JP2002530064A - EGF-like nucleic acids and polypeptides and uses thereof - Google Patents

Abstract

  (57) [Summary] The present invention provides isolated nucleic acid molecules termed ELVIS-1, ELVIS-2 and ELVIS-3 (skin epidermal growth factor-like variants 1, 2 and 3). ELVIS nucleic acid molecules encode fully secreted and transmembrane proteins with homology to EGF and TGF-α. The present invention also provides an antisense nucleic acid molecule, an expression vector containing the nucleic acid molecule of the present invention, a host cell into which the expression vector has been introduced, and a non-human transgenic animal into which the nucleic acid molecule of the present invention has been introduced or disrupted. . The invention further provides isolated polypeptides, fusion polypeptides, antigenic peptides and antibodies. Also provided are diagnostic, screening and therapeutic methods using the compositions of the present invention. The nucleic acids and polypeptides of the invention are useful as modulators in regulating a variety of cellular processes. Accordingly, in one aspect, the invention provides an isolated nucleic acid molecule encoding a polypeptide of the invention or a biologically active portion thereof. The present invention also provides a nucleic acid molecule suitable as a primer or a hybridization probe for detecting a nucleic acid encoding the polypeptide of the present invention.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] background epidermal growth factor of the invention (EGF) and transforming growth factor α (TGF-α) is,
A ligand that interacts with the same cell surface receptor (EGF receptor) and acts as a mitogen for various tissues (Groenen et al. (1994) Growth Factors 11: 235-257
). Based on sequence similarity, mitogenic activity, and the ability to interact with the EGF receptor, several other EGF family members have been identified, including amphiregulin, heparin-binding EGF (HB-EGF) and betacellulin (Car
Penter et al. (1990) Handbook Exptl. Pharmacol. 95 (1): 69-171; Massague et al. (199
3) Ann. Rev. Biochem. 62: 515-541; Shing et al. (1993) Science 259: 1604-1607.
).

EGF and TGF-α are both involved in various processes from wound healing to carcinogenesis (Burgess et al. (1989) Br. Med. Bull. 45: 401-424; Massague et al. (1983) J :
Biol. Chem. 258: 13614-13620; Ozanne et al. (1986) J. Pathol. 149: 9-14).

[0003] In particular, both EGF and TGF-α induce proliferation of dermal fibroblasts (Buckley
-Sturrock et al. (1989) J. Cell. Physiol. 138: 70-78; Laato et al. (1987) J. Biochem.
247: 385-388; Paulsson et al. (1987) Nature 328: 715-717). In addition, EGF
It exerts a proliferative and chemotactic effect on fibroblasts derived from granulation tissue and induces the synthesis of collagenase and hyaluronic acid (Laato (1988) Acta. Chir. S.
cand. Suppl. 546: 4-44). EGF has been reported to promote keratinocyte proliferation and migration in vitro, which indicates that
Further suggests an important role for EGF (Barrandon et al. (1987) Cell 50: 1131-113.
7; Chernoff (1990) Tissue Cell. 22: 123-135).

[0004] Several cancer cell lines show elevated levels of EGF receptors, and TGF-α has been shown to be involved as an autocrine growth factor in many carcinomas (Groenen et al. (1994) Gr.
owth Factors 11: 235-257). Tumor-derived TGF-α is angiogenic, and it has been speculated that antagonists of TGF-α may suppress tumor angiogenesis and / or suppress carcinoma growth (Groenen et al. (1994) Growth Factors 11: 235-2
57; Okamura et al. (1992) Biochem. Biophys. Res. Comm. 186: 1471-1479).

SUMMARY OF THE INVENTION [0005] The present invention relates, at least in part, to epidermal growth factor-like variants in skin proteins herein.
Transforming growth factor α (TGF-α) and epidermal growth factor (EG
F) is based on the discovery of cDNA molecules encoding a novel family of proteins with significant identity to ("ELVIS" family or "ELVIS" proteins). In the following, human ELVIS-1 (also referred to herein as "TANGO 225" or "T225") and its splice variants referred to as ELVIS-2 and ELVIS-3 (herein referred to as "T225vl" and "T225vl", respectively) T225v2 "). These proteins, fragments, derivatives and variants thereof are collectively referred to as "the polypeptide of the present invention" or "the protein of the present invention". Nucleic acid molecules encoding a polypeptide of the invention are collectively referred to as "nucleic acids of the invention".

[0006] The nucleic acids and polypeptides of the present invention are useful as modulators in regulating a variety of cellular processes. Accordingly, in one aspect, the invention provides an isolated nucleic acid molecule encoding a polypeptide of the invention or a biologically active portion thereof. The present invention also provides a nucleic acid molecule suitable as a primer or a hybridization probe for detecting a nucleic acid encoding the polypeptide of the present invention.

[0007] The present invention relates to the nucleotide sequence of SEQ ID NO: 1, 3, 4, 6, 7 or 9, or the cDNA insert of the clone deposited with the ATCC under accession numbers 98981, 98982 and 98983 ("ATCC98981, 98982 and 98983"). At least 45% (or 55%, 65%, 75%) of the nucleotide sequence of any cDNA ") or its complement.
, 85%, 95%, or 98%).

[0008] The invention relates to a nucleotide sequence of SEQ ID NO: 1, 3, 4, 6, 7 or 9, or at least 300 of the nucleotide sequence of the cDNA of any of the ATCC accession numbers 98981, 98982 and 98983, or the complement thereof. 325, 350, 375, 400, 425, 450, 500,
550, 600, 650, 700, 800, 900, 1000 or 1200) nucleotides.

[0009] The present invention also relates to an amino acid sequence of SEQ ID NO: 2, 5 or 8, or at least 45% (or 55%, 65%) to the amino acid sequence encoded by the cDNA of ATCC Accession No. 98981, 98982 and 98983. %, 75%, 85%, 95%, or 98%), characterized by a nucleic acid molecule comprising a nucleotide sequence encoding a protein having an amino acid sequence having the identity of (%, 75%, 85%, 95% or 98%), or a complement thereof.

In a preferred embodiment, the nucleic acid molecule has the nucleotide sequence of SEQ ID NO: 1, 3, 4, 6, 7 or 9 or the nucleotide sequence of any of the cDNAs of ATCC accession numbers 98981, 98982 and 98983.

[0011] The present invention also provides a fragment of a polypeptide having the amino acid sequence of SEQ ID NO: 2, 5 or 8, or SEQ ID NO: 2, 5 or 8 or ATCC Accession No. 98981, 989.
At least 15 of the amino acids encoded by the cDNA of either 82 or 98983 (2
Nucleic acid molecules that encode fragments containing 5, 30, 50, 100, 150, 300 or 400) contiguous amino acids are included.

[0012] The present invention relates to the amino acid sequence of SEQ ID NO: 2, 5 or 8, or the ATCC accession number 98
981, 98982 and 98983 include nucleic acid molecules encoding naturally occurring allelic variants of a polypeptide comprising the amino acid sequence encoded by the cDNA of any of SEQ ID NO: 2, Hybridizing under stringent conditions with a nucleic acid sequence encoding the amino acid sequence of 5 or 8 or the amino acid sequence encoded by the cDNA of any of ATCC Accession Nos. 98981, 98982 and 98983, or a complement thereof. Soy.

The present invention also provides at least about 60%, preferably 65%, of the amino acid sequence of SEQ ID NO: 2, 5 or 8 or the amino acid encoded by the cDNA of any of ATCC Accession Nos. 98981, 98982 and 98983. , 75%, 85%, 95% or 98% identity.

The invention also has a nucleotide sequence that has at least about 60%, preferably 65%, 75%, 85% or 95% identity to the nucleic acid sequence encoding SEQ ID NO: 2, 5, or 8. The isolated polypeptide or protein encoded by the nucleic acid molecule, and the nucleotide sequence of SEQ ID NO: 1, 3, 4, 6, 7, 9 or its complement, or any of ATCC accession numbers 98981, 98982 and 98983 cDN
Included is an isolated polypeptide or protein encoded by a nucleic acid molecule having a nucleotide sequence that hybridizes under stringent hybridization conditions to a nucleic acid molecule having the nucleotide sequence of the non-coding strand of A.

The present invention also provides a naturally occurring allele of a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, 5 or 8, or the amino acid sequence encoded by any of the cDNAs of ATCC Accession Nos. 98981, 98982 and 98983. Included are polypeptides that are genetic variants, wherein the polypeptide hybridizes under stringent conditions to a nucleic acid molecule comprising SEQ ID NO: 1, 3, 4, 6, 7, 9, or a complement thereof. Encoded by a nucleic acid molecule that

The present invention also provides a nucleotide sequence of SEQ ID NO: 1, 3, 4, 6, 7 or 9;
The nucleotide sequence of any of the cDNAs of ATCC accession numbers 98981, 98982 and 98983,
Or a nucleic acid molecule that hybridizes under stringent conditions with a nucleic acid molecule containing the complement thereof. In another embodiment, the nucleic acid molecule is at least 300 (325, 350, 375, 400, 425, 450, 500, 550, 600, 650, 700, 800, 900
, 1000 or 1290) nucleotides in length and comprises the nucleotide sequence of SEQ ID NO: 1, 3, 4, 6, 7 or 9, the nucleotide sequence of any of the ATCC accession numbers 98981, 98982 and 98983, or the complement thereof. Hybridizes with a nucleic acid molecule under stringent conditions.

In one embodiment, the isolated nucleic acid molecule is an extracellular domain (SEQ ID NO: 12), a transmembrane domain (SEQ ID NO: 11), and / or a cytoplasmic domain (SEQ ID NO: 10) of a polypeptide of the invention. Code. In another embodiment, the invention provides an isolated nucleic acid molecule that is antisense to the coding strand of a nucleic acid of the invention.

[0018] Another aspect of the invention provides a vector, eg, a recombinant expression vector, containing a nucleic acid molecule of the invention. In another embodiment, the invention provides a host cell containing such a vector or a nucleic acid molecule of the invention. The present invention also provides a method for producing the polypeptide of the present invention. The method is performed by culturing the host cell of the present invention containing the recombinant expression vector in an appropriate medium to produce the polypeptide.

[0019] Another aspect of the invention features the isolated or recombinant polypeptides and proteins of the invention. Preferred proteins and polypeptides are those that have at least one of the biological activities of the corresponding naturally occurring human polypeptide. The activity, biological activity and functional activity of the polypeptide or nucleic acid of the present invention, when measured in vivo or in vitro by standard methods,
It refers to the activity of a protein, polypeptide or nucleic acid molecule of the present invention on responding cells. Such an activity may be a direct activity (such as an association with a second protein or an enzymatic activity on the second protein) or an indirect activity (by interaction of the protein of the present invention with the second protein). Mediated cell signaling activity). Thus, such activities include, for example, (1) the ability to cause protein-protein interactions with proteins in the signaling pathway of naturally occurring polypeptides; (2) the ability to bind ligands of naturally occurring polypeptides; 3) Interaction between ELVIS protein and receptor. Other activities include
(1) the ability to modulate cell proliferation; (2) the ability to modulate cell migration and chemotaxis; (3) the ability to modulate cell differentiation; (4) the ability to modulate angiogenesis. (5) the ability to modulate proliferation and / or differentiation of epithelial cells; and (6) the ability to modulate keratinocyte proliferation and / or differentiation.

In one embodiment, a polypeptide of the invention has an amino acid sequence that has sufficient identity to an identified domain of a polypeptide of the invention. As used herein, the term "has sufficient identity" refers to a sufficient number of amino acid residues or nucleotides that are identical or equivalent (e.g., having a similar side chain) to a second amino acid or nucleotide sequence. Or a first amino acid or nucleotide sequence containing a minimal number, whereby the first and second amino acid or nucleotide sequences have a common structural domain and / or a common functional activity. For example, about 60% identity, preferably 65% identity, more preferably 75% identity.
Amino acid or nucleotide sequences containing a common structural domain having%, 85%, 95%, 98% or more identity are defined herein as having sufficient identity. In one embodiment, the ELVIS protein comprises an EGF domain. In another embodiment, the ELVIS protein includes a signal sequence. In yet other embodiments, the ELVIS protein comprises an EGF domain and a signal sequence, wherein the ELVIS protein is a secreted protein. In another embodiment, the ELVIS protein comprises a transmembrane domain. In yet other embodiments, the ELVIS protein comprises an EGF domain, a transmembrane domain, a signal sequence, wherein the ELVIS protein is a membrane-bound protein.

[0021] In one embodiment, the isolated polypeptide lacks both the transmembrane domain and the cytoplasmic domain. In other embodiments, the polypeptide lacks both the transmembrane and cytoplasmic domains and is soluble under physiological conditions.

In another embodiment, a nucleic acid molecule of the invention encodes an ELVIS protein comprising an epidermal growth factor (EGF) domain. In another embodiment, a nucleic acid molecule of the invention encodes an ELVIS protein comprising a signal sequence. In other embodiments,
The nucleic acid molecules of the invention encode an ELVIS protein comprising an EGF domain and a signal sequence. In yet another embodiment, a nucleic acid molecule of the invention encodes an ELVIS protein comprising a transmembrane domain.

A polypeptide of the invention, or a biologically active portion thereof, can be operably linked to a heterologous amino acid sequence to form a fusion protein. The invention further features an antibody that specifically binds a polypeptide of the invention, eg, a monoclonal or polyclonal antibody. Further, a polypeptide of the present invention or a biologically active portion thereof can be included in a pharmaceutical composition, which can optionally include a pharmaceutically acceptable carrier.

In another aspect, the invention provides a method for detecting the presence of activity or expression of a polypeptide of the invention in a biological sample. The method is performed by contacting the biological sample with an agent capable of detecting an indicator of activity, and detecting the presence of the activity in the biological sample.

[0025] In another aspect, the invention provides a method of modulating the activity of a polypeptide of the invention. The method includes contacting the cell with an agent that modulates (suppresses or promotes) the activity or expression of the polypeptide of the present invention so that the activity or expression in the cell is modulated. It is. In one embodiment, the agent is an antibody that specifically binds a polypeptide of the invention.

In another embodiment, the agent is an mRNA encoding a polypeptide of the invention.
By modulating the transcription, splicing or translation of the polypeptide of the present 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 a method for treating a subject suffering from a disease characterized by abnormal activity of the polypeptide of the present invention or abnormal expression of the nucleic acid of the present invention. The method is performed by administering to the subject an agent that is a modulator of the activity of the polypeptide of the invention or the expression of the nucleic acid of the invention. In one embodiment, the modulator is a protein of the invention. In another embodiment, the modulator is a nucleic acid of the invention. In another embodiment, the modulator is
A peptide, peptidomimetic, or other small molecule.

The present invention also provides a diagnostic assay for identifying the presence or absence of genetic damage or mutation characterized by at least one of the following (i) to (iii): That is, when the wild-type gene encodes a protein having the activity of the polypeptide of the present invention, (i) abnormal modification or mutation of the gene encoding the polypeptide of the present invention; Misregulation of the gene encoding the peptide, and (iii) abnormal post-translational modification of the gene encoding the polypeptide of the invention.

In another aspect, the invention provides a method for identifying a compound that binds to a polypeptide of the invention or that modulates the activity of a polypeptide of the invention.
Generally, such methods require measuring the biological activity of the polypeptide in the presence or absence of the test compound, and identifying compounds that alter the activity of the polypeptide.

The invention also features a method of identifying a compound that modulates expression of a polypeptide or nucleic acid of the invention. The method is performed by measuring the expression of the polypeptide or nucleic acid in the presence and absence of the compound.

[0031] Other features and advantages of the invention will be apparent from the following detailed description, and from the claims set forth herein.

DETAILED DESCRIPTION The present invention relates, at least in part, to proteins having significant identity to transforming growth factor α and epidermal growth factor, referred to herein as epidermal growth factor-like variants of skin proteins. Based on the discovery of a cDNA molecule encoding a novel family of proteins ("ELVIS" family or "ELVIS" protein). As used herein, ELVIS-1 (SEQ ID NO: 1), ELVIS-2 (SEQ ID NO: 4) and ELVIS-3 (SEQ ID NO: 7) nucleic acid molecules, and the corresponding polypeptides encoded by the nucleic acid molecules (SEQ ID NO: 2, SEQ ID NO: 5 and SEQ ID NO: 8) are described.

[0033] ELVIS proteins and nucleic acid molecules consist of a family of molecules that have certain conserved structural and functional characteristics. As used herein, the term "family" refers to two or more protein or nucleic acid molecules that have a common structural domain, and that have sufficient amino acid or nucleotide sequence identity as defined herein. Shall mean. Family members can be of the same or different origin. For example, a family may include two or more proteins of human origin, or may include one or more proteins of human origin and one or more proteins of non-human origin. Members of the same family may also have common structural domains.

