full length mature human and murine INTERCEPT 400 proteins and immature human INTERCEPT 400 protein, the invention includes fragments, derivatives, and variants of these INTERCEPT 400 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: 61 or some portion thereof or SEQ ID NO: 81 or some portion thereof, such as the portion which encodes mature human or murine INTERCEPT 400 protein, immature human INTERCEPT 400 protein, or a domain of human or murine INTERCEPT 400 protein. These nucleic acids are collectively referred to as nucleic acids of the invention. INTERCEPT 400 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 400 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 a preferred embodiment, a signal sequence contains at least about 10 to 50 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 bilayer. Thus, in one embodiment, a INTERCEPT 400 protein contains a signal sequence corresponding to the portion of the protein from amino acid residue 1 to about amino acid residue 46 of SEQ ID NO: 63 (SEQ ID NO: 64). 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., at residue 44, 45, 46, 47, or 48 of SEQ ID NO: 63). The signal sequence is cleaved during processing of the mature protein.

INTERCEPT 400 proteins can also include an extracellular domain. Human INTERCEPT 400 protein includes extracellular domains located from about amino acid residues 47 to 62, 154 to 164, and 218 to 231 of SEQ ID NO: 63 (i.e., the extracellular domains having the amino acid sequences SEQ ID NOs: 66, 70, and 74, respectively). Murine INTERCEPT 400 protein includes extracellular domains located from about amino acid residues 61 to 71 and 125 to 140 of SEQ ID NO: 83 (i.e., these extracellular domains having the amino acid sequences SEQ ID NOs: 86 and 90, respectively).

In addition, INTERCEPT 400 can include a transmembrane domain. Human INTERCEPT 400 protein includes transmembrane domains corresponding to about amino acid residues 63 to 79, 137 to 153, 165 to 183, 194 to 217, and 232 to 251 of SEQ ID NO: 63 (i.e., the transmembrane domains having the sequences SEQ ID NOs: 67, 69, 71, 73, and 75, respectively). Murine INTERCEPT 400 protein includes transmembrane domains corresponding to about amino acid residues 44 to 60, 72 to 90, 101 to 124, and 141 to 160 of SEQ ID NO: 83 (i.e., the transmembrane domains having the sequences SEQ ID NOs: 85, 87, 89, and 94, respectively).

The present invention includes INTERCEPT 400 proteins having a cytoplasmic domain. Human INTERCEPT 400 cytoplasmic domains are located from about amino acid residue 80 to 136, 184 to 193, and 252 to 265 of SEQ ID NO: 63 (i.e., the cytoplasmic domains having the sequences SEQ ID NOs: 68, 72, and 76, respectively). Murine

INTERCEPT 400 cytoplasmic domains are located from about amino acid residue 1 to 43, 91 to 100, and 161 to 174 of SEQ ID NO: 83 (i.e., the cytoplasmic domains having the sequences SEQ ID NOs: 84, 88, and 95, respectively).

It is recognized that, in one form, murine INTERCEPT 400 protein can include an amino terminal portion approximately 60-120 (likely 80-100) amino acid residues in length. In an alternative embodiment, human INTERCEPT 400 proteins have cytoplasmic domains located from about amino acid residues 47 to 62, 154 to 164, and 218 to 231 of SEQ ID NO: 63 (i.e., the cytoplasmic domains having the amino acid sequences SEQ ID NOs: 66, 70, and 74, respectively); transmembrane domains corresponding to about amino acid residues 63 to 79, 137 to 153, 165 to 183, 194 to 217, and 232 to 251 of SEQ ID NO: 63 (i.e., the transmembrane domains having the sequences SEQ ID NOs: 67, 69, 71, 73, and 75, respectively); and extracellular domains are located from about amino acid residue 80 to 136, 184 to 193, and 252 to 265 of SEQ ID NO: 63 (i.e., the extracellular domains having the sequences SEQ ID NOs: 68, 72, and 76, respectively). In an alternative embodiment, murine INTERCEPT 400 proteins have cytoplasmic domains located from about amino acid residues 61 to 71 and 125 to 140 of SEQ ID NO: 83 (i.e., these cytoplasmic domains having the amino acid sequences SEQ ID NOs: 86 and 90, respectively); transmembrane domains corresponding to about amino acid residues 44 to 60, 72 to 90, 101 to 124, and 141 to 160 of SEQ ID NO: 83 (i.e., the transmembrane domains having the sequences SEQ ID NOs: 85, 87, 89, and 94, respectively); and extracellular domains are located from about amino acid residue 1 to 43, 91 to 100, and 161 to 174 of SEQ ID NO: 83 (i.e., the extracellular domains having the sequences SEQ ID NOs: 84, 88, and 95, respectively).

INTERCEPT 400 proteins typically comprise a variety of potential post- translational modification sites (often within an extracellular domain), such as those described herein in Tables VII (for human INTERCEPT 400) and VIII (for murine INTERCEPT 400), as predicted by computerized sequence analysis of INTERCEPT 400 proteins using amino acid sequence comparison software (comparing the amino acid sequence of INTERCEPT 400 with the information in the PROSITE database {rel. 12.2; Feb, 1995} and the Hidden Markov Models database {Rel. PFAM 3.3}).

Table VII

Table VII (Continued)

N-myristoylation site 77 to 82 GALRTG

93 to 98 GLKQSV

209 to 214 GCVVNY

Table VIII

As used herein, the term "post-translational modification site" refers to a protein domain 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. Exemplary 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 Tables VII and VIII. The signal peptide prediction program SIGNALP (Nielsen et al. (1997) Protein Engineering 10:1-6) predicted that human INTERCEPT 400 protein includes an approximately 46 amino acid signal peptide (amino acid residues 1 to about 46 of SEQ ID NO: 63; SEQ ID NO: 64) preceding the mature INTERCEPT 400 protein (amino acid residues 47 to 265 of SEQ ID NO: 63; SEQ ID NO: 65). Human INTERCEPT 400 protein includes three extracellular domains (amino acid residues 47 to 62, 154 to 164, and 218 to 231 of SEQ ID NO: 63; SEQ ID NOs: 66, 70, and 74, respectively), five transmembrane domains (amino acid residues 63 to 79, 137 to 153, 165 to 183, 194 to 217, and 232 to 251 of SEQ ID NO: 63; SEQ ID NOs: 67, 69, 71, 73, and 75, respectively), and three intracellular domains (amino acid residues 80 to 136, 184 to 193, and 252 to 265 of SEQ ID NO: 63; SEQ ID NOs: 68, 72, and 76, respectively).

Figure 5D depicts a hydrophilicity plot of human INTERCEPT 400 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 46 of SEQ ID NO: 63 is the signal sequence of human INTERCEPT 400 (SEQ ID NO: 64). 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 400 protein from about amino acid residue 218 to about amino acid residue 231 appears to be located at or near the surface of the protein, while the region from about amino acid residue 80 to about amino acid residue 95 appears not to be located at or near the surface.

The predicted molecular weight of human INTERCEPT 400 protein without modification and prior to cleavage of the signal sequence is about 31.4 kilodaltons. The predicted molecular weight of the mature human INTERCEPT 400 protein without modification and after cleavage of the signal sequence is about 25.8 kilodaltons.

Human INTERCEPT 400 exhibits sequence similarity to murine Cig30 protein (GENBANK™ Accession No. U97107), as indicated herein in Figure 5L, which lists an alignment (made using the ALIGN software; paml20.mat scoring matrix; gap penalties -12/-4) of the amino acid sequences of these proteins. Figures 5M through 50 depict an alignment (also made using the ALIGN software; paml20.mat scoring matrix; gap penalties -12/-4) of the nucleotide sequences of the ORFs of human INTERCEPT 400 (SEQ ID NO: 62) and Cig30 (SEQ ID NO: 78). In these alignments (made using the ALIGN software; paml20.mat scoring matrix, gap penalties -12/-4), the amino acid sequences of these two proteins are 43.3% identical and the ORF nucleotide sequences corresponding to these two proteins are 56.8% identical. The cDNAs corresponding to these two proteins were found to be 48.4% identical using the LALIGN software (paml20.mat scoring matrix; gap penalties -12/-4).

The length of the incomplete cDNA encoding the carboxyl-terminal portion of murine INTERCEPT 400 protein (Figure 5; SEQ ID NO: 81) is 2032 nucleotide residues. The ORF of this cDNA, nucleotide residues 3 to 524 (SEQ ID NO: 82), encodes a protein comprising at least 180 amino acid residues (Figure 5; SEQ ID NO: 83). It is recognized that murine INTERCEPT 400 protein has about 60-120, more likely 80-100, additional amino acid residues at the amino terminal end thereof. The signal peptide prediction program SIGNALP (Nielsen et al. (1997) Protein

Engineering 10:1-6) predicted that the portion of murine INTERCEPT 400 protein described herein includes at least two extracellular domains (amino acid residues 61 to 71 and 125 to 140 of SEQ ID NO: 83; SEQ ID NOs: 86 and 90, respectively), at least four transmembrane domains (amino acid residues 44 to 60, 72 to 90, 101 to 124, and 141 to 160 of SEQ ID NO: 83; SEQ ID NOs: 85, 87, 89, and 94, respectively), and at least three cytoplasmic domains (amino acid residue 1 to 43, 91 to 100, and 161 to 174 of SEQ ID NO: 83; SEQ ID NOs: 84, 88, and 95, respectively).

Figure 5G depicts a hydrophilicity plot of murine INTERCEPT 400 protein. Relatively hydrophobic regions are above the dashed horizontal line, and relatively hydrophilic regions are below the dashed horizontal line. Hydrophobic regions corresponds to the identified transmembrane regions of murine INTERCEPT 400. 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 from about amino acid residue 125 to about amino acid residue 140 appears to be located at or near the surface of the protein, while the region from about amino acid residue 14 to about amino acid residue 19 appears not to be located at or near the surface

The predicted molecular weight of the portion of murine INTERCEPT 400 protein described herein is about 20.6 kilodaltons.

Human and murine INTERCEPT 400 proteins exhibit considerable sequence similarity, as indicated herein in Figures 5H through 5K. Figure 5H depicts an alignment of human and murine INTERCEPT 400 amino acid sequences (SEQ ID NOs: 63 and 83, respectively). In this alignment (made using the ALIGN software; paml20.mat scoring matrix; gap penalties -12/-4), the proteins are 94.8% identical in the overlapping region (i.e., 163 identical residues out of 172 residues in the overlapping region, which includes amino acid residues 94-265 of SEQ ID NO: 63 and amino acid residues 1-174 of SEQ ID NO: 83). The human and murine ORFs encoding INTERCEPT 400 are 92.8% identical in the overlapping portions (i.e., nucleotide residues 280-795 of SEQ ID NO: 62 and nucleotide residues 1-522 of SEQ ID NO: 82), as assessed using the same software and parameters and as indicated in Figures 51 through 5K in an alignment made using the ALIGN software (paml20.mat scoring matrix; gap penalties -12/-4).

