WO2000050442A2

WO2000050442A2 - Secreted proteins and uses thereof

Info

Publication number: WO2000050442A2
Application number: PCT/US2000/004784
Authority: WO
Inventors: Sean A. Mccarthy
Original assignee: Millennium Pharmaceuticals, Inc.
Priority date: 1999-02-26
Filing date: 2000-02-25
Publication date: 2000-08-31
Also published as: EP1159413A4; AU3706600A; EP1159413A2; WO2000050442A3

Abstract

The invention provides isolated nucleic acid molecules, designated TANGO 201 and TANGO 223, which encode wholly secreted or membrane-associated proteins. 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.

Description

SECRETED PROTEINS AND USES THEREOF

Cross Reference to Related Applications This application is a continuation-in-part of co-pending United States Patent

Application Serial No. 09/259,388 filed February 26, 1999, the entire contents of which is incorporated herein by reference in its entirety.

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

Many membrane associated proteins are receptors which bind a ligand and transduce an intracellular signal, leading to a variety of cellular responses. The identification and characterization of such a receptor enables one to identify both the ligands which bind to the receptor and the intracellular molecules and signal transduction pathways associated with the receptor, permitting one to identify or design modulators of receptor activity, e.g., receptor agonists or antagonists and modulators of signal transduction.

Summary of the Invention The present invention is based, at least in part, on the discovery of cDNA molecules encoding TANGO 201 and TANGO 223, both of which are predicted to be either wholly secreted or transmembrane proteins. These proteins, fragments, derivatives, and variants thereof are collectively referred to as "polypeptides of the invention" or "proteins of the invention." Nucleic acid molecules encoding the polypeptides or proteins 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, this invention provides isolated nucleic acid molecules encoding a polypeptide of the invention or a biologically active portion thereof. The present invention also provides nucleic acid molecules which are suitable for use as primers or hybridization probes for the detection of nucleic acids encoding a polypeptide of the invention.

The invention features nucleic acid molecules which are at least about 45% (or 55%, 65%, 75%, 85%, 95%, or 98%) identical to the nucleotide sequence of SEQ ID NOs.l, 2, 6, 7, 11, 12, 24, 25, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56 or the nucleotide sequence of the cDNA insert of a clone deposited with ATCC™ as Accession Number 207081 (the "cDNA of ATCC™ 207081"), or a complement thereof.

The invention features nucleic acid molecules which are at least 45% (or 55%, 65%,

5 75%, 85%o, 95%), or 98%) identical to the nucleotide sequence of any of SEQ ID NOs: 1, 2, 6, 7, 11, 12, 24, 25, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56 or the nucleotide sequence of the cDNA of a clone deposited with ATCC™ as Accession Number 207081 (the "cDNA of a clone deposited as ATCC™ 207081"), or a complement thereof, wherein such nucleic acid molecules encode polypeptides or proteins that exhibit at least one

10 structural and/or functional feature of a polypeptide of the invention.

The invention features nucleic acid molecules of at least 615, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700 or 1758 nucleotides of the nucleotide sequence of SEQ ID NO:l, or a complement thereof. The invention also features nucleic acid molecules comprising at least 15, 25, 50, 100, 150, 200, 250 nucleotides of

15 nucleic acids 1 to 252 of SEQ ID NO:l, or a complement thereof. The invention also features nucleic acid molecules comprising at least 15, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 490 nucleotides of nucleic acids 546 to 1041 of SEQ ID NO:l, or a complement thereof. The invention also features nucleic acid molecules comprising at least 25, 50, 75 or 100 nucleotides of nucleic acids 1657 to 1758 of SEQ ID NO:l, or a

20 complement thereof.

The invention features nucleic acid molecules comprising at least 465, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1445 nucleotides of the nucleotide sequence of SEQ ID NO:2, or a complement thereof. The invention also features nucleic acid molecules which include a fragment of at

25 least 15, 25, 50, 75, 100, 150, or 190 nucleotides of nucleic acids 1 to 193 of SEQ ID NO:2, or a complement thereof. The invention also features nucleic acid molecules comprising at least 15, 25, 50, 75, 100, 200, 250, 300, 350, 400, 450 or 480 nucleotides of nucleic acids 497 to 983 of SEQ ID NO:2, or a complement thereof.

The invention features nucleic acid molecules of at least 570, 600, 650, 700, 750,

30 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200 or 2252 nucleotides of the nucleotide sequence of SEQ ID NO:6, the nucleotide sequence of the mouse TANGO 201 cDNA clone, or a complement thereof. The invention also features nucleic acid molecules comprising at least 25, 50, 100, 150, 200, 250, 300, 350 or 370 nucleotides of nucleic acids 1 to 371 of SEQ ID NO:6, or a complement thereof.

35 The invention also features nucleic acid molecules comprising at least 25, 50, 100, 150, 200, 250, 300, 350 or 375 nucleotides of nucleic acids 665 to 1042 of SEQ ID NO:6, or a complement thereof. The invention also features nucleic acid molecules comprising at least 25, 50, 75 or 100 nucleotides of nucleic acids 1657 to 1758 of SEQ ID NO:6, or a complement thereof.

The invention features nucleic acid molecules of at least 300, 350, 400, 450, 500,

5 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, or 1205 nucleotides of the nucleotide sequence of SEQ ID NO: 7, or a complement thereof. The invention also features nucleic acid molecules which include a fragment of at least 15, 25, 50, 75, 100, 150 or 190 nucleotides of the nucleotide sequence of nucleic acids 1 to 192 of SEQ ID NO: 7, or a complement thereof. The invention also features nucleic acid molecules which include a

10 fragment of at least 15, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 995 nucleotides of the nucleotide sequence of nucleic acids 484 to 1483 of SEQ ID NO:7, or a complement thereof.

The invention features nucleic acid molecules of at least 585, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400 or 1470 nucleotides of the nucleotide

15 sequence of SEQ ID NO: 11 , the nucleotide sequence of the human TANGO 223 cDNA clone of ATCC™ deposit 207081, or a complement thereof. The invention also features nucleic acid molecules of at least 15, 25, 50, 75, 100, 150, 200, 250, 300 or 381 nucleotides of nucleic acids 1 to 382 of SEQ ID NO: 11, or a complement thereof.

The invention features nucleic acid molecules of at least 360, 400, 450, 500, 550,

20 600, 650, 700 or 740 nucleotides of the nucleotide sequence of SEQ ID NO: 12, or a complement thereof. The invention also features nucleic acid molecules which include a fragment of at least 15, 25, 50, 75, 100, 150, 200, 250, 300 or 351 nucleotides of nucleic acids 1 to 352 of SEQ ID NO: 12, or a complement thereof.

The invention features nucleic acid molecules of at least 530, 550, 600, 650, 700,

25 750, 800 or 850 nucleotides of the nucleotide sequence of SEQ ID NO:24, or a complement thereof. The invention also features nucleic acid molecules of at least 15, 25, 50, 75, 100 or 152 nucleotides of nucleic acids 1 to 153 of SEQ ID NO:24, or a complement thereof.

The invention features nucleic acid molecules of at least 385, 450, 500, 550, 600, 650 or 685 nucleotides of the nucleotide sequence of SEQ ID NO:25, or a complement

30 thereof. The invention also features nucleic acid molecules which include a fragment of at least 15, 25, 50, 75, 100 or 147 nucleotides of nucleic acids 1 to 148 of SEQ ID NO:25, or a complement thereof.

The invention features nucleic acid molecules which include a fragment of at least 300 (325, 350, 375, 400, 425, 450, 500, 550, 600, 650, 700, 800, 900, 1000, or 1200)

35 nucleotides of the nucleotide sequence of SEQ ID NO:l, 2, 6, 7, 11, 12, 24, 25, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56 or the nucleotide sequence of a cDNA of ATCC™ 207081, or a complement thereof.

The invention features nucleic acid molecules which include a fragment of at least 300 (325, 350, 375, 400, 425, 450, 500, 550, 600, 650, 700, 800, 900, 1000, or 1200) nucleotides of the nucleotide sequence of SEQ ID NO:l, 2, 6, 7, 11, 12, 24, 25, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56 or the nucleotide sequence of a cDNA of ATCC™ 207081, or a complement thereof, wherein such nucleic acid molecules encode polypeptides or proteins that exhibit at least one structural and/or functional feature of a polypeptide of the invention. The invention also features nucleic acid molecules which comprise a nucleotide sequence encoding a protein comprising an amino acid sequence that is at least about 45% (or 55%, 65%, 75%, 85%, 95%, or 98%) identical to the amino acid sequence of SEQ ID NO:3, 8, 13, 26, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, or the amino acid sequence encoded by a cDNA of ATCC™ 207081, or a complement thereof. The invention also features nucleic acid molecules which comprise a nucleotide sequence encoding a protein comprising an amino acid sequence that is at least 45% (or 55%o, 65%>, 75%, 85%, 95%, or 98%>) identical to the amino acid sequence of any of SEQ ID NOs:3, 8, 13, 26, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, or the amino acid sequence encoded by a cDNA of a clone deposited as ATCC™ 207081 , or a complement thereof, wherein the protein encoded by the nucleotide sequence also exhibits at least one structural and/or functional feature of a polypeptide of the invention.

In preferred embodiments, the nucleic acid molecules comprise the nucleotide sequence of SEQ ID NO:l, 2, 6, 7, 11, 12, 24, 25, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, or the nucleotide sequence of a cDNA of ATCC™ 207081, or a complement thereof.

Also within the invention are nucleic acid molecules which encode a fragment of a polypeptide comprising the amino acid sequence of any of SEQ ID NOs:3, 8, 13, 26, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, or the amino acid sequence encoded by a cDNA of a clone deposited as ATCC™ 207081, or a fragment including at least 15 (20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390 or 400) contiguous amino acids of any of SEQ ID NOs:3, 8, 13, 26, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, or the polypeptide encoded by a cDNA of a clone deposited as ATCC™ 207081, or a complement thereof. Also within the invention are nucleic acid molecules which encode a fragment of a polypeptide comprising the amino acid sequence of any of SEQ ID NOs:3, 8, 13, 26, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, or the amino acid sequence encoded by a cDNA of a clone deposited as ATCC™ 207081, or a fragment thereof including at least 15 (20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350,

5 360, 370, 380, 390 or 400) contiguous amino acids of any of SEQ ID NOs:3, 8, 13, 26, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, or the polypeptide encoded by a cDNA of a clone deposited as ATCC™ 207081, wherein the polypeptide fragment thereof exhibits at least one structural and/or functional feature of a polypeptide of the invention.

The invention also features nucleic acid molecules that hybridize under stringent

10 hybridization conditions to a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:l, 2, 6, 7, 11, 12, 24, 25, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, or a cDNA of ATCC™ 207081, or a complement thereof. In other embodiments, the nucleic acid molecules are at least 300 (325, 350, 375, 400, 425, 450, 500, 550, 600, 650, 700, 800, 900, 1000, or 1290) nucleotides in length and hybridize under stringent hybridization conditions

15 to a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 1, 2, 6, 7, 11, 12, 24, 25, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, a cDNA of ATCC™ 207081, or a complement thereof.

The invention also features nucleic acid molecules that hybridize under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence of any of SEQ ID

20 NOs:l, 2, 6, 7, 11, 12, 24, 25, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, of a cDNA of a clone deposited as ATCC™ 207081, or a complement thereof, wherein preferably such nucleic acid molecules encode polypeptides or proteins that exhibit at least one structural and/or functional feature of a polypeptide of the invention. In other embodiments, the nucleic acid molecules are at least 300 (325, 350, 375, 400, 425, 450, 500, 550, 600, 650,

25 700, 800, 900, 1000, or 1290) nucleotides in length and hybridize under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence of any of SEQ ID NOs:l, 2, 6, 7, 11, 12, 24, 25, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, of the cDNA of a clone deposited as ATCC™ 207081 , or a complement thereof, wherein preferably such nucleic acid molecules encode polypeptides or proteins that exhibit at least one structural and/or

30 function feature of a polypeptide of the invention.

The invention includes nucleic acid molecules which encode a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of any of SEQ ID NOs:3, 8, 13, 26, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, or an amino acid sequence encoded by a cDNA of a clone deposited as ATCC™ 207081 , or a complement

35 thereof, wherein the nucleic acid molecule hybridizes under stringent conditions to a nucleic acid molecule comprising a nucleic acid sequence encoding any of SEQ ID NOs:3, 8, 13, 26, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, or a complement thereof.

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

5 NOs:3, 8, 13, 26, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, or an amino acid sequence encoded by a cDNA of a clone deposited as ATCC™ 207081, or a complement thereof, wherein the nucleic acid molecule hybridizes under stringent conditions to a nucleic acid molecule comprising a nucleic acid sequence encoding any of SEQ ID NOs:3, 8, 13, 26, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, or a complement thereof, wherein the

10 polypeptides or proteins exhibit at least one structural and/or functional feature of a polypeptide of the invention.

Also within the invention are isolated polypeptides or proteins having an amino acid sequence that is at least about 45%, preferably 55%>, 65%, 75%, 85%>, 95%, or 98% identical to the amino acid sequence of any of SEQ ID NO:3, 8, 13, 26, 33, 35, 37, 39, 41,

15 43, 45, 47, 49, 51, 53, 55, 57, or the amino acid sequence encoded by a cDNA of ATCC™ 207081, or a complement thereof.

Also within the invention are isolated polypeptides or proteins having an amino acid sequence that is at least about 45%, preferably 55%, 65%, 75%>, 85%, 95%), or 98% identical to the amino acid sequence of any of SEQ ID NOs:3, 8, 13, 26, 33, 35, 37, 39, 41,

20 43, 45, 47, 49, 51, 53, 55, 57, wherein the polypeptides or proteins also exhibit at least one structural and/or functional feature of a polypeptide of the invention.

Also within the invention are isolated polypeptides or proteins which are encoded by a nucleic acid molecule comprising a nucleotide sequence that is at least about 45%>, preferably 55%), 65%), 75%, 85%, 95% or 98%> identical to a nucleic acid sequence encoding

25 SEQ ID NO:3, 8, 13, 26, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, or a cDNA of ATCC™ 207081, and isolated polypeptides or proteins which are encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:l, 2, 6, 7, 11, 12, 24, 25, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, or complement thereof, or

30 the non-coding strand of a cDNA of ATCC™ 207081 , or a complement thereof.

Also within the invention are isolated polypeptides or proteins which preferably are encoded by a nucleic acid molecule comprising a nucleotide sequence that is at least about 45%,, preferably 55%, 65%, 75%, 85%, 95% or 98% identical the nucleic acid sequence encoding any of SEQ ID NOs:3, 8, 13, 26, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57,

35 or a cDNA of ATCC™ 20701, and isolated polypeptides or proteins which are encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule having the sequence of any of SEQ ID NOs:l, 2, 6, 7, 11, 12, 24, 25, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, or a complement thereof, or the non-coding strand of a cDNA of a clone deposited as ATCC™ 207081, wherein the polypeptides or proteins preferably also exhibit at least one structural and/or functional feature of a polypeptide of the invention,.

Also within the invention are polypeptides which are naturally occurring allelic variants of a polypeptide that comprises the amino acid sequence of SEQ ID NO:3, 8, 13, 26, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, or the amino acid sequence encoded by a cDNA of ATCC™ 207081, wherein the polypeptide is encoded by a nucleic acid molecule which hybridizes under stringent conditions to a nucleic acid molecule having the sequence of SEQ ID NO:l, 2, 6, 7, 11, 12, 24, 25, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, a cDNA of ATCC 207081 or a complement thereof.

Also within the invention are polypeptides which are naturally occurring allelic variants of a polypeptide that includes the amino acid sequence of any of SEQ ID NOs:3, 8, 13, 26, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, or the amino acid sequence encoded by the cDNA of a clone deposited as ATCC™ 207081, wherein the polypeptide is encoded by a nucleic acid molecule which hybridizes under stringent conditions to a nucleic acid molecule having the sequence of any of SEQ ID Nos:l, 2, 6, 7, 11, 12, 24, 25, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, or a complement thereof, wherein the polypeptide or proteins exhibit at least one structural and/or functional feature of a polypeptide of the invention.

In other embodiments, the isolated nucleic acid molecules encode an extracellular, transmembrane, cytoplasmic, or cysteine-rich domain of a polypeptide of the invention. In another embodiment, the invention provides an isolated nucleic acid molecule which is antisense to the coding strand of a nucleic acid of the invention.

Another aspect of the invention provides vectors, e.g., recombinant expression vectors, comprising a nucleic acid molecule of the invention. In another embodiment, the invention provides host cells containing such a vector, or engineered to contain a nucleic acid of the invention and/or to express 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 such that a polypeptide is produced.

Another aspect of this invention features isolated or recombinant proteins and polypeptides of the invention. Preferred proteins and polypeptides possess at least one biological activity possessed by the corresponding naturally-occurring human polypeptide. An activity, a biological activity, and a functional activity of a polypeptide of 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 signaling activity mediated by interaction of the protein with a second protein. Thus, such activities include, e.g., (1) the ability to form protein-protein interactions with proteins in the signaling pathway of the naturally-occurring polypeptide; (2) the ability to bind a ligand of the naturally-occurring polypeptide; (3) the ability to bind to an intracellular target of the naturally-occurring polypeptide. Other activities include, e.g., (1) the ability to modulate cellular proliferation; (2) the ability to modulate cellular differentiation; (3) the ability to modulate chemotaxis and/or migration; and (4) the ability to modulate cell death.