For example, EGF / TGF- having significant sequence identity to the ELVIS protein of the present invention
The alpha family is a family of mitogens that contain a conserved cysteine residue pattern. As used herein, a conserved cysteine residue refers to a cysteine residue retained within an ELVIS family member (and / or an EGF / TGF-α family member). This cysteine pattern
It is referred to herein as epidermal growth factor (EGF) domain. These cysteine residues form disulfide bonds that can affect the structural integrity of the protein. Thus, ELVIS proteins having an EGF domain are included within the scope of the present invention. As used herein, an EGF domain is about 25-50,
It preferably refers to an amino acid sequence of about 30-45, 30-40, more preferably about 35, 36-40 amino acids in length. The EGF domain may further comprise at least about 2-10, preferably 3-9
It contains 4-8, or 6-7 conserved cysteine residues. The EGF domain of ELVIS-1 extends from about amino acid 60 of SEQ ID NO: 2 (SEQ ID NO: 15) to about amino acid 95; the EGF domain of ELVIS-2 comprises the amino acid of SEQ ID NO: 5 (SEQ ID NO: 16)
Extending from 51 to about amino acid 87; the EGF domain of ELVIS-3 is SEQ ID NO: 8 (
It extends from amino acid 51 of SEQ ID NO: 17 to around amino acid 86. By aligning the amino acid sequence of TGF-α with the amino acid sequences of ELVIS-1, ELVIS-2 and ELVIS-3, conserved cysteine residues can be found. For example, there is a conserved cysteine residue at amino acid 47 of human TGF-α (SEQ ID NO: 13), which is amino acid 60 of ELVIS-1 (SEQ ID NO: 2), amino acid 51 of ELVIS-2 (SEQ ID NO: 5), And ELVIS-3
(SEQ ID NO: 8) corresponding to the cysteine residue located at amino acid 51;
There is a conserved cysteine residue at amino acid 55 of human TGF-α (SEQ ID NO: 13), which is amino acid 68 of ELVIS-1 (SEQ ID NO: 2) and amino acid 59 of ELVIS-2 (SEQ ID NO: 5).
, And corresponds to the cysteine residue located at amino acid 59 of ELVIS-3 (SEQ ID NO: 8); there is a conserved cysteine residue at amino acid 60 of human TGF-α (SEQ ID NO: 13). Human corresponding to cysteine residue located at amino acid 73 of ELVIS-1 (SEQ ID NO: 2), amino acid 64 of ELVIS-2 (SEQ ID NO: 5) and amino acid 64 of ELVIS-3 (SEQ ID NO: 8); human There is a conserved cysteine residue at amino acid 71 of TGF-α (SEQ ID NO: 13), which is amino acid 84,
Amino acid 75 of LVIS-2 (SEQ ID NO: 5) and amino acid 7 of ELVIS-3 (SEQ ID NO: 8)
Corresponding to the cysteine residue located at position 5; human TGF-α (SEQ ID NO: 13).
) Has a conserved cysteine residue at amino acid 73, which corresponds to ELVIS-1 (SEQ ID NO: 2).
), Amino acid 77 of ELVIS-2 (SEQ ID NO: 5), and cysteine residue located at amino acid 77 of ELVIS-3 (SEQ ID NO: 8); and / or human TGF-α At amino acid 82 of (SEQ ID NO: 13) there is a conserved cysteine residue, which is amino acid 95 of ELVIS-1 (SEQ ID NO: 2), amino acid 87 of ELVIS-2 (SEQ ID NO: 5), and ELVIS-3 (SEQ ID NO: 5). It corresponds to the cysteine residue located at amino acid 86 of No. 8).

In addition, ELVIS proteins having a signal sequence are also included in the scope of the present invention. As used herein, "signal sequence" includes a peptide that is at least about 20 amino acid residues in length, is present at the N-terminus of secreted and membrane-bound proteins, and has at least about 70% hydrophobic amino acid residues. (Eg, alanine, leucine, isoleucine, phenylalanine, proline, tyrosine, tryptophan or valine). In a preferred embodiment, the signal sequence is at least about
It contains 15-25 amino acid residues, preferably about 18-22 amino acid residues, and has at least about 60-80%, more preferably 65-75%, even more preferably at least about 70% hydrophobicity It has a sex residue. Signal sequences serve to direct proteins containing such sequences to lipid bilayers. Thus, in one embodiment, the ELVIS protein contains a signal sequence around amino acids 1-20 of SEQ ID NO: 2, SEQ ID NO: 5 or SEQ ID NO: 8. The signal sequence is cleaved during processing of the mature protein.

The present invention also includes an ELVIS protein having a transmembrane domain. As used herein, a “transmembrane domain” has a length of at least about 20-25 amino acid residues and at least about 65-70% of hydrophobic amino acid residues (eg, alanine, leucine, isoleucine, phenylalanine, Proline, tyrosine, tryptophan or valine). In a preferred embodiment, the transmembrane domain contains at least about 15-30 amino acid residues, preferably about 20-25 amino acid residues, and at least about 60-80%, more preferably 65-75. %
And even more preferably those having at least about 70% hydrophobic residues. An example of a transmembrane domain includes amino acids 111-133 of SEQ ID NO: 2.

[0037] In one embodiment, the ELVIS protein of the present invention contains an EGF domain and a signal sequence and is a secreted protein. In another embodiment, an ELVIS protein of the invention contains an EGF domain and a transmembrane domain and is a membrane-bound protein.
In yet other embodiments, the ELVIS proteins of the present invention contain an EGF domain, a signal sequence and a transmembrane domain.

The various features of human ELVIS-1, ELVIS-2 and ELVIS-3 are outlined below.

Human ELVIS-1 cDNA encoding human ELVIS-1 was identified by screening a human keratinocyte library. Briefly, a cDNA library was prepared using cDNA prepared from RNA extracted from a human keratinocyte library and directionally cloned. Subsequently, ESTs were made from the 5 'end. Clones were selected based on homology to TGF-α as determined by BlastX screening.

One such clone was identified that contained human ELVIS-1. ELVIS-1 contains a 1858 nucleotide cDNA (FIG. 1; SEQ ID NO: 1). Nucleotides 31 to 492 (SEQ ID NO: 3), the open reading frame of this cDNA, encode a putative membrane protein of 154 amino acids (FIG. 1; SEQ ID NO: 2).

SIGNALP, a signal peptide prediction program (Nielsen et al., (1997) Protein
Engineering 10: 1-6), human ELVIS-1 is a 20 amino acid signal peptide (SEQ ID NO: 2) preceding a mature ELVIS-1 protein (SEQ ID NO: 2, corresponding to approximately amino acids 21-154; SEQ ID NO: 18). From about amino acid 1 to about amino acid 20). Human ELVIS-1 has an extracellular domain (amino acids 21 to 110 of SEQ ID NO: 2; SEQ ID NO: 12) and a transmembrane (TM) domain (amino acids 111 to 133 of SEQ ID NO: 2; SEQ ID NO: 11).
), And a cytoplasmic domain (amino acids 134-154 of SEQ ID NO: 2; SEQ ID NO: 10).

The extracellular region of human ELVIS-1 comprises an EGF-domain (approximately amino acids 60-95 of SEQ ID NO: 2).
SEQ ID NO: 15).

Epjthkt074e01, a clone encoding human ELVIS-1, was obtained from American Typ.
e Culture Collection (ATCC, 10801 University Boulevard, Manassas, VA 2011
0-2209) and deposited on November 11, 1998, and assigned accession number 98981. This deposit is maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for Patent Procedure. This deposit is made for the convenience of those skilled in the art only and does not imply that the deposit was made to meet the requirements of 35 USC 112.

FIG. 2 shows a hydropathy plot of human ELVIS-1. Relatively hydrophobic residues are above the horizontal line, and relatively hydrophilic residues are below the horizontal line. As shown in this hydropathy plot, the hydrophobic region at the start of the plot (corresponding to amino acids 1 to 20 of SEQ ID NO: 2) is the signal sequence of ELVIS-1. Further, a hydrophobic region in the center of the plot (amino acid of SEQ ID NO: 2)
111-133) is the transmembrane domain of ELVIS-1. Cysteine residues (cys) and potential N-glycosylation sites (Ngly) are indicated by short vertical lines just below the hydropathy race.

Using the amino acid sequence of human ELVIS-1 (SEQ ID NO: 2), a public database was searched to identify proteins having homology to human ELVIS-1 (BLASTP; Altschul et al., ( 1990) J. Mol. Biol. 215: 403-410). This analysis revealed that human ELVIS-1 has considerable homology to various species of TGF-α (eg, mouse, rat, pig, sheep TGF-α). FIG. 7 shows an alignment of the amino acid sequences of human ELVIS-1 and human TGF-α with 24.4% sequence identity (SEQ ID NO: 13).

Human ELVIS-2 Two additional clones with significant homology to human ELVIS-1 and TGF-α were identified by the screening method described above. Sequencing of both of these clones revealed splice variants of ELVIS-1. The first variant is referred to herein as ELVIS-2. Human ELVIS-2 cDNA (FIG. 3; SEQ ID NO: 4) contains an open reading frame (nucleotides 15-323 of SEQ ID NO: 4; SEQ ID NO: 6) encoding a putative secreted protein of 103 amino acids (FIG. 3; SEQ ID NO: 5). Including. SIGNALP, a signal peptide prediction program (Nielsen et al., (1997) Protein
Engineering 10: 1-6), human ELVIS-2 is a mature protein (SEQ ID NO: 5,
It was predicted to include a signal peptide of 20 amino acids (amino acids 1 to about 20 of SEQ ID NO: 5) preceding about amino acids 21 to 103; SEQ ID NO: 19). Human ELVIS-1 also had an EGF domain (approximately amino acids 51-87 of SEQ ID NO: 5; SEQ ID NO: 16).

The clone jthkt037e06 encoding human ELVIS-2 was obtained from American Type Culture Co.
llection (ATCC, 10801 University Boulevard, Manassas, VA 20110-2209)
Deposited on November 11, 2008 and assigned accession number 98982. This deposit is maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for Patent Procedure. This deposit is made for the convenience of those skilled in the art only and does not imply that the deposit was made to meet the requirements of 35 USC 112.

FIG. 4 shows a hydropathy plot of the splice variant ELVIS-2. Relatively hydrophobic residues are above the horizontal line, and relatively hydrophilic residues are below the horizontal line. As shown in this hydropathy plot, the hydrophobic region (corresponding to amino acids 1 to 20 of SEQ ID NO: 5) at the start of the plot was ELVIS-
2 is a signal sequence. Cysteine residues (cys) and potential N-glycosylation sites
(Ngly) is indicated by a short vertical line immediately below the hydropaseat race.

Human ELVIS-3 A second clone with significant homology to human ELVIS-1 and TGF-α is a splice variant of human ELVIS-1, referred to herein as ELVIS-3. Human ELVI
The S-3 cDNA (FIG. 5; SEQ ID NO: 7) is an open reading frame (nucleotide of SEQ ID NO: 7) encoding a putative secreted protein of 94 amino acids (FIG. 3; SEQ ID NO: 8).
12 to 294; SEQ ID NO: 9). SIGNALP (Signal peptide prediction program
According to Nielsen et al., (1997) Protein Engineering 10: 1-6), human ELVIS-3 is a 20 amino acid signal peptide preceding the mature protein (approximately amino acids 21-94 of SEQ ID NO: 8; SEQ ID NO: 20). (Amino acid 1 to about amino acid 2 of SEQ ID NO: 8
0).

Human ELVIS-3 also contains an EGF domain (approximately amino acids 51-8 of SEQ ID NO: 8).
6; SEQ ID NO: 17).

The clone jthkf064b08 encoding human ELVIS-3 was obtained from American Type Culture Co.
llection (ATCC, 10801 University Boulevard, Manassas, VA 20110-2209)
Deposited on November 11, 2008 and assigned accession number 98983. This deposit is maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for Patent Procedure. This deposit is made for the convenience of those skilled in the art only and does not imply that the deposit was made to meet the requirements of 35 USC 112.

FIG. 6 shows a hydropathy plot of the splice variant ELVIS-3. Relatively hydrophobic residues are above the horizontal line, and relatively hydrophilic residues are below the horizontal line. As shown in the hydropathy plot, the hydrophobic region (corresponding to amino acids 1 to 20 of SEQ ID NO: 8) at the start of the plot was ELVIS-
3 is a signal sequence. Cysteine residues (cys) and potential N-glycosylation sites
(Ngly) is indicated by a short vertical line immediately below the hydropaseat race.

Further sequence analysis of the ELVIS-3 cDNA described above provided the cDNA sequence shown in FIG. 9 (SEQ ID NO: 21).

Comparison of ELVIS-1, ELVIS-2 and ELVIS-3 Human ELVIS-1 (SEQ ID NO: 2), ELVIS-2 (SEQ ID NO: 5) and ELVIS-3 (SEQ ID NO: 8)
FIG. 8 shows an alignment of the amino acid sequences of This alignment was performed using the Clustal algorithm, which is part of the MEGALIGN program. The parameters for multiple alignment are as follows: PAM250 scoring matrix;
Gap penalty = 10; Gap length penalty = 0.05. ELVIS-1 is ELVIS-2
67% for ELVIS-2 and 61% for ELVIS-2, while 9% for ELVIS-2 and ELVIS-3
1.3% identity.

This alignment shows that ELVIS-1 has a signal sequence from amino acids 1-20 of SEQ ID NO: 2, which is the signal sequence of ELVIS-2 (amino acids 1-20 of SEQ ID NO: 5).
) And the signal sequence of ELVIS-3 (amino acids 1 to 20 of SEQ ID NO: 8). Furthermore, this alignment shows that ELVIS-1 has an extracellular, transmembrane, and cytoplasmic domain, while both ELVIS-2 and ELVIS-3 lack the transmembrane and cytoplasmic domains. Show. Thus, it is concluded that ELVIS-2 and ELVIS-3 are themselves fully secreted forms of ELVIS-1, a membrane-bound protein.

Antibodies to ELVIS Polyclonal antibodies were generated in rabbits by standard techniques using the following peptides: LCLEDHNSYCING (SEQ ID NO: 22), GYTGERCEHLTLT (SEQ ID NO: 23)
And LKSPYNVCSGERRPL (SEQ ID NO: 24).

A retroviral expression vector capable of expressing ELVIS-1 or ELVIS-2, which increases the growth rate of fibroblasts by the expression of ELVIS-1, was prepared by using MSCV
It was prepared using a puro retrovirus vector. ELVIS-1 and ELVIS-2 obtained
A retrovirus for infecting NIH3T3 fibroblasts was prepared using the retrovirus expression vector, and ELVIS-1 and ELVIS-2 expressing fibroblasts were created. Infected cells were grown for 8 days under puromycin selection conditions. ELVIS-
The growth rates of 1-expressing fibroblasts, ELVIS-2 expressing fibroblasts and control fibroblasts were
It was measured by thymidine incorporation as follows. Cells were plated at various concentrations in 96-well plates. Twenty-four hours later, 1 μC of H ³ -thymidine was added to each well. Six hours after labeling, cells were harvested and the level of H ³ -thymidine incorporation was measured.

This analysis showed that ELVIS-1 expressing fibroblasts grew twice as fast as control fibroblasts.

ELVIS-2 Stimulates Keratinocyte Proliferation and Differentiation The effect of ELVIS-2 on keratinocyte proliferation and differentiation was measured as follows. In the first experiment, about 50,000 human fetal keratinocytes (strain YF29)
Were plated on irradiated NIH3T3 cells in cFAD medium with various combinations of TGF-α and recombinant ELVIS-2. Cells were fed every three days. On day 10, cells were fixed in formaldehyde and stained with rhodamine B and observed. Cells cultured in the presence of ELVIS-2 overlaid the epithelial layer rather than spreading and growing on the plate. Cells cultured in the presence of 100 ng / mL ELVIS-2 formed raised "blisters" of cells. In a second experiment, approximately 20 human fetal keratinocytes (strain YF29) were plated on irradiated NIH3T3 cells in cFAD medium. Six days later, TGF-α and / or ELVIS-2 were added to the medium. 4 cells
Supplied daily. On day 16, cells were fixed in formaldehyde and rhodamine B
And observed. This experiment shows that in both the presence and absence of TGF-α,
Exposure to ELVIS-2 was found to induce "blister" formation in cell culture. Induction of blister formation by ELVIS-2 suggests that ELVIS can stimulate keratinocyte proliferation and differentiation.

ELVIS-2 and ELVIS-3 are fully secreted proteins. A secretion assay showed that both ELVIS-2 and ELVIS-3 were secreted into the medium.

ELVIS Chromosome Location ELVIS genomic mapping revealed that ELVIS is located on the map at chromosome position 4q12-13.

Northern blot analysis of ELVIS expression Northern blot analysis of ELVIS expression revealed expression in keratinocytes and HeLa cells.

In Situ Analysis of ELVIS Expression ELVIS expression in situ was analyzed using two probes: one for the ELVIS coding region and one for the 3′UTR of ELVIS. With this analysis, ELVIS
Was found to be expressed in stratified squamous epithelium in the cervix. No expression was found in human skin, monkey skin, human esophagus or human tonsils.

ELVIS as EGF Agonists and Antagonists ELVIS, and modulators of ELVIS expression or activity, can act as EGF agonists or antagonists. Such a molecule is the EGF receptor erbB2 (H
It may increase or decrease the activity of the ER-2), erbB3 (HER-3) and / or erbB4 (HER-4) receptors. The effects of ELVIS, and modulators of ELVIS expression or activity, on these receptors can be tested using cells that express one or more of these receptors. For example, A431 cells (ATCC CRL-1555) overexpress the EGF receptor; SKOV-3 cells (ATCC HTB-77) express erbB2; SKBR-3 cells (ATCC HTB-
30) express the EGF receptors erbB2 and erbB3; and MDA MB-453 cells (ATCC HTB-13
1) expresses erbB2, erbB3 and erbB4.