The partial nucleotide sequences of a rat cDNA clone (designated jtmba232bl2; SEQ ID NO: 91) and ORF (SEQ ID NO: 92) encoding INTERCEPT 400 are depicted in Figures 5P and 5Q, together with the amino acid sequence (SEQ ID NO: 93) of the portion of the protein encoded by these nucleic acids. An alignment (made using the ALIGN software; paml20.mat scoring matrix; gap penalties -12/-4) of human, murine and rat INTERCEPT 400 amino acid sequences is listed in Figure 5R.

Biological function of INTERCEPT 400 proteins, nucleic acids, and modulators thereof

INTERCEPT 400 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 400 occurs in a human normal embryonic keratinocyte cDNA library and in a murine brain polysome cDNA library, it is evident that INTERCEPT 400 protein is involved in one or more biological processes which occur in these tissues. In particular, INTERCEPT 400 is involved in modulating one or more of growth, proliferation, survival, differentiation, activity, moφhology, and movement/migration of cells of these tissues. INTERCEPT 400 is involved in modulating the structure of extracellular matrix which contacts or is in fluid communication with cells of these tissues. Thus, INTERCEPT 400 has a role in disorders which affect these cells and one or more of their growth, proliferation, survival, differentiation, activity, moφhology, and movement/migration, as well as the biological function of organs comprising one or more of these tissues.

Exemplary brain disorders 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. Exemplary 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 400 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 the like. INTERCEPT 400 proteins, nucleic acids encoding them, and agents that modulate activity or expression of either of these can be used to prognosticate, diagnose, treat, and inhibit one or more of these disorders.

Exemplary skin disorders with which INTERCEPT 400 can be associated include, by way of example, psoriasis, infections, wounds (and healing of wounds), inflammation, dermatitis, acne, benign and malignant dermatological tumors, and the like. INTERCEPT 400 proteins, nucleic acids encoding them, and agents that modulate activity or expression of either of these can be used to prognosticate, diagnose, treat, and inhibit one or more of these disorders.

Murine Cig30 protein, with which human INTERCEPT 400 shares significant amino acid sequence homology, is an integral membrane protein that is involved in recruitment and thermogenesis in brown adipose tissue in mice (Tvrdik et al., 1997, J. Biol. Chem. 272:31738-31746). Yeast proteins which share significant homology with murine Cig30 and human, murine, and rat INTERCEPT 400 protein include proteins encoded by yeast genes SUR4 {APA1) and FEN1 {GNS1). These proteins are involved in phospholipid metabolism, sterol synthesis, budding, activation of glucose-regulated genes, glucose uptake, and glucan synthesis (Desfarges et al., 1993, Yeast 9:267-277; Silve et al., 1996, Mol. Cell. Biol. 16:2719-2727; Durrens et al., 1995, Curr. Genet. 27:213-216; Garcia-Arranz, 1994, J. Biol. Chem. 269:18076-18082; El-Sherbeini et al., 1995, J. Bacteriol. 177:3227-3234). These activities relate to remodeling of the plasma membrane and actin cytoskeleton in response to growth signals, most likely by modulating interaction between membrane phospholipids and the cytoskeleton. Thus, INTERCEPT 400 protein is involved in one or more of these activities, such as in immune stimulation, proliferation of leukocytes, generation and prolongation of an immune response, control of cellular metabolic processes, and the like. INTERCEPT 400 is involved in generation, accumulation, and regulation of brown adipose tissue and other adipose tissues in humans, and is therefore involved in body temperature regulation, lipid metabolism, carbohydrate metabolism, body weight regulation, and the like. Thus, INTERCEPT 400 is implicated in disorders which relate to aberrance or imbalance in the normal physiological regulation of these processes. INTERCEPT 400 is also involved in disorders which relate to aberrant proliferation and growth of cells. Exemplary disorders in which INTERCEPT 400 is involved include obesity, unusual susceptibility or insensitivity to heat or cold, diabetes, arteriosclerosis, atherosclerosis, cancer, hypo- and hyper-immune disorders (e.g., acquired immune deficiency syndrome and auto-immune disorders), immune proliferation, and the like. INTERCEPT 400 proteins, nucleic acids encoding them, and agents that modulate activity or expression of either of these can be used to prognosticate, diagnose, treat, and inhibit one or more of these disorders.

Chromosomal mapping data have been used to locate the gene encoding human INTERCEPT 400 at chromosome 4, between markers D4S1616 and D4S1611 (115.8-119.6 centimorgans). A form of iris hypoplasia associated with early onset glaucoma has been linked with this chromosomal region. Human INTERCEPT 400 allelic variants can include INTERCEPT 400 nucleotide sequence polymoφhisms (e.g., nucleotide sequences that vary from SEQ ID NO: 61) that map to this chromosomal region. The gene encoding human TANGO 405 has been mapped at chromosome 8, between markers D8S269 and D8S1799 (125.8-132.4 centimorgans). Human TANGO 405 allelic variants can include TANGO 405 nucleotide sequence polymoφhisms (e.g., nucleotide sequences that vary from SEQ ID NO: 101) that map to this chromosomal region.

TANGO 405

A cDNA clone (designated jthLal52h06) encoding at least a portion of human TANGO 405 protein was isolated from a human mixed lymphocyte reaction cDNA library. A corresponding murine cDNA (designatedjtmMa025al 1) was isolated from a long-term bone marrow cDNA library. Human and murine TANGO 405 proteins are secreted proteins.

The full length of the cDNA encoding human TANGO 405 protein (Figure 6; SEQ ID NO: 101) is 3114 nucleotide residues in length. The open reading frame (ORF) of this cDNA, nucleotide residues 154 to 780 of SEQ ID NO: 101 (i.e., SEQ ID NO: 102), encodes a 209-amino acid residue protein (Figure 6; SEQ ID NO: 103), corresponding to a 161-residue secreted protein.

The invention thus includes purified human TANGO 405 protein, both in the form of the immature 209 amino acid residue protein (SEQ ID NO: 103) and in the form of the mature 161 amino acid residue protein (SEQ ID NO: 105). The invention also includes purified murine TANGO 405 protein, both in the form of the immature 178-amino acid residue protein (SEQ ID NO: 113) and in the form of the mature, secreted 136-amino acid residue protein (SEQ ID NO: 115). Mature human or murine TANGO 405 protein can be synthesized without the signal sequence polypeptide at the amino terminus thereof, or they can be synthesized by generating immature TANGO 405 protein and cleaving the signal sequence therefrom.

In addition to full length mature and immature human and murine TANGO 405 proteins, the invention includes fragments, derivatives, and variants of these TANGO 405 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: 101 or some portion thereof or SEQ ID NO: 111 or some portion thereof, such as the portion which encodes mature human or murine TANGO 405 protein, immature human or murine TANGO 405 protein, or a domain of human or murine TANGO 405 protein. These nucleic acids are collectively referred to as nucleic acids of the invention.

TANGO 405 proteins and nucleic acid molecules encoding them comprise a family of molecules having certain conserved structural and functional features.

A common domain present in TANGO 405 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 a preferred embodiment, a signal sequence contains at least about 10 to 50 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 bilayer. Thus, in one embodiment, a TANGO 405 protein contains a signal sequence corresponding to the portion of the protein from amino acid residue 1 to about amino acid residue 48 of SEQ ID NO: 103 (SEQ ID NO: 104) or to the portion of the protein from amino acid residue 1 to about amino acid residue 42 of SEQ ID NO: 113 (SEQ ID NO: 114). 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., at residue 46, 47, 48, 49, or 50 of SEQ ID NO: 103 or at residue 40, 41, 42, 43 or 44 of SEQ ID NO: 113). The signal sequence is cleaved during processing of the mature protein.

TANGO 405 proteins typically comprise a variety of potential post- translational modification sites (often within an extracellular domain), such as those described herein in Table IX (for human TANGO 405) and X (for murine TANGO 405), as predicted by computerized sequence analysis of TANGO 405 proteins using amino acid sequence comparison software (comparing the amino acid sequence of TANGO 405 with the information in the PROSITE database {rel. 12.2; Feb, 1995} and the Hidden Markov Models database {Rel. PFAM 3.3}).

Table IX

Table X

Table X (Continued)

As used herein, the term "post-translational modification site" refers to a protein domain 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. Exemplary 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 Tables IX and X.

Exemplary additional domains present in human and murine TANGO 405 protein include a C-type lectin domain and a corresponding signature sequence. In one embodiment, the protein of the invention has a C-type lectin domain or signature sequence that is at least 55%, preferably at least about 65%, more preferably at least about 75%, yet more preferably at least about 85%, and most preferably at least about 95% identical to one of those described herein in Tables IX and X.

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 incoφorated by reference.

In PCT Publication No. WO 98/28332, a cDNA encoding murine protein, designated dectin-2, was isolated from dendritic cells and described. Human and murine TANGO 405 proteins exhibit amino acid sequence homology with murine dectin-2. As indicated in the alignment in Figure 6M (made using the ALIGN software; paml20.mat scoring matrix; gap penalties -12/-4), human TANGO 405 exhibits about 89.0% sequence identity with murine dectin-2. As indicated in the alignment in Figure 6L (made using the ALIGN software; paml20.mat scoring matrix; gap penalties -12/-4), murine TANGO 405 exhibits about 70.3% sequence identity with murine dectin-2.

Another embodiment of a murine TANGO 405 cDNA is shown in Figures 6N to 6P (the cDNA having the sequence SEQ ID NO: 124 and the ORF having the nucleotide sequence SEQ ID NO: 125). In this embodiment murine TANGO 405 includes a translational frame shift, and the amino acid sequence (SEQ ID NO: 126) of murine TANGO 405 is identical to the amino acid sequence reported for murine dectin-2. These data further confirm that human TANGO 405 is the human ortholog of murine dectin-2. The signal peptide prediction program SIGNALP (Nielsen et al. (1997) Protein

Engineering 10:1-6) predicted that human TANGO 405 protein includes an approximately 48 amino acid signal peptide (amino acid residues 1 to about 48 of SEQ ID NO: 103; SEQ ID NO: 104) preceding the mature TANGO 405 protein (amino acid residues 49 to 209 of SEQ ID NO: 103; SEQ ID NO: 105). It is recognized that both human and murine TANGO 405 can, at least transiently, exist in an integral membrane form, at least until cleavage of the corresponding signal sequence (i.e., either during or following translation of the complete TANGO 405 protein).

Figure 6D depicts a hydrophilicity plot of human TANGO 405 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 48 of SEQ ID NO: 103 is the signal sequence of human TANGO 405 (SEQ ID NO: 104). 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 405 protein from about amino acid residue 90 to about amino acid residue 105 appears to be located at or near the surface of the protein, while the region from about amino acid residue 110 to about amino acid residue 120 appears not to be located at or near the surface. The predicted molecular weight of human TANGO 405 protein without modification and prior to cleavage of the signal sequence is about 24.0 kilodaltons. The predicted molecular weight of the mature human TANGO 405 protein without modification and after cleavage of the signal sequence is about 18.6 kilodaltons.

The full length of the cDNA encoding murine TANGO 405 protein (Figure 6; SEQ ID NO: 111) is 821 nucleotide residues, although this cDNA sequence is incomplete. The ORF of this cDNA, nucleotide residues 174 to 707 of SEQ ID NO: 111 (i.e. SEQ ID NO: 112), encodes a protein comprising at least 178 amino acid residues (Figure 6; SEQ ID NO: 113).