In particular, for TANGO 201, biological activities include, e.g., (1) the ability to form protein-protein interactions with proteins in the signaling pathway of the naturally- occurring polypeptide; (2) the ability to bind a ligand of the naturally-occurring polypeptide; (3) the ability to interact with a TANGO 201 receptor; (4) the ability to modulate chemotaxis and/or migration; and (5) the ability to modulate cell death. Other activities include (1) the ability to modulate function, survival, morphology, proliferation and/or differentiation of cells of tissues in which it is expressed (e.g., pancreas, adrenal medulla, thyroid, adrenal cortex, testis, stomach, heart, brain, placenta, lung, liver, kidney, skeletal muscle, or small intestine); and (2) the ability to function in the amplification of cellular oncogenes.

For TANGO 223, biological activities include, e.g., (1) the ability to form protein- protein interactions with proteins in the signaling pathway of the naturally-occurring polypeptide; (2) the ability to bind a ligand of the naturally-occurring polypeptide; (3) the ability to interact with a TANGO 223 receptor; and (4) the ability to modulate chemotaxis and/or migration; and (5) the ability to modulate cell death. Other activities include: (1) the ability to modulate function, survival, morphology, proliferation and/or differentiation of cells of tissues in which it is expressed (e.g., heart, brain, liver, kidney, testis, prostate, ovary, colon, peripheral blood leukocytes, and the small intestine).

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 structural domain and/or common functional activity. For example, amino acid or nucleotide sequences which contain a common structural domain having about 45% identity, preferably 55% identity, more preferably 65%, 75%, 85%, 95%, 98% or more identity are defined herein as sufficiently identical.

In one embodiment, a TANGO 201 polypeptide of the invention includes a signal sequence. In another embodiment, a nucleic acid molecule of the invention encodes a 5 TANGO 201 polypeptide which includes a signal sequence.

In another embodiment, a TANGO 223 polypeptide of the invention includes one or more of the following domains: (1) a signal sequence; (2) an extracellular domain; (3) a transmembrane domain; and (4) a cytoplasmic domain.

In another embodiment, a nucleic acid molecule of the invention encodes a TANGO 10 223 polypeptide with one or more of the following domains: (1) a signal sequence; (2) an extracellular domain; (3) a transmembrane domain; and (4) a cytoplasmic domain.

In yet another embodiment, a TANGO 201 or TANGO 223 polypeptide of the invention includes a cysteine-rich domain.

In yet another embodiment, a nucleic acid molecule of the invention encodes a 15 TANGO 201 or TANGO 223 polypeptide of the invention which includes a cysteine-rich domain.

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 antibodies that specifically bind a polypeptide of the invention 0 such as monoclonal or polyclonal antibodies.

In addition, the polypeptides of the invention or biologically active portions thereof, or antibodies of the invention, can be incorporated into pharmaceutical compositions, which optionally include pharmaceutically acceptable carriers.

In another aspect, the present invention provides methods for detecting the presence 5 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 0 (inhibits or stimulates) 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 5 polypeptide of the invention. In yet another embodiment, the agent is a nucleic acid molecule having a nucleotide sequence that is antisense 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 abeπant activity of a polypeptide of the invention or aberrant expression of a nucleic acid of the invention by administering an agent which is a modulator of the activity of a polypeptide of the invention or a modulator of the expression of a nucleic acid of the invention to the subject. In one embodiment, the modulator is a protein of the invention. In another embodiment, the modulator is a nucleic acid of the invention. In other embodiments, the modulator is a peptide, peptidomimetic, or other small 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 compound.

In yet a further aspect, the invention provides antibodies, including substantially purified antibodies, or fragments thereof, which antibodies or fragments specifically bind to a polypeptide comprising an amino acid sequence selected from the group consisting of: the amino acid sequence of SEQ ID NO:3, 8, 13, 26, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, or 57, or the amino acid sequence encoded by the cDNA insert of the plasmid deposited with ATCC™ as Accession Number 207081 ; a fragment of at least 15 amino acid residues of the amino acid sequence of SEQ ID NO:3, 8, 13, 26, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, or 57; an amino acid sequence which is at least 95% identical to the amino acid sequence of SEQ ID NO:3, 8, 13, 26, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, or 57, 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 an amino acid sequence which is encoded by a nucleic acid molecule which hybridizes to the nucleic acid molecule consisting of SEQ ID NO:l, 2, 6, 7, 11, 12, 24, 25, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, or 56 under conditions of hybridization of 6X SSC at 45°C and washing in 0.2 X SSC, 0.1% SDS at 65°C. In various embodiments, the antibodies of the invention, or fragments thereof, can be human, non-human (e.g., goat, mouse, sheep, horse, chicken, rabbit, or rat antibodies), chimeric and/or humanized antibodies. In addition, the 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 to a polypeptide comprising an amino acid sequence selected from the group consisting of: the amino acid sequence of SEQ ID NO:3, 8, 13, 26, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, or 57, or the amino acid sequence encoded by the cDNA insert of the plasmid deposited with ATCC™ as Accession Number 207081; a fragment of at least 15 amino acid residues of the amino acid sequence of SEQ ID NO:3, 8, 13, 26, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, or 57; an amino acid sequence which is at least 95% identical to the amino acid sequence of SEQ ID NO: 3 or 16, 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 an amino acid sequence which is encoded by a nucleic acid molecule which hybridizes to the nucleic acid molecule consisting of SEQ ID NO:l, 2, 6, 7, 11, 12, 24, 25, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, or 56 under conditions of hybridization of 6X SSC 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.

In a particularly preferred embodiment, the antibodies or fragments thereof, the non- human antibodies or fragments thereof, and/or the monoclonal antibodies or fragments thereof, of the invention specifically bind to an extracellular domain of the amino acid sequence of SEQ ID NO: 16 or 29. Preferably, the extracellular domain to which the antibody, or fragment thereof, binds comprises amino acid residues 30 to 215 of SEQ ID NO: 16 or amino acid residues 30 to 198 of SEQ ID NO:29.

Any of the antibodies of the invention can be conjugated to a therapeutic moiety or to a detectable substance. Non-limiting examples of detectable substances that can be conjugated to the antibodies of the invention are an enzyme, a prosthetic group, a fluorescent material, a luminescent material, a bioluminescent material, and a radioactive material.

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

Figures 1A-1B depict the cDNA sequence (SEQ ID NO:l) and the predicted amino acid sequence (SEQ ID NO:3) of murine TANGO 201. The open reading frame of SEQ ID NO:l extends from nucleotide 60 to nucleotide 1508 of SEQ ID NO:l (SEQ ID NO:2). Figure 2 depicts a hydropathy plot of murine TANGO 201. Relatively hydrophobic regions are above the dashed horizontal line, and relatively hydrophilic regions are below the dashed horizontal line. The cysteine residues (cys) are indicated by short vertical lines just below the hydropathy trace. The dashed vertical line separates the signal sequence on the left from the mature protein on the right. Figures 3A-3B depict the cDNA sequence (SEQ ID NO:6) and the predicted amino acid sequence (SEQ ID NO:8) of human TANGO 201. The open reading frame of SEQ ID NO:6 extends from nucleotide 179 to nucleotide 1387 of SEQ ID NO:6 (SEQ ID NO:7).

Figure 4 depicts a hydropathy plot of human TANGO 201. Relatively hydrophobic regions are above the dashed horizontal line, and relatively hydrophilic regions are below the dashed horizontal line. The cysteine residues (cys) are indicated by short vertical lines just below the hydropathy trace. The dashed vertical line separates the signal sequence on the left from the mature protein on the right.

Figures 5A-5C depict an alignment of the nucleotide sequence of murine TANGO 201 (nucleotides 1-1758 of SEQ ID NO:l) and human TANGO 201 (nucleotides 101-1660 of SEQ ID NO:6). An identity of 84.8% was obtained using the program GAP (Needleman and Wunsch (1970) J. Mol Biol 48:443-453) in GCG (Wisconsin Package Version 9.1, Genetics Computer Group, Madison WI) with the following settings: score matrix nwsgapdna, gap penalty 50, and gap extension penalty 3.

Figure 6 depicts an alignment of the amino acid sequences of murine TANGO 201 (amino acids 1-483 of SEQ ID NO:3) and human TANGO 201 (amino acids 1-403 of SEQ ID NO:8). An identity of 97%> was obtained using the program GAP (Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453) in GCG (Wisconsin Package Version 9.1, Genetics Computer Group, Madison WI) with the following settings: score matrix blosum62, gap penalty 12, and gap extension penalty 4. Figure 7 depicts an alignment of a portion of murine TANGO 201 amino acid sequence (amino acids 78-264 of SEQ ID NO:3) and a portion of human TANGO 201 amino acid sequence (amino acids 78-264 of SEQ ID NO: 8) with a portion of OS-9 (SEQ ID NO:58), a human protein referred to as OS-9 (amino acids 73-250 of SwissProt Accession No. Q 13438). This alignment defines a cysteine-rich domain that is conserved between TANGO 201 and OS-9. Figure 8 depicts the cDNA sequence (SEQ ID NO:l 1) and the predicted amino acid sequence (SEQ ID NO:13) of human TANGO 223. The open reading frame of human TANGO 223 extends from nucleotide 30 to nucleotide 770 of SEQ ID NO:l 1 (SEQ ID NO: 12).

Figure 9 depicts a hydropathy plot of human TANGO 223. Relatively hydrophobic regions are above the dashed horizontal line, and relatively hydrophilic regions are below the dashed horizontal line. The cysteine residues (cys) and potential N-glycosylation sites (Ngly) are indicated by short vertical lines just below the hydropathy trace. The dashed vertical line separates the signal sequence on the left from the mature protein on the right. Figure 10 depicts an alignment of a portion of human TANGO 223 amino acid sequence (amino acids 82-180 of SEQ ID NO:13) with a portion of a putative C. elegans (SEQ ID NO:59) protein belonging to the family of DNA/RNA nonspecific endonucleases (amino acids 288-378 of Swiss-Prot Accession No. O01975). This alignment reveals a cysteine-rich domain that is conserved between TANGO 223 and the C. elegans protein. Figure 11 depicts the cDNA sequence (SEQ ID NO:24) and the predicted amino acid sequence (SEQ ID NO:26) of murine TANGO 223. The open reading frame of murine TANGO 223 extends from nucleotide 5 to nucleotide 694 of SEQ ID NO:24 (SEQ ID NO:25).

Description of the Prefeπed Embodiments The present invention is based, at least in part, on the discovery of cDNA molecules encoding TANGO 201 and TANGO 223, each of which is predicted to be either wholly secreted or transmembrane proteins.

The proteins and nucleic acid molecules of the present invention 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 structural domain and having sufficient amino acid or nucleotide sequence identity as defined herein. Family members can be from either the same or different species. For example, a family can comprise two or more proteins of human origin, or can comprise one or more proteins of human origin and one or more of non- human origin. Members of the same family may also have common structural domains. For example, a TANGO 201 or TANGO 223 family member includes a signal sequence. As used herein, a "signal sequence" includes a peptide of at least about 15 amino acid residues in length which occurs at the N-terminus of secretory and membrane-bound proteins and which contains at least about 70% hydrophobic amino acid residues such as alanine, leucine, isoleucine, phenylalanine, proline, tyrosine, tryptophan, or valine. In a preferred embodiment, a signal sequence contains at least about 15 to 40 amino acid residues, preferably about 25-35 amino acid residues, and has at least about 60-80%>, more preferably 65-75%), and more preferably at least about 70% hydrophobic residues. A signal sequence serves to direct a protein containing such a sequence to a lipid bilayer. Thus, in one embodiment, a murine TANGO 201 protein contains a signal sequence of about amino acids 1-33 of SEQ ID NO:3 (SEQ ID NO:4). In another embodiment, a human TANGO 201 protein contains a signal sequence of about amino acids 1-33 of SEQ ID NO:8 (SEQ ID NO:9). In another embodiment, a human TANGO 223 protein contains a signal sequence of about amino acids 1-29 of SEQ ID NO:13 (SEQ ID NO:14). The signal sequence is cleaved during processing of the mature protein.

In another example, a TANGO 223 family member also includes one or more of the following domains: (1) an extracellular domain; (2) a transmembrane domain; and (3) a cytoplasmic domain. Thus, in one embodiment, a TANGO 223 protein contains an extracellular domain of about amino acids 30-215 of SEQ ID NO: 13 (SEQ ID NO: 16). In another embodiment, a TANGO 223 protein contains a transmembrane domain of about amino acids 216-238 of SEQ ID NO:13 (SEQ ID NO:17). In another embodiment, a TANGO 223 protein contains a cytoplasmic domain of about amino acids 239-247 of SEQ ID NO: 13 (SEQ ID NO: 18). Alternatively, in another embodiment, a TANGO 223 protein contains an extracellular domain at amino acid residues 239 to 247 of SEQ ID NO: 13 ( SEQ ID NO: 18), a transmembrane domain at amino acid residues 216 to 238 of SEQ ID NO: 13 (SEQ ID NO: 17), and a cytoplasmic domain at amino acid residues 30 to 215 of SEQ ID NO:13 (SEQ ID NO:16).

In another embodiment, a TANGO 223 protein contains a 169 amino acid extracellular domain (amino acids 30-198 of SEQ ID NO:26; SEQ ID NO:29), a single 23 amino acid transmembrane domain (amino acids 199-221 of SEQ ID NO:26; SEQ ID NO:30), and a nine amino acid cytoplasmic domain (amino acids 222-230 of SEQ ID NO:26; SEQ ID NO:31). Alternatively, in another embodiment, the TANGO 223 protein contains an extracellular domain at amino acid residues 222 to 230 of SEQ ID NO:26 ( SEQ ID NO:31), a transmembrane domain at amino acid residues 199 to 221 of SEQ ID NO:26 (SEQ ID NO:30), and a cytoplasmic domain at amino acid residues 30 to 198 of SEQ ID NO:26 (SEQ ID NO:29).

Various features of human and mouse TANGO 201 and TANGO 223 are summarized below.

Murine TANGO 201

A cDNA clone, AtmMa41h08, encoding full-length murine TANGO 201 was identified by analysis of EST sequences from a bone marrow stromal cell cDNA library. The cDNA of this clone is 1758 nucleotides long (Figure 1; SEQ ID NO:l). The open reading frame of this cDNA, nucleotides 60 to 1508 of SEQ ID NO:l (SEQ ID NO:2), encodes a 483 amino acid secreted protein (Figure 1; SEQ ID NO:3). It is noted that the nucleotide sequence depicted in SEQ ID NO: 1 contains a Sail adapter sequence on the 5' end (GTCGACCCACGCGTCCG (SEQ ID NO:22)). Thus, it is to be understood that the nucleic acid molecules of the invention include not only those sequences with an adaptor sequence but also the nucleic acid sequences described herein lacking the adaptor sequence. The signal peptide prediction program SIGNALP (Nielsen et al. (1997) Protein Engineering 10:1-6) predicted that murine TANGO 201 includes a 33 amino acid signal peptide (amino acid 1 to about amino acid 33 of SEQ ID NO:3; SEQ ID NO:4) preceding the mature murine TANGO 201 protein (corresponding to about amino acid 34 to amino acid 483 of SEQ ID NO:3; SEQ ID NO:5). Murine TANGO 201 is predicted to have a molecular weight of 54.9 kDa prior to cleavage of its signal peptide and a molecular weight of 51.7 kDa subsequent to cleavage of its signal peptide. The presence of a methionine residue at positions 69, 154, 185, 193, 212, and 449 of SEQ ID NO:3 indicate that there can be alternative forms of murine TANGO 201 of 415 amino acids, 330 amino acids, 299 amino acids, 291 amino acids, 272 amino acids, and 35 amino acids, respectively. In certain embodiments, a TANGO 201 family member has the amino acid sequence of SEQ ID NO:3, and the signal sequence is located at amino acids 1 to 31, 1 to 32, 1 to 33, 1 to 34 or 1 to 35. In such embodiments of the invention, the domains and the mature protein resulting from cleavage of such signal peptides are also included herein. For example, the cleavage of a signal sequence consisting of amino acids 1 to 33 of SEQ ID NO:3 (SEQ ID NO:4) results in a mature TANGO 201 protein corresponding to amino acids 34 to 483 of SEQ ID NO:3 (SEQ ID NO:5). The signal sequence is normally cleaved during processing of the mature protein.

The present invention contemplates mutations, which are either naturally occurring or targeted mutations, in the nucleotide sequence resulting in changes in the polypeptide amino acid sequence. More particularly, mutations can be conservative substitutions of an amino acid or amino acids wherein the resulting polypeptide retains essentially the same functional activity. For example, in one embodiment the TANGO 201 nucleotide at position 65 is a cytosine (C) (SEQ ID NO: 32). In this embodiment, the amino acid at position 2 is aspartate (D) (SEQ ID NO:33). In another embodiment of a nucleotide sequence of mouse TANGO 201, the nucleotide at position 74 is a guanine (G) (SEQ ID NO:34). In this embodiment, the amino acid at position 5 is glutamate (E)(SEQ ID NO:35) In another embodiment of a nucleotide sequence of mouse TANGO 201, the nucleotide at position 81 is a guanine (G) (SEQ ID NO:36). In this embodiment, the amino acid at position 8 is a valine (V) (SEQ ID NO:37). In another embodiment of a nucleotide sequence of mouse TANGO 201, the nucleotide at position 93 is an adenine (A) (SEQ ID NO:38). In this embodiment, the amino acid at position 12 is a isoleucine (I) (SEQ ID NO:39).