EGF, TGF-α, or neuregulin and ELVIS polypeptide (
Standard media (eg, DME supplemented with 10% fetal calf serum) in the presence or absence of the extracellular domain of ELVIS-1 or a modulator of ELVIS expression and activity
Propagate the cells in M). The activity of the receptor expressed by the cells can be measured directly using anti-phosphotyrosine antibodies or indirectly using standard cell proliferation assays. For example, MDA-MB453, SKBR-3 and SKOV-3 cells are grown to 80% density in DMEM supplemented with 10% FCS in a humidified atmosphere of 37 ° C., 5% CO ₂ . The cells were then replated in serum-free medium for 16 hours, followed by NDF (100 ng / mL).
), EGF (100 ng / mL) or various concentrations of ELVIS at 37 ° C. for 15 minutes. Prepare cell lysate and separate comparable amounts of protein on a 10% SDS PAGE gel. After transfer to nitrocellulose, immunodetection of phosphorylated proteins was performed using monoclonal antibody 4G10 (Upstate Biotechnology, NY) according to the manufacturer's instructions and ECL (A
mersham). NDF and EGF can be purchased from R & D Systems (Minneapolis, MN). For immunoprecipitation assays, erbB4, EGFR, erbB3 or 1 mg / ml concentration
Incubate 1 mg of cell extract with antibody to erbB2 (Santa Cruz, Ca) at 4 ° C. for 2 hours. Then, transfer the sample to Protein G agarose.
After binding for 12 hours at C, wash four times in lysis buffer. The immunoprecipitated sample is resuspended in sample buffer, boiled for 5 minutes and then electrophoresed on an SDS PAGE gel. The gel is subsequently transferred and blotted as detailed above.

[0066]

[Table 1]

[0067]

[Table 2]

Use of ELVIS Nucleic Acids, Polypeptides and ELVIS Agonists or Antagonists The ELVIS proteins of the present invention include a family of proteins having significant identity to TGF-α, a member of the EGF / TGF-α family of proteins. . Amphiregulin, heparin-binding EGF (HB-EGF) and betacellulin (
betacellulin), other members of the EGF / TGF-α family have sequence similarities,
It has been identified based on its blastogenic activity and its ability to interact with the EGF receptor. ELVIS
Based on the sequence similarity between the protein and TGF-α, the ELVIS protein is EGF / TGF-
It is thought that they function in a similar manner as members of the alpha family. Thus,
ELVIS modulators could be used for the treatment of any EGF / TGF-α related disorder.

For example, the ELVIS protein may play a role in wound healing. In vitro studies have shown that EGF and TGF-α affect a number of cellular processes in soft tissue repair and are classified as wound hormones for wound healing. For example, both EGF and TGF-α induce dermal fibroblast proliferation (Buckley-Sturrock et al., (1989) J. Cell. Biol. 138: 70-78; Laato et al., (19987)
J. Biochem. 247: 385-388), EGF exerts the proliferative and chemotactic effects of fibroblasts derived from granulation tissue and induces collagenase and hyaluronic acid synthesis.
(Lato et al., (1988) Acta.Chir.Scand.Suppl. 546: 4-44), and EGF stimulates keratinocyte proliferation and migration (Barrandon et al., (1987) Cell 50: 1131-1137,
Chernoff (1990) Growth Factors 11: 235-257). TGF-α is also present in large amounts in self-renewing tissues, such as skin, and can thus provide germ-line signals to self-renewing cells.

Clinical studies on EGF have also shown that EGF hastens the treatment of burn patients who have undergone skin transplantation (Brown et al., (1989) N. Engl. J. Med. 321: 76-79). ). The ELVIS nucleic acid molecule, protein or modulator thereof of the present invention can be used for various cells (
For example, it may act on various skin cells involved in wound healing. The effects of ELVIS nucleic acid molecules, proteins or their modulators on various cells (eg, skin cells) include proliferation and migration. ELVIS-1 is isolated from a keratinocyte library, expressed in keratinocytes, and has homology to TGF-α, and thus may play a role in keratinocyte proliferation and / or migration. Accordingly, the ELVIS proteins, nucleic acids and / or modulators of the present invention are useful for wound healing such as burns.

Furthermore, the action of the ELVIS protein of the present invention on various skin cells can be applied to cosmetics. For example, the ELVIS protein plays a role in the growth and / or migration of skin cells, such as hair follicles, and is therefore useful for regulating (eg, stimulating or promoting) hair growth. Accordingly, the ELVIS proteins, nucleic acids and / or
Or modulators have cosmetic use for the treatment of hair loss or the treatment of unwanted hair growth.

A number of different cancer cells show elevated EGF-receptor levels (Groenen et al. (1994) Gro
wth Factors 11: 235-257). In addition, TGF-α is associated with the growth and maintenance of tumors, including tumors of the lung, breast, head and neck, prostate, brain, liver, bladder, endometrium, kidney and gastrointestinal tract ( Oppenheim et al., Clinical Applications of Cytokin
es: Role in Pathogenesis, Diagnosis and Therapy (Oxford University Press,
New York, NY, 1993, pp 263-265)). TGF-α toxin to EGF-receptor, such as P
TGF- fused to seudomonas exotoxin (PE), diphtheria toxin (DT) or ricin
For α, experimental trials for cancer treatment have begun. Further, it is believed that antagonists of TGF-α can compete with autocrine TGF-α and thus inhibit cancer growth.

[0073] ELVIS nucleic acid molecules, proteins or modulators thereof also modulate cell proliferation and are therefore useful for modulating proliferative disorders such as cancer. Thus, ELVIS nucleic acid molecules, proteins or modulators thereof (such as antibodies) can be used in epithelial cancer cells (tumor cells; e.g., cancerous lung, breast, head and neck, prostate, liver, brain, bladder, child). Receptors such as EGF receptors (e.g., EGFr, HER-2, HER-3, or HER-4) that are present on cancer cells such as cervix, endometrium, kidney, epithelial cells of the gastrointestinal or ovarian tissues Can be targeted and fused with a therapeutic component (such as a toxin) and administered in combination with a therapeutic component (such as PE, DT or lysine). In addition, ELVIS
Modulators (such as antagonists and antibodies) associated with abnormal ELVIS expression,
For example, it can be used to modulate (eg, suppress or reverse) a proliferative disorder such as cancer. Thus, modulators of ELVIS activity (such as antagonists) and ELVIS proteins used as toxin targeting agents can be used to treat proliferative disorders such as cancer.

In particular, the ELVIS protein may play a role in the proliferation and migration of various skin cells. Thus, abnormal ELVIS expression and / or ELVIS activity may be involved in various skin growth disorders. Therefore, modulators of ELVIS expression and / or activity (ELVIS
Antagonists and the like) are useful in treating skin proliferative disorders such as psoriasis. Modulators of ELVIS expression and / or activity (such as ELVIS antagonists)
) Are useful in the treatment of contact dermatitis.

The locus MHW1 (603663) on the 4p of D4S2949 is strongly associated with good mental health status (eg, absence of bipolar affective disorder) in the Old Order Amish pedigree. (Ginns et al., (1998)). These findings were consistent with the hypothesis that certain alleles could prevent or alter the clinical manifestations of bipolar affective disorder, and possibly other related affective disorders. Thus, modulators of ELVIS expression or activity are useful for the treatment of bipolar affective disorder and other related affective disorders.

Various aspects of the invention are described in further detail in the following subsections.

I. Isolated Nucleic Acid Molecules One aspect of the invention is an isolated nucleic acid molecule encoding a polypeptide of the invention or a biologically active portion thereof, as well as encoding a polypeptide of the invention. And nucleic acid molecules sufficient to be used as hybridization probes to identify fragments of the above nucleic acid molecules suitable for use as PCR primers for amplification or mutation of the nucleic acid molecule. As used herein, the term "nucleic acid molecule" is intended to include DNA molecules (eg, cDNA or genomic DNA) and RNA molecules (eg, mRNA) and analogs of DNA or RNA made 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 a nucleic acid molecule that has been separated from other nucleic acid molecules that are present in the natural source of the nucleic acid molecule. Preferably, an “isolated” nucleic acid molecule is a sequence that is naturally adjacent to the nucleic acid in the genomic DNA of the organism from which the nucleic acid is derived (ie, the sequences located at the 5 ′ and 3 ′ ends of the nucleic acid). (Preferably a sequence encoding a protein). For example, in various embodiments, an isolated nucleic acid molecule has a nucleotide sequence that is naturally adjacent to the nucleic acid molecule in the genomic DNA of the cell from which the nucleic acid molecule is derived is approximately 5 kB, 4 kB, 3 kB, 2 kB
, 1 kB, 0.5 kB or less than 0.1 kB. Further, an "isolated" nucleic acid molecule, such as a cDNA molecule, is substantially free of other cellular material or media when made by recombinant techniques, or when chemically synthesized. It is substantially free of chemical precursors or other chemicals.

The nucleic acid molecules of the invention, for example, nucleic acid molecules having the nucleotide sequence of SEQ ID NO: 1, 3, 4, 6, 7, 9, or the complement thereof, can be prepared using standard molecular biology techniques and the present specification. Can be isolated using the sequence information provided in the textbook. Using all or part of the nucleic acid sequence of SEQ ID NO: 1, 3, 4, 6, 7 or 9 as a hybridization probe, nucleic acid molecules of the invention can be prepared using standard hybridization and cloning techniques (eg, Sambrook et al.). Ed., “Molecular Cloning: Laboratory Manual (Mo
lecular Cloning: A Laboratory Manual), 2nd edition, Cold Spring Harbor Labor
atory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989
)).

The nucleic acid molecules of the invention can be amplified according to standard PCR amplification techniques using cDNA, mRNA or genomic DNA as a template and appropriate oligonucleotide primers. The nucleic acid thus amplified can be cloned into an appropriate vector and analyzed by DNA sequence analysis. In addition, oligonucleotides corresponding to all or a portion of the nucleic acid molecules of the invention can be prepared by standard synthetic techniques, for example, using an automated DNA synthesizer.

[0081] In another preferred embodiment, the isolated nucleic acid molecule of the invention comprises SEQ ID NO: 1
, 3, 4, 6, 7 or 9 nucleotide sequences. A nucleic acid molecule that is complementary to a given nucleotide sequence is one that is sufficiently complementary to that nucleotide sequence to hybridize to the given nucleotide sequence to form a stable duplex. A nucleic acid molecule.

Further, the nucleic acid molecule of the present invention may be a part of a nucleic acid sequence encoding the full length of the polypeptide of the present invention (for example, a fragment that can be used as a probe or a primer or the biological activity of the polypeptide of the present invention). (The fragment that encodes the part). The nucleotide sequence determined from one cloned gene may be used to identify and / or clone homologs in other cell types (eg, from other tissues) and from other mammals Probes and primers designed to do so. Typically,
The probe / primer consists essentially of a purified oligonucleotide.
Typically, the oligonucleotide is a naturally occurring of the sense or antisense sequence of SEQ ID NO: 1, 3, 4, 6, 7 or 9 or of SEQ ID NO: 1, 3, 4, 6, 7 or 9 At least about 12, preferably about 25, more preferably about 50,
It consists of a region of nucleotide sequence that hybridizes under stringent conditions to 75, 100, 125, 150, 175, 200, 250, 300, 350 or 400 contiguous nucleotides.

[0083] Probes based on the sequences of the nucleic acid molecules of the invention can be used to detect transcripts or genomic sequences that encode similar protein molecules encoded by the selected nucleic acid molecule. The probe consists of a label group attached to it, for example, a radioisotope, a fluorescent compound, an enzyme, or a cofactor for an enzyme. Such probes can be used to determine the level of a nucleic acid molecule encoding the protein in a cell sample of a subject (eg, to detect mRNA levels, or when the gene encoding the protein is mutated or deleted). To determine if a cell or tissue mis-expresses a protein.

The nucleic acid fragment encoding the “biologically active portion” of the polypeptide of the present invention can be obtained by isolating a part of any one of SEQ ID NOs: 3, 6, and 9, and replacing the portion encoding the polypeptide protein with the nucleic acid fragment. It can be expressed (eg, by recombinant expression in vitro) and prepared by measuring the activity of the polypeptide-encoding moiety.

The present invention further relates to SEQ ID NO: 1, 3, 4, 6, 7 or 9 due to the degeneracy of the genetic code.
But nucleic acid molecules that encode proteins similar to those encoded by the nucleotide sequence of SEQ ID NOs: 3, 6 or 9.

Those skilled in the art will recognize that, in addition to the nucleotide sequences of SEQ ID NOs: 3, 6, and 9, polymorphisms in the DNA sequence that result in amino acid sequence changes are present in the population (eg, the human population). Would. Such genetic polymorphisms exist among individuals within a population due to natural allelic variation. An allele is one of a group of genes that occur alternatively at a given locus. As used herein, "allelic variants"
The term refers to a nucleotide sequence that occurs at a given locus or the polypeptide encoded by this nucleotide sequence. As used herein, "gene"
And the term "recombinant gene" refers to a nucleic acid molecule consisting of an open reading frame encoding a polypeptide of the invention. Such natural allelic variations typically result in 1-5% variation in the nucleotide sequence of a given gene. Alternative alleles can be identified by sequencing the gene of interest in many different individuals. This can be easily accomplished by using hybridization probes to identify the same locus in different individuals. All such nucleotide variations and consequent amino acid polymorphisms or variations that are the result of natural allelic variation and do not alter functional activity are intended to be within the scope of the invention.

In addition, nucleic acid molecules (homologues) encoding a protein of the invention from another species having a nucleotide sequence that differs from the nucleotide sequence of the human protein described herein are within the scope of the invention. Shall be included. Nucleic acid molecules corresponding to natural allelic variants and homologues of the cDNAs of the present invention can be prepared using human cDNA or a portion thereof as a hybridization probe, under stringent hybridization conditions, according to standard hybridization techniques. And can be isolated based on their identity to the human nucleic acid molecules disclosed herein. For example, a cDNA encoding a soluble form of the membrane-bound protein of the invention
Was isolated based on its hybridization to a nucleic acid molecule encoding all or part of the membrane-bound form. Similarly, 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.

Thus, in another embodiment, the isolated nucleic acid molecule of the invention has a length of at least 300 (325, 350, 375, 400, 425, 450, 500, 550, 600, 650, 700, 800, 900,
1000 or 1290) nucleotides and hybridizes under stringent conditions to a nucleic acid molecule consisting of a nucleotide sequence, preferably the coding sequence of SEQ ID NO: 1, 3, 4, 6, 7 or 9 or a complement thereof. I do.

As used herein, the term “hybridizes under stringent conditions” typically refers to nucleotide sequences that are at least 60% (65%, 70%, preferably 75%) identical to each other. Are intended to describe the conditions of hybridization and washing so that they remain hybridized to each other. Such stringent conditions are known to those skilled in the art and are described in "Current Protocols in Molecular Biology", John Wiley & Sons, NY (1
989), 6.3.1-6.3.6. A preferred example of stringent hybridization conditions is 6X sodium chloride / sodium citrate (SSC) at about 45 ° C.
After washing in the medium, the cells are washed one or more times in 0.2X SSC, 0.1% SDS at 50-65 ° C, but not limited thereto. Preferably, an isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to the sequence of SEQ ID NO: 1, 3, 4, 6, 7 or 9 or a complement thereof is a naturally occurring nucleic acid. Corresponds to a molecule. As used herein, a “naturally occurring” nucleic acid molecule is an RNA or DNA having a naturally occurring nucleotide sequence (eg, encoding a naturally occurring protein).
Refers to a molecule.

In addition to naturally occurring allelic variants of a nucleic acid molecule of the sequences of the present invention, which may be present in a population, one of skill in the art will further appreciate that mutations can introduce changes to the encoded protein. It will be appreciated that the amino acid sequence of can be changed without altering the biological activity of the protein. For example, nucleotide substitutions can be made that result in amino acid substitutions at "non-essential" amino acid residues. “Non-essential” amino acid residues are those residues that can be changed from the wild-type sequence without altering the biological activity, while “essential” amino acid residues are required for biological activity It is. For example, amino acid residues that are not conserved or only partially conserved among homologs of various species may be targeted for alteration since they may not be essential for activity. Alternatively, amino acid residues conserved between homologues of various species (eg, mouse and human) may not be targets for alteration since they appear to be essential for activity.

Thus, another aspect of the invention pertains to nucleic acid molecules encoding a polypeptide of the invention having changes in amino acid residues that are not essential for activity. Such polypeptides differ in amino acid sequence from SEQ ID NOs: 2, 5, or 8, but still retain biological activity. In one embodiment, the isolated nucleic acid molecule is at least about 45% identical to the amino acid sequence of SEQ ID NO: 2, 5, or 8, or 65%, 75%, 85%, 9%
Includes nucleotide sequences encoding proteins containing amino acid sequences that are 5% or 98% identical.

[0092] An isolated nucleic acid molecule encoding a variant protein can be prepared by introducing one or more amino acid substitutions, additions or deletions into the encoded protein.
It can be made by introducing one or more nucleotide substitutions, additions or deletions into the 4, 6, 7 or 9 nucleotide sequence. 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 putative non-essential amino acid residues. "Conservative amino acid substitutions" are those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. The family of amino acid residues having similar side chains is clear in the art. These strains include amino acids with basic side chains (eg, lysine, arginine, histidine), amino acids with acidic side chains (eg, aspartic acid, glutamic acid), amino acids with uncharged polar side chains (eg, For example, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids having nonpolar side chains (eg, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side Amino acids having a chain (eg, threonine,
Valine, isoleucine) and amino acids with aromatic side chains (eg, tyrosine, phenylalanine, tryptophan, histidine). Alternatively, the mutations may be introduced randomly, for example by saturation mutagenesis, into all or part of the coding sequence, and the resulting mutants screened for biological activity and abruptly retaining the activity Identify the mutant. After mutagenesis, the encoded protein can be expressed recombinantly and the activity of the protein measured.

In a preferred embodiment, a mutant polypeptide that is a variant of the polypeptide of the invention comprises (1) the ability of the polypeptide of the invention to form a protein: protein interaction with a protein in a signal pathway. (2) the ability of the polypeptides of the invention to bind to ligands; (3) the polypeptides of the invention can be assayed for their ability to bind to intracellular target proteins. In yet another preferred embodiment, the mutant polypeptide can be assayed for its ability to modulate cell proliferation, cell migration or chemotaxis, or cell differentiation.