The signal peptide prediction program SIGNALP (Nielsen et al. (1997) Protein Engineering 10:1-6) predicted that murine TANGO 405 protein includes an approximately 42 amino acid signal peptide (amino acid residues 1 to about 42 of SEQ ID NO: 113; SEQ ID NO: 114) preceding the mature TANGO 405 protein (amino acid residues 43 to 178 of SEQ ID NO: 113; SEQ ID NO: 115). Murine TANGO 405 protein is a secreted protein.

Figure 6G depicts a hydrophilicity plot of murine TANGO 405 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 42 of SEQ ID NO: 113 is the signal sequence of murine TANGO 405 (SEQ ID NO: 114). 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 murine TANGO 405 protein from about amino acid residue 95 to about amino acid residue 110 appears to be located at or near the surface of the protein, while the region from about amino acid residue 110 to about amino acid residue 120 appears not to be located at or near the surface

The predicted molecular weight of murine TANGO 405 protein without modification and prior to cleavage of the signal sequence is about 20.0 kilodaltons. The predicted molecular weight of the mature murine TANGO 405 protein without modification and after cleavage of the signal sequence is about 25.3 kilodaltons. Human and murine TANGO 405 proteins exhibit considerable sequence similarity, as indicated herein in Figure 6H. Figure 6H depicts an alignment of human and murine TANGO 405 amino acid sequences (SEQ ID NOs: 103 and 113, respectively). In this alignment (made using the ALIGN software {Myers and Miller (1989) CABIOS, ver. 2.0}; paml20.mat scoring matrix; gap penalties -12/-4), the proteins are 51.7% identical in the overlapping region (i.e. amino acid residues 1-209 of SEQ ID NO: 103 and amino acid residues 1-178 of SEQ ID NO: 113). The human and murine ORFs encoding TANGO 405 are 74.5% identical in the 541 nucleotide residue overlapping region, as assessed using the same software and parameters and as indicated in Figures 61 through 6K. The nucleotide sequences encoding human and murine TANGO 405 (i.e. SEQ ID NOs: 101 and 111) are about 71.2% identical in the 838 nucleotide residue overlapping region, as assessed using the L ALIGN software (Myers and Miller (1989) CABIOS, ver. 2.0; paml20.mat scoring matrix; gap penalties -12/-4).

Biological function of TANGO 405 proteins, nucleic acids, and modulators thereof TANGO 405 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 405 occurs in a human mixed lymphocyte reaction cDNA library and in a murine long-term bone marrow cDNA library, it is evident that TANGO 405 protein is involved in one or more biological processes which occur in these tissues (i.e., in blood-related tissues, such as tissues containing lymphocytes). In particular, TANGO 405 is involved in modulating one or more of growth, proliferation, survival, differentiation, activity, moφhology, and movement/migration of cells of these tissues. TANGO 405 is involved in modulating the structure of extracellular matrix which contacts or is in fluid communication with cells of these tissues. Thus, TANGO 405 has a role in disorders which affect these cells and one or more of their growth, proliferation, survival, differentiation, activity, moφhology, and movement/migration, as well as the biological function of tissues comprising one or more of these types of cells.

Presence of a C-type lectin domain in TANGO 405 is an indication that this protein is capable of specifically recognizing particular surfaces, such as the surface of cells of a particular type. Further supportive of this observation is the fact that human TANGO 405 protein exhibits significant sequence homology with murine dectin-2 protein. Murine dectin-2 has been shown to be expressed by murine dendritic cells, and has also been shown to be involved in activation of naive T cells. Murine dectin-2 can also be involved in inflammatory and non-T cell-mediated immune responses. Thus, human and murine TANGO 405 are also involved in activating or inhibiting one or more types of lymphocytes, thereby modulating T cell-mediated immune responses, non-T cell-mediated immune responses, inflammatory responses, and other components of the immune response in mammals. It is recognized that the amino acid sequence differences among murine dectin-2, human TANGO 405, and murine TANGO 405 can lead to different lymphocyte-activating capacities for these three proteins. Human and murine TANGO 405 proteins are involved both in normal activation of lymphocytes (e.g., in response to the presence of a pathogen in a tissue) and in aberrant activation of lymphocytes (e.g., as in auto-immune and immune inflammatory disorders (e.g., asthma), in disorders characterized by an insufficient immune response, and in disorders characterized by un-controlled proliferation of lymphocytes). TANGO 405 proteins are thus involved in a variety of disorders relating to aberrant lymphocyte activation or proliferation. Exemplary disorders include leukemias (e.g., ALL, CML, CLL, and myelodysplastic syndrome), lymphomas (e.g., Hodgkin's disease, non-Hodgkin's lymphoma, Burkitt's lymphoma, and mycosis fungoides), plasma cell dyscrasias, auto-immune disorders such as multiple sclerosis, bacterial and viral infections (e.g., acquired immune deficiency syndrome), leukopenias, eosinophilic disorders such as idiopathic hypereosinophilic syndrome, and the like. TANGO 405 proteins, nucleic acids encoding them, and agents that modulate activity or expression of either of these can be used to prognosticate, diagnose, treat, and inhibit one or more of these disorders.

Tables Al and Bl summarize sequence data corresponding to the human nucleic acids and proteins herein designated TANGO 210, TANGO 364, TANGO 366, INTERCEPT 394, INTERCEPT 400, and TANGO 405. Tables A2 and B2 summarize sequence data corresponding to the murine nucleic acids and proteins herein designated TANGO 210 and INTERCEPT 400.

Table Al Human Nucleic Acids and Proteins

Table A2 Murine Nucleic Acids and Proteins

Table A3 Rat Nucleic Acids and Protein

Table Bl

Human Proteins

Note: It is recognized that the carboxyl terminal boundary of the signal sequence can be ± 1 or 2 residues

Table B2

Murine Proteins

Protein Desig. Signal Mature Protein Extracellular Transmembrane Cytoplasmic Domain(s) Sequence¹ Domain(s) Domain(s)

SEQ ID NOs

TANGO 210 1-24 14 25-518 15 N/A N/A N/A

INTERCEPT N/A N/A 61-71 86 44-60 85 1-43 84

I 400 125-140 90 72-90 87 91-100 88 O

I 101-124 89 161-174 95

141-160 94

TANGO 405 1-42 114 43-178 115 43-178 115

Amino Acid Residues

Note: 'It is recognized that the carboxyl terminal boundary of the signal sequence can be ± 1 or 2 residues

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, but preferably is double-stranded DNA.

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 kilobases, 4 kilobases, 3 kilobases, 2 kilobases, 1 kilobases, 0.5 kilobases, 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, 31, 32, 41, 42, 61, 62, 81, 82, 91, 92, 101, 102, 121, and 122, 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 a nucleic acid having a nucleotide sequence of any of SEQ ID NOs: 1, 2, 11, 12, 21, 22, 31, 32, 41, 42, 61, 62, 81, 82, 91, 92, 101, 102, 121, and 122 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 preferred 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, 31, 32, 41, 42, 61, 62, 81, 82, 91, 92, 101, 102, 121, and 122, or a portion thereof. A nucleic acid molecule which is complementary to a given nucleotide sequence is one which is sufficiently complementary to the given nucleotide sequence that it can hybridize to the given nucleotide sequence thereby forming a stable duplex.

Moreover, a nucleic acid molecule of the invention can comprise only a portion of a nucleic acid sequence encoding a full length polypeptide of the invention for example, a fragment which can be used as a probe or primer or a fragment encoding a biologically active portion of a polypeptide of the invention. The nucleotide sequence determined from the cloning one gene allows for the generation of probes and primers designed for use in 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 having a nucleotide sequence such that it hybridizes under stringent conditions with a polynucleotide region having a sequence which comprises 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 nucleotide residues of the sense or anti-sense sequence of any of SEQ ID NOs: 1, 2, 11, 12, 21, 22, 31, 32, 41, 42, 61, 62, 81, 82, 91, 92, 101, 102, 121, and 122, or of a naturally occurring mutant of any of SEQ ID NOs: 1, 2, 11, 12, 21, 22, 31, 32, 41, 42, 61, 62, 81, 82, 91, 92, 101, 102, 121, and 122.

Probes based on the sequence of a nucleic acid molecule of the invention can be used to detect transcripts or genomic sequences encoding the same protein molecule encoded by a selected nucleic acid molecule. The probe comprises a label group attached thereto, e.g., a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. Such probes can be used as part of a diagnostic test kit for identifying cells or tissues which mis-express the protein, such as by measuring levels of a nucleic acid molecule encoding the protein 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 NO: 2, 10, 34, 39, 47, 55, and 60, 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, 31, 32, 41, 42, 61, 62, 81, 82, 91, 92, 101, 102, 121, and 122 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, 32, 42, 62, 82, 102, and 112.

In addition to the nucleotide sequences of any of SEQ ID NOs: 2, 12, 22, 32, 42, 62, 82, 102, and 112, it will be appreciated by those skilled in the art that DNA sequence polymoφhisms that lead to changes in the amino acid sequence can exist within a population (e.g., the human population). Such genetic polymoφhisms 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. 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, chromosomal mapping data have been used to locate the gene encoding human INTERCEPT 394 at chromosome 2, near marker D2S126 (228.8 centimorgans). Human INTERCEPT 394 allelic variants can include INTERCEPT 394 nucleotide sequence polymoφhisms (e.g., nucleotide sequences that vary from SEQ ID NO: 41) that map to this chromosomal region. Chromosomal mapping data have also been used to locate the gene encoding human INTERCEPT 400 gene at chromosome 4, between markers D4S1616 and D4S1611 (115.8-119.6 centimorgans). A form of iris hypoplasia associated with early onset glaucoma has been linked with this chromosomal region. Human INTERCEPT 400 allelic variants can include INTERCEPT 400 nucleotide sequence polymoφhisms (e.g., nucleotide sequences that vary from SEQ ID NO: 61) that map to this chromosomal region. The gene encoding human TANGO 405 has been mapped at chromosome 8, between markers D8S269 and D8S1799 (125.8-132.4 centimorgans). Human TANGO 405 allelic variants can include TANGO 405 nucleotide sequence polymoφhisms (e.g., nucleotide sequences that vary from SEQ ID NO: 101) that map to this chromosomal region.

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 polymoφhisms 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 specific proteins described herein are intended to be 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 homology with human nucleic acid molecules using the specific 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 isolated based on its hybridization to a nucleic acid molecule encoding all or part of the membrane-bound form. Likewise, a cDNA encoding a membrane-bound form can be isolated based on its hybridization to a nucleic acid molecule encoding all or part of the soluble form.

Accordingly, 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, 31, 32, 41, 42, 61, 62, 81, 82, 91, 92, 101, 102, 121, and 122, 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 to 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 preferred, non-limiting example of stringent hybridization conditions are hybridization in 6 x sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2 x SSC, 0.1% SDS at 50-65°C. Preferably, an isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to the sequence of any of SEQ ID NOs: 1, 2, 11, 12, 21, 22, 31, 32, 41, 42, 61, 62, 81, 82, 91, 92, 101, 102, 121, and 122, 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: 3-5, 13-15, 23-28, 33-38, 43-54, 63-76, 83-92, 93, 103-105, and 123, 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: 3-5, 13-15, 23-28, 33-38, 43-54, 63-76, 83-92, 93, and 103- 105.