In another embodiment of a nucleotide sequence of mouse TANGO 201, the nucleotide at position 124 is a thymidine (T) (SEQ ID NO:40). In this embodiment, the amino acid at position 22 is a phenylalanine (F) (SEQ ID NO:41).

In another embodiment of a nucleotide sequence of mouse TANGO 201, the nucleotide at position 172 is a cytosine (C) (SEQ ID NO:42). In this embodiment, the amino acid at position 38 is a threonine (T) (SEQ ID NO:43).

In another embodiment of a nucleotide sequence of mouse TANGO 201, the nucleotide at position 244 is a guanine (G) (SEQ ID NO:44). In this embodiment, the amino acid at position 62 is an arginine (R) (SEQ ID NO:45).

In another embodiment of a nucleotide sequence of TANGO 201, the nucleotide at position 1092 is a thymidine (T) (SEQ ID NO:l). In this embodiment, the amino acid at position 345 is phenylalanine (F) (SEQ ID NO:3) In another embodiment of a nucleotide sequence of mouse TANGO 201, the nucleotide at position 1092 is a cytosine (C) (SEQ ID NO:46). In this embodiment, the amino acid at position 345 is leucine (L) (SEQ ID NO:47) In another embodiment of a nucleotide sequence of mouse TANGO 201, the nucleotide at position 1092 is adenine (A)(SEQ ID NO:48). In this embodiment, the amino acid at position 345 is a isoleucine (I) (SEQ ID NO:49). In another embodiment of a nucleotide sequence of mouse TANGO 201, the nucleotide at position 1092 is guanine (G)(SEQ ID NO:50). In this embodiment, the amino acid at position 345 is a valine (V) (SEQ ID NO:51).

A glycosaminoglycan attachment site having the sequence SGGG is found from amino acids 28 to 31 of SEQ ID NO:3. A cAMP- and cGMP-dependent protein kinase C (PKC) phosphorylation site having the sequence KKNT is found from amino acids 391 to 394. A PKC phosphorylation site having the sequence SYR is found from amino acids 114 to 116. A second PKC phosphorylation site having the sequence SLK is found from amino acids 200 to 202. A third PKC phosphorylation site having the sequence TLR is found from amino acids 273 to 275. A fourth PKC phosphorylation site having the sequence SAK is found from amino acids 298 to 300. A fifth PKC phosphorylation site having the sequence TAR is found from amino acids 394 to 396. A sixth PKC phosphorylation site having the sequence TVR is found from amino acids 407 to 409. A seventh PKC phosphorylation site having the sequence TDK is found from amino acids 424 to 426. An eighth PKC phosphorylation site having the sequence TVK is found from amino acids 431 to 433.

A casein kinase II (CKII) phosphorylation site having the sequence TSGD is found from amino acids 85 to 88. A second CKII phosphorylation site having the sequence SKHE is found from amino acids 219 to 222. A third CKII phosphorylation site having the sequence SVAE is found from amino acids 225 to 228. A fourth CKII phosphorylation site having the sequence TTCE is found from amino acids 230 to 233. A fifth CKII phosphorylation site having the sequence SAKE is found from amino acids 298 to 301. A sixth CKII phosphorylation site having the sequence TADE is found from amino acids 472 to 475.

An N-myristoylation (N-MRTL) site having the sequence GGLRSL is found from amino acids 6 to 11. A second N-MRTL site having the sequence GLLEAS is found from amino acids 23 to 28. A third N-MRTL site having the sequence GGGRAL is found from amino acids 29 to 34. A fourth N-MRTL site having the sequence GTEFSL is found from amino acids 49 to 54. A fifth N-MRTL site having the sequence GQKVNI is found from amino acids 141 to 146. A sixth N-MRTL site having the sequence GNMLAK is found from amino acids 152 to 157. A seventh N-MRTL site having the sequence GMGNGT is found from amino acids 192 to 197.

Figure 2 depicts a hydropathy plot of murine TANGO 201. Relatively hydrophobic regions are above the horizontal line, and relatively hydrophilic regions are below the horizontal line. The cysteine residues (cys) are indicated by short vertical lines just below the hydropathy trace. The dashed vertical line separates the signal sequence (amino acids 1- 33 of SEQ ID NO:3; SEQ ID NO:4) on the left from the mature protein (amino acids 34- 483 of SEQ ID NO:3; SEQ ID NO:5) on the right.

Human TANGO 201 A cDNA clone, Athbb012c06, encoding human TANGO 201 was identified using mouse TANGO 201 cDNA probes on a pituitary library. The human TANGO 201 clone is 2252 nucleotides long (Figure 3; SEQ ID NO:6). The open reading frame of the cDNA is nucleotides 179 to 1387 of SEQ ID NO:6 (SEQ ID NO:7) which encodes a 403 amino acid protein shown in Figure 3 (SEQ ID NO:8). It is noted that the nucleotide sequence depicted in SEQ ID NO:6 contains Sal I and Not I adapter sequences on the 5' and 3' ends, respectively ((5' GTCGACCCACGCGTCCG 3' (SEQ ID NO:22), and 5' GGGCGGCCGC 3' (SEQ ID NO:23), respectively). Thus, it is to be understood that the nucleic acid molecules of the invention include not only those sequences with such adaptor sequences but also the nucleic acid sequences described herein lacking the adaptor sequences.

The signal peptide prediction program SIGNALP (Nielsen, et al. (1997) Protein Engineering 10:1-6) predicted that human TANGO 201 includes a 33 amino acid signal peptide (amino acid 1 to about amino acid 33 of SEQ ID NO:8; SEQ ID NO:9) preceding the mature human TANGO 201 protein (corresponding to about amino acid 34 to amino acid 403 of SEQ ID NO:8; SEQ ID NO:10). Human TANGO 201 is predicted to have a molecular weight of 45.9 kDa prior to cleavage of its signal peptide and a molecular weight of 42.8 kDa subsequent to cleavage of its signal peptide. The presence of a methionine residue at positions 69, 154, 185, 193, and 212 of SEQ ID NO:8 indicate that there can be alternative forms of human TANGO 201 of 335 amino acids, 250 amino acids, 219 amino acids, 211 amino acids, and 192 amino acids, respectively.

In certain embodiments, a TANGO 201 family member has the amino acid sequence of SEQ ID NO:8, and the signal sequence is located at amino acids 1 to 31, 1 to 32, 1 to 33, 1 to 34 or 1 to 35. In such embodiments of the invention, the domains and the mature protein resulting from cleavage of such signal peptides are also included herein. For example, the cleavage of a signal sequence consisting of amino acids 1 to 33 of SEQ ID NO:8 (SEQ ID NO:9) results in a mature TANGO 201 protein corresponding to amino acids 34 to 403 of SEQ ID NO:8 (SEQ ID NO:9). The signal sequence is normally cleaved during processing of the mature protein.

A glycosaminoglycan attachment site having the sequence SGGG is found from amino acids 28 to 31 of SEQ ID NO:8. A cAMP- and cGMP-dependent protein kinase phosphorylation site having the sequence KKNT is found from amino acids 337 to 340. A protein kinase C (PKC) phosphorylation site having the sequence SYR is found from amino acids 114 to 116. A second PKC phosphorylation site having the sequence SLK is found from amino acids 200 to 202. A third PKC phosphorylation site having the sequence TLR is found from amino acids 273 to 275. A fourth PKC phosphorylation site having the sequence SGK is found from amino acids 317 to 319. A fifth PKC phosphorylation site having the sequence TAR is found from amino acids 340 to 342. A sixth PKC phosphorylation site having the sequence TNR is found from amino acids 353 to 355. A casein kinase II (CKII) phosphorylation site having the sequence TSGD is found from amino acids 85 to 88. A second CKII phosphorylation site having the sequence SKHE is found from amino acids 219 to 222. A third CKII phosphorylation site having the sequence SNAE is found from amino acids 225 to 228. A fourth CKII phosphorylation site having the sequence TTCE is found from amino acids 230 to 233. A fifth CKII phosphorylation site having the sequence TADE is found from amino acids 392 to 395. An Ν- myristoylation (Ν-MRTL) site having the sequence GGNRSL is found from amino acids 6 to 11. A second Ν-MRTL site having the sequence GLLEAS is found from amino acids 23 to 28. A third Ν-MRTL site having the sequence GGGRAL is found from amino acids 29 to 34. A fourth Ν-MRTL site having the sequence GTEFSL is found from amino acids 49 to 54. A fifth Ν-MRTL site having the sequence GQKIΝI is found from amino acids 141 to 146. A sixth Ν-MRTL site having the sequence GΝMLAK is found from amino acids 152 to 157. A seventh Ν-MRTL site having the sequence GMGNGT is found from amino acids 192 to 197. Clone Athbb012c06, which encodes human TANGO 201 , was deposited as a composite deposit with the American Type Culture Collection (10801 University Boulevard, Manassas, VA 20110-2209) on January 22, 1999 and assigned Accession Number 207081. This deposit will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. This deposit was made merely as a convenience for those of skill in the art and is not an admission that a deposit is required under 35 U.S.C. §112.

Figure 4 depicts a hydropathy plot of human TANGO 201. Relatively hydrophobic regions are above the horizontal line, and relatively hydrophilic regions are below the horizontal line. The cysteine residues (cys) are indicated by short vertical lines just below the hydropathy trace. The dashed vertical line separates the signal sequence (amino acids 1- 33 of SEQ ID NO:8; SEQ ID NO:9) on the left from the mature protein (amino acids 34- 403 of SEQ ID NO:8; SEQ ID NO: 10) on the right.

Tissue Distribution of TANGO 201 mRNA Tissue distribution of TANGO 201 mRNA was determined by Northern blot hybridization performed under standard conditions and washed under stringent conditions, i.e., 0.2x SSS at 65°C. RNA from various human tissues were as provided in Multiple Tissue Northern Blots (MTN Blots, Clontech Laboratories, Inc., Palo Alto CA). The results indicated that human TANGO 201 mRNA is expressed in multiple human tissues, including pancreas, testis, adrenal medulla, adrenal cortex, kidney, liver, thyroid, brain, skeletal muscle, placenta, heart, lung, and stomach. The detection of TANGO 201 mRNA in a wide range of human normal tissues suggests that TANGO 201 has an essential cellular function. Two transcripts were observed of approximately 2.0 and 2.5 kb, consistent with the suggestion of alternative splicing raised by the sequence alignment. Furthermore, the ratios of these two forms differs among the tissues. For example, the 2.0 kb transcript predominates in adrenal medulla whereas the 2.5 kb form predominates in thyroid. This suggests tissue specific expression of spliced forms of human TANGO 201.

In situ tissue screening was performed on mouse adult and embryonic tissue to analyze the expression of mouse TANGO 201 mRNA. Expression in the adult mouse was not detected in any tissues tested.

Similarities Between Murine and Human TANGO 201 and to Other Sequences

An alignment of the nucleotide sequences of murine TANGO 201 (nucleotides 1- 1758 of SEQ ID NO:l) and human TANGO 201 (nucleotides 101-1660 of SEQ ID NO:6) using the program GAP (Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453) in GCG (Wisconsin Package Version 9.1, Genetics Computer Group, Madison WI) with a score matrix of nwsgapdna, a gap penalty 50, and a gap extension penalty 3 resulted in an identity of 84.8%o. The mouse sequence differs from the human sequence by the presence of two inserted sequences. The first is a 162 base insertion from nucleotide 938 to 1100 of SEQ ID NO:l and the second is 78 bases from nucleotide 1286 to 1363 of SEQ ID NO:l. This alignment is shown in Figures 5A-5C.

The amino acid sequences of murine TANGO 201 (amino acids 1-483 of SEQ ID NO:3) and human TANGO 201 (amino acids 1-403 of SEQ ID NO:8) were aligned and analyzed using the program GAP (Needleman and Wunsch (1970) J. Mol. Biol. 48:443- 453) in GCG (Wisconsin Package Version 9.1, Genetics Computer Group, Madison WI). An identity of 97%> was seen in which the program settings were a score matrix of blosum 62, a gap penalty 12, and a gap extension penalty 4. The mouse sequence differs from the human sequence by the presence of two inserted sequences. The first is a 54 residue insertion from amino acid 294 to 347 of SEQ ID NO:3 and the second is 26 residues from amino acid 410 to 435 of SEQ ID NO:3. This alignment is shown in Figure 6. In one embodiment, the invention contemplates alternative splicing of the mRNA encoding a TANGO 201 protein. For example, one embodiment of the invention includes a human TANGO 201 nucleotide sequence further comprising exons which encode for a polypeptide sequence which is similar to the mouse TANGO 201 polypeptide sequence between amino acids 294 to 247 and 410 to 435 of SEQ ID NO:3, in the same relative position of the polypeptide. Further, the invention also features splicing of the mouse TANGO 201, that is, mouse TANGO 201 is alternatively spliced so that the mRNA encoding the polypeptide has deletions corresponding to amino acids 294 to 247 and 410 to 435 of SEQ ID NO:3.

Murine and human TANGO 201 show homology to OS-9, a putative secreted human protein believed to be involved in cell growth (Su, et al, (1966) Mol. Carcinogenesis 15:270-275; Kimura, et al, (1998) J. Biochem. 123:876-882). Figure 7 depicts an alignment of a portion of murine TANGO 201 amino acid sequence (amino acids 78 to 264 of SEQ ID NO:3) and a portion of human TANGO 201 amino acid sequence (amino acids 78 to 264 of SEQ ID NO:8) with a portion of OS-9 (amino acids 73 to 250 of SwissProt Accession No. Q 13438). The alignment reveals that the homology is restricted to the N-terminus in which a conserved cysteine-rich domain as defined below is found. The conserved cysteine residues are highlighted in boldface type.

An alignment of human or mouse TANGO 201 with the above-described portion of the OS-9 protein sequence (Q13438) reveals 39.0%> identity between human TANGO 201 and OS-9, and 42.2% identity between mouse TANGO 201 and OS-9. This alignment was performed using the ALIGN alignment program with a BLOSUM62 scoring matrix, a gap length penalty of 10, and a gap penalty of 0.05.

As used herein, a cysteine-rich domain of a TANGO 201 polypeptide includes about 140-215 amino acid residues, preferably about 150-205 amino acid residues, more preferably about 155-200 amino acid residues, and most preferably about 165-190 amino acid residues. Typically, a cysteine-rich domain is found at the N-terminal half of TANGO 201 and includes a cluster of about 4-12 cysteine residues conserved in TANGO 201 protein family members, more preferably about 6-10 cysteine residues, and still more preferably about 8 cysteine residues. In addition, a cysteine-rich domain includes at least the following consensus sequence: C-Xaa(nl)-C-Xaa(n2)-C-Xaa(n3)-C-Xaa(n4)-C-Xaa(n4)-C- Xaa(n2)-C-Xaa(n4)-C, wherein C is a cysteine residue, Xaa is any amino acid, nl is about 20-50 amino acid residues, more preferably about 25-45 amino acid residues, and more preferably 30-40 amino acid residues in length, n2 is about 2-20 amino acid residues, more preferably 5-15 amino acid residues, and more preferably 11-12 amino acid residues in length, n3 is about 40-90 amino acid residues, more preferably about 50-80 amino acid residues, and more preferably 55-75 amino acid residues in length, and n4 is about 5-25 amino acid residues, more preferably 8-20 amino acid residues, and more preferably 13-21 amino acid residues in length.

In one embodiment, a TANGO 201 family member includes a cysteine-rich domain having an amino acid sequence that is at least about 55%>, preferably at least about 65%>, more preferably at least about 75%, yet more preferably at least about 85 >, and most preferably at least about 95% identical to amino acids 79 to 261 of SEQ ID NO:3 (SEQ ID NO: 19), to amino acids 79 to 261 of SEQ ID NO: 8 (SEQ ID NO:20) or to amino acids 68 to 178 of SEQ ID NO: 13 (SEQ ID NO:21), which are the cysteine-rich domains of murine and human TANGO 201, respectively.

In another embodiment, a TANGO 201 family member includes a cysteine-rich

5 domain having an amino acid sequence that is at least about 55%, preferably at least about 65%, more preferably at least about 75%>, yet more preferably at least about 85%>, and most preferably at least about 95% identical to amino acids 79 to 261 of SEQ ID NO:3 or SEQ ID NO:8 (SEQ ID NO: 19 or SEQ ID NO:20, respectively), includes a conserved cluster of 8 cysteine residues, and a cysteine-rich domain consensus sequence as described herein. In

10 yet another embodiment, a TANGO 201 family member includes a cysteine-rich domain having an amino acid sequence that is at least 55%, preferably at least about 65%>, more preferably at least about 75%, yet more preferably at least about 85%), and most preferably at least about 95% identical to amino acids 79 to 261 of SEQ ID NO:3 (SEQ ID NO: 19) or SEQ ID NO:8 (SEQ ID NO:20), includes a conserved cluster of 8 cysteine residues, a

15 cysteine-rich consensus sequence as described herein, and has at least one TANGO 201 biological activity as described herein.

In a preferred embodiment, a TANGO 201 family member has the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:8 wherein the cluster of conserved cysteine residues is located within amino acid residues 79 to 261 (at positions 79, 113, 126, 199,

20 215, 232, 244, and 261 of SEQ ID NO:3 and SEQ ID NO:8), and the cysteine-rich domain consensus sequence is located at amino acid residues 79 to 261.