The present invention relates to antisense nucleic acid molecules, ie, molecules complementary to a sense nucleic acid encoding a polypeptide of the invention (eg, a molecule complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence). Molecule). Thus, an antisense nucleic acid can hydrogen bond to a sense nucleic acid. An antisense nucleic acid can be complementary to the entire coding strand or to only a portion thereof. For example, an antisense nucleic acid can be complementary to all or part of a protein coding region (or open reading frame). An antisense nucleic acid molecule may be antisense to all or a portion of the non-coding region of the coding strand of a nucleotide sequence encoding a polypeptide of the present invention. Non-coding regions ("5 'and
"3 'untranslated regions") are the 5' and 3 'sequences adjacent to the coding region and are not translated into amino acids.

Antisense oligonucleotides are, for example, about 5,10,15,20,25,30
, 35, 40, 45 or 50 nucleotides. The antisense nucleic acids of the present invention can be constructed by chemical synthesis and enzymatic ligation using methods known to those skilled in the art. For example, antisense nucleic acids (e.g., antisense oligonucleotides) can be formed using naturally occurring nucleotides or to increase the biological stability of the molecule, or between antisense and sense nucleic acids. It can be chemically synthesized using a variety of modified nucleotides designed to increase the physical stability of the strand. For example, phosphorothioic acid derivatives and acridine substituted nucleotides can be used. Examples of modified nucleotides that can be used to make antisense nucleic acids include 5-fluorouracil,
5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyhydroxymethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylamino Methyluracil, dihydrouracil, β-D-galactosylkeosin (galactosy
lqueosine), inosine, N6-isopentenyl adenine, 1-methyl guanine, 1-methyl inosine, 2,2-dimethyl guanine, 2-methyl adenine, 2-methyl guanine,
3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D-mannosylqueosine, 5'- Methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5
-Oxyacetic acid (v), wybutoxosine, pseudouracil, keosin (queosine), 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4
-Thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3- (3-amino-3-N-2-carboxypropyl ) Uracil, (acp3) w, and 2,6-diaminopurine. Alternatively, antisense nucleic acids are subcloned in a nucleic acid in an antisense orientation (ie, RNA transcribed from the inserted nucleic acid is in an antisense orientation to the target nucleic acid of interest; further described below). It can be produced biologically using an expression vector.

[0096] The antisense nucleic acid molecules of the invention are typically administered to a subject or made in situ, where they encode a selected polypeptide of the invention. Hybridization or binding to DNA inhibits expression, for example, by inhibiting transcription and / or translation. Hybridization is due to normal nucleotide complementarity forming a stable duplex, or, for example, in the case of an antisense nucleic acid molecule binding to a DNA duplex, in the major groove of the duplex. This is due to a specific interaction. An example of a route of administration of the antisense nucleic acid molecules of the invention is direct injection into a tissue site. Alternatively, the antisense nucleic acid molecule can be modified to target selected cells prior to systemic administration. As an example, for systemic administration, an antisense molecule can be expressed on a selected cell surface by linking the antisense nucleic acid molecule to a peptide or antibody that binds the cell surface receptor or antigen, for example. It can be modified to specifically bind to the body or antigen.
Antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve a sufficient intracellular concentration of the antisense molecule, a vector construct in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter is preferred.

[0097] The antisense nucleic acid molecules of the invention can be α-anomeric nucleic acid molecules. α-anomeric nucleic acid molecules form specific double-stranded hybrids with complementary RNA, in which the strands run parallel to one another, contrary to the normal β-unit (Gaultier et al., (1987) Nuc
leic Acids Res. 15: 6625-6641). Antisense nucleic acid molecules can also be 2'-o-methyl ribonucleotides (Inoue et al., (1987) Nucleic Acids Res. 15: 6131-6148) or chimeric RNA-DNA analogs (Inoue et al., (1987) FEBS Lett. 327-330).

The present invention also encompasses ribozymes. Ribozymes are catalytic RNA molecules with ribonuclease activity that can cleave single-stranded nucleic acids (eg, mRNA) that have complementary regions. Thus, ribozymes, such as hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature 334: 585-591), are encoded by the mRNA by catalytically cleaving the mRNA transcript. It can be used to inhibit protein translation. Ribozymes having specificity for a nucleic acid molecule encoding a polypeptide of the present invention can be designed based on the nucleotide sequence of the cDNA disclosed herein. For example, Cech
Et al., US Pat. No. 4,987,071 and Cech et al., US Pat. No. 5,116,742, construct derivatives of Tetrahymena L-19 IVS RNA such that the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved. can do. Alternatively, mRNA encoding a polypeptide of the invention can be used to select a catalytic RNA having specific ribonuclease activity from a pool of RNA molecules. See, for example, Bartel and Szostak (1993) Science 261: 1411-1418.

The present invention also encompasses nucleic acid molecules that form a triple helix structure. For example, expression of a polypeptide of the present invention can target a nucleotide sequence complementary to a regulatory region (eg, a promoter and / or enhancer) of the gene encoding the polypeptide, such that the expression of the polypeptide is such that it prevents transcription of the gene in target cells. It can be inhibited by forming a helical structure. In general, Helene (1991) Anticanc
er Drug Des. 6 (6): 569-84; Helene (1992) Ann. NY Acad. Sci. 660: 27-36;
And Maher (1992) Bioassays 14 (12): 807-15.

In various embodiments, the nucleic acid molecules of the present invention may be, for example, those having molecular stability,
The base moiety, sugar moiety or phosphate backbone can be modified to improve hybridization, or solubility. For example, the deoxyribose phosphate backbone of nucleic acids can be modified to create peptide nucleic acids (Hyrup et al., (19
96) Bioorganic & Medicinal Chemistry 4 (1): 5-23). As used herein, the term "peptide nucleic acid" or "PNA" refers to a nucleic acid mimetic in which the deoxyribose phosphate backbone has been replaced by a pseudopeptide backbone and only four natural nucleobases have been retained, e.g., a DNA mimic. Point to. The neutral backbone of PNA has been shown to allow specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomers is described by Hyrup et al. (1996) supra; Perry-O'Keefe et al. (1996) Proc. Natl. Acad. Sci. USA 93: 14670-675. It can be performed using a peptide synthesis protocol.

The PNA has therapeutic and diagnostic applications. As an example, PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression, for example, by inducing transcription or translation arrest or inhibiting replication. PNAs can also be used, for example, in the analysis of single base pair mutations in genes, for example, by PCR clamping directed to the PNA;
As an artificial restriction enzyme when used in combination with other enzymes, such as S1 nuclease (Hyrup (1996), supra); or as a probe or primer for DNA sequencing and hybridization (Hyrup (1996), supra; Perry- O'Keefe et al. (1
Natl. Acad. Sci. USA 93: 14670-675).

In another embodiment, the PNA is provided by attaching a lipophilic or other auxiliary group to the PNA, eg, to increase its stability or cellular uptake.
-Can be modified by formation of a DNA chimera or by the use of liposomes or other drug delivery techniques known to those skilled in the art. For example, a PNA-DNA chimera can be created that combines the advantageous properties of PNA and DNA. Such a chimera has a DNA
While allowing interaction with DNA moieties by recognition enzymes, such as RNase H and DNA polymerase, PNA moieties can provide high binding affinity and specificity. PNA
-DNA chimeras can be ligated using linkers of appropriate length selected in terms of base stacking, number of nucleobase linkages, and orientation (Hyrup (1
996), above). The synthesis of PNA-DNA chimeras is described in Hyrup (1996), supra, and Finn et al., (
1996) Nucleic Acids Res. 24 (17): 3357-63. For example, DNA strands can be synthesized on a solid support using standard phosphoramidite coupling chemistry and modified nucleoside analogs. 5 '-(4-
Compounds such as methoxytrityl) amino-5′-deoxythymidine-phosphoramidite can be used as a link between the PNA and the 5 ′ end of DNA (Mag et al., (19
89) Nucleic Acids Res. 17: 5973-88). The PNA monomers are then linked in sequence to produce a chimeric molecule having a 5 ′ PNA segment and a 3 ′ DNA segment (Fin
n et al., (1996) Nucleic Acids Res. 24 (17): 3357-63). Alternatively, the chimeric molecule is
Can be synthesized with 5 'DNA segment and 3' PNA segment
(Peterser et al., (1975) Bioorganic Med. Chem. Lett. 5: 1119-11124).

[0103] In other embodiments, the oligonucleotide is a peptide (eg, in vivo)
Or a reagent that enhances transport across the cell membrane (eg, Letsinger et al., (1989) Proc. Natl. Acad. Sci. USA 86: 6553-6).
556; Lemaitre et al., (1987) Proc. Natl. Acad. Sci. USA 84: 648-652; PCT Publication No. WO 88/09810), or reagents that enhance transport across the blood-brain barrier (eg, (See PCT Publication No. WO 89/10134). In addition, oligonucleotides can be used with hybridization-triggered cleavage reagents (see, eg, Krol et al., (1988) Bio / Techniques 6: 958-976) or intercalating reagents (eg, Zon (1988) Pharm. Res. 5: 5
39-549). At this end, the oligonucleotide may be linked to another molecule, such as a peptide, a hybridization-triggered crosslinker, a transport reagent, a hybridization-triggered cleavage reagent, and the like.

II. Isolated Proteins and Antibodies One embodiment of the present invention relates to isolated proteins, and biologically active portions thereof,
And a polypeptide fragment suitable for use as an immunogen for producing an antibody against the polypeptide of the present invention. In one embodiment, the native polypeptide can be isolated from a cell or tissue source by a suitable purification step using standard protein purification methods. In another embodiment, the polypeptide of the invention is:
Produced by recombinant DNA technology. As an alternative to recombinant expression, the polypeptides of the invention can be synthesized chemically using standard peptide synthesis methods.

An “isolated” or “purified” protein or biologically active portion thereof is
Chemical precursors or other chemicals when the protein is substantially free of cellular material or other contaminating proteins from cells or tissue sources obtained therefrom or chemically synthesized Is not substantially contained. The term "substantially free of cellular material" means that the protein was isolated or recombinantly produced therefrom.
And a protein preparation separated from the cellular components of the cell. That is, a protein that is substantially free of cellular matter is about 30%, 20%, 10%, or
Includes protein preparations containing less than 5% heterologous protein (also referred to herein as "contaminating proteins"). Also, when the protein or biologically active portion thereof is produced recombinantly, it is substantially free of culture medium, ie, the culture medium is about 20%, 10%, or 5% of the volume of the protein preparation. It is preferably less than. When a protein is produced by chemical synthesis, it may be substantially free of chemical precursors or other chemicals, i.e., separated from chemical precursors or other chemicals involved in the synthesis of the protein. preferable. Thus, such protein preparations contain about 30%, 20%, 10%, 5% by dry weight of chemical precursors or compounds other than the polypeptide of interest.

The biologically active portion of a polypeptide of the present invention may be substantially identical to the amino acid sequence of a full-length protein, comprising fewer amino acids than the full-length protein and exhibiting at least one activity of the corresponding full-length protein. Or a polypeptide comprising an amino acid sequence derived from or derived therefrom (eg, the amino acid sequence shown in any of SEQ ID NOs: 2, 5, or 8). Typically, a biologically active portion comprises a domain or motif having at least one activity of the corresponding protein. Biologically active portions of the proteins of the invention can be, for example, 10, 25, 50,
The polypeptide can be 100 amino acids or more. In addition, other biologically active portions that lack other regions of the protein can be produced by recombinant techniques and can be evaluated for one or more functional activities of a native form of a polypeptide of the invention.

[0107] Preferred polypeptides have the amino acid sequence of SEQ ID NO: 2, 5, or 8. Other useful proteins are substantially identical (eg, at least about 45%, preferably 55%, 65%, 75%, 85%, 95%, or 99%) to any of SEQ ID NOs: 2, 5, or 8. )
And retains the protein's functional activity of the corresponding natural protein,
Amino acid sequences differ due to natural allelic variation or mutagenesis.

To determine the percent identity between two amino acid sequences or two nucleic acids, the sequences are aligned for optimal comparison purposes (eg, the first amino acid or the second amino acid or nucleic acid sequence is optimally aligned with the second amino acid or nucleic acid sequence). Gaps can be introduced in the sequence of the nucleic acid sequence). Then, the amino acid residue or nucleotide at the corresponding amino acid position or nucleotide position is 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 two sequences is a function of the number of identical positions shared by the sequences (ie,% identity = number of identical positions / total number of positions (eg, overlapping Position) x 100). In some embodiments, the two sequences are the same length.

Determining the percent identity between two sequences can be accomplished using a mathematical algorithm. Non-limiting preferred examples of mathematical algorithms used to compare two sequences are described in Karlin and Altschul (1993, Proc. Natl. Acad. Sci. USA 90: 5873-5.
877), the algorithm of Karlin and Altschul (1990, Proc. N
atl. Acad. Sci. USA 87: 2264-2268). Such an algorithm is called Alts
(1990, J. Mol. Biol. 215: 403-410), incorporated into the NBLAST and XBLAST programs. BLAST nucleotide search, NBLAST program, score = 1
Performed using a word length = 12, one can obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. BLAST protein search, XBLAST program, score = 5
The procedure is performed using 0, word length = 3 to obtain an amino acid sequence homologous to the protein molecule of the present invention. Gapped alignment for comparison purposes
To obtain s), Gapped BLAST can be used as described in Altschul et al. (1997, Nucleic Acids Res. 25: 3389-3402). Alternatively, PSI-Blast
Can be used to perform an iterated search that detects distance relationships between molecules. That is, when using BLAST, Gapped BLAST, and PSI-Blast programs, default parameters of each program (for example, XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov. Another preferred, non-limiting example of a mathematical algorithm used for sequence comparison is Myers
And Miller's algorithm (1988, CABIOS 4: 11-17). Such an algorithm has been incorporated into the ALIGN program (version 2.0) which is part of the CGC sequence alignment software package. When using the ALIGN program for comparing amino acid sequences, the PAM120 weight residue table (weight residue table) is used.
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.

[0111] The present invention also provides chimeric or fusion proteins. As used herein, a "chimeric protein" or "fusion protein" refers to all or a portion of a polypeptide of the invention operably linked to a heterologous polypeptide (ie, a polypeptide that is not identical to a polypeptide of the invention). (Preferably biologically active) moieties. In a fusion protein, the term "operably linked" is intended to indicate that the polypeptide of the invention and the heterologous polypeptide are fused together in-frame. A 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 a polypeptide of the invention is fused to the C-terminus of a GST sequence. Such a fusion protein can facilitate the purification of the 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 secretion sequence of the outer membrane protein of baculovirus can be used as a heterologous signal sequence (Current Protocols in Molecular Biology, Au
edited by subel et al., published by John Wiley & Sons, 1992). Other examples of eukaryotic heterologous signal sequences include the secretory sequences of melittin and human placental alkaline phosphatase (St.
ratagene; La Jolla, CA). In yet another example,
Useful heterologous prokaryotic signal sequences include the phoA secretion signal (see Sambrook, supra).
Et al.) And protein A secretion signal (Pharmacia Biotech; Piscataway, NJ).

[0113] In yet another embodiment, the fusion protein is an immunoglobulin fusion protein in which all or a portion of a polypeptide of the invention is fused to a sequence derived from a member of the immunoglobulin protein family. . An immunoglobulin fusion protein of the invention is incorporated into a pharmaceutical composition and administered to a subject to inhibit the interaction between a ligand (soluble or membrane-bound) and a cell surface protein (receptor). Thus, signal transduction in vivo can be suppressed. An immunoglobulin fusion protein can be used to affect the bioavailability of a cognate ligand of a polypeptide of the invention. Inhibition of ligand / receptor interactions is therapeutically useful both for treating proliferative and differentiation disorders and for modulating (eg, promoting or inhibiting) cell viability. Furthermore, using the immunoglobulin fusion protein of the present invention as an immunogen, an antibody against the polypeptide of the present invention in a subject is produced, the ligand is purified, and a molecule that inhibits the interaction between the receptor and the ligand in a screening assay is produced. Can be identified.

The chimeric and fusion proteins of the present invention can be produced by standard DNA recombination techniques. In another embodiment, the fusion gene can be synthesized by conventional methods, including automatic DNA synthesizers. Alternatively, then annealed,
PCR amplification of gene fragments can be performed using anchor primers that cause a complementary overhang between two consecutive gene fragments that can be reamplified to produce a chimeric gene sequence (e.g., See Ausubel et al.)
. In addition, many expression vectors are commercially available that previously encode a fusion moiety (eg, a GST polypeptide). A nucleic acid encoding a polypeptide of the invention can be cloned into an expression vector such that the fusion moiety is linked in-frame to the polypeptide of the invention.

The signal sequence of a polypeptide of the invention (SEQ ID NO: 14) can be used to facilitate secretion and isolation of a secreted protein or other protein of interest.
The signal sequence is typically characterized by a core of hydrophobic amino acids that are normally cleaved from the mature protein at one or more cleavage events during secretion. Such signal peptides contain processing sites that allow cleavage of the signal sequence from the mature protein as they pass through the secretory pathway. Thus, the present invention relates to the above-mentioned polypeptides having a signal sequence, as well as the signal sequence itself and the polypeptide without the signal sequence (ie, the cleavage product). In certain embodiments, a nucleic acid encoding a signal sequence of the invention can be operably linked to a protein of interest (eg, a protein that is not normally secreted or otherwise difficult to isolate) in an expression vector. . The signal sequence directs secretion of a protein from, for example, a eukaryotic host into which the expression vector is introduced, and the signal sequence is subsequently or simultaneously cleaved. This protein can then be readily purified from the extracellular media by art-recognized methods. Alternatively, the signal sequence can be linked to the protein of interest using a sequence that facilitates purification (eg, having a GST domain).