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, 31, 32, 41, 42, 61, 62, 81, 82, 91, 92, 101, 102, 121, and 122, 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 a preferred embodiment, a mutant polypeptide that is a variant of a polypeptide of the invention can be assayed for: (1) the ability to form proteimprotein interactions with one or more proteins (e.g., a protein involved in a signaling pathway); (2) the ability to bind a ligand of the polypeptide of the invention (e.g., another protein identified herein); (3) the ability to bind to an intracellular target protein (e.g., 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 (e.g., ability to modulate cellular proliferation, cellular migration, chemotaxis, or cellular differentiation).

The present invention encompasses antisense nucleic acid molecules, i.e., molecules which are complementary to a sense nucleic acid encoding a polypeptide of the invention, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence. Accordingly, an antisense nucleic acid can hydrogen bond to a sense nucleic acid. The antisense nucleic acid can be complementary to an entire coding strand, or to only a portion thereof, e.g., all or part of the protein coding region (or open reading frame). An antisense nucleic acid molecule can be antisense to all or part of a non-coding region of the coding strand of a nucleotide sequence encoding a polypeptide of the invention. The non-coding regions ("5' and 3' untranslated 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 to cellular mRNA and/or genomic DNA encoding a selected polypeptide of the invention to thereby inhibit expression, e.g., by inhibiting transcription and/or translation. The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule which binds to DNA duplexes, through specific interactions in the major groove of the double helix. An example of a route of administration of antisense nucleic acid molecules of the invention includes direct injection at a tissue site. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For example, for systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies which bind to cell surface receptors or antigens. The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of the antisense molecules, vector constructs 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 alpha-anomeric nucleic acid molecule. An alpha-anomeric nucleic acid molecule forms specific double- stranded hybrids with complementary RNA in which, contrary to the usual beta-units, the strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids Res. 15:6625-6641). The antisense nucleic acid molecule can also comprise a 2'-o-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 with ribonuclease activity which are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region. Thus, ribozymes (e.g., hammerhead ribozymes 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 active site is complementary to the nucleotide sequence to be cleaved in a Cech et al. U.S. Patent No. 4,987,071 ; and Cech et al. U.S. Patent No. 5,116,742. Alternatively, an mRNA encoding a polypeptide of the invention can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel and Szostak (1993) Science 261 :1411-1418. The invention also encompasses nucleic acid molecules which form triple helical structures. For example, expression of a polypeptide of the invention can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the gene encoding the polypeptide (e.g., the promoter and/or enhancer) to form triple helical structures that prevent transcription of the gene 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.

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 for specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described 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 transcription or translation arrest or inhibiting replication. PNAs can also be used, e.g., in the analysis of single base pair mutations in a gene by, e.g., PNA directed PCR clamping; as artificial restriction enzymes when used in combination with other enzymes, e.g., SI nucleases (Hyrup (1996), supra; or as probes or primers for DNA sequence and hybridization (Hyrup (1996), supra; Perry-O'Keefe et al. (1996) Proc. Natl. Acad. Sci. USA 93: 14670-675).

In another embodiment, PNAs can be modified, e.g., to enhance their stability or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known 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 would provide high binding affinity and specificity. PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (Hyrup (1996), supra). The synthesis of PNA-DNA chimeras can be performed as described 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 to another molecule, e.g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc. II. Isolated Proteins and Antibodies

One aspect of the invention pertains to isolated proteins, and biologically active portions thereof, as well as polypeptide fragments suitable for use as immunogens to raise antibodies directed against a polypeptide of the invention. In one embodiment, the native polypeptide can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques. In another embodiment, polypeptides of the invention are produced by recombinant DNA techniques. Alternative to recombinant expression, a polypeptide of the invention can be synthesized chemically using standard peptide synthesis techniques. An "isolated" or "purified" protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the protein is derived, or substantially free of chemical precursors or other chemicals when chemically synthesized. The language "substantially free of cellular material" includes preparations of protein 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 polypeptides comprising amino acid sequences 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: 3-5, 13-15, 23-28, 33-38, 43-54, 63-76, 83-92, 93, and 103-105), 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 br more amino acids in length. Moreover, other biologically active portions, in which other regions of the protein are deleted, can be prepared by recombinant techniques and evaluated for one or more of the functional activities of the native form of a polypeptide of the invention.

Preferred polypeptides have the amino acid sequence of any of SEQ ID NOs: 3- 5, 13-15, 23-28, 33-38, 43-54, 63-76, 83-92, 93, and 103-105. 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: 3-5, 13-15, 23-28, 33-38, 43-54, 63-76, 83-92, 93, and 103- 105, and retain the functional activity of the protein of the corresponding naturally-occurring protein yet differ in amino acid sequence due to natural allelic variation or mutagenesis. To determine the percent identity of two amino acid sequences or of two nucleic acids, the sequences are aligned for optimal comparison puφoses (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 incoφorated 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 puφoses, 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 algorithm of Myers and Miller, (1988) CABIOS 4:X 1-17. Such an algorithm is incoφorated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, only exact matches are counted. The 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 to 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 to each other. The heterologous polypeptide can be fused to 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 to the carboxyl terminus of GST sequences. Such fusion proteins can facilitate the purification of a recombinant polypeptide of the invention. In another embodiment, the fusion protein contains a heterologous signal sequence at its 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 to sequences derived from a member of the immunoglobulin protein family. The immunoglobulin fusion proteins of the invention can be incoφorated 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 carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments 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, 13, 23, 33, 43, 63, 83, and 103) can be used to facilitate secretion and isolation of the secreted protein or other proteins 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 to a protein of interest, such as a protein which is ordinarily not secreted or is otherwise difficult to isolate. The signal sequence directs secretion of the protein, such as from a eukaryotic host into which the expression vector is transformed, and the signal sequence is subsequently or concurrently cleaved. The protein can then be readily purified from the extracellular medium by art recognized methods. Alternatively, the signal sequence can be linked to the protein of interest using a sequence which facilitates purification, such as with a GST domain.

In another embodiment, the signal sequences of the present invention can be used to identify regulatory sequences, e.g., promoters, enhancers, repressors. Since signal sequences are the most amino-terminal sequences of a peptide, it is expected that the nucleic acids which flank the signal sequence on its amino-terminal side are 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 to a downstream or upstream member of a cellular signaling cascade which includes the protein of interest. Thus, specific biological effects can be elicited by treatment with a variant of limited function. Treatment of a subject with a variant having a subset of the biological activities of the naturally occurring form of the protein can have fewer side effects 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 (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 is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display). There are a variety of methods which can be used to produce libraries of potential variants of the polypeptides of the invention from a degenerate oligonucleotide sequence. Methods for synthesizing degenerate oligonucleotides are known 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. X X :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: 3-5, 13-15, 23-28, 33-38, 43-54, 63-76, 83-92, 93, 103-105, and 123, and encompasses an epitope of the protein such that an antibody raised against the peptide forms a specific immune complex with the protein.

Preferred epitopes encompassed by the antigenic peptide are regions that are located on the surface of the protein, e.g., hydrophilic regions. Figures IE, 1 J, 2F, 3E, 4G, 5D, 5G, 6D, and 6G 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 (e.g., an epitope of a polypeptide of the invention). A molecule which specifically binds to a given polypeptide of the invention is a molecule which binds the polypeptide, but does not substantially bind other molecules 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. A substantially purified antibody composition, in this context, means an antibody sample that 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 No. WO 92/18619; PCT Publication No. WO 91/17271; PCT Publication No. WO 92/20791; PCT Publication No. WO 92/15679; PCT Publication No. WO 93/01288; PCT Publication No. WO 92/01047; PCT Publication No. WO 92/09690; PCT Publication No. WO 90/02809; Fuchs et al. (1991) Bio/Technology 9: 1370-1372; Hay et al. (1992) Hum. Antibod. Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffiths et al. (1993) EMBOJ. 12:725-734.

Additionally, recombinant antibodies, such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the invention. 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. (Fremont, 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 ¹²⁵I, ¹³¹I, ³⁵S or ³H.

Further, an antibody substance can be conjugated with a therapeutic moiety such as a cytotoxin, a therapeutic agent, or a radioactive metal ion. Cytotoxins and cytotoxic agents include any agent that is detrimental to cells. Examples 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, puromycin, and analogs or homologs of these compounds. Therapeutic agents include, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, and decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine {BSNU}, lomustine {CCNU}, cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin {formerly daunomycin} and doxorubicin), antibiotics (e.g., dactinomycin {formerly actinomycin}, bleomycin, mithramycin, and anthramycin {AMC}), and anti-mitotic agents (e.g., vincristine and vinblastine).

The conjugates of the invention can be used to modify a biological response; the drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety can be a protein or polypeptide which exhibits 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 (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6), granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), and other growth factors.

Techniques for conjugating a therapeutic moiety with an antibody substance are well known (see, e.g., Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies and Cancer Therapy. Reisfeld et al., eds., pp. 243-256, Alan R. Liss, Inc., 1985; Hellstrom et al., "Antibodies For Drug Delivery", in Controlled Drug Deliverv. 2nd Ed., Robinson et al., eds., pp. 623-653, Marcel Dekker, Inc., 1987; Thoφe, "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, 1985; "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., pp. 303-316, Academic Press, 1985; and Thoφe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev. 62:119-58, 1982). Alternatively, an antibody can be conjugated with 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: 3-5, 13-15, 23-28, 33-38, 43-54, 63-76, 83-92, 93, 103-105, and 123; (ii) the amino acid sequence encoded by a cDNA of a clone deposited as any of

ATCC^® Accession Nos. PTA-424, PTA-425, and PTA-438;

(iii) at least 15 amino acid residues of the amino acid sequence of any of SEQ ID NOs: 3-5, 13-15, 23-28, 33-38, 43-54, 63-76, 83-92, 93, 103-105, and 123;

(iv) an amino acid sequence which is at least 95% identical to the amino acid sequence of any of SEQ ID NOs: 3-5, 13-15, 23-28, 33-38, 43-54, 63-76, 83-92, 93, 103-105, and 123, 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) an amino acid sequence which is encoded by a nucleic acid molecule which hybridizes with a nucleic acid having a sequence selected from the group consisting of: SEQ ID NOs: 1, 2, 11, 12, 21, 22, 31, 32, 41, 42, 61, 62, 81, 82, 91, 92, 101, 102, 121, and 122 under conditions of hybridization of 6 x SSC (standard saline citrate) at 45°C and washing in 0.2 x 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: 3-5, 13-15, 23-28, 33-38, 43-54, 63-76, 83-92, 93, 103-105, and 123; (ii) the amino acid sequence encoded by a cDNA of a clone deposited as any of ATCC^® Accession Nos. PTA-424, PTA-425, and PTA-438; (iii) at least 15 amino acid residues of the amino acid sequence of any of SEQ ID NOs: 3-5, 13-15, 23-28, 33-38, 43-54, 63-76, 83-92, 93, 103-105, and 123;

(v) an amino acid sequence which is encoded by a nucleic acid molecule which hybridizes with a nucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 1, 2, 11, 12, 21, 22, 31, 32, 41, 42, 61, 62, 81, 82, 91, 92, 101, 102, 121, and 122 under conditions of hybridization of 6 x SSC (standard saline citrate) at 45°C and washing in 0.2 x 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:

ATCC^® Accession Nos. PTA-424, PTA-425, and PTA-438;

(iv) an amino acid sequence which is at least 95% identical to the amino acid sequence of any of SEQ ID NOs: 3-5, 13-15, 23-28, 33-38, 43-54, 63-76, 83-92, 93, 103-105, and 123, 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) an amino acid sequence which is encoded by a nucleic acid molecule which hybridizes with a nucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 1, 2, 11, 12, 21, 22, 31, 32, 41, 42, 61, 62, 81, 82, 91, 92, 101, 102, 121, and 122 under conditions of hybridization of 6 x SSC (standard saline citrate) at 45°C and washing in 0.2 x 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 210, TANGO 364, TANGO 366, INTERCEPT 394, INTERCEPT 400, and TANGO 405. Preferably, the extracellular domain with which the antibody substance binds has an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 26, 36, 48, 52, 66, 70, 74, and 105.