Uses of TANGO 201 Nucleic Acids. Polypeptides. and Modulators Thereof

TANGO 201 polypeptides, nucleic acids, and modulators thereof, can be used to 25 modulate the function, morphology, proliferation and/or differentiation of cells in the tissues in which it is expressed. Such molecules can be used to treat disorders associated with abnormal or aberrant metabolism or function of cells in the tissues in which it is expressed. Tissues in which TANGO 201 is expressed include, for example, pancreas, adrenal medulla, adrenal cortex, kidney, thyroid, testis, stomach, heart, brain, liver, 30 placenta, lung, skeletal muscle, or small intestine.

For example, such molecules can be used to treat proliferative disorders, i.e., neoplasms or tumors (e.g., a carcinoma, a sarcoma, adenoma, or myeloid leukemia).

In another example, TANGO 201 polypeptides, nucleic acids, or modulators thereof, can be used to treat pancreatic disorders, such as pancreatitis (e.g., acute hemorrhagic 35 pancreatitis and chronic pancreatitis), pancreatic cysts (e.g., congenital cysts, pseudocysts, and benign or malignant neoplastic cysts), pancreatic tumors (e.g., pancreatic carcinoma and adenoma), diabetes mellitus (e.g. , insulin- and non-insulin-dependent types, impaired glucose tolerance, and gestational diabetes), or islet cell tumors (e.g., insulinomas, adenomas, Zollinger-Ellison syndrome, glucagonomas, and somatostatinoma).

In another example, TANGO 201 polypeptides, nucleic acids, or modulators thereof, can be used to treat disorders of the adrenal cortex, such as hypoadrenalism (e.g., primary chronic or acute adrenocortical insufficiency, and secondary adrenocortical insufficiency), hyperadrenalism (Cushing's syndrome, primary hyperaldosteronism, adrenal virilism, and adrenal hyperplasia), or neoplasia (e.g., adrenal adenoma and cortical carcinoma).

In another example, TANGO 201 polypeptides, nucleic acids, or modulators thereof, can be used to treat disorders of the adrenal medulla, such as neoplasms (e.g., pheochromocytomas, neuroblastomas, and ganglioneuromas).

In another example, TANGO 201 polypeptides, nucleic acids, or modulators thereof, can be used to treat disorders of the thyroid gland, such as hyperthyroidism (e.g., diffuse toxic hyperplasia, toxic multinodular goiter, toxic adenoma, and acute or subacute thyroiditis), hypothyroidism (e.g., cretinism and myxedema), thyroiditis (e.g., Hashimoto's thyroiditis, subacute granulomatous thyroiditis, subacute lymphocytic thyroiditis, Riedel's thryroiditis), Graves' disease, goiter (e.g., simple diffuse goiter and multinodular goiter), or tumors (e.g., adenoma, papillary carcinoma, follicular carcinoma, medullary carcinoma, undifferentiated malignant carcinoma, Hodgkin's disease, and non-Hodgkin's lymphoma). In another example, TANGO 201 polypeptides, nucleic acids, or modulators thereof, can be used to treat gastric disorders, such as congenital anamolies (e.g. , diaphragmatic hernias, pyloric stenosis, gastric diverticula, and gastric dilatation), gastritis (e.g., acute mucosal inflammation, chronic fundal gastritis, chronic antral gastritis, hypertrophic gastritis, granulomatous gastritis, eosinophilic gastritis), ulcerations (e.g., peptic ulcers, gastric ulcers, and duodenal ulcers), or tumors (e.g., benign polyps, malignant carcinoma, argentaffmomas, carcinoids, gastrointestinal lymphomas, carcomas, and metastatic carcinoma).

In another example, TANGO 201 polypeptides, nucleic acids, or modulators thereof, can be used to treat placental disorders, such as toxemia of pregnancy (e.g., preeclampsia and eclampsia), placentitis, or spontaneous abortion.

In another example, TANGO 201 polypeptides, nucleic acids, or modulators thereof, can be used to treat pulmonary disorders, such as atelectasis, pulmonary congestion or edema, chronic obstructive airway disease (e.g., emphysema, chronic bronchitis, bronchial asthma, and bronchi ectasis), diffuse interstitial diseases (e.g., sarcoidosis, pneumoconiosis, hypersensitivity pneumonitis, Goodpasture's syndrome, idiopathic pulmonary hemosiderosis, pulmonary alveolar proteinosis, desquamative interstitial pneumonitis, chronic interstitial pneumonia, fibrosing alveolitis, hamman-rich syndrome, pulmonary eosinophilia, diffuse interstitial fibrosis, Wegener's granulomatosis, lymphomatoid granulomatosis, and lipid pneumonia), or tumors (e.g., bronchogenic carcinoma, bronchio- alveolar carcinoma, bronchial carcinoid, and mesenchymal tumors). In another example, TANGO 201 polypeptides, nucleic acids, or modulators thereof, can be used to treat disorders of skeletal muscle, such as 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, Debrancher enzyme deficiency, mitochondrial myopathy, carnitine deficiency, carnitine palmityl transferase deficiency, phosphoglycerate kinase deficiency, phosphoglycerate mutase deficiency, lactate dehydrogenase deficiency, and myoadenylate deaminase deficiency). In another example, TANGO 201 polypeptides, nucleic acids, or 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), or myocardial disease (e.g., myocarditis, congestive cardiomyopathy, and hypertrophic cariomyopathy).

In another example, TANGO 201 polypeptides, nucleic acids, or modulators thereof, can be used to treat disorders of the brain, such as cerebral edema, hydrocephalus, brain hemiations, iatrogenic disease (due to, e.g., infection, toxins, or drugs), inflammations (e.g., bacterial and viral meningitis, encephalitis, and cerebral toxoplasmosis), cerebrovascular diseases (e.g., hypoxia, ischemia, and infarction, intracranial hemoπhage and vascular malformations, and hypertensive encephalopathy), and tumors (e.g., neuroglial tumors, neuronal tumors, tumors of pineal cells, meningeal tumors, primary and secondary lymphomas, intracranial tumors, and meduUoblastoma), and to treat injury or trauma to the brain. In another example, TANGO 201 polypeptides, nucleic acids, or modulators thereof, can be used to treat hepatic disorders, such as jaundice, hepatic failure, hereditary hyperbihruinemias (e.g., Gilbert's syndrome, Crigler-Naijar syndromes and Dubin- Johnson and Rotor's syndromes), hepatic circulatory disorders (e.g., hepatic vein thrombosis and portal vein obstruction and thrombosis) hepatitis (e.g., chronic active hepatitis, acute viral hepatitis, and toxic and drug-induced hepatitis) cirrhosis (e.g., alcoholic crrrhosis, biliary cirrhosis, and hemochromatosis), or malignant tumors (e.g., primary carcinoma, hepatoblastoma, and angiosarcoma).

In another example, TANGO 201 polypeptides, nucleic acids, or modulators thereof, can be used to treat renal disorders, such as glomerular diseases (e.g., acute and chronic glomerulonephritis, rapidly progressive glomerulonephritis, nephrotic syndrome, focal proliferative glomerulonephritis, glomerular lesions associated with systemic disease, such as systemic lupus erythematosus, Goodpasture's syndrome, multiple myeloma, diabetes, neoplasia, sickle cell disease, and chronic inflammatory diseases), tubular diseases (e.g. , acute tubular necrosis and acute renal failure, polycystic renal diseasemedullary sponge kidney, medullary cystic disease, nephrogenic diabetes, and renal tubular acidosis), tubulointerstitial diseases (e.g., pyelonephritis, drug and toxin induced tubulointerstitial nephritis, hypercalcemic nephropathy, and hypokalemic nephropathy) acute and rapidly progressive renal failure, chronic renal failure, nephrolithiasis, vascular diseases (e.g., hypertension and nephrosclerosis, microangiopathic hemolytic anemia, atheroembolic renal disease, diffuse cortical necrosis, and renal infarcts), or tumors (e.g. , renal cell carcinoma and nephroblastoma).

In another example, TANGO 201 polypeptides, nucleic acids, or modulators thereof, can be used to treat testicular disorders, such as unilateral testicular enlargement (e.g., nontuberculous, 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).

In another example, TANGO 201 polypeptides, nucleic acids, or modulators thereof, can be used to treat intestinal disorders, such as ischemic bowel disease, infective enterocolitis, Crohn's disease, benign tumors, malignant tumors (e.g., argentaffmomas, lymphomas, adenocarcinomas, and sarcomas), malabsorption syndromes (e.g., celiac disease, tropical sprue, Whipple's disease, and abetalipoproteinemia), obstructive lesions, hernias, intestinal adhesions, intussusception, or volvulus. Human TANGO 223

A clone, Athua075b02, encoding full-length human TANGO 223 was identified by use of a partial clone encoding a signal peptide and obtained by use of a yeast signal trap method. This methodology, described, for example, in WO99/24616 dated May 20, 1999, takes advantage of the fact that molecules such as TANGO 223 have an amino terminal signal sequence that directs certain secreted and membrane-bound proteins through the cellular secretory apparatus.

Briefly, a cDNA library from human fetal kidney was prepared in pBOSSl and transformed into the yeast strain Yscreen2 as described in WO99/24616. CDNA inserts of plasmids rescued from the resulting yeast colonies after selection on glucose were sequenced. The initial signal trap clone obtained, ZmhKy398, was shown to encode a 29 amino acid signal peptide, followed by a 13 amino acid open reading frame. This clone was then fused to a yeast KRE9 gene lacking a functional signal sequence and used to search proprietary databases for a full length clone. A clone representing an extension of the initial signal sequence positive clone was identified in a human fetal lung library. The cDNA of this clone is 1473 nucleotides long (Figure 6; SEQ ID NO:l 1). The open reading frame of this cDNA, nucleotides 30 to 770 of SEQ ID NO:l 1 (SEQ ID NO: 12), encodes a 247 amino acid protein (Figures 8; SEQ ID NO: 13). TANGO 223 is predicted to be a transmembrane protein having a 186 amino acid extracellular domain (amino acids 30-215 of SEQ ID NO:13; SEQ ID NO: 16), a single 23 amino acid transmembrane domain (amino acids 216-238 of SEQ ID NO: 13; SEQ ID NO: 17), and a nine amino acid cytoplasmic domain (amino acids 239-247 of SEQ ID NO: 13; SEQ ID NO: 18). Alternatively, in another embodiment, the TANGO 223 protein contains an extracellular domain at amino acid residues 239 to 247 of SEQ ID NO: 13 ( SEQ ID NO:18), a transmembrane domain at amino acid residues 216 to 238 of SEQ ID NO:13 (SEQ ID NO: 17), and a cytoplasmic domain at amino acid residues 30 to 215 of SEQ ID NO: 13 (SEQ ID NO: 16). In addition, there are 15 cysteines in the extracellular domain at positions 68, 74, 81, 84, 90, 100, 108, 125, 128, 138, 144, 149, 158, 166, and 178 and two in the signal peptide sequence at positions 15 and 25 of SEQ ID NO: 13.

The signal peptide prediction program SIGN ALP (Nielsen et al. (1997) Protein Engineering 10:1-6) predicted that TANGO 223 includes a 29 amino acid signal peptide (amino acid 1 to about amino acid 29 of SEQ ID NO: 13; SEQ ID NO: 14) preceding the mature TANGO 223 protein (coπesponding to about amino acid 30 to amino acid 247 of SEQ ID NO: 13 ; SEQ ID NO: 15). Human TANGO 223 is predicted to have a molecular weight of 27.2 kDa prior to cleavage of its signal peptide and a molecular weight of 24 kDa subsequent to cleavage of its signal peptide. The presence of a methionine residue at positions 66, 123, 145, and 175 of SEQ ID NO: 13 indicate that there can be alternative forms of TANGO 223 of 182 amino acids, 125 amino acids, 103 amino acids, and 73 amino acids, respectively. In certain embodiments, a TANGO 223 family member has the amino acid sequence of SEQ ID NO:3, and the signal sequence is located at amino acids 1 to 27, 1 to 28, 1 to 29, 1 to 30 or 1 to 31. In such embodiments of the invention, the domains and the mature protein resulting from cleavage of such signal peptides are also included herein. For example, the cleavage of a signal sequence consisting of amino acids 1 to 29 of SEQ ID NO:13 (SEQ ID NO:14) results in a mature TANGO 223 protein corresponding to amino acids 30 to 247 of SEQ ID NO: 13 (SEQ ID NO: 15). The signal peptide sequence is normally cleaved during processing of the mature protein.

In one embodiment of a nucleotide sequence of human TANGO 223, the nucleotide at position 98 is guanine (G)(SEQ ID NO:l 1). In this embodiment, the amino acid at position 57 is a glutamate (E) (SEQ ID NO: 13). In another embodiment of a nucleotide sequence of human TANGO 223, the nucleotide at position 98 is a thymidine (T) (SEQ ID NO:52). In this embodiment, the amino acid at position 57 is stop codon resulting in a truncated protein of 57 amino acids length (SEQ ID NO:53) In another embodiment of a nucleotide sequence of human TANGO 223, the nucleotide at position 98 is a cytosine (C) (SEQ ID NO:54). In this embodiment, the amino acid at position 57 is glutamine (Q) (SEQ ID NO: 55) In another embodiment of a nucleotide sequence of human TANGO 223, the nucleotide at position 98 is adenine (A)(SEQ ID NO:56). In this embodiment, the amino acid at position 57 is a lysine (K) (SEQ ID NO:57).

An N-glycosylation (N-GCL) site having the sequence NFSC is found from amino acids 87 to 90 of SEQ ID NO: 13. A second N-GCL site having the sequence NMTC is found from amino acids 122 to 125. A third N-GCL site having the sequence NSTS is found from amino acids 140 to 143. A fourth N-GCL site having the sequence NCTV is found from amino acids 157 to 160. A fifth N-GCL site having the sequence NRTF is found from amino acids 169 to 172. A sixth N-GCL site having the sequence NWTG is found from amino acids 179 to 182. A protein kinase C (PKC) phosphorylation site having the sequence SIK is found from amino acids 39 to 41. A second PKC phosphorylation site having the sequence SQK is found from amino acids 115 to 117. A third PKC phosphorylation site having the sequence TCR is found from amino acids 124 to 126. A fourth PKC phosphorylation site having the sequence TNR is found from amino acids 159 to 161. A casein kinase II (CKII) phosphorylation site having the sequence SGGE is found from amino acids 28 to 31. A second CKII phosphorylation site having the sequence SIKD is found from amino acids 39 to 42. A third CKII phosphorylation site having the sequence TCVD is found from amino acids 107 to 110. A fourth CKII phosphorylation site having the sequence TYDE is found from amino acids 134 to 137. A fifth CKII phosphorylation site having the sequence TVRD is found from amino acids 159 to 162. A sixth CKII

5 phosphorylation site having the sequence TLID is found from amino acids 226 to 229. An N-myristoylation site having the sequence GGEQSQ is found from amino acids 29 to 34. A second N-myristoylation site having the sequence GGFGAD is found from amino acids 197 to 202.

Figure 9 depicts a hydropathy plot of TANGO 223. Relatively hydrophobic regions

10 are above the horizontal line, and relatively hydrophilic regions are below the horizontal line. The cysteine residues (cys) and potential N-glycosylation sites (Ngly) are indicated by short vertical lines just below the hydropathy trace. The dashed vertical line separates the signal sequence (amino acids 1-29 of SEQ ID NO: 13; SEQ ID NO: 14) on the left from the mature protein (amino acids 30-247 of SEQ ID NO:13; SEQ ID NO: 15) on the right.

15 The human TANGO 223 gene was mapped on radiation hybrid panels to chromosome 15, in the region q26. Flanking markers for this region are WI-3162 and WI-4919. The OTS (otosclerosis) locus also maps to this region of the human chromosome. The ALDH6 (aldehyde dehydrogenase 6), CHRM5 (cholinergic receptor), STX (sialyltransferase X), and IDDM3 (insulin-dependent diabetes mellitus 3) genes also

20 map to this region of the human chromosome. This region is syntenic to mouse chromosome 7. The tp (taupe) locus also maps to this region of the mouse chromosome. The age (shhtrvsn), hf (hepatic fusion), sur (sulfonylurea receptor), and fah (fumarylacetoacetate hyrdrolase) genes also map to this region of the mouse chromosome. Clone Athua075b02, which encodes TANGO 223, was deposited as a composite

25 deposit with the American Type Culture Collection (10801 University Boulevard,

Manassas, VA 20110-2209) on January 22, 1999 and assigned Accession Number 207081. This deposit will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. This deposit was made merely as a convenience for those of skill in the art and is not an

30 admission that a deposit is required under 35 U.S.C. § 112.

Mouse TANGO 223

A mouse TANGO 223 clone, Aompa001h06, was identified using the cDNA of the human TANGO 223 as a probe in a screen of a mouse pancreatic library. Mouse TANGO 35 223 is 854 nucleotides long (Figure 11 ; SEQ ID NO:24). The open reading frame of this cDNA, nucleotides 5 to 694 of SEQ ID NO:24 (SEQ ID NO:25), encodes a 230 amino acid protein (Figures 12; SEQ ID NO:26).