In another embodiment, the signal sequences of the present invention can be used to identify regulatory sequences (eg, promoters, enhancers, repressors). Since the signal sequence is the most amino-terminal sequence of the peptide, the nucleic acid adjacent to the signal sequence at the amino-terminal side is expected to be a regulatory sequence that affects transcription. Thus, a nucleic acid sequence encoding all or part of a signal sequence can be used as a probe to identify and isolate signal sequences and their flanking regions, and by studying these flanking regions, Regulatory sequences can be identified.

The invention also relates to variants of the polypeptide of the invention. Such variants have an altered amino acid sequence that can function as either agonists (mimics) or antagonists. Mutants can be produced by mutagenesis (eg, discrete point mutation or truncation). An agonist can retain substantially the same biological activity as the native form of the protein, or a subset thereof. Antagonists of a protein can inhibit one or more activities of a native form of the protein, for example, by competitively binding to a downstream or upstream member of a cellular signaling cascade containing the protein of interest. Thus, specific biological effects can be elicited by treatment with a variant having a limited function. Treatment of a subject with a variant having a subset of the biological activity of the native form of the protein can result in fewer side effects in the subject than treatment with the native form of the protein.

Variants of the proteins of the present invention that function as either agonists (mimics) or antagonists can be obtained by combining combinatorial libraries of mutants of the proteins of the present invention (eg, truncation mutants) for agonist or antagonist activity. It can be identified by screening. In one embodiment, a variegated variant library is created by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library. For example, a degenerate set of potential protein sequences can be used as individual polypeptides or as a larger set of fusion proteins (eg, phage display).
By enzymatically linking a mixture of synthetic oligonucleotides into a gene sequence so that it can be expressed as a library of variants, one can generate a library of varied variants. There are a variety of methods that can be used to produce libraries of potential variants of the polypeptides of the present invention from degenerate oligonucleotide sequences. Methods for synthesizing degenerate oligonucleotides are known in the art (see, for example, Narang, (198
3) Tetrahedron 39: 3; Itakura et al., Annu. Rev. Biochem. 53; 323; Itakura et al., (1
984) Science 198: 1056; see Ike et al., (1983) Nucleic Acid Res. 11: 477).

In addition, a library consisting of fragments of the coding sequence of a polypeptide of the present invention can be used to produce a varied population of polypeptides for screening and subsequent selection of variants. For example, a library of coding sequence fragments can be used for double-stranded PCR of the coding sequence of interest under conditions where nicking occurs only once per molecule.
The fragment is treated with nuclease to denature the double-stranded DNA, regenerate the DNA,
Double-stranded D that can contain sense / antisense pairs from different nick products
1 from the duplex re-formed by forming NA and treating with S1 nuclease
It can be produced by removing the main chain portion and ligating the resulting fragment library into an expression vector. By this method, expression libraries encoding N-terminal and internal fragments of various sizes of the protein of interest can be derived.

Several techniques are known in the art for screening gene products of combinatorial libraries made by point mutation or truncation, and for screening cDNA libraries for gene products having selected properties. is there. The most widely used, high through-p
ut) The amenable method of screening large gene libraries generally involves cloning the gene library into a replicable expression vector, characterizing the appropriate cells with the resulting vector library. Includes the expression of combinatorial genes under conditions that facilitate conversion and isolation of the vector encoding the gene whose gene product is detected by detection of the desired activity. Recursive ensemble mutagenesis (REM), a technique for increasing the frequency of functional mutants in libraries, is used in conjunction with screening assays to identify variants of the proteins of the invention. (Arkin & Yourvan (1992) Proc. Natl. Acad. Sci. USA 89: 7811-7815).
Delgrave et al. (1993) Protein Engineering 6 (3): 327-331).

Antibodies can be produced using the isolated polypeptides of the present invention, or fragments thereof, as immunogens and using standard methods for the preparation of poroclonal and monoclonal antibodies. Either full length polypeptides or proteins can be used, or the invention provides antigenic peptide fragments for use as immunogens. The antigenic peptide of the protein of the present invention contains at least 8 (preferably 10, 15, 20, or 30) amino acid residues of the amino acid sequence represented by SEQ ID NO: 2, 5, or 8, and the peptide Antibodies raised against include an epitope of the protein such that it forms a specific immune complex with the protein.

Preferred epitopes included in antigenic peptides are regions located on the surface of the protein (eg, hydrophilic regions). FIGS. 2, 4 and 6 are hydrophobicity plots of the proteins of the invention. These plots or similar analytical methods can be used to identify hydrophilic regions.

In general, immunogens are used to prepare antibodies by immunizing a suitable subject (eg, a rabbit, goat, mouse or other mammal). Suitable immunogenic preparations can contain, for example, recombinantly expressed or chemically synthesized polypeptides. The preparation can further include an adjuvant (eg, Freund's complete or incomplete adjuvant), or a similar immunostimulant.

Thus, another aspect of the present invention pertains to antibodies to the polypeptides of the present invention.
As used herein, the term "antibody" refers to an immunoglobulin molecule and an immunologically active portion of an immunoglobulin (ie, a molecule comprising an antigen binding site that specifically binds an antigen, such as a polypeptide of the invention). ). A molecule that specifically binds to a given polypeptide of the invention will bind to the polypeptide but will be substantially different from other molecules in a sample (eg, a biological sample) that naturally contains the polypeptide. A molecule that does not bind specifically. Examples of immunologically active portions of immunoglobulin molecules include F (ab) and F (ab '), which can be produced by treating an antibody with an enzyme such as pepsin.
) ₂ fragments. The present invention provides poroclonal and monoclonal antibodies. As used herein, the term "monoclonal antibody" or "monoclonal antibody composition" refers to a population of antibody molecules that contains only one antigen binding site that can immunoreact with a particular epitope.

[0125] Poroclonal antibodies can be prepared as described above by immunizing a suitable subject with a polypeptide of the invention as an immunogen. Antibody titer in an immunized subject can be monitored over time by standard methods (eg, enzyme-linked immunosorbent assay (ELISA) using immobilized polypeptide). If desired, the antibody molecule can be isolated from a mammal (eg, from blood) and further purified by well-known methods, such as protein A chromatography, to obtain an IgG fraction. At an appropriate time after immunization, e.g., when the titer of the specific antibody is maximized, antibody-producing cells can be obtained from the subject and used to produce monoclonal antibodies by standard methods. . Standard methods include, for example, the hybridoma technology first reported by Kohler and Milstein ((1975) Nature 256: 495-497), the human B cell hybridoma technology (Kozbor et al. (1983) I
mmunol. Today 4:72), EBV-hybridoma technology (Cole et al. (1985) Monoclonal Antibodies and Cancer Therapy), Alan R. Li
ss, Inc., pp. 77-96) or trioma technology. Hybridoma production methods are well known (generally, the latest protocols in immunology (Cu
rrent Protocols in Immunology) (1994) Edited by Coligan et al., John Wiley & Sons,
Inc., New York, NY). Hybridoma cells producing the monoclonal antibodies of the invention are screened in hybridoma culture supernatants for antibodies that bind the polypeptide of interest (eg, standard EL).
(Using an ISA assay).

Instead of producing a monoclonal antibody secreting hybridoma, a monoclonal antibody against a polypeptide of the invention is screened against a recombinant combinatorial immunoglobulin library (eg, an antibody phage display library) using the polypeptide of interest. Can be identified and isolated. Kits for producing and screening phage display libraries are commercially available (eg, Pharmacia Recombinant Phage Antibodies).
dy System, Catalog No. 27-9400-01; and Stratagene SurfZAP ™ Phage Di
splay Kit, Catalog No. 240612). In addition, examples of methods and reagents that are particularly preferably used in the production and screening of antibody display libraries include, for example, US Pat. No. 5,223,409; International Patent Publication No. WO92 / 18619; International Patent Publication No. WO91 / 17271. International Publication No. WO92 / 20791; International Patent Publication No. WO92 / 15679; International Patent Publication No. WO93 / 01288; International Patent Publication No. WO92
/ 01047; International Patent Publication No. WO92 / 09690; International Patent Publication No. WO90 / 02809; Fuche et al. (1991) Bio / Technology 9: 1370-1372; Hay et al. (1992) Hum. Antib
od. Hybridomas 3: 81-85; Huse et al. (1989) Science 246: 1275-1281; Griffiths et al.
(1993) in EMBO J. 12: 725-734.

In addition, recombinant antibodies containing human and non-human moieties, such as chimeric and humanized monoclonal antibodies, that can be made using standard DNA recombination techniques are included within the scope of the invention. Such chimeric and humanized monoclonal antibodies can be produced by DNA recombination methods known in the art. Examples of the method include International Patent Publication No. WO87 / 02671; European Patent Application No. 184,187; and European Patent Application No. 171.4.
No. 96; European Patent Application No. 173,494; International Patent Publication No. WO86 / 01533; US Patent No. 4,816,567; European Patent Application No. 125,023; Better et al. (1988) Science 240: 1041.
Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84: 3439-3443; Liu et al. (1987)
) J. Immunol. 139: 3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. USA 84:
214-218; Nishimura et al. (1987) Canc. Res. 47: 999-1005; Wood et al. (1985) Nature
314: 446-449; and Shaw et al. (1988) J. Natl. Cancer Inst. 80: 1553-1559; Morr.
ison (1985) Science 229: 1202-1207; Oi et al. (1986) Bio / Techniques 4: 214; U.S. Patent No. 5,225,539; Jones et al. (1986) Nature 321: 552-525; Verhoeyan et al. (198
8) Science 239: 1534; and the method described in Beidler et al. (1988) J. Immunol. 141: 4053-4060.

[0128] Fully human antibodies are particularly desirable for therapeutic treatment of human patients. Such antibodies can be produced using transgenic mice that cannot express the endogenous immunoglobulin heavy and light chain genes but can express the human heavy and light chain genes. The transgenic mice are immunized in a conventional manner with a selected antigen (eg, all or a portion of a polypeptide of the present invention). Monoclonal antibodies to this antigen can be obtained using conventional hybridoma technology. The human immunoglobulin transgenes carried by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation. That is, using such a technique, it is possible to produce therapeutically useful IgG, IgA and IgE antibodies. For a review of technologies for producing human antibodies, see Lonberg
And Huszar (1995, Int. Rev. Immunol. 13: 65-93). For a detailed discussion of this technology for the production of human and human monoclonal antibodies and protocols for the production of such antibodies, see, for example, US Pat.
5,126; U.S. Patent No. 5,633,425; U.S. Patent No. 5,569,825; U.S. Patent No. 5,661,0
See No. 16 and U.S. Pat. No. 5,545,806. In addition, companies such as Abgenix, Inc. (Fremont, CA) provide human antibodies to selected antigens using techniques similar to those described above.

[0129] Fully human antibodies that recognize a selected epitope are described in "Guided Selection".
Section) ". This method uses selected non-human monoclonal antibodies (eg, murine antibodies) to guide the selection of fully human antibodies that recognize the same epitope (Jespers et al. (1994) Bio / Technolog).
y 12: 899-903).

[0130] Antibodies to the polypeptides of the invention (eg, monoclonal antibodies) can be used to isolate the polypeptide by standard methods (eg, affinity chromatography or immunoprecipitation). In addition, proteins (eg, in cell lysates or cell supernatants) can be detected using such antibodies to assess the amount and pattern of polypeptide expression. Antibodies can also be used diagnostically to monitor protein levels in tissues as part of a clinical trial method (eg, to determine the effectiveness of a given treatment regimen). Detection can be facilitated by coupling the antibody to a detectable substance. Examples of detection substances include various enzymes, prosthetic groups, fluorescent substances, luminescent substances, bioluminescent substances, and radioactive substances. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic complexes include streptavidin / biotin and avidin / biotin. Examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; examples of luminescent materials include luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and the like, and examples of suitable radioactive material include ^{12 5 I, 131 I, 35} S or ³ H.

In addition, the antibody (or fragment thereof) can be conjugated to a therapeutic moiety (eg, a cytotoxin, therapeutic or radiometal ion). The cytotoxin or cytotoxin agent includes any drug harmful to cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetin, mitomycin, etoposide, tenoposide, vincristine, vinblastine,
Colchicine, doxorubicin, daunorubicin, dihydroxyanthracindione, mitozantrone, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoid, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. No. Therapeutic agents include, but are not limited to, antimetabolites (eg, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (eg, mechlorethamine) , Thioepachlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclotosfamide (
cyclothosphamide), busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamineplatinum (II) (DDP) cisplatin), anthracyclines (eg, daunorubicin (formerly called daunomycin), and doxorubicin), antibiotics Materials include, for example, dactinomycin (formerly referred to as actinomycin), bleomycin, mithramycin, and anthramycin (AMC), and antimitotic agents, such as vincristine and vinblastine.

[0132] The conjugates of the invention can be used to modify a given biological response. The drug moiety should not be construed as limited to classical chemotherapeutic agents. For example, the drug moiety can be a protein or polypeptide having the desired biological activity. Such proteins include, for example, toxins (eg,
Abrin, ricin A, Pseudomonas exotoxin, or diphtheria toxin); proteins (eg, tumor necrosis factor, α-interferon, β-interferon,
Nerve growth factor, platelet-derived growth factor, tissue plasminogen activator) or biological response modifier (eg, lymphokine, interleukin-1 (IL-1))
, Interleukin-2 (IL-2), interleukin-6 (IL-6), granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF),
Or other growth factors).

[0133] Methods for coupling such therapeutic moieties to antibodies are well known and include, for example, Arnon
Et al., Monoclonal Antibodies and Cancer Thera
py), Monoclonal Antibodies for Immunotargeting of Drags in Cancer Thera
py) ", edited by Reisfeld et al., pp. 243-56 (Alan R. Liss, Inc., 1985); Hel.
lstorm et al., "Controlled Drug Delivery (2nd Edition)"
Antibodies for Drug Delivery, ”edited by Robinson et al.
pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, monoclonal antibody
1984 edition: Biological and clinical applications (Monoclonal Antibodies '84: Biological an
d Clinical Applications), “Antibody Carriers for Cytotoxic Agents in Cancer Treatment:
Review (Antibody Carriers of Cytotoxic Agents in Cancer Therapy: A Review
) ", Edited by Pinchera et al., Pp. 475-506 (1985);" Monoclonal Antibodies for Cancer Detection and Therapy "," Analysis of the therapeutic use of radiolabeled antibodies in cancer therapy, Analysis, Results, and Future Prospective of The Therapeutic Use of Ra
diolabeled Antibody In Cancer Therapy), edited by Baldwin et al., pp. 303-16 (Ac
ademic Press, 1985), and Thorpe et al., "The Preparation and Cytotoxic Properties of Antibody-Toxin Con
jugates) ", 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 US Pat. No. 4,676,980.

Further, an antibody of the invention, either conjugated or unconjugated to a therapeutic moiety, can be associated with a therapeutic moiety (eg, a cytotoxin, therapeutic, or radiometal ion) or It can be administered in combination therewith. The order of administration of the antibody and therapeutic moiety can vary. For example, in some embodiments, the antibody is administered simultaneously (by the same or different delivery device, eg, a syringe) with the therapeutic agent portion.
Alternatively, the antibody can be administered separately from and prior to the therapeutic moiety. Furthermore, the therapeutic moiety is administered separately and before the antibody. In many embodiments, these regimens will last for days, months, or years.

III. Recombinant Expression Vectors and Host Cells Another aspect of the invention relates to a vector, preferably an expression vector, 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 carrying another nucleic acid to which the nucleic acid molecule 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 the vector is introduced (eg, bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (eg, 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. In addition, certain vectors, which are expression vectors, are capable of directing the expression of genes to which they are operably linked. Generally, expression vectors utilized in DNA recombination techniques are often in the form of a plasmid (vector). However, the invention is intended to include other forms of expression vectors, such as viral vectors (eg, replication defective retroviruses, adenoviruses and adeno-associated viruses), and provide equivalent functions.

A recombinant expression vector of the invention comprises 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 vector contains one or more regulatory sequences, selected based on the host cell used for expression, and operably linked to the nucleic acid sequence to be expressed. In a recombinant expression vector, “operably linked” means that the nucleotide sequence of interest is capable of expressing the nucleotide sequence (eg, when the vector is introduced into a host cell, vit
ro (in a transcription / translation system or in a host cell) is intended to mean linked to regulatory sequence (s). The term “regulatory sequence” is intended to include promoters, enhancers and other expression control sequences (eg, polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185 (Expression Technology: Methods in Enzymology 18).
5), published by Academic Press, Inc., San Diego, CA (1990). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of hosts and those that direct expression of the nucleotide sequence only in certain host cells (eg, tissue-specific regulatory sequences). Those skilled in the art will appreciate that the design of an expression vector depends on such factors as the choice of the host to be transformed and the level of expression of the desired protein. An expression vector of the invention can be introduced into a host cell, thereby producing a protein or peptide, including a fusion protein or peptide, encoded by a nucleic acid described herein.

[0137] The recombinant expression vector of the present invention may be a prokaryotic cell (eg, E. coli) or a eukaryotic cell (eg, E. coli).
For example, it can be designed for expression of a polypeptide of the invention in insect cells (using yeast cells or mammalian cells using baculovirus expression vectors). Further, suitable host cells are discussed in Goeddel, supra. Alternatively, the recombinant expression vector can be transcribed and translated in vitro (eg, using a T7 promoter regulatory sequence and T7 polymerase).