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).

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. Still another aspect of the invention is a method of making an antibody that specifically recognizes one of TANGO 210, TANGO 364, TANGO 366, INTERCEPT 394, INTERCEPT 400, and TANGO 405. 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: 3-5, 13-15, 23-28, 33-38, 43-54, 63-76, 83-92, 93, 103-105, and 123; (ii) the amino acid sequence encoded by a cDNA of a clone deposited as any of ATCC^® Accession Nos. PTA-424, PTA-425, and PTA-438; (iii) at least 15 amino acid residues of the amino acid sequence of any of SEQ ID NOs:

3-5, 13-15, 23-28, 33-38, 43-54, 63-76, 83-92, 93, 103-105, and 123;

(v) an amino acid sequence which is encoded by a nucleic acid molecule which hybridizes with a nucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 1, 2, 11, 12, 21, 22, 31, 32, 41, 42, 61, 62, 81, 82, 91, 92, 101, 102, 121, and 122 under conditions of hybridization of 6 x SSC (standard saline citrate) at 45°C and washing in 0.2 χ 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.

- I l l III. Recombinant Expression Vectors and Host Cells

Another aspect of the invention pertains to vectors, preferably expression vectors, containing a nucleic acid encoding a polypeptide of the invention (or a portion thereof). As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a

"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, expression vectors, are capable of directing the expression of genes to 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 to 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 to the regulatory sequence(s) in a manner which allows for 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, the level of expression of protein desired, and the like. 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 puφoses: 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 RNA polymerase transcription from a hybrid tφ-lac fusion promoter. Target gene expression from the pET 1 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 HMS174(DE3) from a resident lambda 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 with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 119-128). Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (Wada et al. (1992) Nucleic Acids Res. 20:2111-2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.

In another embodiment, the expression vector is a yeast expression vector. Examples of vectors for expression in yeast S. cerevisiae include pYepSecl (Baldari et al. (1987) EMBO J 6:229-234), pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al. (1987) Gene 54:113-123), pYES2 (Invitrogen Coφoration, San Diego, CA), and pPicZ (Invitrogen Coφ, 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) EMBO J. 6:187-195). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook et al., supra.

In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al. (1987) Genes Dev.

X :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) EMBOJ. 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 to a regulatory sequence in a manner which allows for expression (by transcription of the DNA molecule) of an RNA molecule which is antisense to the mRNA encoding a polypeptide of the invention. Regulatory sequences operably linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the 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 chosen 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 may 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, or 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 may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., for resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Preferred selectable markers include those which confer resistance to drugs, such as G418, hygromycin and methotrexate. Cells stably transfected with the introduced nucleic acid can be identified by drag selection (e.g., cells that have incoφorated the selectable marker gene will survive, while the other cells die).

In another embodiment, the expression characteristics of an endogenous nucleic acid within a cell, cell line, or microorganism (e.g., a TANGO 210, TANGO 364, TANGO 366, INTERCEPT 394, INTERCEPT 400, or TANGO 405 nucleic acid, as described herein) can be modified by inserting a heterologous DNA regulatory element (i.e., one that is heterologous with respect to the endogenous gene) into the genome of the cell, stable cell line, or cloned microorganism. The inserted regulatory element can be operatively linked with the endogenous gene (e.g., TANGO 210, TANGO 364, TANGO 366, INTERCEPT 394, INTERCEPT 400, or TANGO 405) and thereby control, modulate, or activate the endogenous gene. For example, an endogenous TANGO 210, TANGO 364, TANGO 366, INTERCEPT 394, INTERCEPT 400, or TANGO 405 gene which is normally "transcriptionally silent" (i.e., a TANGO 210, TANGO 364, TANGO 366, INTERCEPT 394, INTERCEPT 400, or TANGO 405 gene which is normally not expressed, or is normally expressed only at only a very low level) can be activated by inserting a regulatory element which is capable of promoting expression of the gene in the cell, cell line, or microorganism. Alternatively, a transcriptionally silent, endogenous TANGO 210, TANGO 364, TANGO 366, INTERCEPT 394, INTERCEPT 400, or TANGO 405 gene can be activated by inserting a promiscuous regulatory element that works across cell types. A heterologous regulatory element can be inserted into a stable cell line or cloned microorganism such that it is operatively linked with and activates expression of an endogenous TANGO 210, TANGO 364, TANGO 366, INTERCEPT 394, INTERCEPT 400, or TANGO 405 gene, using techniques, such as targeted homologous recombination, which are well known to those of skill in the art (described e.g., in Chappel, U.S. Patent No. 5,272,071; PCT Publication No. WO 91/06667, published May 16, 1991).

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 also 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 the 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 nucleic acid encoding a polypeptide of the invention (or a homologue thereof) into the male pronuclei of a fertilized oocyte, e.g., by microinjection, 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 to the transgene to direct expression of the 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 can be used to produce 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 then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying the transgene can further be bred to other transgenic animals carrying other transgenes.

To create 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 a preferred embodiment, the vector is designed such that, upon homologous recombination, the endogenous gene is functionally disrupted (i.e., no longer encodes a functional protein; also referred to as a "knock out" vector). Alternatively, the vector can be designed such that, upon homologous recombination, the endogenous gene is mutated or otherwise altered but still encodes functional protein (e.g., the upstream regulatory region can 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 also 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 incoφorated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier. As used herein the language "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absoφtion 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 incoφorated 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 other than a nucleic acid, polypeptide, or antibody of the invention. 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. Exemplary 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). Exemplary 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). 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 drug 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 with 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). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid 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 antibacterial and antifungal 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 absoφtion of the injectable compositions can be brought about by including in the composition an agent which delays absoφtion, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incoφorating 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 incoφorating the active compound into a sterile vehicle which contains a basic dispersion medium, and then incoφorating the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred 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 puφose of oral therapeutic administration, the active compound can be incoφorated 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, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches, and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as 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 Coφoration and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes which can be targeted to bind with virus-infected cells using a monoclonal antibody which binds specifically with a viral antigen) 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.

For antibodies, the preferred dosage is 0.1 mg/kg to 100 mg/kg of body weight (generally 10 mg/kg to 20 mg/kg). If the antibody is to act in the brain, a dosage of 50 mg/kg to 100 mg/kg is usually appropriate. Generally, partially human antibodies and fully human antibodies have a longer half-life 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 to 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 puφoses identified herein in portions of the disclosure relating to individual types of protein of the invention (e.g., TANGO 210 proteins, TANGO 364 proteins, TANGO 366 proteins, INTERCEPT 394 proteins,

INTERCEPT 400 proteins, and TANGO 405 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 drugs or compounds which modulate activity or expression of a polypeptide of the invention as well as to treat disorders characterized by insufficient or excessive production of a protein of the invention or production of a form of a protein of the invention which has decreased or aberrant activity compared to the wild type protein. In addition, the antibodies of the invention can be used to detect and isolate a protein of the and modulate activity of a protein of the invention. This invention further pertains to novel agents identified by the above- described screening assays and uses thereof for treatments as described herein. A. Screening Assays

The invention provides a method (also refeπed to herein as a "screening assay") for identifying modulators, i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) which bind to polypeptide of the invention or have a stimulatory or inhibitory effect on, for example, expression or activity of a polypeptide of the invention.

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

Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad. Sci. USA 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al. (1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061; and Gallop et al. (1994) J. Med. Chem. 37:1233.

Libraries of compounds can be presented in solution (e.g., Houghten (1992) Bio/Techniques 13:412-421), or on beads (Lam (1991) Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (U.S. Patent No. 5,223,409), spores (Patent 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. Mol. Biol. 222:301-310). In one embodiment, an assay is a cell-based assay in which a cell which expresses a membrane-bound form of a polypeptide of the invention, or a biologically active portion thereof, on the cell surface is contacted with a test compound and the ability of the test compound to bind to the polypeptide determined. The cell, for example, can be a yeast cell or a cell of mammalian origin. Determining the ability of the test compound to bind to the polypeptide can be accomplished, for example, by coupling the test compound with a radioisotope or enzymatic label such that binding of the test compound to the polypeptide or biologically active portion thereof can be determined by detecting the labeled compound in a complex. For example, test compounds can be labeled with ¹²⁵1, ³⁵S, ¹⁴C, or ³H, either directly or indirectly, and the radioisotope detected by direct counting of 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 a preferred embodiment, the assay comprises contacting a cell which expresses a membrane-bound form of a polypeptide of the invention, or a biologically active portion thereof, on the cell surface with a known compound which binds the polypeptide to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with the polypeptide, wherein determining the ability of the test compound to interact with the polypeptide comprises determining the ability of the test compound to preferentially bind to the polypeptide or a biologically active portion thereof as compared to the known compound.

In another embodiment, 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 to 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 to or interact with a target molecule can be accomplished by one of the methods described above for determining direct binding. As used herein, a "target molecule" is a molecule with which a selected polypeptide (e.g., a polypeptide of the invention binds or interacts with 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 the association of downstream signaling molecules with a polypeptide of the invention. Determining the ability of a polypeptide of the invention to bind to or interact with a target molecule can be accomplished by determining the activity of the target molecule. For example, the activity of the target molecule can be determined by detecting induction of a cellular second messenger of the target (e.g., an mRNA, intracellular Ca²⁺, diacylglycerol, IP3, and the like), detecting catalytic/enzymatic activity of the target on an appropriate substrate, detecting the induction of a reporter gene (e.g., a regulatory element that is responsive to a polypeptide of the invention operably linked to a nucleic acid encoding a detectable marker, e.g., luciferase), or detecting a cellular response, for example, cellular differentiation, or cell proliferation.