The signal peptide prediction program SIGNALP (Nielsen et al. (1997) Protein Engineering 10:1-6) predicted that TANGO 223 includes a 29 amino acid signal peptide (amino acid 1 to about amino acid 29 of SEQ ID NO:26; SEQ ID NO:27) preceding the mature TANGO 223 protein (corresponding to about amino acid 30 to amino acid 230 of SEQ ID NO:26; SEQ ID NO:28). Murine TANGO 223 is predicted to have a molecular weight of 25.6 kDa prior to cleavage of its signal peptide and a molecular weight of 22.4 kDa subsequent to cleavage of its signal peptide. The presence of a methionine residue at positions 48, 106 and 128 of SEQ ID NO:26 indicate that there can be alternative forms of TANGO 223 of 183 amino acids, 125 amino acids and 103 amino acids, respectively.

In certain embodiments, a TANGO 223 family member has the amino acid sequence of SEQ ID NO:26, and the signal sequence is located at amino acids 1 to 27, 1 to 28, 1 to 29, 1 to 30 or 1 to 31. In such embodiments of the invention, the domains and the mature protein resulting from cleavage of such signal peptides are also included herein. For example, the cleavage of a signal sequence consisting of amino acids 1 to 29 of SEQ ID NO:26 (SEQ ID NO:27) results in a mature TANGO 223 protein corresponding to amino acids 30 to 247 of SEQ ID NO:26 (SEQ ID NO:28). The signal peptide sequence is normally cleaved during processing of the mature protein. TANGO 223 is predicted to be a transmembrane protein having a 169 amino acid extracellular domain (amino acids 30-198 of SEQ ID NO:26; SEQ ID NO:29), a single 23 amino acid transmembrane domain (amino acids 199-221 of SEQ ID NO:26; SEQ ID NO:30), and a nine amino acid cytoplasmic domain (amino acids 222-230 of SEQ ID NO:26; SEQ ID NO:31). Alternatively, in another embodiment, the TANGO 223 protein contains an extracellular domain at amino acid residues 222 to 230 of SEQ ID NO:26 ( SEQ ID NO:31), a transmembrane domain at amino acid residues 199 to 221 of SEQ ID NO:26 (SEQ ID NO:30), and a cytoplasmic domain at amino acid residues 30 to 198 of SEQ ID NO:26 (SEQ ID NO:29). There are 14 cysteines in the extracellular domain at positions 51, 64, 67, 73, 83, 91, 108, 111, 121, 127, 132, 141, 149 and 161 and one in the signal peptide sequence at position 24 of SEQ ID NO:24.

An N-glycosylation (N-GCL) site having the sequence NVSC is found in TANGO 223 from amino acids 70 to 73 of SEQ ID NO:24. A second N-GCL site having the sequence NMTC is found from amino acids 105 to 108. A third N-GCL site having the sequence NSTT is found from amino acids 123 to 126. A fourth N-GCL site having the sequence NCTV is found from amino acids 140 to 143. A fifth N-GCL site having the sequence NRTF is found from amino acids 152 to 155. A sixth N-GCL site having the sequence NWTG is found from amino acids 162 to 165.

A protein kinase C (PKC) phosphorylation site having the sequence SVR is found from amino acids 10 to 12. A second PKC phosphorylation site having the sequence TVK 5 is found from amino acids 84 to 86. A third PKC phosphorylation site having the sequence TCR is found from amino acids 107 to 109. A fourth PKC phosphorylation site having the sequence TVR is found from amino acids 142 to 144.

A casein kinase II (CKII) phosphorylation site having the sequence SGDE is found from amino acids 28 to 31. A second CKII phosphorylation site having the sequence 10 TCVD is found from amino acids 90 to 93. A third CKII phosphorylation site having the sequence TDYE is found from amino acids 117 to 120. A fourth CKII phosphorylation site having the sequence TVRD is found from amino acids 142 to 145. A fifth CKII phosphorylation site having the sequence TLID is found from amino acids 209 to 212.

An N-myristoylation site having the sequence GGFGAD is found from amino acids 15 180 to 185.

Tissue Distribution of TANGO 223 mRNA

Tissue distribution of TANGO 223 mRNA was determined by Northern blot hybridization performed under standard conditions and washed under stringent conditions, 0 i.e., .2x SSS at 65°C. RNA from various human and murine tissues were as provided in

Multiple Tissue Northern Blots (MTN Blots, Clontech Laboratories, Inc., Palo Alto CA). TANGO 223 is expressed in multiple human tissues and hybridizes to nucleic acids in murine tissues, including heart, brain, liver, kidney, testis, prostate, ovary, small intestine, colon, and peripheral blood leukocytes. TANGO 223 mRNA has highest 5 expression in adult brain and the submandibular gland. Expression was also observed in the testes in a pattern that outlined the seminiferous vesicles. A single transcript of approximately 1 kb was detected in these tissues. The detection of TANGO 223 mRNA in a wide range of normal tissues suggests that TANGO 223 has an essential cellular function.

Embryonic mouse tissues also had a ubiquitous signal. 0 In situ tissue screening was performed on mouse adult and embryonic tissue to analyze the expression of TANGO 223 mRNA.

In the case of adult expression, the following results were obtained: For the testis, a signal outlining some seminiferous tubules was detected. In the placenta, a signal was very weak. In the ovaries, a very weak signal was detected. A weak signal was detected from 5 the adrenal gland. A moderate, ubiquitous signal was detected in the submandibular gland.

A moderate signal was detected in the brain. A weak signal was detected in the spinal cord. A weak signal was detected in the lymph node. And a moderate signal was observed in the stomach. No signal was detected in the following tissues: eye and harderian gland, white and brown fat, heart, lung, liver, kidney, colon, small intestine, thymus, spleen, pancreas, skeletal muscle, and bladder.

5 Embryonic expression was seen in a number of tissues. The highest expressing tissue was the brain and spinal cord which was seen at El 3.5 and continues to PI.5. At El 5.5, the strongest signal observed was in the brain, spinal cord, lung and kidney. At El 6.5, the signal was the same as in El 5.5. At El 8.5, the signal is highest in the brain, spinal cord, eye and submaxillary gland and kidney. At PI.5, the signal pattern is identical

10 to E18.5.

Similarity of TANGO 223 to Other Polypeptides

The orientation of the N-terminus toward the extracellular domain indicates TANGO 223 as being a type I transmembrane protein. A BLASTp search (version

15 1.4.10MP-WashU, Altschul, et al, (1990) J. Mol. Biol 215:403-410) of the amino acid sequence of TANGO 223 revealed similarity to two Caenorhabditis elegans proteins. One protein, Swiss-Prot accession number O01975 and gene name C41D11.5, is a putative 85.1 kDa nuclease belonging to the family of DNA/RNA nonspecific endonucleases. However, the domain characteristic of this family of proteins is not seen in TANGO 223. Another

20 protein, Swiss-Prot accession number P34280 and gene name C02F5.3, is a putative 64.3 kd GTP-binding protein in chromosome III belonging to the GTP1/OBG family.

TANGO 223 contains a cysteine-rich domain in which multiple N-glycosylation sites are also present. A homologous cysteine-rich domain is found in the polypeptide sequence of SwissProt O01975. Figure 10 depicts an alignment of a portion of human

25 TANGO 223 amino acid sequence (amino acids 83 to 178 of SEQ ID NO: 13) with amino acids 258 to 376 of SwissProt O01975. The conserved cysteine residues are highlighted in boldface type. A double dot between two residues indicates a complete identity, and a single dot indicates a conservative substitution.

Human TANGO 223 aligned with SwissProt O01975 reveals a sequence identity of

30 37.5%o over a portion polypeptides corresponding to amino acids 82 to 180 of SEQ ID NO: 13. This alignment was performed using the ALIGN alignment program with a BLOSUM62 scoring matrix, a gap length penalty of 10, and a gap penalty of 0.05.

As used herein, a cysteine-rich domain of a TANGO 223 polypeptide includes about 60-140 amino acid residues, preferably about 70-130 amino acid residues, more preferably

35 about 80-120 amino acid residues, and most preferably about 95-105 amino acid residues. Typically, a cysteine-rich domain includes a cluster of about 5-25 cysteine residues conserved in TANGO 223 protein family members, more preferably about 10-18 cysteine residues, and still more preferably about 15 cysteine residues. In addition, a cysteine-rich domain includes at least the following consensus sequence: C-Xaa(nl)-C-Xaa(nl)-C- Xaa(n4)-C-Xaa(nl)-C-Xaa(nl)-C-Xaa(n2)-C-Xaa(nl)-C-Xaa(n3)-C-Xaa(n4)-C-Xaa(n2)-C- Xaa(nl)-C-Xaa(n3)-C-Xaa(nl)-C-Xaa(n3)-C, wherein C is a cysteine residue, Xaa is any amino acid, nl is about 2-12 amino acid residues, more preferably about 3-10 amino acid residues, and more preferably 4-8 amino acid residues in length, n2 is about 5-15 amino acid residues, more preferably 7-12 amino acid residues, and more preferably 9-10 amino acid residues in length, n3 is about 6-22 amino acid residues, more preferably about 8-20 amino acid residues, and more preferably 10-17 amino acid residues in length, and n4 is about 1-7 amino acid residues, more preferably 1-5 amino acid residues, and more preferably 2-3 amino acid residues in length. In one embodiment, a TANGO 223 family member includes a cysteine-rich domain having an amino acid sequence that is at least about 55%>, preferably at least about 65%>, more preferably at least about 75%, yet more preferably at least about 85%>, and most preferably at least about 95%> identical to amino acids 68 to 178 of SEQ ID NO: 13 (SEQ ID NO:20), which is the cysteine-rich domain of TANGO 223. In another embodiment, a TANGO 223 family member includes a cysteine- rich domain having an amino acid sequence that is at least about 55%>, preferably at least about 65%o, more preferably at least about 75%, yet more preferably at least about 85%>, and most preferably at least about 95% identical to amino acids 68 to 178 of SEQ ID NO: 13, includes a conserved cluster of 15 cysteine residues, and a cysteine-rich domain consensus sequence as described herein. In yet another embodiment, a TANGO 223 family member includes a cysteine-rich domain having an amino acid sequence that is at least 55%>, preferably at least about 65%, more preferably at least about 75%, yet more preferably at least about 85%>, and most preferably at least about 95%> identical to amino acids 68 to 178 of SEQ ID NO: 13 (SEQ ID NO:20), includes a conserved cluster of 15 cysteine residues, a cysteine-rich consensus sequence as described herein, and has at least one TANGO 223 biological activity as described herein.

In a preferred embodiment, a TANGO 223 family member has the amino acid sequence of SEQ ID NO: 13 wherein the cluster of conserved cysteine residues is located within amino acid residues 68 to 178 (at positions 68,74, 81, 84, 90, 100, 108, 125, 128, 138, 144, 149, 158, 166, and 178), and the cysteine-rich domain consensus sequence is located at amino acid residue 68 to amino acid residue 178.

Uses of TANGO 223 Nucleic Acids. Polypeptides and Modulators Thereof TANGO 223 polypeptides, nucleic acids, and modulators thereof, can be used to modulate the function, morphology, proliferation and/or differentiation of cells in the tissues in which it is expressed. Such molecules can be used to treat disorders associated with abnormal or aberrant metabolism or function of cells in the tissues in which it is expressed. Tissues in which TANGO 223 is expressed include, for example, heart, brain, liver, kidney, testis, prostate, ovary, small intestine, colon, and peripheral blood leukocytes.

In one example, TANGO 223 polypeptides, nucleic acids, or 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), or myocardial disease (e.g., myocarditis, congestive cardiomyopathy, and hypertrophic cariomyopathy). In another example, TANGO 223 polypeptides, nucleic acids, or modulators thereof, can be used to treat disorders of the brain, such as cerebral edema, hydrocephalus, brain hemiations, iatrogenic disease (due to, e.g., infection, toxins, or drugs), inflammations (e.g., bacterial and viral meningitis, encephalitis, and cerebral toxoplasmosis), cerebrovascular diseases (e.g., hypoxia, ischemia, and infarction, intracranial hemoπhage and vascular malformations, and hypertensive encephalopathy), and tumors (e.g., neuroglial tumors, neuronal tumors, tumors of pineal cells, meningeal tumors, primary and secondary lymphomas, intracranial tumors, and meduUoblastoma), and to treat injury or trauma to the brain.

In another example, TANGO 223 polypeptides, nucleic acids, or modulators thereof, can be used to treat hepatic disorders, such as jaundice, hepatic failure, hereditary hyperbihruinemias (e.g., Gilbert's syndrome, Crigler-Naijar syndromes and Dubin- Johnson and Rotor's syndromes), hepatic circulatory disorders (e.g., hepatic vein thrombosis and portal vein obstruction and thrombosis) hepatitis (e.g., chronic active hepatitis, acute viral hepatitis, and toxic and drug-induced hepatitis) cirrhosis (e.g. , alcoholic cirrhosis, biliary cirrhosis, and hemochromatosis), or malignant tumors (e.g., primary carcinoma, hepatoblastoma, and angiosarcoma).

In another example, TANGO 223 polypeptides, nucleic acids, or modulators thereof, can be used to treat renal disorders, such as glomerular diseases (e.g., acute and chronic glomerulonephritis, rapidly progressive glomerulonephritis, nephrotic syndrome, focal prohferative glomerulonephritis, glomerular lesions associated with systemic disease, such as systemic lupus erythematosus, Goodpasture's syndrome, multiple myeloma, diabetes, neoplasia, sickle cell disease, and chronic inflammatory diseases), tubular diseases (e.g., acute tubular necrosis and acute renal failure, polycystic renal diseasemedullary sponge kidney, medullary cystic disease, nephrogenic diabetes, and renal tubular acidosis), tubulointerstitial diseases (e.g., pyelonephritis, drug and toxin induced tubulointerstitial nephritis, hypercalcemic nephropathy, and hypokalemic nephropathy) acute and rapidly progressive renal failure, chronic renal failure, nephrolithiasis, vascular diseases (e.g. , hypertension and nephrosclerosis, microangiopathic hemolytic anemia, atheroembolic renal disease, diffuse cortical necrosis, and renal infarcts), or tumors (e.g., renal cell carcinoma and nephroblastoma). In another example, TANGO 223 polypeptides, nucleic acids, or modulators thereof, can be used to treat testicular disorders, such as unilateral testicular enlargement (e.g. , nontuberculous, 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). In another example, TANGO 223 polypeptides, nucleic acids, or modulators thereof, can be used to treat prostate disorders, such as inflammatory diseases (e.g. , acute and chronic prostatitis and granulomatous prostatitis), hyperplasia (e.g., benign prostatic hypertrophy or hyperplasia), or tumors (e.g., carcinomas).

In another example, TANGO 223 polypeptides, nucleic acids, or modulators thereof, can be used to treat ovarian disorders, such as non-neoplastic cysts (e.g. , follicular and luteal cysts and polycystic ovaries) and tumors (e.g., tumors of surface epithelium, germ cell tumors, sex cord-stromal tumors, and metastatic carcinomas.

In another example, TANGO 223 polypeptides, nucleic acids, or modulators thereof, can be used to treat intestinal disorders, 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, or volvulus.

In another example, TANGO 223 polypeptides, nucleic acids, or 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 colistis, hemoπhoids, angiodysplasia), inflammatory diseases (e.g. , idiopathic ulcerative colitis, pseudomembranous colitis, and lymphopathia venereum), tumors (e.g., hyperplastic polyps, adenomatous polyps, bronchogenic cancer, colonic carcinoma, squamous cell carcinoma, adenoacanthomas, sarcomas, lymphomas, argentaffinomas, carcinoids, and melanocarcinomas). In another example, TANGO 223 polypeptides, nucleic acids, or modulators thereof, can be used to treat leukocytic disorders, such as leukopenias (e.g., neutropenia, monocytopenia, lymphopenia, and granulocytopenia), leukocytosis (e.g., granulocytosis, lymphocytosis, eosinophilia, monocytosis, acute and chronic lymphadenitis), malignant lymphomas (e.g., Non-Hodgkin's lymphomas, Hodgkin's lymphomas, leukemias, agnogenic myeloid metaplasia, multiple myeloma, plasmacytoma, Waldenstrom's macroglobulinemia, heavy-chain disease, monoclonal gammopathy, histiocytoses, eosinophilic granuloma, and angioimmunoblastic lymphadenopathy).

Tables 1 and 2 below provide summaries of TANGO 201 and TANGO 223 sequence information.

TABLE 1: Summary of Sequence Information of TANGO 201 and TANGO 223.

TABLE 2:Summary of Domains of TANGO 201 and TANGO 223.

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 kB, 4 kB, 3 kB, 2 kB, 1 kB, 0.5 kB or 0.1 kB of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. As used herein, the term "isolated," when referring to a nucleic acid molecule excludes an isolated chromosome. 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 SEQ ID NO:l, 2, 6, 7, 11, 12, 24, 25, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, or a complement thereof, can be isolated using standard molecular biology techniques and the sequence information provided herein. Using all or a portion of the nucleic acid sequences of SEQ ID NO:l, 2, 6, 7, 11, 12, 24, 25, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56 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 SEQ ID NO:l, 2, 6, 7, 11, 12, 24, 25, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 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, e.g., in identifying and/or cloning homologues in other cell types, e.g. , from other tissues, as well as homologues from other mammals. The probe/primer typically comprises substantially purified oligonucleotide. The oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, preferably about 25, more preferably about 50, 75, 100, 125, 150, 175, 200, 250, 300, 350 or 400 consecutive nucleotides of the sense or anti-sense sequence of SEQ ID NO:l, 2, 6, 7, 11, 12, 24, 25, 32,

34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, or of a naturally occurring mutant of SEQ ID

NO:l, 2, 6, 7, 11, 12, 24, 25, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, or 56.