Expression of proteins in prokaryotes is often performed in E. coli by vectors containing a constitutive or inducible promoter that directs 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 usually serve three purposes: 1) increase the expression of the recombinant protein;
3) increase the solubility of the recombinant protein; and 3) aid in the purification of the recombinant protein by acting as a ligand in the affinity purification. Often, fusion expression vectors introduce a proteolytic cleavage site at the junction of the fusion moiety and the recombinant protein, allowing purification of the fusion protein followed by separation of the recombinant protein from the fusion moiety. 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 (19) which fuses glutathione S-transferase (GST), maltose E binding protein, or protein A to the target recombinant protein, respectively.
88) Gene 67: 31-40), pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ).

An example of a suitable inducible non-fused E. coli expression vector is pTrc (Amann et al., (1988)
Gene 69: 301-315) and pET 11d (Sundier et al., Gene Expression Technology; Methods in Enzymology 185), Academic Pres
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 11d vector is mediated by co-expressed viral RNA polymerase (T7 gn1)
It depends on transcription from the gn 10-lac fusion promoter. This viral polymerase is provided by the host strain BL21 (DE3) or HMS174 (DE3). Supplied from a resident λ prophage carrying the T7 gnl gene under the transcriptional control of the lacUV5 promoter.

One strategy for maximizing recombinant protein expression in E. coli is to express the protein in a host bacterium that has impaired its ability to proteolytically cleave the recombinant protein (Gottesman, Gene Expression Technology: Enzymology Method 185 (Gene E
xpression Technology; Methods in Enzymology 185), Academic Press, San Diego, CA (1990) 119-128). Another strategy is to modify the nucleic acid sequence of the nucleic acid inserted into the expression vector so that individual codons for each amino acid are preferentially utilized in E. coli (Wada et al. (
1992) Nucleic Acids Res. 20: 2111-2118). Such modification of the nucleic acid sequence of the present invention can be performed by a standard DNA synthesis method.

[0141] In another embodiment, the expression vector is a yeast expression vector. Yeast S. Examples of vectors for expression in S. cerevisae include pYepSec1 (B
aldari et al. (1987) EMBO J. 6: 229-234), pMFa (Kurijan and Herskowitz, (1982)
Cell 30: 933-943), pJRY88 (Schultz et al. (1987) Gene 54: 113-123), pYES2 (I
nvitrogen Corporation, San Diego, CA, and pPicZ (In
vitrogen Corporation, San Diego, CA).

[0142] Alternatively, the expression vector is a baculovirus expression vector. Baculovirus vectors that can be used for expression of proteins in cultured insect cells (eg, Sf9 cells) include the pAc series (Smith et al. (1983) Mol. Cell Biol.
3: 2156-2165) and pVL series (Lucklow and Summers (1989) Virology 170: 31-
39).

In yet another embodiment, the nucleic acids of the invention are 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).
Is mentioned. When used in mammalian cells, the control functions on the expression vectors are often provided by viral regulatory sequences. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus and simian virus 40. See Sambrook et al., Supra, Chapters 16 and 17 for other expression systems suitable for both prokaryotic and eukaryotic cells.

In another embodiment, a recombinant mammalian expression vector is capable of directing the expression of a nucleic acid preferentially in a particular cell type (eg, using a tissue-specific regulatory element to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al. (1987) Genes Dev. 1: 268-277), the lymph-specific promoter (Calame and Eaton (1988) Adv. Immunol. 43: 235-275), especially the promoter of the T cell receptor (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), a neuron-specific promoter (eg, a neurofilament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci. USA 86: 5473-5477).
), A pancreas-specific promoter (Edlund et al. (1985) Science 230: 912-916), and a mammary gland-specific promoter (eg, the milk whey promoter; US Pat. No. 4,873,
316 and EP-A-264,166). Developmentally regulated (
Also included are developmentally-regulated promoters, such as the murine hox promoter (Kessel and Gruss (1990) Science 249: 374-379) and the α-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3: 537-546). is there.

The present invention further provides a recombinant expression vector comprising a DNA molecule of the present invention cloned into the expression vector in an antisense orientation. That is, a DNA molecule is operably linked to a regulatory sequence so that expression of an RNA molecule that is antisense to mRNA encoding a polypeptide of the present invention is possible (by transcription of the DNA molecule). Selecting a regulatory sequence (eg, a viral promoter and / or enhancer) operably linked to the nucleic acid cloned in the antisense orientation that directs continuous expression of the antisense RNA molecule in a variety of cell types; Alternatively, select a regulatory sequence that directs constitutive, tissue-specific or cell type-specific expression of the antisense RNA. Antisense expression vectors are recombinant plasmids, phagemids or attenuated vectors in which the antisense nucleic acid is produced under the control of highly efficient regulatory regions, the activity of which is determined by the cell type into which the vector is introduced. Virus. For a discussion of the regulation of gene expression using antisense genes, see Weintraub et al. (Reviews-Trends in Genetics).
), Vol. 1 (1) 1986).

[0146] Another embodiment of the present invention relates to a host cell into which the recombinant expression vector of the present invention has been introduced. The terms "host cell" and "recombinant host cell" are used interchangeably herein. The term is understood to refer not only to the particular cell of interest, but also to the progeny or potential progeny of such a cell. The progeny may not actually be the same as the parent cell, since certain modifications may occur in subsequent generations due to mutations or environmental influences, but nonetheless, the scope of terms used herein Within.

[0147] A host cell can be any prokaryotic cell (eg, E. coli) or can be a eukaryotic cell (eg, insect cells, yeast or mammalian cells).

The DNA of the vector can be introduced into prokaryotic or eukaryotic cells by conventional transformation or transfection methods. As used herein, the terms "transformation" and "transfection" refer to exogenous nucleic acid introduced into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran mediated transformation, lipofection, or electroporation. It is intended to refer to a variety of art-recognized methods of implementation. 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 on the expression vector and transfection method used, only a small fraction of the cells integrates foreign DNA into their genome. These intrinsics (integ
To identify and select rants, generally, a gene encoding a selectable marker (eg, a selectable marker for resistance to an antibiotic) is introduced into the host cell along with the gene of interest. Preferred selectable markers include drugs (eg, G418
, Hygromycin and methotrexate). Stably transfected cells with the introduced nucleic acid can be identified by drug selection (eg, cells that have incorporated the selectable marker gene will survive, but other cells will die).

The polypeptides of the invention can be produced using host cells of the invention, such as prokaryotic and eukaryotic host cells in culture. Therefore, the present invention further provides a method for producing the polypeptide of the present invention using the host cell of the present invention. In one embodiment, the method comprises culturing a host cell of the invention (in which a recombinant expression vector encoding a polypeptide of the invention has been introduced) in a suitable medium in which the polypeptide is produced. Including. In another embodiment, the method further comprises isolating the polypeptide from the medium or the host cell.

In addition, non-human transgenic animals can be produced using the host cells of the present invention. For example, in one embodiment, a host cell of the invention is a fertilized egg or embryonic stem cell into which a sequence encoding a polypeptide of the invention has been introduced.
Then, using such a host cell, the non-human transgenic animal in which the exogenous sequence encoding the polypeptide of the present invention has been introduced into its genome, or the endogenous sequence encoding the polypeptide of the present invention has been modified. The resulting homologous recombinant animal can be created. 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, "transgenic animal" refers to the
Non-human animals, preferably mammals, more preferably rodents such as rats or mice, wherein one or more cells contain the transgene. Examples of other transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, and the like. The transgene is exogenous DNA that is integrated into the genome of the cell from which the transgenic animal develops, and which remains in the genome of the mature animal, thereby resulting in one or more cell types of the transgenic animal or The expression of the encoded gene product in the tissue is dictated. As used herein, “homologous recombinant animal” refers to an exogenous DNA in which an endogenous gene has been introduced into the endogenous gene and cells of the animal (eg, embryonic cells of the animal prior to the development of the animal). Non-human animals, preferably mammals, more preferably mice, which have been modified by homologous recombination with the molecule.

A transgenic animal of the invention comprises introducing a nucleic acid encoding a polypeptide of the invention (or a homolog thereof) into the male pronucleus of a fertilized egg (eg, by microinjection, retroviral infection), The eggs can be produced by developing them in pseudopregnant female foster animals. To increase the expression efficiency of the transgene, an intron sequence and a polyadenylation signal may be introduced into the transgene. The tissue-specific regulatory sequence (s) can be operably linked to the transgene to direct specific cells to express a polypeptide of the invention. Methods for producing transgenic animals, particularly animals such as mice, by embryo manipulation and microinjection have become common in the art and are described, for example, in US Pat.
Nos. 4,736,866 and 4,870,009, U.S. Pat. No. 4,873,191 and Hogan, Manipulating the Mouse Embryo, (Cold Spring Harbor Laborato
ry Press, Cold Spring Harbor, NY, 1986). Similar methods for producing other transgenic animals are used. A transgenic founder can be identified based on the presence of the transgene in its genome and / or expression of the mRNA encoding the transgene in tissues or cells of the animal. The transgenic founder can then be used to breed additional animals with the transgene. In addition, transgenic animals having a transgene can be further propagated to other transgenic animals having other transgenes.

To create a homologous recombinant animal, a polypeptide of the invention in which a deletion, addition or substitution has been introduced, whereby the gene has been altered, eg, functionally disrupted, can be obtained. A vector containing at least a part of the gene to be encoded is prepared. In a preferred embodiment, the vector is designed such that upon homologous recombination, the endogenous gene is functionally disrupted (ie, no longer encodes a functional protein; also referred to as a "knockout" vector). Alternatively, upon homologous recombination, the endogenous gene is mutated or otherwise modified, but still encodes a functional protein (eg, an upstream regulatory region is modified, thereby altering the endogenous gene). (Which can alter the expression of a sex protein). In a homologous recombination vector, an additional nucleic acid of a gene that causes homologous recombination between an exogenous gene carried by the vector and an endogenous gene in embryonic stem cells has a modified portion of the gene of 5%. Flanking the 'and 3' ends. The additional flanking nucleic acid sequence is a sequence of sufficient length for successful homologous recombination with the endogenous gene. Generally, several kilobases of flanking (at both the 5 'and 3' ends)
DNA is introduced into the vector (for example, see T
homas and Capecchi (1987) Cell 51: 503). The vector is introduced (eg, by electroporation) into an embryonic stem cell line and selects for cells in which the introduced gene has been homologously recombined with the endogenous gene (eg, Li et al. (
1992) Cell 69: 915). The selected cells are then injected into the blastocyst of an animal (eg, a mouse) to form an aggregation chimera (eg, Bradley, teratocarcinoma and embryonic stem cells: a practical approach (Teratocarcinomas and Embryonic Stem).
m Cells: A Practical Approach), edited by Robertson (IRL, Oxford, 1987) pp. 113
-152). The chimeric embryo can then be implanted into a suitable pseudopregnant female developing animal and the embryo allowed to grow to term. Homologous recombinant DN in germ cells
Germline transmission of the transgene using progeny with A
) Allows the breeding of animals containing DNA in which all cells of the animal have been homologously recombined. Furthermore, methods for constructing homologous recombination vectors and animals are described in Bradley (1991) Current Opinion in Bio / Technology 2: 823-8.
29 and International Patent Publication Nos. WO90 / 11354, WO91 / 01140, WO92 / 0968, and WO93 / 04169.

In another embodiment, transgenic non-human animals can be generated that contain a selection system that allows for regulated expression of the transgene. One example of such a system is the cre / loxP recombinase system of bacteriophage P1. For a description of the cre / loxP recombinase system, see, eg, Lakso et al. (1992) Proc. Natl. Acad.
ci. USA 89: 6232-3236. Another example of a recombinase system is the Saccharomyces cerevisiae FLP recombinase system (O'Gorman et al. (1991) Science
251: 1351-1355). When regulating the expression of a transgene using the cre / loxP recombinase system, an animal containing a transgene encoding both the Cre recombinase and the selected protein is required. Such animals can be "double", for example, by mating two transgenic animals, one containing a transgene encoding a selection protein and the other containing a transgene encoding a recombinase. ) "Can be provided by the construction of transgenic animals.

Also, the clones of the non-human transgenic animals described herein are:
It can be prepared according to the method described in Wilmut et al. (1997) Nature 385: 810-813 and International Patent Publication Nos. WO97 / 07668 and WO97 / 07669.

IV. Pharmaceutical Compositions The nucleic acid molecules, polypeptides, and antibodies of the invention (herein "active compounds")
) Can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically include the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" refers to any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorptions which are compatible with the administration of the medicament. It is intended to include retarders and the like.
The use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, its use in the composition is contemplated. Supplementary active compounds can also be incorporated into these compositions.

The present invention includes a method for preparing a pharmaceutical composition for modulating the expression or activity of a polypeptide or nucleic acid of the present invention. Such methods include combining a pharmaceutically acceptable carrier with an agent that modulates the expression or activity of a polypeptide or nucleic acid of the present invention. Such compositions may further include additional active agents. Accordingly, the present invention further provides a method of preparing a pharmaceutical composition by combining a pharmaceutically acceptable carrier with an agent that modulates the expression or activity of a polypeptide or nucleic acid of the invention and one or more additional active compounds. Including.

A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration.
Examples of routes of administration include parenteral, eg, intravenous, intradermal, subcutaneous, oral (eg, inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous use can include the following components: sterile diluents, such as water for injection, saline, fixed oils, polyethylene glycols, glycerin, Propylene glycol or other synthetic solvents; antimicrobial agents, such as benzyl alcohol or methyl paraben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetate, citric acid Salts or phosphates and agents for adjusting tonicity, such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. Parenteral preparations 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 dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cr
emophor EL ™ (BASF; Parsippany, NJ) or phosphate buffered saline (
PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like) and suitable mixtures thereof. Proper fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersion, and by using a surfactant. 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 agents that delay absorption, for example,
This can be achieved by including aluminum monostearate and gelatin in the composition.

A sterile injectable solution is obtained by incorporating the active compound (eg, polypeptide or antibody) in the required amount in a suitable solvent with one or a combination of the above-listed components, followed by filtration. It can be prepared by sterilizing.
Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for preparing sterile injectable solutions, the preferred methods of preparation are vacuum drying and lyophilization, the latter being the addition of the active ingredient plus any additional desired ingredient powders from their pre-sterile filtered solutions. Occurs.

[0161] Oral compositions generally include an inert diluent or an edible carrier. These 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 flowable carrier for use as a gargle, wherein the compound in the flowable carrier is applied in the mouth and is exhaled or swallowed.

Pharmaceutically compatible binding agents, and / or auxiliary substances can be included as part of the composition. Tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds having similar properties: binders, for example, microcrystalline cellulose, tragacanth gum or gelatin; excipients, for example, starch or Lactose; disintegrants, such as alginic acid, Primogel, or corn starch; lubricants, such as magnesium stearate or Sterotes; glidants, such as colloidal silicon dioxide; sweeteners, such as sucrose or Saccharin; or a flavor, such as peppermint, methyl salicylate, or an orange flavor.

For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressurized containers or dispensers which contain a suitable propellant, eg, a gas such as carbon dioxide, or a nebulizer.

[0164] Systemic administration may 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 treated with an ointment, as generally known in the art.
ointments), salves, gels, or creams.

The compounds can also be prepared in the form of suppositories (eg, 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 such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoester,
And polyacetic acid can be used. Methods for preparing such formulations will be apparent to those skilled in the art. These materials are available from Alza Corporation and Nova Pharmaceuti
It is also commercially available from cals, Inc. Liposomal suspensions (including liposomes targeting 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 US Pat. 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 suitable for unit administration to a subject to be treated; each unit has been previously calculated to produce the desired therapeutic effect. A determined amount of the active compound is included with the required pharmaceutical carrier. The specifications for the dosage unit forms of the present invention are determined by the unique characteristics of the active compounds, the particular therapeutic effect sought to be achieved, and the limitations inherent in the art of formulating such active compounds for treatment of an individual, and Depends directly on

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 works in the brain, 50mg / kg ~
A dose of 100 mg / kg is usually appropriate. Generally, partially human antibodies and fully human antibodies have a longer half-life in the human body than other antibodies. Thus, lower dosages and less frequent administration is often possible. Modifications such as lipidation can be used to stabilize antibodies and enhance uptake and tissue penetration (eg, into the brain). Methods for lipidating antibodies are described by Cruikshank et al. ((199
7) J. Acquired Immune Deficiency Syndromes and Human Retrovirology 14:19
3).

A nucleic acid molecule of the present invention can be inserted into a vector and used as a gene therapy vector. Gene therapy vectors can be administered, for example, by intravenous injection, topical administration (US Pat. No. 5,328,470), or by stereotactic injection (see, eg, Chen et al.
(1994) Proc. Natl. Acad. Sci. USA 91: 3054-3057). Pharmaceutical formulations of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can include a slow release matrix in which the gene delivery vehicle is embedded. Alternatively, if a complete gene delivery vector, e.g., a retroviral vector, can be produced intact from recombinant cells, the pharmaceutical formulation should include one or more cells producing the gene delivery system. Can be.

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

V. Uses and Methods of the Invention The nucleic acid molecules, proteins, protein homologs, and antibodies described herein can be used in one or more of the following methods: a) screening assays; b) detection assays (eg, Chromosomal mapping, tissue typing, forensic biology); c) predictive medicine (eg, diagnostic assays, prognostic assays, monitoring of clinical trials, and pharmacogenomics); and d) treatment methods (eg, treatment and prevention). For example, the polypeptides of the invention may be used for (i) regulating cell proliferation; (ii) regulating cell migration and chemotaxis; (iii) regulating cell differentiation; and / or (iv) regulating angiogenesis. be able to. The isolated nucleic acid molecules of the invention can be used to detect protein expression (e.g., by recombinant expression vectors in host cells in gene therapy applications), mRNA (e.g., in biological samples) or genetic damage,
And the activity of the polypeptide of the present invention. In addition, the polypeptide of the present invention, in addition to screening for drugs or compounds that modulate the activity or expression of the polypeptide of the present invention, has reduced activity compared to the protein of the present invention or the wild-type protein. Alternatively, it can be used to treat disorders characterized by insufficient or overproduction of an abnormal form of the protein of the invention. In addition, the antibodies of the invention can be used for detecting and isolating the proteins of the invention, and for modulating the activity of the proteins of the invention.