In yet another embodiment, an assay of the present invention is a cell-free assay comprising contacting a polypeptide of the invention or biologically active portion thereof with a test compound and determining the ability of the test compound to bind to the polypeptide or biologically active portion thereof. Binding of the test compound to the polypeptide can be determined either directly or indirectly as described above. In a prefeπed embodiment, the assay includes contacting the polypeptide of the invention or biologically active portion thereof with a known compound which binds the polypeptide to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with the polypeptide, wherein determining the ability of the test compound to interact with the polypeptide comprises determining the ability of the test compound to preferentially bind to the polypeptide or biologically active portion thereof as compared to the known compound.

In another embodiment, an assay is a cell-free assay comprising contacting a polypeptide of the invention or biologically active portion thereof with a test compound and determining the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of the polypeptide or biologically active portion thereof. Determining the ability of the test compound to modulate the activity of the polypeptide can be accomplished, for example, by determining the ability of the polypeptide to bind to a target molecule by one of the methods described above for determining direct binding. In an alternative embodiment, determining the ability of the test compound to modulate the activity of the polypeptide can be accomplished by determining the ability of the polypeptide of the invention to further modulate the target molecule. For example, the catalytic 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, wherein determining the ability of the test compound to interact with the polypeptide comprises determining the ability of the polypeptide to preferentially bind to or modulate the activity of a target molecule.

The cell-free assays of the present invention are amenable to use of both a soluble form or the membrane-bound form of a polypeptide of the invention. In the case of cell-free assays comprising the membrane-bound form of the polypeptide, it can be desirable to utilize a solubilizing agent such that the membrane-bound form of the polypeptide is maintained in solution. Examples of such solubilizing agents include non-ionic detergents such as n-octylglucoside, n-dodecylglucoside, n-octylmaltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton X-100, Triton X-114, Thesit, Isotridecypoly(ethylene glycol ether)n, 3-[(3-cholamidopropyl) dimethylamminio]-l -propane sulfonate (CHAPS), 3- [(3-cholamidopropyl) dimethylamminio]-2-hydroxy-l -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 to facilitate separation of complexed from non-complexed forms of one or both of the proteins, as well as to accommodate automation of the assay. Binding of a test compound to the polypeptide, or interaction of the polypeptide with a target molecule in the presence and absence of a candidate compound, can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and micro- centrifuge tubes. In one embodiment, a fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix. For example, glutathione-S- transferase fusion proteins or glutathione-S-transferase fusion proteins can be adsorbed onto glutathione SEPHAROSE™ beads (Sigma Chemical; St. Louis, MO) or glutathione derivatized microtiter plates, which are then combined with the test compound or the test compound and either the non-adsorbed target protein or A polypeptide of the invention, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components and complex formation is measured either directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of binding or activity of the polypeptide of the invention can be determined using standard techniques.

Other techniques for immobilizing proteins on matrices can also be used in the screening assays of the invention. For example, either the polypeptide of the invention or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin. Biotinylated polypeptide of the invention or target molecules can be prepared from biotin- NHS (N-hydroxy-succinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals; Rockford, IL), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). Alternatively, antibodies reactive with the polypeptide of the invention or target molecules but which do not interfere with binding of the polypeptide of the invention to its target molecule can be derivatized to the wells of the plate, and unbound target or polypeptide of the invention 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 the expression of the selected mRNA or protein (i.e., the mRNA or protein coπesponding 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 to 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, when 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, the candidate compound is identified as a stimulator of the selected mRNA or protein expression. Alternatively, when expression of the selected mRNA or protein is less (i.e., statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of the selected mRNA or protein expression. The level of the selected mRNA or protein expression in the cells can be determined by methods described herein. In yet another aspect of the invention, a polypeptide of the inventions can be used as "bait proteins" 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 to or interact with the polypeptide of the invention and modulate activity of the polypeptide of the invention. Such binding proteins are also likely to be involved in the propagation of signals by the polypeptide of the inventions as, for example, upstream or downstream elements of a signaling pathway involving the polypeptide of the invention. This invention further pertains to novel agents identified by the above- described screening assays and uses thereof for treatments as described herein.

B. Detection Assays

Portions or fragments of the cDNA sequences identified herein (and the coπesponding 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 coπesponding genes on a chromosome. The mapping of the sequences to chromosomes is an important first step in coπelating these sequences with genes associated with disease.

Briefly, genes can be mapped to chromosomes by preparing PCR primers (preferably 15-25 base pairs 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, thus complicating the amplification process. These primers can then be used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene coπesponding to the gene sequences will yield an amplified fragment. For a review of this technique, see D'Eustachio et al. ((1983) Science 220:919-924).

PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular sequence to a particular chromosome. Three or more sequences can be assigned per day using a single thermal cycler. Using the nucleic acid sequences of the invention to design oligonucleotide primers, sub-localization can be achieved with panels of fragments from specific chromosomes. Other mapping strategies which can similarly be used to map a gene to its chromosome include 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 to chromosome specific cDNA libraries. Fluorescence in situ hybridization (FISH) of a DNA sequence to a metaphase chromosomal spread can further 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 that chromosome, or panels of reagents can be used for marking multiple sites and/or multiple chromosomes. Reagents coπesponding to non-coding regions of the genes actually are prefeπed for mapping puφoses. Coding sequences are more likely to be conserved within gene families, thus increasing the chance of cross hybridizations during chromosomal mapping.

Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be coπelated 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 through 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 unaffected 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 unaffected individuals, then the mutation is likely to be the causative agent of the particular disease. Comparison of affected and unaffected individuals generally involves first looking for structural alterations 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 polymoφhisms.

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.

In the instant case, the human gene for INTERCEPT 394 is located on chromosome 2 near marker D2S126 (228.8 centimorgans). A form of familial paroxysmal dyskinesia has been linked with this chromosomal location. The gene encoding human INTERCEPT 400 is located on chromosome 4, between markers D4S1616 and D4S1611 (115.8-119.6 centimorgans). A form of iris hypoplasia associated with early onset glaucoma has been linked with this chromosomal region.

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 polymoφhism (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 cuπent limitations of "Dog Tags" which can be lost, switched, or stolen, making positive identification difficult. The sequences of the present invention are useful as additional DNA markers for RFLP (described 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. Thus, the nucleic acid sequences described herein can be used to prepare two PCR primers from the 5' and 3' ends of the sequences. These primers can then be used to amplify an individual's DNA and subsequently sequence it.

Panels of coπesponding DNA sequences from individuals, prepared in this manner, can provide unique individual identifications, as each individual will have a unique set of such DNA sequences due to allelic differences. The sequences of the present invention can be used to obtain such identification sequences from individuals and from tissue. The nucleic acid sequences of the invention uniquely represent portions of the human genome. Allelic variation occurs to some degree 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 each 500 bases. Each of the sequences described herein can, to some degree, be used as a standard against which DNA from an individual can be compared for identification puφoses. Because greater numbers of polymoφhisms occur in the non-coding regions, fewer sequences are necessary to differentiate individuals. The non-coding sequences of any of SEQ ID NOs: 1, 11, 21, 31, 41, 61, 81, 101, and 111 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, 32, 42, 62, 82, 102, and 112 are used, a more appropriate number of primers for positive individual identification would be 500-2,000. If a panel of reagents from the nucleic acid sequences described herein is used to generate a unique identification database for an individual, those same reagents can later be used to identify tissue from that individual. Using the unique identification database, positive identification of the individual, living or dead, can be made from extremely small tissue samples.

3. Use of Partial Gene Sequences in Forensic Biology

DNA-based identification techniques can also 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 then be compared to a standard, thereby allowing identification of the origin of the biological sample.

The sequences of the present invention can be used to provide polynucleotide reagents, e.g., PCR primers, targeted to specific loci 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 base sequence information can be used for identification as an accurate alternative to patterns formed by restriction enzyme generated fragments. Sequences targeted to non-coding regions are particularly appropriate for this use as greater numbers of polymoφhisms occur in the 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 bases.

The nucleic acid sequences described herein can further be used to provide polynucleotide reagents, e.g., labeled or labelable probes which can be used 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 clinical trials are used for prognostic (predictive) piuposes to thereby treat an individual prophylactically.

Accordingly, one aspect of the present invention relates to diagnostic assays for determining expression of a polypeptide or nucleic acid of the invention and/or activity of a polypeptide of the invention (e.g., expression or activity of one of the TANGO 210, TANGO 364, TANGO 366, INTERCEPT 394, INTERCEPT 400, or TANGO 405 genes or proteins), 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 abeπant expression or activity of a polypeptide of the invention. The invention also provides for prognostic (or predictive) assays for determining whether an individual is at risk of developing a disorder associated with abeπant 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 puφose to thereby prophylactically treat an individual prior to the onset of a disorder characterized by or associated with abeπant expression or activity of a polypeptide of the invention. As an alternative to making determinations based on the absolute expression level of a selected gene, determinations can be based on normalized expression levels of the gene. A gene expression level is normalized by coπecting the absolute expression level of the gene (e.g., a TANGO 210, TANGO 364, TANGO 366, INTERCEPT 394, INTERCEPT 400, or TANGO 405 gene as described herein) by comparing its expression to expression of a gene for which expression is not believed to be co-regulated with the gene of interest, e.g., a housekeeping gene that is constitutively expressed. Suitable genes for normalization include housekeeping genes such as the actin gene. Such normalization allows comparison of the expression level in one sample, e.g., a patient sample, with the expression level in another sample, e.g., a sample obtained from a patient known not to be afflicted with a disease or condition, or between samples obtained from different sources.

Alternatively, the expression level can be assessed as a relative expression level. To assess a relative expression level for a gene (e.g., a TANGO 210, TANGO 364, TANGO 366, INTERCEPT 394, INTERCEPT 400, or TANGO 405 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 abeπant 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 abeπant expression of one of the TANGO 210, TANGO 364, TANGO 366, INTERCEPT 394, INTERCEPT 400, or TANGO 405 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 abeπant 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 (refeπed 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 (e.g., one or more of the TANGO 210, TANGO 364, TANGO 366, INTERCEPT 394, INTERCEPT 400, and TANGO 405 proteins) in clinical trials. These and other agents are described in further detail in the following sections.

1. Diagnostic Assays

An exemplary 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 such that the presence of a polypeptide or nucleic acid of the invention is detected in the biological sample. A prefeπed agent for detecting mRNA or genomic DNA encoding a polypeptide of the invention is a labeled nucleic acid probe capable of hybridizing to mRNA or genomic DNA encoding a polypeptide of the invention. The nucleic acid probe can be, for example, a full-length cDNA, such as the nucleic acid of any of SEQ ID NOs: 1, 11, 21, 31, 41, 61, 81, 101, and 111, 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 to 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.

A prefeπed agent for detecting a polypeptide of the invention is an antibody capable of binding to a polypeptide of the invention, preferably an antibody with a detectable label. Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab')₂) can be used. The term "labeled", with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with 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 hybridizations and in situ hybridizations. In vitro techniques for detection of a polypeptide of the invention include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofiuorescence. 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 whose presence and location in a subject can be detected by standard imaging techniques.