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 any of SEQ ID NO:3, 8, 13, 26, 33,

35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57 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 SEQ ID NO:l, 2, 6, 7, 11, 12, 24, 25, 32, 34, 36, 38, 40, 42, 44, 46, 5 48, 50, 52, 54, or 56, due to degeneracy of the genetic code and thus encode the same protein as that encoded by the nucleotide sequence of SEQ ID NO:l, 2, 6, 7, 11, 12, 24, 25, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56.

In addition to the nucleotide sequences of SEQ ID NO:l, 2, 6, 7, 11, 12, 24, 25, 32,

34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, or 56, it will be appreciated by those skilled in the art that DNA sequence polymorphisms that lead to changes in the amino acid sequence may exist within a population (e.g., the human population). Such genetic polymorphisms may 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. For example, a human TANGO 201 allele is one that maps to human chromosome 2 between markers 15

D2S123 and D2S378. Likewise, a human TANGO 223 allele is one that maps to human chromosome 15q26 between flanking markers WI-3162 and WI-4919.

Furthermore, the nucleic acid sequences disclosed herein can be used to perform searches against "mapping databases", such that the chromosome position of the gene is 20 identified by sequence homology with known sequence fragments which have been mapped to chromosomes.

Analysis reveals that human TANGO 201 maps to chromosome 2 between markers D2S123 and D2S378.

In addition, the human gene for TANGO 223 was mapped on radiation hybrid

25 panels to chromosome 15, in the region q26. Flanking markers for this region are WI-3162 and WI-4919. The OTS (otosclerosis) locus also maps to this region of the human chromosome. The ALDH6 (aldehyde dehydrogenase 6), CHRM5 (cholinergic receptor),

STX (sialyltransferase X), and IDDM3 (insulin-dependent diabetes mellitus 3) genes also map to this region of the human chromosome. This region is syntenic to mouse

30 chromosome 7. The tp (taupe) locus also maps to this region of the mouse chromosome.

The age (shhtrvsn), hf (hepatic fusion), sur (sulfonylurea receptor), and fah

(fumarylacetoacetate hydrolase) genes also map to this region of the mouse chromosome.

As used herein, the phrase "allelic variant" refers to a nucleotide sequence which -, - occurs at a given locus or to a polypeptide encoded by the nucleotide sequence. As used herein, the terms "gene" and "recombinant gene" refer to nucleic acid molecules comprising an open reading frame encoding a polypeptide of the invention. Such natural allelic variations can typically result in l-5%> variance in the nucleotide sequence of a given gene. Alternative alleles can be identified by sequencing the gene of interest in a number of different individuals. This can be readily carried out by using hybridization probes to identify the same genetic locus in a variety of individuals. Any and all such nucleotide variations and resulting amino acid polymorphisms or variations that are the result of natural allelic variation and that do not alter the functional activity are intended to be within the scope of the invention. A human TANGO 201 allele is one that maps to human chromosome 2 between markers D2S123 and D2S378. Likewise, a human TANGO 223 allele is one that maps to human chromosome 15q26 between flanking markers WI-3162 and WI-4919.

Moreover, nucleic acid molecules encoding proteins of the invention from other species (homologues), which have a nucleotide sequence which differs from that of the human protein described herein are intended to be within the scope of the invention. Nucleic acid molecules corresponding to natural allelic variants and homologues of a cDNA of the invention can be isolated based on their identity to the human nucleic acid molecule disclosed herein using the human cDNAs, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions. For example, a cDNA encoding a soluble form of a membrane-bound protein of the invention 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 300 (325, 350, 375, 400, 425, 450, 500, 550, 600, 650, 700, 800, 900, 1000, or 1290) nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising the nucleotide sequence, preferably the coding sequence, of SEQ ID NO:l, 2, 6, 7, 11, 12, 24, 25, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 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 prefeπed, non-limiting example of stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45 °C, followed by one or more washes in 0.2 X SSC, 0.1%> SDS at 50-65°C. Preferably, an isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to the sequence of SEQ ID NO:l, 2, 6, 7, 11, 12, 24, 25, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 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).

Representative species that hybridize under such conditions to one or more of the sequences above include, but are not limited to, SEQ ID NOs:32, 34, 36, 38, 40, 42, 44, 46, 48, or 50, which in particular hybridize to the TANGO 201 sequences listed above (SEQ ID NO:l). Likewise, representative species that hybridize under such conditions to one or more of the sequences above include, but are not limited to, SEQ ID NOs:52, 54, or 56, which in particular hybridize to the TANGO 223 sequences listed above (SEQ ID NO: 11).

In addition to naturally-occurring allelic variants of a nucleic acid molecule of the invention sequence that may 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 homologues of various species can be non-essential for activity and thus would be likely targets for alteration. Alternatively, amino acid residues that are conserved among the homologues of various species (e.g., murine and human) can be essential for activity and thus would not be likely targets for alteration. For example, representative species of the mouse TANGO 201 presented for illustrative purposes only and not by way of limitation, include but are not limited to, SEQ ID NOs:32, 34, 36, 38, 40, 42, 44, 46, 48, 50. Likewise, representative species that hybridize under such conditions to one or more of the sequences above include, but are not limited to, SEQ ID NOs:52, 54, or 56, which in particular hybridize to the TANGO 223 sequences listed above (SEQ ID NO:l 1).

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 SEQ ID

NO:3, 8, 13, 26, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 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 45 %> identical, 65%, 75%, 85%, 95%, or 98% identical to the amino acid sequence of SEQ ID NO:3, 8, 13, 26, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57.

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 SEQ ID NO:l, 2, 6, 7, 11, 12, 24, 25, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Preferably such variant proteins retain or exhibit at least one structural or biological activity of the polypeptides of the invention. 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), nonpolar 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 protei protein interactions with proteins in a signaling pathway of the polypeptide of the invention; (2) the ability to bind a ligand of the polypeptide of the invention; or (3) the ability to bind to an intracellular target protein of the polypeptide of the invention. In yet another preferred embodiment, the mutant polypeptide can be assayed for the ability to modulate cellular proliferation, cellular migration or 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 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 subcloned 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 -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 (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. Set 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 antigene agents for sequence-specific modulation of gene expression by, e.g., inducing transcription or translation arrest or inhibiting replication.

PNAs can also be used, e.g., in the analysis of single base pair mutations in a gene by, e.g.,

PNA directed PCR clamping; as artificial restriction enzymes when used in combination with other enzymes, e.g., SI nucleases (Hyrup (1996), supra; or as probes or primers for

DNA sequence and hybridization (Hyrup (1996), supra; Perry-O'Keefe et al. (1996) Proc.

Natl. Acad. Sci. USA 93: 14670-675).

In another embodiment, PNAs can be modified, e.g., to enhance their stability or cellular uptake, by attaching Hpophilic 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 may combine the advantageous properties of PNA and DNA. Such chimeras allow DNA recognition enzymes, e.g. , RNAse H and DNA polymerases, to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity. PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (Hyrup (1996), supra). The synthesis of PNA-DNA chimeras can be performed as described 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 stepwise 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 may 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. W0 89/10134). In addition, oligonucleotides can be modified with hybridization-triggered cleavage agents (see, e.g., Krol et al. (1988) Bio/Techniques 6:958-976) or intercalating agents (see, e.g., Zon (1988) Pharm. Res. 5:539-549). To this end, the oligonucleotide 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, be., 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 NO:3, 8, 13, 26, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, or 57), 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 coπesponding protein. A biologically active portion of a protein of the invention can be a polypeptide which is, for example, 10, 25, 50, 100 or more amino acids in length. Moreover, other biologically active portions, in which other regions of the protein are deleted, can be prepared by recombinant techniques and evaluated for one or more of the functional activities of the native form of a polypeptide of the invention.

Preferred polypeptides have the amino acid sequence of SEQ ID NO:3, 8, 13, 26,

33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57. Other useful proteins are substantially identical (e.g., at least about 45%, preferably 55%, 65%, 75%, 85%, 95%, or 99%) to any of

SEQ ID NO: :3, 8, 13, 26, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, or 57 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 purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = # of identical positions/total # of positions (e.g., overlapping positions) x 100). In one embodiment the two sequences are the same length. The determination of percent identity between two sequences can be accomplished using a mathematical algorithm. A preferred, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul, et al. (1990) J. Mol Biol. 215:403-410. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to a nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to a protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-Blast can be used to perform an iterated search which detects distant relationships between molecules (Id.). When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov. Another preferred, non- limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, (1988) CABIOS 4:11-17. Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing 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 N-terminus or C-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 C-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 N-terminus. For example, the native signal sequence of a polypeptide of the invention can be removed and replaced with a signal sequence from another protein. For example, the gp67 secretory sequence of the baculovirus envelope protein can be used as a heterologous signal sequence (Current Protocols 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 incorporated into pharmaceutical compositions and administered to a subject to inhibit an interaction between a ligand (soluble or membrane-bound) and a protein on the surface of a cell (receptor), to thereby suppress signal transduction in vivo. The immunoglobulin fusion protein can be used to affect the bioavailability of a cognate ligand of a polypeptide of the invention. Inhibition of ligand/receptor interaction can be useful therapeutically, both for treating prohferative 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 reamplified to generate a chimeric gene sequence (see, e.g., Ausubel et al, supra). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide). A nucleic acid encoding a polypeptide of the 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 (SEQ ID NO:4, 9, 14 or 27) 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 will be regulatory sequences which affect transcription. Thus, a nucleotide sequence which encodes all or a portion of a signal sequence can be used as a probe to identify and isolate signal sequences and their flanking regions, and these flanking regions can be studied to identify regulatory elements therein.

The present invention also pertains to variants of the polypeptides of the invention. Such variants have an altered 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. 11:477).

In addition, libraries of fragments of the coding sequence of a polypeptide of the invention can be used to generate a variegated population of polypeptides for screening and subsequent selection of variants. For example, a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of the coding sequence of interest with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing 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 N-terminal and internal fragments of various sizes of the protein of interest.

Several techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property. The most widely used techniques, which are amenable to high through-put analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates isolation of the vector encoding the gene whose product was detected. Recursive ensemble mutagenesis (REM), a technique which enhances the frequency of functional mutants 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) amino acid residues of the amino acid sequence of SEQ ID NO:3, 8, 13, 26, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, or 57 and encompasses an epitope of the protein such that an antibody raised against the peptide forms a specific immune complex with the protein.

Preferred epitopes encompassed by the antigenic peptide are regions that are located on the surface of the protein, e.g., hydrophilic regions. Figures 2, 4, and 9 are hydropathy plots of the proteins of the invention. These plots or similar analyses can be used to identify hydrophilic regions.

An immunogen typically is used to prepare antibodies by immunizing a suitable subject, (e.g., rabbit, goat, mouse or other mammal). An appropriate immunogenic preparation can contain, for example, recombinantly expressed or chemically synthesized polypeptide. The preparation can further include an adjuvant, such as Freund's complete or incomplete adjuvant, or similar immunostimulatory agent.

Accordingly, another aspect of the invention pertains to antibodies directed against a polypeptide of the invention. The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site which specifically binds an antigen, such as a polypeptide of the invention, 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 a polypeptide or polypeptides of the invention. Particularly preferred polyclonal antibody preparations are ones that contain only antibodies directed against a polypeptide or 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 isolated from the mammal (e.g., from the blood) and further purified by well-known techniques, such as protein A chromatography to obtain the IgG fraction. Alternatively, antibodies specific for a protein or polypeptide of the invention can be selected for (e.g., partially purified) or purified by, e.g. , affinity chromatography. For example, a recombinantly expressed and purified (or partially purified) protein of the invention is produced as described herein, and covalently or non-covalently coupled to a solid support such as, for example, a chromatography column. The column can then be used to affinity purify antibodies specific for the proteins of the invention from a sample containing antibodies directed against a large number of different epitopes, thereby generating a substantially purified antibody composition, i.e., one that is substantially free of contaminating antibodies. By a substantially purified antibody composition is meant, in this context, that the antibody sample contains at most only 30%> (by dry weight) of contaminating antibodies directed against epitopes other than those on the desired protein or polypeptide of the invention, and preferably at most 20%), yet more preferably at most 10%), and 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) EMBO J. 12:725-734.

Additionally, recombinant antibodies, such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the invention. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region. (See, e.g., Cabilly et al., U.S. Patent No. 4,816,567; and Boss et al., U.S. Patent No. 4,816397, which are incorporated herein by reference in their entirety.) Humanized antibodies are antibody molecules from non-human species having one or more complementarily determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule. (See, e.g., Queen, U.S. Patent No. 5,585,089, which is incorporated herein by reference in its entirety.) 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 αb (1987) Proc. Natl. Acad. Sci. USA 84:214- 218; Nishimura et al. (1987) Cane. Res. 47:999-1005; Wood et al. (1985) Nature 314:446- 449; and Shaw et al. (1988) J. Natl. Cancer Inst. 80:1553-1559); Morrison (1985) 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 mouse 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, beta-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include ¹²⁵1, ¹³¹1, ³⁵S or ³H.

Further, an antibody (or fragment thereof) can be conjugated to a therapeutic moiety such as a cytotoxin, a therapeutic agent or a radioactive metal ion. A cytotoxin or cytotoxic agent includes 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, and puromycin and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly 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 for modifying a given biological response, the drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, . alpha. -interferon, .beta.-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator; or, biological response modifiers such as, for example, lymphokines, interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"), granulocyte macrophase colony stimulating factor ("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or other growth factors.

Techniques for conjugating such therapeutic moiety to antibodies 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. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "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. 303-16 (Academic Press 1985), and Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev., 62:119-58 (1982).

Alternatively, an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Patent No. 4,676,980.

Accordingly, in one aspect, the invention provides substantially purified antibodies or fragment thereof, and non-human antibodies or fragments thereof, which antibodies or fragments specifically bind to a polypeptide comprising an amino acid sequence selected from the group consisting of: the amino acid sequence of any one of SEQ ID NOs:3, 8, 13,

26, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, or an amino acid sequence encoded by the cDNA of a clone deposited as ATCC™ 207081 , respectively; a fragment of at least 15 amino acid residues of the amino acid sequence of any one of SEQ ID NOs:3, 8, 13, 26, 33,

35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, an amino acid sequence which is at least 95% identical to the amino acid sequence of any one of SEQ ID NOs:3, 8, 13, 26, 33, 35, 37, 39,

41, 43, 45, 47, 49, 51, 53, 55, 57, 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 an amino acid sequence which is encoded by a nucleic acid molecule which hybridizes to the nucleic acid molecule consisting of any one of SEQ ID NOs:l, 3, 4, 6, 8, 10, 11, 13, 14, 16, 17, 19, 20, 22, 38, 39, 53, 55, 56, 58,

59, 61, 62, 64, 65, 67, 68, 70, 71, 73, or the cDNA of a clone deposited as ATCC™ 207081, or a complement thereof, under conditions of hybridization of 6X SSC at 45 °C and washing in 0.2 X SSC, 0.1%o SDS at 65°C. In various embodiments, the antibodies of the invention, or fragments thereof, can be human, non-human, chimeric and/or humanized antibodies.

In another aspect, the invention provides non-human antibodies or fragments thereof, which antibodies or fragments specifically bind to a polypeptide comprising an amino acid sequence selected from the group consisting of: the amino acid sequence of any one of SEQ ID NOs:3, 8, 13, 26, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, or an amino acid sequence encoded by the cDNA of a clone deposited as ATCC™ 207081, respectively; a fragment of at least 15 amino acid residues of the amino acid sequence of any one of SEQ ID NOs:3, 8, 13, 26, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, or 57, an amino acid sequence which is at least 95%> identical to the amino acid sequence of any one of SEQ ID NOs:3, 8, 13, 26, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, or 57, 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 an amino acid sequence which is encoded by a nucleic acid molecule which hybridizes to the nucleic acid molecule consisting of any one of SEQ ID NOs:l, 3, 4, 6, 8, 10, 11, 13, 14, 16, 17, 19, 20, 22, 38, 39, 53, 55, 56, 58, 59, 61, 62, 64, 65, 67, 68, 70, 71, 73, or the cDNA of a clone deposited as ATCC™ 207081, or a complement thereof, under conditions of hybridization of 6X SSC 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 to a polypeptide comprising an amino acid sequence selected from the group consisting of: the amino acid sequence of any one of SEQ ID NOs:3, 8, 13, 26, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, or an amino acid sequence encoded by the cDNA of a clone deposited as ATCC™ 207081, respectively; a fragment of at least 15 amino acid residues of the amino acid sequence of any one of SEQ ID NOs:3, 8, 13, 26, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, or 57, an amino acid sequence which is at least 95%> identical to the amino acid sequence of any one of SEQ ID NOs:3, 8, 13, 26, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, or 57, 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 an amino acid sequence which is encoded by a nucleic acid molecule which hybridizes to the nucleic acid molecule consisting of any one of SEQ ID NOs:l, 3, 4, 6, 8, 10, 11, 13, 14, 16, 17, 19, 20, 22, 38, 39, 53, 55, 56, 58, 59, 61, 62, 64, 65, 67, 68, 70, 71, 73, or the cDNA of a clone deposited as ATCC™ 207081, or a complement thereof, under conditions of hybridization of 6X SSC 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 antibodies or fragments thereof specifically bind to 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 antibodies or fragments thereof, the non-human antibodies or fragments thereof, and/or the monoclonal antibodies or fragments thereof, of the invention specifically bind to a secreted sequence or an extracellular domain of the amino acid sequence of SEQ ID NOs:3, 8, 13, 26, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, or 57. Preferably, the secreted sequence or extracellular domain to which the antibody, or fragment thereof, binds comprises from about amino acids 33-483 of SEQ ID NO:3 (SEQ ID NO:5), from amino acids 33-403 of SEQ ID NO:8 (SEQ ID NO: 10), from about amino acids 30-247 of SEQ ID NO:13 (SEQ ID NO:15) and from about amino acids 30-198 of SEQ ID NO:26 (SEQ ID NO:29).