The present invention further relates to novel agents identified by the above screening assays and their use in the treatments described herein.

A. Screening Assays The invention binds to a modulator, ie, a polypeptide of the invention, or
For example, a method for identifying a candidate or test compound or agent (eg, a peptide, peptidomimetic, small molecule, or other drug) that has a stimulatory or inhibitory effect on the expression or activity of a polypeptide of the invention (eg, herein). Provides a “screening assay”).

In one embodiment, the present invention provides assays for screening for candidate or test compounds that bind to or modulate the activity of a polypeptide of the invention or a biologically active portion thereof in a membrane-bound form. I will provide a. The test compounds of the present invention include biological libraries; spatially addressable parallel solid or liquid phase libraries; synthetic library methods requiring deconvolution; "1 compound per bead (
can be obtained using any of a number of approaches to combinatorial library methods known in the art, including "one-bead one-compound" library methods; and synthetic library methods using affinity chromatography selection. it can. Biological library approaches are limited to peptide libraries, whereas the other four approaches are applicable to small molecule libraries of peptides, non-peptide oligomers or compounds (Lam (1997) Anticance
r Drug Des. 12: 145).

Examples of methods for synthesizing molecular libraries are described in the art, for example: DeWitte
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. E
d. Engl. 33: 2061; and Gallop et al. (1994) J. Med. Chem. 37: 1233.

Compound libraries can be prepared in solution (eg, in Houghten (1992) Bio / Techniques).
13: 412-421) or on beads (Lam (1991) Nature 354: 82-84), chip (
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) Scie.
nce 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, the assay is a cell-based assay, wherein cells expressing a polypeptide of the invention in a membrane-bound form or a biologically active portion thereof on the cell surface are tested with a test compound. And determine the ability of the test compound to bind to the polypeptide. The cell can be, for example, a yeast cell or a cell of mammalian origin. Determining the ability of a test compound to bind to a polypeptide can include, for example,
Coupling the test compound to a radioisotope or enzyme label such that binding of the test compound to the polypeptides or biologically active portions thereof can be determined by detecting the labeled compound in the complex. Can be achieved by: For example, a test compound can be labeled directly or indirectly with ¹²⁵ I, ³⁵ S, ¹⁴ C, or ³ H, and its radioisotope detected by direct counting of radioemission or scintillation counting. Alternatively, the test compound can be enzymatically labeled, for example, with horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzyme label detected by determining the conversion of the appropriate substrate to product. In a preferred embodiment, the assay involves contacting a cell that expresses a polypeptide of the invention in a membrane-bound form or a biologically active portion thereof on the cell surface with a known compound that binds the polypeptide. Forming a mixture and contacting the assay mixture with a test compound to determine 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 a known compound.

In another embodiment, the assay comprises contacting a cell that expresses a polypeptide of the invention in a membrane-bound form or a biologically active portion thereof on the cell surface with a test compound, wherein the polypeptide is Alternatively, a cell-based assay comprising determining the ability of a test compound to modulate (eg, stimulate or inhibit) the activity of a biologically active portion thereof. Determining the ability of a test compound to modulate the activity of a polypeptide or biologically active portion thereof can be performed, for example, by determining the ability of the polypeptide protein to bind to or interact with a target molecule. Can be achieved.

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 to which a selected polypeptide (eg, a polypeptide of the invention) naturally binds or interacts, eg, a cell that expresses a selected protein. Molecule on the surface, number
2 Molecules on the surface of cells, molecules in the extracellular environment, molecules associated with the inner surface of the cell membrane or plasma membrane. The target molecule can be a polypeptide of the invention or another polypeptide or protein. For example, a target molecule may be an extracellular signal to a cell or a second intercellular protein having catalytic activity or a protein that facilitates the association of a downstream signaling molecule with a polypeptide of the present invention through the cell membrane (eg, the present invention). A signal generated by the binding of a compound to a polypeptide of the present invention. Determining the ability of a polypeptide of the invention to bind to or interact with a target molecule can be achieved by determining the activity of the target molecule. For example, the activity of a target molecule may be determined by detecting the induction of a cellular second messenger of the target (eg, intracellular Ca ²⁺ , diacylglycerol, IP3, etc.), thereby determining the catalytic / enzymatic activity of the target on a suitable substrate. By detecting the induction of a reporter gene (eg, a regulatory element responsive to a polypeptide of the invention operably linked to a nucleic acid encoding a detectable marker, eg, luciferase),
Alternatively, it can be determined by detecting a cellular response, eg, cell differentiation or cell proliferation.

In yet another embodiment, the assay of the invention comprises contacting a polypeptide of the invention or a biologically active portion thereof with a test compound, wherein the polypeptide or a biologically active portion thereof is A cell-free assay that involves determining the ability of a test compound to bind to a moiety. The binding of the test compound to the polypeptide can be determined either directly or indirectly, as described below. In a preferred embodiment, the assay comprises contacting a polypeptide of the invention, or a biologically active portion thereof, with a known compound that binds the polypeptide to form an assay mixture, and combining 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 comparing the polypeptide or polypeptide with the known compound. Determining the ability of test compounds to bind preferentially to their biologically active moieties.

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

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

The cell-free assays of the present invention are suitable for using both soluble or membrane-bound forms of the polypeptides of the present invention. For cell-free assays involving a membrane-bound form of the polypeptide, it may be desirable to use a solubilizing agent so that the membrane-bound form of the polypeptide is maintained in solution. Examples of such solubilizers include non-ionic surfactants such as n-octylglucoside, n-dodecylglucoside, n-octylmaltoside, octanoyl-N-methylglucamide, decanoyl-N-methylgluca. Mid, Triton X-100, Triton X-114, Thesit, isotridecipoly (ethylene glycol ether) _n , 3-[(3-cholamidopropyl) dimethylamminio] -1-propanesulfonate (CHAPS), 3-[(3 -Colamidopropyl) dimethylamminio] -2-hydroxy-1-propanesulfonate (CHAPSO) or N-dodecyl = N, N-dimethyl-3-ammonio-1
-Includes propane sulfonate.

In two or more embodiments of the above-described assay methods of the invention, in addition to facilitating the separation of the complex form from the uncomplexed form of one or both of the proteins, to accommodate the automation of the assay, It may be desirable to immobilize either the polypeptide or the target molecule of the present invention. Binding of the test compound to the polypeptide in the presence and absence of the candidate compound, or interaction of the polypeptide with the target molecule, can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and microcentrifuge tubes. In one embodiment, a fusion protein can be provided that adds a domain that binds one or both proteins to a matrix. For example, a glutathione-S-transferase fusion protein or a glutathione-S-transferase fusion protein is adsorbed to glutathione-Sepharose beads (Sigma Chemical; St. Louis, MO) or a glutathione-derivatized microtiter plate, which is then adsorbed to a test compound or The test compound and either the non-adsorbed target protein or the polypeptide of the present invention can be combined and the mixture can be incubated under conditions that favor complex formation (eg, physiological conditions for salt and pH). Following incubation, the beads or microtiter plate are washed away of unbound components and complex formation is measured directly or indirectly, for example, as described above. Alternatively, the complex can be separated from the matrix and the level of binding or activity of the polypeptide of the invention can be assessed using standard methods.

[0185] Other techniques for immobilizing proteins on matrices can also be used in the screening assays of the present invention. For example, either the polypeptide of the present invention or its target molecule can be immobilized using the binding of biotin and streptavidin. Biotinylated polypeptides or target molecules of the invention can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques known in the art (eg, biotinylation kits, Pierce Chemicals; Rockford, IL).
) And streptavidin-coated 96-well plate (Pierce Chemi
cal) can be immobilized in the wells. Alternatively, an antibody that reacts with the polypeptide or target molecule of the present invention but does not interfere with the binding of the polypeptide of the present invention to its target molecule is derivatized to the wells of a plate to convert unbound target or polypeptide of the present invention. It can be captured in the well by antibody binding. Methods for detecting such complexes include those described above for GST-immobilized complexes, as well as immunodetection of the complexes using antibodies reactive with a polypeptide or target molecule of the invention, as well as with the present invention. Includes enzyme binding assays by detecting enzyme activity associated with a polypeptide of the invention or a target molecule.

In another embodiment, a modulator of the expression of a polypeptide of the invention is
A cell is contacted with a candidate compound and identified in a method for assessing expression of a selected mRNA or protein (ie, an mRNA or protein corresponding to a polypeptide or nucleic acid of the invention) in the cell. 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. Thereafter, based on this comparison, the candidate compound can be identified as a modulator of expression of a polypeptide of the invention. For example, if the expression of a selected mRNA or protein is higher in the presence of a candidate compound than in its absence (statistically significantly higher), the candidate compound is identified as a stimulator of the expression of the selected mRNA or protein. Is done. Alternatively, if the expression of the selected mRNA or protein is lower (statistically significantly lower) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of the expression of the selected mRNA or protein. Is done. The level of expression of the selected mRNA or protein in the cell can be determined by the methods described herein.

In still another embodiment of the present invention, wherein the polypeptide of the present invention is used in a two-hybrid assay or a three-hybrid assay to determine the "bait pr
oteins) "(eg, US Patent No. 5,283,317; Zervos et al. (1993) Cell 7
2: 223-232; Madura et al. (1993) J. Biol. Chem. 268: 12046-2054; Bartel et.
al. (1993) Bio / Techniques 14: 920-924; Iwabuchi et al. (1993) Oncogene 8
1693-1696; and PCT Publication WO 94/10300) to identify other proteins that bind to or interact with a polypeptide of the invention and modulate the activity of the polypeptide of the invention. be able to. Such a binding protein is also likely to be involved in signal transmission by the polypeptide of the present invention, for example, as a downstream or upstream element of a signaling pathway involved in the polypeptide of the present invention.

The present invention further relates to novel agents identified by the above screening assays and their use in the treatments described herein.

B. Detection Assays The portions or fragments of the cDNA (and corresponding complete gene sequences) identified herein can be used in a variety of ways as polynucleotide reagents. For example, these sequences provide (i) the mapping of each gene on the chromosome, and thus the location of gene regions associated with genetic diseases; (ii) the identification of individuals from small biological samples (tissue typing); It can also be used to assist in forensic identification of biological samples. These uses are described in the following subsections.

[0190] 1. Chromosome Mapping The sequence of a gene (or a portion of a sequence) can be isolated and the sequence used to map gene locations on a chromosome. Thus, using the nucleic acid molecules or fragments thereof described herein, the location of the corresponding gene on the chromosome can be mapped.
Mapping of sequences to chromosomes is an important first step in correlating these sequences with genes associated with disease.

Briefly, by preparing PCR primers (preferably 15-25 bp in length) from the sequence of the gene of the present invention, the gene can be mapped to a chromosome. Because of the complexity of the amplification process, computer analysis of the gene sequences of the present invention allows rapid selection of primers that do not span more than one exon in genomic DNA. These primers can then be used to PCR screen somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the gene sequence will yield an amplified fragment. For confirmation of this technique, see D'Eustachio et al. (1983) Science 220: 919-914.

PCR mapping of somatic cell hybrids is a rapid technique for assigning specific sequences to specific chromosomes. More than two sequences can be assigned per day using a single thermal cycler. Oligonucleotide primers can be designed using the nucleic acid sequences of the present invention, and a panel of fragments derived from a particular chromosome can be used to achieve a determination of sublocalization. Similarly, other mapping methods that can be used to map a gene to its chromosome include in situ hybridization (Fan et al. (1990) Proc.
Acad. Sci. USA 87: 6223-27), labeled flow sorted (flow so
rted) chromosome-based preliminary screening, as well as preliminary selection by hybridization with a chromosome-specific cDNA library. In addition, accurate chromosome localization can be performed in one step using fluorescence in situ hybridization (FISH) with metaphase chromosome spreads. For confirmation of this technique, see Verma et al. (Human Chromosomes: A Manual of Basic Techniques (Pergam
on Press, New York, 1988)).

A single chromosome or a single site on the chromosome can be labeled using reagents for chromosome mapping. Alternatively, a panel of reagents can be used to label multiple sites and / or multiple chromosomes. In practice, for mapping, reagents corresponding to non-coding regions of the gene are preferred. The coding sequence is believed to be conserved within the gene family, which increases the likelihood of cross-hybridization during chromosome mapping.

Once a sequence has been mapped to a precise chromosomal location, the physical location of the sequence on the chromosome can be correlated with genetic map data (such data can be found, for example, in V. McKusick, Mendelian Inheritance in Man and is available online from the John Hopkins University Welch Medical Library). Next, the relationship between the gene and the disease mapped to the same chromosomal region is described, for example, by Egeland et al.
987) It can be confirmed by linkage analysis (simultaneous inheritance of physically adjacent genes) described in Nature 325: 785-787.

In addition, differences in DNA sequence between individuals afflicted with a disease associated with the gene of the present invention and unaffected individuals can be determined. If the mutation is found in some or all of the affected individuals but not in unaffected individuals, the mutation is:
It is thought to be a factor causing certain diseases. Comparisons between affected and unaffected individuals are visible in chromosome spreads or DNA
Includes looking for structural changes in the chromosome such as deletions or translocations that can be detected using sequence-based PCR. Finally, by performing a complete sequencing of the genes from multiple individuals, the presence of the mutation can be confirmed and the mutation from the polymorphism can be identified.

[0196] 2. Tissue Typing It is also possible to identify individuals from small biological samples using the nucleic acid sequences of the invention. For example, the United States Army is considering the use of restriction fragment length polymorphisms (RFLPs) to identify its personnel. In this technique, a unique band is obtained by digesting an individual's genomic DNA with one or more restriction enzymes and probing with a Southern blot. This method overcomes the real limitations of "Dog Tags" (which can be lost, replaced or stolen, making clear identification difficult).
The sequences of the present invention are useful as additional DNA markers for RFLP (described in US Pat. No. 5,272,057).

Furthermore, the use of the sequences of the present invention allows for the actual base-by-base
An alternative method for determining the DNA sequence is provided. Thus, using the nucleic acid sequences described herein, two PCR primers can be generated from the 5 'and 3' ends of the sequence. Next, the DNA of an individual is amplified using these primers, and the sequence is determined.

The panel of corresponding DNAs from the individuals thus generated allows each individual to be uniquely identified, since each individual has a unique set of such DNA sequences due to allelic differences To Such discriminating sequences can be obtained from individuals and tissues using the sequences of the present invention. The nucleic acid sequences of the present invention represent unique portions of the human genome. Allelic variations are present to some extent in the coding regions of these sequences, and to a greater extent in non-coding regions. Allelic variation between human individuals is estimated to occur at a frequency of about 1 in 500 bases. Each of the sequences described herein can be used as a basis for comparison to identify DNA from an individual to some extent. Because of the large number of polymorphisms in non-coding regions, only a small number of sequences are needed to identify an individual. The non-coding sequence of SEQ ID NOs: 1, 4 or 7 is approximately 10-1,000 with each primer providing a 100 base non-coding amplified sequence.
Distinct individual identification can easily be provided on a panel consisting of as many primers. When using a deduced coding sequence such as the sequence of SEQ ID NOs: 3, 6 or 9, the number of primers more suitable for unambiguous individual identification is 500-2,000.

When an individual-specific identification database is created using a panel of reagents derived from a nucleic acid sequence described herein, the same reagents may be used later to identify tissue from the individual. Can be. The use of a unique identification database allows for the clear identification of individuals from very small tissue samples, whether living or dead.

[0200] 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 discipline that uses genotyping of biological evidence found at crime scenes, for example, as a means to unambiguously identify criminals. To perform such identification, PCR techniques were used to remove tissues found at crime scenes, such as hair and skin, or body fluids, such as very small biological samples such as blood, saliva, or semen. Amplify the DNA. Next, the origin of the biological sample is identified by comparing the amplified sequence with a reference.

The sequences of the present invention can be used to provide a polynucleotide reagent, eg, a PCR primer, that targets a particular locus in the human genome, thereby, for example, providing another “discriminating marker” (ie, DNA sequence specific to a particular individual)
Can improve the reliability of forensic identification based on DNA. As described above, the actual nucleotide sequence information can be used for identification as an accurate alternative to the pattern formed by the restriction enzyme-generated fragments. Sequences targeting non-coding regions are particularly suitable for this application, as there are numerous polymorphisms in the non-coding regions, which facilitate identification of individuals using the above techniques. Examples of polynucleotide reagents include nucleic acids of the invention or portions thereof, eg, fragments derived from non-coding regions at least 20 or 30 bases in length.

Further, the nucleic acid sequences described herein can be used to provide polynucleotide reagents, eg, labeled or labelable probes, which include, for example, in s
The use of the itu hybridization technique allows identification of a specific tissue, for example, brain tissue. This is very useful when a forensic pathologist presents tissue of unknown origin. Using such a panel of probes,
Tissues can be identified by species and / or organ type.