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

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

The invention also encompasses kits for detecting the presence of a polypeptide or nucleic acid of the invention in a biological sample (a test sample). Such kits can be used to determine if a subject is suffering from or is at increased risk of developing a disorder associated with abeπant 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 and means for determining the amount of the polypeptide or mRNA in the sample (e.g., an antibody which binds the polypeptide or an oligonucleotide probe which binds to DNA or mRNA encoding the polypeptide). Kits can also include instructions for observing that the tested subject is suffering from or is at risk of developing a disorder associated with abeπant 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 binds to a polypeptide of the invention; and, optionally, (2) a second, different antibody which binds to either the polypeptide or the first antibody and is conjugated to a detectable agent.

For oligonucleotide-based kits, the kit can comprise, for example: (1) an oligonucleotide, e.g., a detectably labeled oligonucleotide, which hybridizes to a nucleic acid sequence encoding a polypeptide of the invention or (2) a pair of primers useful for amplifying a nucleic acid molecule encoding a polypeptide of the invention. The kit can also comprise, e.g., 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 also contain a control sample or a series of control samples which can be assayed and compared to the test sample contained. Each component of the kit is usually enclosed within an individual container and all of the various containers are within a single package along with instructions for observing whether the tested subject is suffering from or is at risk of developing a disorder associated with abeπant expression of the polypeptide.

2. Prognostic Assays

The methods described herein can furthermore be utilized as diagnostic or prognostic assays to identify subjects having or at risk of developing a disease or disorder associated with abeπant expression or activity of a polypeptide of the invention. For example, the assays described herein, such as the preceding diagnostic assays or the following assays, can be utilized to identify a subject having or at risk of developing a disorder associated with abeπant 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). Alternatively, the prognostic assays can be utilized to identify a subject having or at risk for developing such a disease or disorder. Thus, the present invention provides a method 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 of the polypeptide or nucleic acid is diagnostic for a subject having or at risk of developing a disease or disorder associated with abeπant expression or activity of the polypeptide. As used herein, a "test sample" refers to a biological sample obtained from a subject of interest. For example, a test sample can be a biological fluid (e.g., serum), cell sample, or tissue.

Furthermore, the prognostic assays described herein can be used to determine whether a subject can be administered an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate) to treat a disease or disorder associated with abeπant expression or activity of a polypeptide of the invention. For example, such methods can be used to determine whether a subject can be effectively treated with a specific agent or class of agents (e.g., agents of a type which decrease activity of the polypeptide). Thus, the present invention provides methods for determining whether a subject can be effectively treated with an agent for a disorder associated with abeπant expression or activity of a polypeptide of the invention in which a test sample is obtained and the polypeptide or nucleic acid encoding the polypeptide is detected (e.g., wherein the presence of the polypeptide or nucleic acid is diagnostic for a subject that can be administered the agent to treat a disorder associated with abeπant expression or activity of the polypeptide).

The methods of the invention can also be used to detect genetic lesions or mutations in a gene of the invention, thereby determining if a subject with the lesioned gene is at risk for a disorder characterized abeπant expression or activity of a polypeptide of the invention. In prefeπed embodiments, the methods include detecting, in a sample of cells from the subject, the presence or absence of a genetic lesion or mutation characterized by at least one of an alteration affecting the integrity of a gene encoding the polypeptide of the invention, or the mis-expression of the gene encoding the polypeptide of the invention. For example, such genetic lesions or mutations can be detected by ascertaining the existence of at least one of: 1) a deletion of one or more nucleotides from the gene; 2) an addition of one or more nucleotides to the gene; 3) a substitution of one or more nucleotides of the gene; 4) a chromosomal reaπangement of the gene; 5) an alteration in the level of a messenger RNA transcript of the gene; 6) an abeπant modification of the gene, such as of the methylation pattern of the genomic DNA; 7) the presence of a non- wild type splicing pattern of a messenger RNA transcript of the gene; 8) a non- wild type level of 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 lesions in a gene.

In certain embodiments, detection of the lesion involves the use of a probe/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 to the selected gene under conditions such that hybridization and amplification of the gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. PCR and/or LCR can be desirable to use 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 techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers. In an alternative embodiment, mutations in a selected gene from a sample cell can be identified by 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 mutations in the sample DNA. Moreover, the use of sequence specific ribozymes {see, e.g., U.S. Patent No. 5,498,531) can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site.

In other embodiments, genetic mutations can be identified by hybridizing a sample and control nucleic acids, e.g., DNA or RNA, to high density arrays containing hundreds or thousands of oligonucleotides probes (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 in two-dimensional arrays containing light-generated DNA probes 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 aπays of sequential overlapping probes. This step allows the identification of point mutations. This step is followed by a second hybridization array that allows the characterization of specific mutations by using smaller, specialized probe aπays complementary to all variants or mutations detected. Each mutation array is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene.

In yet another embodiment, any of a variety of sequencing reactions known 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 coπesponding wild-type (control) sequence. Examples of sequencing reactions include those based on techniques developed by Maxim and Gilbert ((1977) Proc. Natl. Acad. Sci. USA 74:560) or Sanger ((1977) Proc. Natl. Acad. Sci. USA 74:5463). It is also contemplated that any of a variety of automated sequencing procedures can be utilized when performing the diagnostic assays ((1995) Bio/Techniques 19:448), including sequencing by mass spectrometry {see, e.g., PCT Publication No. WO 94/16101; Cohen et al. (1996) Adv. Chromatogr. 36:127-162; and 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 which cleaves single-stranded regions of the duplex such as which will 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, either 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 then separated by size on denaturing polyacrylamide gels to determine the site of mutation. See, e.g., Cotton et al. (1988) Proc. Natl. Acad. Sci. USA 85:4397; Saleeba et al. (1992) Methods Enzymol. 217:286-295. In a prefeπed embodiment, the control DNA or RNA can be labeled for detection. In still another embodiment, the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs 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 A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T at G/T mismatches (Hsu et al. (1994) Carcinogenesis 15:1657-1662). According to an exemplary embodiment, a probe based on a selected sequence, e.g., a wild-type sequence, is hybridized to a cDNA or other DNA product from a test cell(s). The duplex is treated with a DNA mismatch repair enzyme, and the cleavage products, if any, can be detected from electrophoresis protocols or the like. See, e.g., U.S. Patent No. 5,459,039. In other embodiments, alterations in electrophoretic mobility will be used to identify mutations in genes. For example, single strand conformation polymoφhism (SSCP) 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 will be denatured and allowed to re- nature. The secondary structure of single-stranded nucleic acids varies according to sequence, and the resulting alteration in electrophoretic mobility enables the detection of even a single base change. The DNA fragments can be labeled or detected with labeled probes. The sensitivity of the assay can be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a change in sequence. In a prefeπed embodiment, the subject method utilizes 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) (Myers et al. (1985) Nature 313:495). When DGGE is used as the method of analysis, DNA will be modified to insure that it does not completely denature, for example by adding a 'GC clamp' of approximately 40 base pairs of high-melting GC-rich DNA by PCR. 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, or selective primer extension. For example, oligonucleotide primers can be prepared in which the known mutation is placed centrally and then hybridized to target DNA under conditions which permit hybridization only if a perfect match is found (Saiki et al. (1986) Nature 324:163); Saiki et al. (1989) Proc. Natl. Acad. Sci. USA 86:6230). Such allele specific oligonucleotides are hybridized to PCR amplified target DNA or a number of different mutations when the oligonucleotides are attached to the hybridizing membrane and hybridized with labeled target DNA.

Alternatively, allele specific amplification technology which depends on selective PCR amplification can be used in conjunction with the instant invention. Oligonucleotides used as primers for specific amplification can carry the mutation of interest in the center of the molecule (so that amplification depends on differential hybridization; 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 reduce polymerase extension (Prossner (1993) Tibtech X 1 :238). In addition, it can be desirable to introduce a novel restriction site in the region of the mutation to create cleavage-based detection (Gasparini et al. (1992) Mol. Cell Probes 6:1). Amplification can also be performed using Taq ligase for amplification (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 making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification.

The methods described herein can be performed, for example, using prepackaged diagnostic kits comprising at least one probe nucleic acid or antibody reagent described herein, which can be conveniently used, e.g., in clinical settings to diagnose patients exhibiting symptoms or family history of a disease or illness involving a gene encoding a polypeptide of the invention. Furthermore, any cell type or tissue, preferably peripheral blood leukocytes, in which the polypeptide of the invention is expressed can be utilized in the prognostic assays described herein.

3. Pharmacogenomics

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

Pharmacogenomics deals with clinically significant hereditary variations in the response to drags due to altered drag disposition and abnormal action in affected persons. See, e.g., Linder (1997) Clin. Chem. 43(2):254-266. In general, two types of pharmacogenetic conditions can be differentiated. Genetic conditions transmitted as a single factor altering the way drags act on the body are refeπed to as "altered drag action." Genetic conditions transmitted as single factors altering the way the body acts on drags are refeπed to as "altered drag metabolism". These pharmacogenetic conditions can occur either as rare defects or as polymoφhisms. 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 drags (anti-malarials, sulfonamides, analgesics, nitrofurans) and consumption of fava beans.

As an illustrative embodiment, the activity of drug metabolizing enzymes is a major determinant of both the intensity and duration of drag action. The discovery of genetic polymoφhisms of drug metabolizing enzymes (e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymes CYP2D6 and CYP2C19) has provided an explanation as to why some patients do not obtain the expected drag effects or show exaggerated drug response and serious toxicity after taking the standard and safe dose 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 coding for CYP2D6 is highly polymoφhic and several mutations have been identified in PM, which all lead to the absence of functional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quite 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. The other extreme are the so called ultra-rapid metabolizers who do not respond to standard doses. Recently, the molecular basis of ultra-rapid metabolism has been identified to be due to CYP2D6 gene amplification. Thus, the activity of a polypeptide of the invention, expression of a nucleic acid encoding the polypeptide, or mutation content of a gene encoding the polypeptide in an individual can be determined to thereby select appropriate agent(s) for therapeutic or prophylactic treatment of the individual. In addition, pharmacogenetic studies can be used to apply genotyping of polymoφhic alleles encoding drug-metabolizing enzymes to the identification of an individual's drug responsiveness phenotype. This knowledge, when applied to dosing or drug 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 exemplary screening assays described herein.

4. Monitoring of Effects During Clinical Trials

Monitoring the influence of agents (e.g., drag compounds) on the expression or activity of a polypeptide of the invention (e.g., the ability to modulate abeπant cell proliferation chemotaxis, and or differentiation) can be applied not only in basic drag 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 preferably, that of other polypeptide that have been implicated in for example, a cellular proliferation disorder, can be used as a marker of the immune responsiveness of a particular cell.

For example, and not by way of limitation, genes, including those of the invention, that are modulated in cells by treatment with an agent (e.g., compound, drug or small molecule) which modulates activity or expression of a polypeptide of the invention (e.g., as identified 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 RNA prepared and analyzed for the levels of expression of a gene of the invention and 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 RT-PCR, as described herein, or alternatively by measuring the amount of protein produced, by one of the methods as described herein, or by measuring the levels of activity of a gene of the invention or other genes. In this way, the gene expression pattern can serve as a marker, indicative of the physiological response of the cells to the agent. Accordingly, this response state can be determined before, and at various points during, treatment of the individual with the agent. In a prefeπed embodiment, the present invention provides a method for monitoring the effectiveness of treatment of a subject with an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate identified by the screening assays described herein) comprising the steps of (i) obtaining a pre-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 samples; (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 or samples; 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 higher levels than 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 lower levels than detected, i.e., to decrease the effectiveness of the agent.