Any of the antibodies of the invention can be conjugated to a therapeutic moiety or to a detectable substance. Non- limiting examples of detectable substances that can be conjugated to the antibodies of the invention are an enzyme, a prosthetic group, a fluorescent material, a luminescent material, a bioluminescent material, and a radioactive material.

The invention also provides a kit containing an antibody of the invention conjugated

10 to a detectable substance, and instructions for use. Still another aspect of the invention is a pharmaceutical composition comprising an antibody of the invention and a pharmaceutically acceptable carrier. In preferred embodiments, the pharmaceutical composition contains an antibody of the invention, a therapeutic moiety, and a pharmaceutically acceptable carrier.

15

Still another aspect of the invention is a method of making an antibody that specifically recognizes TANGO 201 and TANGO 223, the method comprising immunizing a mammal with a polypeptide. The polypeptide used as an immungen comprises an amino acid sequence selected from the group consisting of: the amino acid sequence of any one of

20 SEQ ID NOs:3, 8, 13, 26, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, or an amino acid sequence encoded by the cDNA of a clone deposited as ATCC™ 207081, respectively; a fragment of at least 15 amino acid residues of the amino acid sequence of any one of SEQ ID NOs:3, 8, 13, 26, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, an amino acid sequence which is at least 95% identical to the amino acid sequence of any one of SEQ ID

25 NOs:3, 8, 13, 26, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 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 an amino acid sequence which is encoded by a nucleic acid molecule which hybridizes to the nucleic acid molecule comprising SEQ ID NOs:l, 2, 6, 7, 11, 12, 24, 25, 32, 34, 36, 38, 40,

30 42, 44, 46, 48, 50, 52, 54, 56, or the cDNA of a clone deposited as ATCC™ 207081, or a complement thereof, under conditions of hybridization of 6X SSC at 45 °C and washing in 0.2 X SSC, 0.1%) SDS at 65°C. After immunization, a sample is collected from the mammal that contains an antibody that specifically recognizes the polypeptide encoded by SEQ ID NOs:l, 2, 6, 7, 11, 12, 24, 25, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, or

35 the cDNA of a clone deposited as ATCC™ 207081, or a complement thereof. Preferably, the polypeptide is recombinantly produced using a non-human host cell. Optionally, the antibodies can be further purified from the sample using techniques well known to those of skill in the art. The method can further comprise producing a monoclonal antibody-producing cell from the cells of the mammal. Optionally, antibodies are collected from the antibody-producing cell.

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, etc. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein.

The recombinant expression vectors of the invention can be designed for expression of a polypeptide of the invention in prokaryotic (e.g., E. coli) or eukaryotic cells (e.g., insect cells (using baculovirus expression vectors), yeast cells or mammalian cells). Suitable host cells are discussed further in Goeddel, supra. Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.

Expression of proteins in prokaryotes is most often carried out in E. coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non- fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson (1988) Gene 67:31-40), pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.

Examples of suitable inducible non- fusion E. coli expression vectors include pTrc (Amann et al, (1988) Gene 69:301-315) and pET l id (Studier et al, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 60-89). Target gene expression from the pTrc vector relies on host RNA polymerase transcription from a hybrid trp-lac fusion promoter. Target gene expression from the pET l id vector relies on transcription from a T7 gnlO-lac fusion promoter mediated by a coexpressed viral RNA polymerase (T7 gnl). This viral polymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from a resident 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 αb (1992) Nucleic Acids Res. 20:2111-2118). Such alteration ofnucleic 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 cerivisae include pYepSecl (Baldari et al. (1987) EMBOJ. 6:229-234), pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al. (1987) Gene 54:113-123), pYES2 (Invitrogen Corporation, San Diego, CA), and pPicZ (Invitrogen Corp, San Diego, CA).

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

In yet another embodiment, a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed (1987) Nature 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 Vims 40. For other suitable expression systems for both prokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook et al, supra.

In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue- specific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Νon- limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al. (1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol. 43:235- 275), in particular promoters of T cell receptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) and immunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen and Baltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477), pancreas- specific promoters (Edlund et al. (1985) Science 230:912-916), and mammary gland- specific promoters (e.g., milk whey promoter; U.S. Patent No. 4,873,316 and European Application Publication No. 264,166). Developmentally-regulated promoters are also encompassed, for example the murine hox promoters (Kessel and Gmss (1990) Science 249:374-379) and the a-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 vims 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, DEAE-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 dmgs, such as G418, hygromycin and methotrexate. Cells stably transfected with the introduced nucleic acid can be identified by d g selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).

In another embodiment, the expression characteristics of an endogenous (e.g., TANGO 201 and TANGO 223) nucleic acid within a cell, cell line or microorganism may be modified by inserting a DNA regulatory element heterologous to the endogenous gene of interest into the genome of a cell, stable cell line or cloned microorganism such that the inserted regulatory element is operatively linked with the endogenous gene (e.g., TANGO 201 and TANGO 223) and controls, modulates or activates the endogenous gene. For example, endogenous TANGO 201 and TANGO 223 which are normally "transcriptionally silent", i.e., TANGO 201 and TANGO 223 genes which are normally not expressed, or are expressed only at very low levels in a cell line or microorganism, may be activated by inserting a regulatory element which is capable of promoting the expression of a normally expressed gene product in that cell line or microorganism. Alternatively, transcriptionally silent, endogenous TANGO 201 and TANGO 223 genes may be activated by insertion of a promiscuous regulatory element that works across cell types.

A heterologous regulatory element may be inserted into a stable cell line or cloned microorganism, such that it is operatively linked with and activates expression of endogenous TANGO 201 and TANGO 223 genes, using techniques, such as targeted homologous recombination, which are well known to those of skill in the art, and 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 nonhuman 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, retro viral 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 and in Hogan, Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986) and Wakayama et al, (1999), Proc. Natl. Acad. Sci. USA, 96:14984- 14989. Similar methods are used for production of other transgenic animals. A transgenic founder animal can be identified based upon the presence of the transgene in its genome and/or expression of mRNA encoding the transgene in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying the transgene can further be bred to other transgenic animals carrying other transgenes.

To create an homologous recombinant animal, a vector is prepared which contains at least a portion of a gene encoding a polypeptide of the invention into which a deletion, addition or substitution has been introduced to thereby alter, e.g., functionally disrupt, the gene. In a preferred embodiment, the vector is designed such that, upon homologous recombination, the endogenous gene is functionally dismpted (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 constmction 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 refeπed to herein as "active compounds") of the invention can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier. As used herein the language "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

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

Such methods comprise formulating a pharmaceutically acceptable carrier with an agent which modulates expression or activity of a polypeptide or nucleic acid of the invention.

Such compositions can further include additional active agents. Thus, the invention further includes methods for preparing a pharmaceutical composition by formulating a pharmaceutically acceptable carrier with an agent which modulates expression or activity of a polypeptide or nucleic acid of the invention and one or more addtional active compounds. 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 ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for 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 polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size 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, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound (e.g., a polypeptide or antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and 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 purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed.

Pharmaceutically compatible binding agents, 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 com starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

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

Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery. In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled 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 Cruikshank 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. The pharmaceutical compositions can be included in a container, pack, or dispenser together with instmctions for administration.

V. Uses and Methods of the Invention

The nucleic acid molecules, proteins, protein homologues, 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, the TANGO 201 and TANGO 223 polypeptides of the invention can to used to modulate cellular function, survival, morphology, proliferation, and/or differentiation of the cells in which they are expressed. 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 dmgs or compounds which modulate activity or expression of a polypeptide of the invention as well as to treat disorders characterized by insufficient or excessive production of a protein of the invention or production of a form of a protein of the invention which has decreased or aberrant activity compared to the wild type protein. In addition, the antibodies of the invention can be used to detect and isolate a protein of the and modulate activity of a protein of the invention.

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

A. Screening Assays

The invention provides a method (also referred to herein as a "screening assay") for identifying modulators, i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other dmgs) 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; Cwirla et al. (1990) Proc. Natl Acad. Sci. USA 87:6378-6382; and Felici (1991) J. Mol. Biol. 222:301-310).

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

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

Determining the ability of a polypeptide of the invention to bind to or interact with a target molecule can be accomplished by one of the methods described above for determining direct binding. As used herein, a "target molecule" is a molecule with which a selected polypeptide (e.g., a polypeptide of the invention binds or interacts with 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., intracellular Ca²⁺, diacylglycerol, IP3, etc.), detecting catalytic/enzymatic activity of the target on an appropriate substrate, detecting the induction of a reporter gene (e.g., a regulatory element that is responsive to a polypeptide of the invention operably linked to a nucleic acid encoding a detectable marker, e.g. luciferase), or detecting a cellular response, for example, cdlular differentiation, or cell proliferation. In yet another embodiment, an assay of the present invention is a cell-free assay comprising contacting a polypeptide of the invention or biologically active portion thereof with a test compound and determining the ability of the test compound to bind to the polypeptide or biologically active portion thereof. Binding of the test compound to the polypeptide can be determined either directly or indirectly as described above. In a preferred embodiment, the assay includes contacting the polypeptide of the invention or biologically active portion thereof with a known compound which binds the polypeptide to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with the polypeptide, wherein determining the ability of the test compound to interact with the polypeptide comprises determining the ability of the test compound to preferentially bind to the polypeptide or biologically active portion thereof as compared to the known compound.

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

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

The cell-free assays of the present invention are amenable to use of both a soluble form or the membrane-bound form of a polypeptide of the invention. In the case of cell-free assays comprising the membrane-bound form of the polypeptide, it 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-l 14, 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-l -propane sulfonate.

In more than one embodiment 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 uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay. Binding of a test compound to the polypeptide, or interaction of the polypeptide with a target molecule 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 microtitre plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix. For example, glutathione-S-transferase fusion proteins or glutathione-S-transferase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical; St. Louis, MO) or glutathione derivatized microtitre plates, which are then combined with the test compound or the test compound and either the non-adsorbed target protein or A polypeptide of the invention, and the mixture incubated under conditions conducive to complex formation (e.g. , at physiological conditions for salt and pH). Following incubation, the beads or microtitre plate wells are washed to remove any unbound components and complex formation is measured either directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of binding or activity of the polypeptide of the invention can be determined using standard techniques.

Other techniques for immobilizing proteins on matrices can also be used 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 corresponding to a polypeptide or nucleic acid of the invention) in the cell is determined. The level of expression of the selected mRNA or protein in the presence of the candidate compound is compared 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 (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 (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.

B. Detection Assays

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

1. Chromosome Mapping

Once the sequence (or a portion of the sequence) of a gene has been isolated, this sequence can be used to map the location of the gene on a chromosome. Accordingly, nucleic acid molecules described herein or fragments thereof, can be used to map the location of the corresponding genes on a chromosome. The mapping of the sequences to chromosomes is an important first step in correlating these sequences with genes associated with disease. Briefly, genes can be mapped to chromosomes by preparing PCR primers

(preferably 15-25 bp 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 corresponding to the gene sequences will yield an amplified fragment. For a review of this technique, see D'Eustachio et al. ((1983) Science 220:919-924).

PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular sequence to a particular chromosome. Three or more sequences can be assigned per day using a single thermal cycler. Using the nucleic acid sequences of the invention to design oligonucleotide primers, sublocalization can be achieved with panels of fragments from specific chromosomes. Other mapping strategies which can similarly be used to map a gene to its chromosome include in situ hybridization (described in Fan et al. (1990) Proc. Natl.

Acad. Sci. USA 87:6223-27), pre-screening with labeled flow-sorted chromosomes (CITE), 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 corresponding to noncoding regions of the genes actually are preferred for mapping purposes. Coding sequences are more likely to be conserved within gene families, thus increasing the chance of cross hybridizations during chromosomal mapping. In addition to nucleic acids, polypeptides and fragments and sequences thereof and antibodies specific thereto can be used to map the location of the gene encoding the polypeptide on a chromosome. This mapping can be carried out by specifically detecting the presence of the polypeptide in members of a panel of somatic cell hybrids between cells of a first species of animal from which the protein originates and cells from a second species of animal and then determining which somatic cell hybrid(s) expresses the polypeptide and noting the chromosome(s) from 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, for example, enzymatic activity, as described in Bordelon-Riser et al. (1979; Somatic Cell Genetics 5:597-613 and Owerbach et al. (1978; Proc. Natl. Acad. Sci. USA 75:5640-5644.

Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. (Such data are found, for example, in V. McKusick, Mendelian Inheritance in Man, available on-line through Johns Hopkins University Welch Medical Library). The relationship between genes and disease, mapped to the same chromosomal region, can then be identified 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 stmctural alterations in the chromosomes such as deletions or translocations that are visible from chromosome spreads or detectable using PCR based on that DNA sequence. Ultimately, complete sequencing of genes from several individuals can be performed to confirm the presence of a mutation and to distinguish mutations from polymorphisms. Furthermore, the nucleic acid sequences disclosed herein can be used to perform searches against "mapping databases", such that the chromosome position of the gene is identified by sequence homology with known sequence fragments which have been mapped to chromosomes.

In the instant case, the human gene for TANGO 223 was mapped on radiation hybrid panels to chromosome 15, in the region q26. Flanking markers for this region are WI-3162 and WI-4919. The OTS (otosclerosis) locus also maps to this region of the human chromosome. The ALDH6 (aldehyde dehydrogenase 6), CHRM5 (cholinergic receptor), STX (sialyltransferase X), and IDDM3 (insulin-dependent diabetes mellitus 3) genes also

10 map to this region of the human chromosome. This region is syntenic to mouse chromosome 7. The tp (taupe) locus also maps to this region of the mouse chromosome. The age (shhtrvsn), hf (hepatic fusion), sur (sulfonylurea receptor), and fah (fumarylacetoacetate hydrolase) genes also map to this region of the mouse chromosome.

15

2. Tissue Typing

The nucleic acid sequences of the present invention can also be used to identify individuals from minute biological samples. The United States military, for example, is considering the use of restriction fragment length polymorphism (RFLP) for identification

20 of its personnel. In this technique, an individual's genomic DNA is digested with one or more restriction enzymes, and probed on a Southern blot to yield unique bands for identification. This method does not suffer from the current limitations of "Dog Tags" which can be lost, switched, or stolen, making positive identification difficult. The sequences of the present invention are useful as additional DNA markers for RFLP

25 (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 corresponding 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 35 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 noncoding regions. It is estimated that allelic variation between individual humans occurs with a frequency of about once per each 500 bases. Each of the sequences described herein can, to some degree, be used as a standard against which DNA from an individual can be compared for identification purposes. Because greater numbers of polymorphisms occur in the noncoding regions, fewer sequences are necessary to differentiate individuals. The noncoding sequences of SEQ ID NO:l, 2, 6, 7, 11, 12, 24, 25, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, or 56 can comfortably provide positive individual identification with a panel of perhaps 10 to 1,000 primers which each yield a noncoding amplified sequence of 100 bases. If predicted coding sequences, such as those in SEQ ID NO:2, 7, or 12 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 perpetrator of a crime. To make such an identification, PCR technology can be used to amplify DNA sequences taken from very small biological samples such as tissues, e.g., hair or skin, or body fluids, e.g., blood, saliva, or semen found at a crime scene. The amplified sequence can 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 noncoding regions are particularly appropriate for this use as greater numbers of polymorphisms occur in the noncoding 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 noncoding 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.

0

C. Predictive Medicine

The present invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, pharmacogenomics, and monitoring clinical trails are used for prognostic (predictive) purposes to thereby treat an individual prophylactically. Accordingly, one aspect of the present invention relates to diagnostic assays for determining expression of a polypeptide or nucleic acid of the invention and/or activity of a polypeptide of the invention, in the context of a biological sample (e.g., blood, semm, cells, tissue) to thereby determine whether an individual is afflicted with a disease or disorder, or is at risk of developing a disorder, associated with aberrant expression or activity of a polypeptide of o the invention. The invention also provides for prognostic (or predictive) assays for determining whether an individual is at risk of developing a disorder associated with aberrant expression or activity of a polypeptide of the invention. For example, mutations in a gene of the invention can be assayed in a biological sample. Such assays can be used for prognostic or predictive purpose to thereby prophylactically treat an individual prior to the 5 onset of a disorder characterized by or associated with aberrant expression or activity of a polypeptide of the invention.

Another aspect of the invention provides methods for expression of a nucleic acid or polypeptide of the invention or activity of a polypeptide of the invention in an individual to thereby select appropriate therapeutic or prophylactic agents for that individual (referred to 0 herein as "pharmacogenomics"). Pharmacogenomics allows for the selection of agents (e.g., dmgs) 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 5 (e.g., dmgs or other compounds) on the expression or activity of a polypeptide of the invention in clinical trials. These and other agents are described in further detail in the following sections.

1. Diagnostic Assays

An 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

10 invention such that the presence of a polypeptide or nucleic acid of the invention is detected in the biological sample. A preferred agent for detecting mRNA or genomic DNA encoding a polypeptide of the invention is a labeled nucleic acid probe capable of hybridizing to mRNA or genomic DNA encoding a polypeptide of the invention. The nucleic acid probe can be, for example, a full-length cDNA, such as the nucleic acid of SEQ ID NO:l, 2, 6, 7,

₁₅ 11, 12, 24, 25, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 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. 0 A preferred agent for detecting a polypeptide of the invention is an antibody capable of binding to a polypeptide of the invention, preferably an antibody with a detectable label. Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab')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 5 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" 0 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 5 polypeptide of the invention include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence. In vitro techniques for detection of genomic DNA include Southern hybridizations. Furthermore, in vivo techniques for detection of a polypeptide of the invention include introducing into a subject a labeled antibody directed against the polypeptide. For example, the antibody can be labeled with a radioactive marker 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 preferred biological sample is a peripheral blood leukocyte sample isolated by conventional means from a subject.

In another embodiment, the methods further involve obtaining a control biological sample from a control subject, contacting the control sample with a compound or agent capable of detecting a polypeptide of the invention or mRNA or genomic DNA encoding a polypeptide of the invention, such that the presence of the polypeptide or mRNA or genomic DNA encoding the polypeptide is detected 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 aberrant expression of a polypeptide of the invention (e.g., a prohferative disorder, e.g., psoriasis or cancer). 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 aberrant expression of the polypeptide if the amount of the polypeptide or mRNA encoding the polypeptide is above or below a normal level.

For antibody-based kits, the kit can comprise, for example: (1) a first antibody (e.g., attached to a solid support) which 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 instmctions for observing whether the tested subject is suffering from or is at risk of developing a disorder associated with aberrant expression of the polypeptide.

2. Prognostic Assays

The methods described herein can furthermore be utilized as diagnostic or prognostic assays to identify subjects having or at risk of developing a disease or disorder associated with aberrant expression or activity of a polypeptide of the invention. For example, the assays described herein, such as the preceding diagnostic assays or the following assays, can be utilized to identify a subject having or at risk of developing a disorder associated with aberrant expression or activity of a polypeptide of the invention. Alternatively, the prognostic assays can be utilized to identify a subject having or at risk for developing such a disease or disorder. Thus, the present invention provides a method 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 aberrant expression or activity of the polypeptide. As used herein, a "test sample" refers to a biological sample obtained from a subject of interest. For example, a test sample can be a biological fluid (e.g., semm), 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 d g candidate) to treat a disease or disorder associated with aberrant expression or activity of a polypeptide of the invention. For example, such methods can be used to determine whether a subject can be effectively treated with a specific agent or class of agents (e.g., agents of a type which decrease activity of the polypeptide). Thus, the present invention provides methods for determining whether a subject can be effectively treated with an agent for a disorder associated with aberrant expression or activity of a polypeptide of the invention 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 aberrant 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 aberrant expression or activity of a polypeptide of the invention. In preferred 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 rearrangement of the gene; 5) an alteration in the level of a messenger RNA transcript of the gene; 6) an aberrant modification of the gene, such as of the methylation pattern of the genomic DNA; 7) the presence of a non-wild type splicing pattern of a messenger RNA transcript of the gene; 8) a non-wild type level of a the protein encoded by the gene; 9) an allelic loss of the gene; and 10) an inappropriate post-translational modification of the protein encoded by the gene. As described herein, there are a large number of assay techniques known 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. It is anticipated that 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, amplified (optionally), 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 arrays of sequential overlapping probes. This step allows the identification of point mutations. This step is followed by a second hybridization array that allows the characterization of specific mutations by using smaller, specialized probe arrays complementary to all variants or mutations detected. Each mutation array is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene.

In yet another embodiment, any of a variety of sequencing reactions known in the art can be used to directly sequence the selected gene and detect mutations by comparing the sequence of the sample nucleic acids with the corresponding wild-type (control) sequence. Examples of sequencing reactions include those based on techniques developed by Maxim and Gilbert ((1977) Proc. Natl. Acad. Sci. USA 74:560) or Sanger ((1977) Proc. Natl. Acad. Sci. USA 74:5463). It is also contemplated that any of a variety of automated sequencing procedures can be 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 basepair mismatches between the control and sample strands. RNA/DNA duplexes can be treated with RNase to digest mismatched regions, and DNA/DNA hybrids can be treated with SI nuclease to digest mismatched regions.

In other embodiments, either DNA DNA or RNA/DNA duplexes can be treated with 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 preferred embodiment, the control DNA or RNA can be labeled for detection. In still another embodiment, the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs 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 polymorphism (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 renature. 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 preferred 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 bp 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 may 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, mismatch can prevent or reduce polymerase extension (Prossner (1993) Tibtech 11 :238). In addition, it can be desirable to introduce a novel restriction site in the region of the mutation to create cleavage-based detection (Gasparini et al. (1992) Mol. Cell Probes 6:1). It is anticipated that in certain embodiments amplification may 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, by utilizing 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. Pharmaco genomics

Agents, or modulators which have a stimulatory or inhibitory effect on activity or expression of a polypeptide of the invention as identified by a screening assay described herein can be administered to individuals to treat (prophylactically or therapeutically) disorders associated with aberrant activity of the polypeptide. In conjunction with such treatment, the pharmacogenomics (i.e., the study of the relationship between an individual's genotype and that individual's response to a foreign compound or dmg) of the individual can be considered. Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration of the pharmacologically active dmg. Thus, the pharmacogenomics of the individual permits the selection of effective agents (e.g., dmgs) 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 dmgs due to altered dmg 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 dmgs act on the body are referred to as "altered dmg action." Genetic conditions transmitted as single factors altering the way the body acts on dmgs are referred to as "altered dmg metabolism". These pharmacogenetic conditions can occur either as rare defects or as polymorphisms. For example, glucose-6-phosphate dehydrogenase deficiency (G6PD) is a common inherited enzymopathy in which the main clinical complication is haemolysis after ingestion of oxidant drugs (anti-malarials, sulfonamides, analgesics, nitrofurans) and consumption of fava beans.

As an illustrative embodiment, the activity of dmg metabolizing enzymes is a major determinant of both the intensity and duration of dmg action. The discovery of genetic polymorphisms of dmg metabolizing enzymes (e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymes CYP2D6 and CYP2C19) has provided an explanation as to why some patients do not obtain the expected drug effects or show exaggerated dmg response and serious toxicity after taking the standard and safe dose of a dmg. These polymorphisms are expressed in two phenotypes in the population, the extensive metabolizer (EM) and poor metabolizer (PM). The prevalence of PM is different among different populations. For example, the gene coding for CYP2D6 is highly polymorphic 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 dmg response and side effects when they receive standard doses. If a metabolite is the active therapeutic moiety, a PM will show no therapeutic response, as demonstrated for the analgesic effect of codeine mediated by its CYP2D6-formed metabolite morphine. The other extreme are the so called ultra-rapid metabolizers who do not respond to standard doses. Recently, the molecular basis of ultra-rapid metabolism has been identified to be due to CYP2D6 gene amplification.

Thus, the activity of a polypeptide of the invention, expression of a nucleic acid encoding the polypeptide, or mutation content of a gene encoding the polypeptide 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 geno typing of polymorphic alleles encoding drug-metabolizing enzymes to the identification of an individual's dmg responsiveness phenotype. This knowledge, when applied to dosing or dmg 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.

Monitoring of Effects During Clinical Trials Monitoring the influence of agents (e.g., dmgs, compounds) on the expression or activity of a polypeptide of the invention (e.g., the ability to modulate aberrant cell proliferation chemotaxis, and/or differentiation) can be applied not only in basic dmg 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, dmg 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 preferred embodiment, the present invention provides a method for monitoring the effectiveness of treatment of a subject with an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other dmg 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 preadministration 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, be., 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

10

The present invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with aberrant expression or activity of a polypeptide of the invention. For example, disorders characterized by abberant expression or activity of the polypeptides of the invention include 15 metabolic disorders. Moreover, the polypeptides of the invention can be used to modulate cellular function, survival, morphology, proliferation and/or differentiation (e.g., to treat tumors).

1. Prophylactic Methods

20

In one aspect, the invention provides a method for preventing in a subject, a disease or condition associated with an aberrant expression or activity of a polypeptide of the invention, by administering to the subject an agent which modulates expression or at least one activity of the polypeptide. Subjects at risk for a disease which is caused or contributed

2_<- to by aberrant expression or activity of a polypeptide of the invention can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein. Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the aberrancy, such that a disease or disorder is prevented or, alternatively, delayed in its progression. Depending on the type of aberrancy, for example, an agonist or

-₀ antagonist agent can be used for treating the subject.

2. Therapeutic Methods

Another aspect of the invention pertains to methods of modulating expression or activity of a polypeptide of the invention for therapeutic purposes. The modulatory method

35 of the invention involves contacting a cell with an agent that modulates one or more of the activities of the polypeptide. An agent that modulates activity can be an agent as described herein, such as a nucleic acid or a protein, a naturally-occurring cognate ligand of the polypeptide, a peptide, a peptidomimetic, or other small molecule. In one embodiment, the agent stimulates one or more of the biological activities of the polypeptide. Examples of such stimulatory agents include the active polypeptide of the invention and a nucleic acid

5 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

10 to a subject). As such, the present invention provides methods of treating an individual afflicted with a disease or disorder characterized by aberrant expression or activity of a polypeptide of the invention. In one embodiment, the method involves administering an agent (e.g., an agent identified by a screening assay described herein), or combination of agents that modulates (e.g., upregulates or downregulates) expression or activity. In another

15 embodiment, the method involves administering a polypeptide of the invention or a nucleic acid molecule of the invention as therapy to compensate for reduced or aberrant expression or activity of the polypeptide.

Stimulation of activity is desirable in situations in which activity or expression is abnormally low or downregulated and/or in which increased activity is likely to have a 20 beneficial effect. Conversely, inhibition of activity is desirable in situations in which activity or expression is abnormally high or upregulated and/or in which decreased activity is likely to have a beneficial effect.

All publications, patents and patent applications mentioned in this specification are herein incorporated by reference in to the specification to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference.

Examplification

30

Deposit of Clones

Clones containing cDNA molecules encoding human TANGO 201 and TANGO 223 were deposited on January 22, 1999 with the American Type Culture Collection (Manassas, VA) under accession number ATCC™ 207081, from which each cDNA clone is ^_c- obtainable. This deposit is a mixture of two strains, each carrying one recombinant plasmid. To distinguish the strains and isolate a strain harboring a particular cDNA clone, one can first streak out an aliquot of the mixture to single colonies on nutrient medium (e.g., LB plates) supplemented with 100 g/ml ampicillin, grow single colonies, and then extract the plasmid DNA using a standard minipreparation procedure. Next, one can digest a sample of the DNA minipreparation with a combination of the restriction enzymes Sal I and Not I and resolve the resultant products on a 0.8% agarose gel using standard DNA electrophoresis conditions. The digest will liberate fragments as follows:

TANGO 201 (EpT201), 2.2 kb

TANGO 223 (EpT223), 1.45 kb

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.

MICROORGANISMS

Optional Sheet in connection with the microorganism referred to on pages 9₂ and 93 . lines 31-37 and 1-7 of the description '

A. IDENTIFICATION OF DEPOSIT '

Further deposits are identified on an additional sheet

Name of depositary institution ' American Type Culture Collection

Address of depositary institution (including postal code and country) '

10801 University Blvd. Manassas, VA 201 10-2209 US

Date of deposit ' January 22, 1999 Accession Number ^• 207081

B. ADDITIONAL INDICATIONS ' (leave blank if not applicable). This information is continue- on a separate attached sheet

C. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE ' or -. ----*.,-- -,-,- -,--. *--->

D. SEPARATE FURNISHING OF INDICATIONS ' (leave blank if not applicable)

The Indications listed below will be submitted to the International Bureau later ' (Specify the general nature of the indications e.g., "Accession Number of Deposit")

E. DH This sheet was received with the International application when filed (to be checked by the receiving Office)

^• rvl McDowell

PCT/ln ΆteΓ ariΛppItea

. r-wd-ZO--USc'β "J,rv.;

D The date of receipt (from the applicant) by the International Bureau "

(Authorized Officer) Form PCT/RO/134 (January 1981 )

-93. 1 -

Claims

What is claimed is:

1. An isolated nucleic acid molecule selected from the group consisting of:

5 a) a nucleic acid molecule comprising a nucleotide sequence which is at least

55% identical to the nucleotide sequence of SEQ ID NO:l, 2, 6, 7, 11, 12, 24, 25, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56,or the cDNA insert of the plasmid deposited with the ATCC™ as Accession Number 207081, or a complement thereof; b) a nucleic acid molecule comprising a fragment of at least 300 nucleotides of 10 the nucleotide sequence of SEQ ID NO:l, 2, 6, 7, 11, 12, 24, 25, 32, 34, 36, 38, 40, 42, 44,

46, 48, 50, 52, 54, 56, or the cDNA insert of the plasmid deposited with the ATCC™ as Accession Number 207081, or a complement thereof; c) a nucleic acid molecule which encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:3, 8, 13, 26, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, or 57, or the amino acid sequence encoded by the cDNA insert of the plasmid deposited with the ATCC™ as Accession Number 207081 ; d) a nucleic acid molecule which encodes a fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO:3, 8, 13, 26, 33, 35, 37, 39, 41, 43, 45, ._rs 47, 49, 51, 53, 55, or 57, or the amino acid sequence encoded by the cDNA insert of the plasmid deposited with the ATCC™ as Accession Number 207081, wherein the fragment comprises at least 15 contiguous amino acids of SEQ ID NO:3, 8, 13, 26, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, or 57, or the amino acid sequence encoded by the cDNA insert of the plasmid deposited with the ATCC™ as Accession Number 207081 ; and

25 e) a nucleic acid molecule which encodes a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO:3, 8, 13, 26, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, or 57, or the amino acid sequence encoded by the cDNA insert of the plasmid deposited with the ATCC™ as Accession Number 207081, wherein the nucleic acid molecule hybridizes to a nucleic acid molecule comprising SEQ ID NO:l,

30 2, 6, 7, 11, 12, 24, 25, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, or a complement thereof, under stringent hybridization conditions.

2. The isolated nucleic acid molecule of claim 1, which is selected from the group consisting of:

35 a) a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 1, 2, 6, 7, 11, 12, 24, 25, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, or the cDNA insert of the

- 94 - plasmid deposited with the ATCC™ as Accession Number 207081, or a complement thereof; and b) a nucleic acid molecule which encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:3, 8, 13, 26, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, or -> 57, or the amino acid sequence encoded by the cDNA insert of the plasmid deposited with the ATCC™ as Accession Number 207081.

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

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

15

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

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

20

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

2₄- 8. An isolated polypeptide selected from the group consisting of: a) a fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO:3, 8, 13, 26, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, or 57, wherein the fragment comprises at least 15 contiguous amino acids of SEQ ID NO:3, 8, 13, 26, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, or 57;

30 b) a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO:3,

8, 13, 26, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, or 57, or the amino acid sequence encoded by the cDNA insert of the plasmid deposited with the ATCC™ as Accession Number 207081, wherein the polypeptide is encoded by a nucleic acid molecule which hybridizes to a nucleic acid molecule comprising SEQ ID

35 NO:l, 2, 6, 7, 11, 12, 24, 25, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, or a complement thereof under stringent hybridization conditions; and

- 95 c) a polypeptide which is encoded by a nucleic acid molecule comprising a nucleotide sequence which is at least 55% identical to a nucleic acid comprising the nucleotide sequence of SEQ ID NO:l, 2, 6, 7, 11, 12, 24, 25, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, or a complement thereof.

9. The isolated polypeptide of claim 8 comprising the amino acid sequence of

SEQ ID NO:3, 8, 13, 26, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, or 57.

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

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

12. A method for producing a polypeptide selected from the group consisting of: a) a polypeptide comprising the amino acid sequence of SEQ ID NO:3, 8, 13, 26, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, or the amino acid sequence encoded by the cDNA insert of the plasmid deposited with the ATCC™ as Accession Number 207081; b) a polypeptide comprising a fragment of the amino acid sequence of SEQ ID NO:3, 8, 13, 26, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, or the amino acid sequence encoded by the cDNA insert of the plasmid deposited with the ATCC™ as Accession Number 207081 , wherein the fragment comprises at least 15 contiguous amino acids of SEQ ID NO:3, 8, 13, 26, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, or the amino acid sequence encoded by the cDNA insert of the plasmid deposited with the ATCC™ as Accession Number 207081; and c) a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO:3, 8, 13, 26, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, or the amino acid sequence encoded by the cDNA insert of the plasmid deposited with the ATCC™ as Accession Number 207081, wherein the polypeptide is encoded by a nucleic acid molecule which hybridizes to a nucleic acid molecule comprising SEQ ID NO:l, 2, 6, 7, 11, 12, 24, 25, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, or a complement thereof under stringent hybridization conditions; comprising culturing the host cell of claim 5 under conditions in which the nucleic acid molecule is expressed.

- 96 -

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

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

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

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

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

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

19. A method for identifying a compound which binds to a polypeptide of claim

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

97

20. The method of claim 19, wherein the binding of the test compound to the polypeptide is detected by a method selected from the group consisting of: a) detection of binding by direct detecting of test compound/polypeptide binding; . . . b) detection of binding using a competition binding assay; c) detection of binding using an assay for TANGO 201- or TANGO 223- mediated signal transduction.

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

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

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