C. Methods of Treatment

The present invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with abeπant expression or activity of a polypeptide of the invention and/or in which the polypeptide of the invention is involved. Disorders characterized by abeπant expression or activity of the polypeptides of the invention are described elsewhere in this disclosure. 1. Prophylactic Methods

In one aspect, the invention provides a method for preventing in a subject, a disease or condition associated with an abeπant expression or activity of a polypeptide of the invention, by administering to the subject an agent which modulates expression or at least one activity of the polypeptide. Subjects at risk for a disease which is caused or contributed to by abeπant expression or activity of a polypeptide of the invention can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein. Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the abeπance, such that a disease or disorder is prevented or, alternatively, delayed in its progression. Depending on the type of abeπance, 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-occuπing cognate ligand of the polypeptide, a peptide, a peptidomimetic, or other small molecule. In one embodiment, the agent stimulates one or more of the biological activities of the polypeptide. Examples of such stimulatory agents include the active polypeptide of the invention and a nucleic acid molecule encoding the polypeptide of the invention that has been introduced into the cell. In another embodiment, the agent inhibits one or more of the biological activities of the polypeptide of the invention. Examples of such inhibitory agents include antisense nucleic acid molecules and antibodies. These modulatory methods can be performed 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 abeπant 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 for reduced or abeπant expression or activity of the polypeptide.

Stimulation of activity is desirable in situations in which activity or expression is abnormally low or down-regulated and/or in which increased activity is likely to have a beneficial effect, e.g., in wound healing. Conversely, inhibition of activity is desirable in situations in which activity or expression is abnormally high or up-regulated and/or in which decreased activity is likely to have a beneficial effect.

The contents of all references, patents, and published patent applications cited throughout this application are hereby incoφorated by reference.

Deposit of Clones Clones containing cDNA molecules encoding TANGO 366, TANGO 405, and

INTERCEPT 394 (clones Aped, 405, and 394, respectively), were deposited with the American Type Culture Collection^® (ATCC^®; 10801 University Blvd. Manassas, VA 20110- 2209) on July 23, 1999 as Accession No. PTA-424, as part of a composite deposit representing a mixture of five 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 single colonies on nutrient medium (e.g., LB plates) supplemented with 100 micrograms per milliliter ampicillin, single colonies grown, and then plasmid DNA extracted using a standard mini-preparation procedure. Next, a sample of the DNA mini-preparation can be digested using a combination of the restriction enzymes Sal I and Not I and the resultant products resolved on a 0.8% agarose gel using standard DNA electrophoresis conditions. The digest liberates fragments as follows: TANGO 366 (EpT366): 2.6 kilobase pairs TANGO 405 (405): 3.1 kilobase pairs INTERCEPT 394 (394): 3.7 kilobase pairs

The identity of the strains can be infeπed from the fragments liberated. Clones containing cDN A molecules encoding TANGO 210 and INTERCEPT

400 (clones Aped and 400, respectively), were deposited with the American Type Culture Collection^® (Manassas, VA) on July 29, 1999 as Accession No. PTA-438, as part of a composite deposit representing a mixture of five 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 single colonies on nutrient medium (e.g., LB plates) supplemented with 100 micrograms per milliliter ampicillin, single colonies grown, and then plasmid DNA extracted using a standard mini-preparation procedure. Next, a sample of the DNA mini-preparation can be digested using a combination of the restriction enzymes Sal I and Not I and the resultant products resolved on a 0.8% agarose gel using standard DNA electrophoresis conditions. The digest liberates fragments as follows: TANGO 210 (EpT210): 1.7 kilobase pairs INTERCEPT 400 (400): 3.0 kilobase pairs

The identity of the strains can be infeπed from the fragments liberated. Clones containing cDNA molecules encoding TANGO 364 (clones Aped), were deposited with the American Type Culture Collection^® (Manassas, VA) on July 23, 1999 as Accession No. PTA-425, 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 single colonies on nutrient medium (e.g., LB plates) supplemented with 100 micrograms per milliliter ampicillin, single colonies grown, and then plasmid DNA extracted using a standard mini-preparation procedure. Next, a sample of the DNA mini-preparation can be digested using a combination of the restriction enzymes Sal I and Not I and the resultant products resolved on a 0.8% agarose gel using standard DNA electrophoresis conditions. The digest liberates fragments as follows: TANGO 364 (Aped): 3.5 kilobase pairs

The identity of the strain containing TANGO 364 can be infeπed from the liberation of a fragment of the above identified size.

All publications, patents, and patent applications referenced in this specification are incoφorated by reference into the specification to the same extent as if each individual publication, patent, or patent application had been specifically and individually indicated to be incoφorated herein by reference.

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 intended to be 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, 31, 32, 41, 42, 61, 62, 81, 82, 91, 92, 101, 102, 121, and 122, the nucleotide sequence of a cDNA clone deposited with ATCC^® as any of Accession Nos. PTA-424, PTA-425, and PTA-438, 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, 31, 32, 41, 42, 61, 62, 81, 82, 91, 92, 101, 102, 121, and 122, the nucleotide sequence of a cDNA clone deposited with ATCC^® as any of Accession Nos. PTA- 424, PTA-425, and PTA-438, or a complement thereof; c) a nucleic acid molecule which encodes a polypeptide comprising the amino acid sequence of any of SEQ ID NOs: 3-5, 13-15, 23-28, 33-38, 43-54, 63-76, 83-92, 93, 103-105, and 123, or the amino acid sequence encoded by a cDNA clone deposited with ATCC^® as any of Accession Nos. PTA-424, PTA-425, and PTA-438, or a complement thereof; d) a nucleic acid molecule which encodes a polypeptide having an amino acid sequence comprising at least 8 consecutive amino acid residues of any of SEQ ID NOs: 3-5, 13-15, 23-28, 33-38, 43-54, 63-76, 83-92, 93, 103-105, and 123, or the amino acid sequence encoded by a cDNA clone deposited with ATCC^® as any of Accession Nos. PTA-424, PTA- 425, and PTA-438; and e) a nucleic acid molecule which encodes a naturally occurring allelic variant of a polypeptide having an amino acid sequence comprising any of SEQ ID NOs: 3-5, 13-15, 23- 28, 33-38, 43-54, 63-76, 83-92, 93, 103-105, and 123, wherein the nucleic acid molecule hybridizes with a nucleic acid molecule having the nucleotide sequence of any of SEQ ID NOs: 1, 2, 11, 12, 21, 22, 31, 32, 41, 42, 61, 62, 81, 82, 91, 92, 101, 102, 121, and 122, the nucleotide sequence of a cDNA clone deposited with ATCC^® as any of Accession Nos. PTA- 424, PTA-425, and PTA-438, 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, 31, 32, 41, 42, 61, 62, 81, 82, 91, 92, 101, 102, 121, and 122, the nucleotide sequence of a cDNA clone deposited with ATCC^® as any of Accession Nos. PTA-424, PTA-425, and PTA-438, 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: 3-5, 13-15, 23-28, 33-38, 43-54, 63-76, 83-92, 93, 103-105, and 123, or the amino acid sequence encoded by a cDNA clone deposited with ATCC^® as any of Accession Nos. PTA-424, PTA-425, and PTA-438, 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 having an amino acid sequence comprising at least 8 contiguous amino acid residues of any of SEQ ID NOs: 3-5, 13-15, 23-28, 33-38, 43-54, 63- 76, 83-92, 93, 103-105, and 123, or the amino acid sequence encoded by a cDNA clone deposited with ATCC^® as any of Accession Nos. PTA-424, PTA-425, and PTA-438; b) a naturally occuπing allelic variant of a polypeptide having an amino acid sequence comprising any of SEQ ID NOs: 3-5, 13-15, 23-28, 33-38, 43-54, 63-76, 83-92, 93, 103-105, and 123, or the amino acid sequence encoded by a cDNA clone deposited with ATCC^® as any of Accession Nos. PTA-424, PTA-425, and PTA-438, wherein the polypeptide is encoded by a nucleic acid molecule which hybridizes with a nucleic acid molecule having the nucleotide sequence of any of SEQ ID NOs: 1, 2, 11, 12, 21, 22, 31, 32, 41, 42, 61, 62, 81, 82, 91, 92, 101, 102, 121, and 122, the nucleotide sequence of a cDNA clone deposited with ATCC^® as any of Accession Nos. PTA-424, PTA-425, and PTA-438, or a complement thereof under stringent conditions; and c) a polypeptide which is encoded by a nucleic acid molecule having a nucleotide sequence which is at least 40% identical to any of SEQ ID NOs: 1, 2, 11, 12, 21, 22, 31, 32, 41, 42, 61, 62, 81, 82, 91, 92, 101, 102, 121, and 122, the nucleotide sequence of a cDNA clone deposited with ATCC^® as any of Accession Nos. PTA-424, PTA-425, and PTA-438, or a complement thereof.

9. The isolated polypeptide of claim 8, having the amino acid sequence of any of SEQ ID NOs: 3-5, 13-15, 23-28, 33-38, 43-54, 63-76, 83-92, 93, 103-105, and 123, or the amino acid sequence encoded by a cDNA clone deposited with ATCC^® as any of Accession Nos. PTA-424, PTA-425, and PTA-438, 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 having the amino acid sequence of any of SEQ ID NOs: 3-5, 13-15, 23-28, 33-38, 43-54, 63-76, 83-92, 93, 103-105, and 123, or the amino acid sequence encoded by a cDNA clone deposited with ATCC^® as any of Accession Nos. PTA-424, PTA-425, and PTA-438, or a complement thereof; b) a polypeptide having an amino acid sequence which comprises at least 8 contiguous amino acid residues of any of SEQ ID NOs: 3-5, 13-15, 23-28, 33-38, 43-54, 63-76, 83-92, 93, 103-105, and 123, or the amino acid sequence encoded by a cDNA clone deposited with ATCC^® as any of Accession Nos. PTA-424, PTA-425, and PTA-438; and c) a naturally occuπing allelic variant of a polypeptide having an amino acid sequence comprising any of SEQ ID NOs: 3-5, 13-15, 23-28, 33-38, 43-54, 63-76, 83-92, 93, 103-105, and 123, or the amino acid sequence encoded by a cDNA clone deposited with ATCC^® as any of Accession Nos. PTA-424, PTA-425, and PTA-438, or a complement thereof, wherein the polypeptide is encoded by a nucleic acid molecule which hybridizes with a nucleic acid molecule having the nucleotide sequence of any of SEQ ID NOs: 1, 2, 11, 12, 21, 22, 31, 32, 41, 42, 61, 62, 81, 82, 91, 92, 101, 102, 121, and 122, the nucleotide sequence of a cDNA clone deposited with ATCC^® as any of Accession Nos. PTA-424, PTA-425, and PTA-438, 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 with 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.