C. Prognostic Medicine The present invention also relates to the field of prognostic medicine for prophylactic treatment of individuals by monitoring diagnostic assays, prognostic assays, pharmacogenetics, and clinical trials for prognostic purposes. Therefore, one embodiment of the present invention provides a biological sample (
For example, by measuring the expression of the polypeptide or nucleic acid of the present invention and / or the activity of the polypeptide of the present invention with respect to blood, serum, cells, or tissues), an individual can express abnormal activity or activity of the polypeptide of the present invention. Diagnostic assays for determining whether a subject is afflicted with a disease or disorder associated with, or at risk of developing such a disorder. The present invention further provides prognostic (or prognostic) assays for determining whether an individual is at risk of developing a disorder associated with aberrant expression or activity of a polypeptide of the present invention. For example, mutations in a gene of the invention can be assayed in a biological sample. Using such assays for prognostic or prognostic purposes, one can prevent or treat the individual prior to the onset of seizures characterized by, or associated with, abnormal expression or activity of a polypeptide of the invention. .

Another aspect of the present invention is a method of selecting an appropriate therapeutic or prophylactic agent for an individual by measuring the expression of the nucleic acid or polypeptide of the present invention or the activity of the polypeptide of the present invention in the individual. (Here we call it “pharmacogenetics”)
). Pharmacological genetics provides an agent (eg, a drug) for treating or preventing an individual based on the genotype of the individual (eg, the genotype of the individual being tested to determine the ability of the individual to respond to a particular agent). ) Can be selected.

Yet another aspect of the present invention relates to monitoring the effect of an agent (eg, a drug or other compound) on the expression or activity of a polypeptide of the present invention in a clinical trial. These and other agents are described in further detail in the sections that follow.

[0206] 1. One example of a method for detecting the presence or absence of a polypeptide or nucleic acid of the invention in a diagnostic assay biological sample is to collect a biological sample from a subject,
Detecting the presence of the polypeptide or nucleic acid of the present invention in the biological sample by contacting with a compound or agent capable of detecting the polypeptide or nucleic acid (eg, mRNA, genomic DNA). Preferred agents for detecting mRNA or genomic DNA encoding the polypeptide of the present invention are labeled nucleic acid probes that can hybridize to mRNA or genomic DNA encoding the polypeptide of the present invention. The nucleic acid probe may be, for example, at least 15, 30, 50, 100 of a full-length cDNA such as SEQ ID NO: 1, 3, 4, 6, 7 or 9, or a portion thereof.
, 250 or 500 nucleotides in length, which can specifically hybridize under stringent conditions to mRNA or genomic DNA encoding the polypeptide of the present invention. Other probes suitable for use in the diagnostic assays of the invention are described herein.

A preferred agent for detecting a polypeptide of the invention is an antibody capable of binding to a polypeptide of the invention, preferably an antibody with a detectable label. Antibodies are
It may be polyclonal, but is more preferably monoclonal. An antibody in its native form, or a fragment thereof (eg, Fab or F (ab ') ₂ ) can be used. With respect to a probe or antibody, the term "label" refers to the direct labeling of the probe or antibody by binding (ie, physical binding) of the detectable substance to the probe or antibody, as well as the reactivity of another directly labeled reagent with another reagent. And indirect labeling of the probe or antibody caused by the above. 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 that can be detected with fluorescently labeled streptavidin. The term "biological sample" is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present in a subject. That is, using the detection method of the present invention, mRNA, protein, or genomic DNA in a biological sample in vitro and in vivo can be detected. For example, in mRNA detection
In vitro methods include Northern hybridizations and in situ hybridizations. In vivo methods for detecting a polypeptide of the invention include enzyme-linked immunosorbent assays (ELISA), western blots, immunoprecipitations and immunofluorescence. In vitro methods for genomic DNA detection include Southern hybridizations. Further, an in vivo method for detecting a polypeptide of the present invention comprises introducing into a subject a labeled antibody against the polypeptide. For example, the antibody can be labeled with a radioactive marker and its presence and location within the subject detected with conventional imaging techniques.

In one embodiment, the biological sample contains protein molecules from the subject. Alternatively, the biological sample may be a subject-derived mRNA molecule or a subject-derived genomic DN.
A molecule may be included. A preferred biological sample is a peripheral blood leukocyte sample isolated from a subject by conventional means.

In another embodiment, the method further comprises obtaining a control biological sample from the control subject, and obtaining a polypeptide of the invention, or an mRNA encoding a polypeptide of the invention.
Alternatively, by contacting the control sample with a compound or agent capable of detecting genomic DNA, the presence of the polypeptide or mRNA or genomic DNA encoding the polypeptide in the control sample is detected. Presence of the polypeptide or mRNA or genomic DNA encoding the polypeptide in the test sample and encoding the polypeptide or the polypeptide in a test sample
comparing to the presence of mRNA or genomic DNA.

[0210] The present invention also includes kits for detecting the presence of a polypeptide or nucleic acid of the present invention in a biological sample (test sample). Using such a kit,
Whether the subject has, or is likely to develop, a disorder associated with aberrant expression of a polypeptide of the invention (eg, a proliferative disorder, eg, psoriasis or cancer). Can be determined. For example, the kit includes a labeling compound or an agent capable of detecting a polypeptide or mRNA encoding the polypeptide in a biological sample, and a tool for measuring the amount of the polypeptide or mRNA in the sample (for example, a polypeptide and the like). An antibody that binds, or an oligonucleotide probe that binds to DNA or mRNA encoding the polypeptide). The kit also includes a subject having a disorder associated with aberrant expression of the polypeptide if the amount of the polypeptide, or mRNA encoding the polypeptide, is above or below normal levels. Alternatively, it may include an explanatory item for observing a possibility that such a disorder may occur.

For kits based on antibodies, the kit may include, for example, (1) a primary antibody that binds to a polypeptide of the invention (eg, immobilized on a solid support); optionally, (2)
It includes a different secondary antibody that binds to either the polypeptide or the primary antibody and is conjugated to a detectable agent.

For kits based on oligonucleotides, the kits may include, for example, (1) an oligonucleotide that hybridizes to a nucleic acid sequence encoding a polypeptide of the invention, such as a detectably labeled oligonucleotide, or (2) ) Comprises a pair of primers useful for amplifying a nucleic acid molecule encoding a polypeptide of the invention. The kit may also include, for example, buffers, preservatives, or protein stabilizers. The kit may further comprise a detectable agent (eg, an enzyme or a substrate)
May be included. The kit may also include a control sample or a series of control samples, which can be assayed and compared to the test samples included in the kit. Each component of the kit is usually sealed in an individual container, and the various containers are all contained in a single package, which contains the subject with a disorder associated with abnormal expression of the polypeptide. An explanation is provided for the opinion that one is affected or may develop such a disorder.

[0213] 2. Prognostic Assays The methods described herein may further be used as a diagnostic or prognostic assay to determine whether a disease or disorder associated with aberrant expression or activity of a polypeptide of the invention has been or has been obtained. Subjects at risk of developing can be identified. For example, a diagnostic assay performed before or an assay performed later,
Using the assays described herein, a subject suffering from a disorder associated with aberrant expression or activity of a polypeptide of the invention (eg, a proliferative disorder, eg, psoriasis or cancer, or an angiogenic disorder) Alternatively, subjects at risk of developing such a disorder can be identified. Alternatively, prognostic assays can be used to identify subjects suffering from or at risk of developing such a disorder. Therefore, the present invention
A test sample is taken from a subject and a polypeptide or nucleic acid of the invention (eg,
mRNA or genomic DNA), a subject suffering from or possibly having a disease or disorder associated with abnormal expression or activity of the polypeptide due to the presence of the polypeptide or nucleic acid A method for diagnosing is provided. As used herein, the term "test sample" refers to a biological sample taken from a subject of interest. For example, a test sample can be a biological fluid (eg, serum), a cell sample, or a tissue.

Furthermore, a subject can be administered a drug (eg, an agonist, antagonist, mimetic peptide, protein, peptide, nucleic acid, etc.) using the prognostic assays described herein.
It is possible to determine whether or not a disease or disorder associated with abnormal expression or activity of the polypeptide of the present invention can be treated by administering a low molecular substance or other candidate drug. For example, using such methods to determine whether a subject can be effectively treated with a particular agent or a particular class of agents (eg, a type of drug that reduces the activity of the polypeptide). Can be. Accordingly, the present invention is a method of determining whether a subject can be effectively treated with a drug for a disorder associated with abnormal expression or activity of a polypeptide of the present invention, comprising collecting a test sample. Detecting the polypeptide or a nucleic acid encoding the polypeptide (eg, by administering the agent due to the presence of the polypeptide or nucleic acid, is associated with abnormal expression or activity of the polypeptide) Diagnosing a subject that can treat the disorder).

The method of the present invention can also be used to detect a genetic defect or mutation in a gene of the present invention so that a subject having the defective gene has aberrant expression or activity of a polypeptide of the present invention. It is possible to determine whether or not there is a risk of developing a disorder characterized by the following. In a preferred embodiment, the method comprises modifying, in a sample of cells from the subject, an alteration that affects the integrity of the gene encoding the polypeptide of the invention, or at least the erroneous expression of the gene encoding the polypeptide of the invention. And detecting the presence or absence of a genetic defect or mutation characterized by one. For example, such a genetic defect or mutation may be caused by: 1) deletion of one or more nucleotides from the gene; 2) addition of one or more nucleotides to the gene; 3) one or more nucleotides of the gene. 4) Chromosomal rearrangement of gene; 5
) Fluctuations in the level of the messenger RNA transcript of the gene; 6) abnormal modification of the gene, such as the methylation pattern of genomic DNA; 7) presence of a non-wild-type splicing pattern of the messenger RNA transcript of the gene; 9) deletion of alleles of the gene; and 10) detection by confirming the presence of at least one inappropriate post-translational modification of the protein encoded by the gene. can do. As described herein, a number of assay methods that can be used to detect defects in genes are known to those of skill in the art.

In one embodiment, the detection of the defect is performed by polymerase chain reaction (PCR) such as anchor PCR or RACE PCR (see, eg, US Pat. Nos. 4,683,195 and 4,683,302), or ligation chain reaction (LCR). (See, for example, Landegran et al. (1988) Science 2
41: 1077-1080; and Nakazawa et al. (1994) Proc. Natl. Acad. Sci. USA 91
: 360-364) using probes / primers (see, for example, US Pat.
83,195 and 4,683,202), the latter being particularly useful in detecting point mutations in genes (Abravaya et al. (1995) Nucleic Acids Res.
23: 675-682). This method involves taking a sample of cells from a patient and isolating nucleic acids (eg, genomic, mRNA or both) from the cells of the sample, followed by hybridization and amplification of a selected gene (if present). Under the conditions, the nucleic acid sample is contacted with one or more primers that specifically hybridize to the gene, and the presence or absence of an amplification product is detected, or the size of the amplification product is examined. Comparing the length with the sample. It may be desirable to use PCR and / or LCR as a preliminary amplification step in conjunction with any of the techniques used to detect mutations described herein.

Another amplification method is self-sustaining sequence replication (Guatelli et al. (1990) Proc. Natl. Acad.
Sci. USA 87: 1874-1878), transcription amplification system (Kwoh et al. (1989) Proc. Natl. Acad. Sc).
i. USA 86: 1173-1177), Q-β replicase (Lizardi et al. (1988) Bio / Technolog).
y 6: 1197), or other nucleic acid amplification methods, followed by detection of the amplified molecule using techniques known to those skilled in the art. These detection means are particularly useful when detecting very small numbers of nucleic acid molecules.

In another embodiment, mutations in a selection gene from a sample cell can be identified by altering the restriction enzyme cleavage pattern. For example, sample and control DNA is isolated, amplified (if appropriate), digested with one or more restriction endonucleases, fragment lengths are measured by gel electrophoresis, and the two are compared. Samples and controls
DNA fragment length differences indicate mutations in the sample DNA. In addition, sequence-specific ribozymes (see, eg, US Pat. No. 5,498,531) can be used to assess the presence or absence of a ribozyme cleavage site to determine the presence of a particular mutation.

In other embodiments, genomic mutations can be identified by hybridizing sample and control nucleic acids, eg, DNA or RNA, to a high-density array containing hundreds or thousands of oligonucleotide probes. (Cronin et al., (1996) Huma
n Mutation 7: 244-255; Kozal et al., (1996) Nature Medicine 2: 753-759). For example, gene mutations can be identified in two-dimensional arrays containing light-generated DNA probes, as described in Cronin et al., Supra.
Briefly, a first hybridization probe array is used to scan over long portions of DNA in samples and controls to confirm base changes between sequences by creating a linear array of sequence overlapping probes. be able to. This step allows the identification of point mutations. Following this step, using a second hybridization array to confirm the particular mutation by using a smaller specialty probe array that is complementary to all mutants or mutations detected Can be. Each mutation is composed of a corresponding set of probes, one complementary to the wild-type gene and the other complementary to the mutated gene.

In yet another embodiment, the selection gene is sequenced directly using any of a variety of sequencing reactions known to those skilled in the art, and the sequence of the sample nucleic acid is replaced with the corresponding wild-type (control) sequence. By comparing with, the mutation can be detected. As an example of a sequencing reaction, see Maxim and Gilbert ((1977) Proc. Natl. Acad. Sci. USA).
74: 560) or Sanger ((1977) Proc. Natl. Acad. Sci. USA 74: 5463). In addition, diagnostic assays ((1995) Bio / Tech
niques 19: 448), any of a variety of automated sequencing procedures could be used, but these procedures include sequencing by mass spectrometry (see, eg, PCT Publication No. WO94 / 16101; Cohen et al. (1996) Adv. Chromatogr. 36: 1
27-162; and Griffin et al. (1993) Appl. Bioche. Biotechnol. 38: 147-15.
9).

Another method of detecting mutations in a selection gene is to detect mismatched bases in RNA / RNA or RNA / DNA heteroduplexes using protection from cleavage drugs (Mye
rs et al. (1985) Science 230: 1242). In general, the method of "mismatch cleavage" involves heterologous 2 formation formed by hybridizing (labeled) RNA or DNA containing a wild-type sequence to potentially mutant RNA or DNA obtained from a tissue sample. It is necessary to prepare the main strand. The duplex is treated with a drug that cleaves a single-stranded region of the duplex, such as that present due to base pair mismatch between the control and sample strands. For an RNA / DNA duplex, the mismatch region can be digested by treatment with RNase, and for a DNA / RNA hybrid, the mismatch region can be digested by treatment with S1 nuclease.

In another embodiment, the mismatched region can be digested by treating either the DNA / DNA or RNA / DNA duplex with hydroxylamine or osmium tetroxide, and piperidine. After digestion of the mismatch region, the resulting material is separated by size on a denaturing polyacrylamide gel to determine the site of mutation. See, eg, Cotton et al., (1988) Proc. Natl. Acad. Sci. USA 85: 4397; Saleeba.
See (1992) Methods Enzymol. 217: 286-295. In a preferred embodiment, control DNA or RNA may be labeled for detection.

In yet another embodiment, the mismatch cleavage reaction recognizes mismatched base pairs in double-stranded DNA in a defined system for detecting and mapping point mutations in cDNA obtained from a sample of cells. One or more proteins (so-called "DNA
Mismatch repair "enzyme). For example, the E. coli enzyme mutY cleaves A at a G / A mismatch, and HeLa cell-derived thymidine DNA glycosylase cleaves T at a G / T mismatch (Hsu et al. (1994) Carcinogenesis 15: 1657-1662). According to one example of the embodiment, a probe based on the selected sequence, e.g., a wild-type sequence,
Hybridize to cDNA or other DNA products from the cells to be tested. After treating the resulting double strand with a DNA mismatch repair enzyme, the cleavage product, if present, can be detected by electrophoresis or the like. See, for example, U.S. Patent No. 5,459,039.

In another embodiment, alterations in electrophoretic mobility are used to identify mutations in genes. For example, single-stranded conformational polymorphism (SSCP) may be used to detect differences in electrophoretic mobility between mutant and wild-type nucleic acids (Orita et al. (1989) Proc. Nat.
l. Acad. Sci. USA 86: 2766; Also, Cotton (1993) Mutat. Res. 285: 125-144.
Hayashi (1992) Genet. Anal. Tech. Appl. 9: 73-79). The single-stranded DNA fragments of the sample and control nucleic acids are denatured and then regenerated. The secondary structure of single-stranded nucleic acids differs depending on the sequence, and the resulting change in electrophoretic mobility allows detection of even a single base change. DNA fragments can be labeled or detected with a labeled probe. By using RNA (as opposed to DNA), the sensitivity of this assay can be enhanced, where the secondary structure is more sensitive to sequence changes. In a preferred embodiment, the method uses heterogeneous duplex analysis to classify heterogeneous, double-stranded molecules based on changes in electrophoretic mobility (Keen et al. (1991) Trends Genet. 7 :Five).

In yet another embodiment, migration of mutant or wild-type fragments in polyacrylamide gels containing denaturing gradients is assayed using denaturing gradient gel electrophoresis (DGGE) (Myers). (1985) Nature 313: 495). When DGGE is used as an analysis method, in order to prevent complete denaturation of DNA, the above DNA is added by PCR to add a `` GC clamp '' consisting of approximately 40 bp DNA having a high content of high-temperature melting GC. Qualify. In yet another embodiment, the difference in mobility between control and sample DNA is measured by using a temperature gradient instead of a denaturant gradient (Rosenbaum and
Reissner (1987) Biophys. Chem. 265: 12753).

Examples of other methods for detecting point mutations include, but are not limited to, selective oligonucleotide hybridization, selective amplification, or selective primer extension. For example, an oligonucleotide primer with a known mutation located in the center is prepared and the primer is hybridized to the target DNA under conditions that will cause hybridization only if a perfect binding partner 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
It hybridizes to the amplified target DNA, or a number of different mutations, wherein the oligonucleotide binds to a hybridizing membrane and hybridizes to a labeled target DNA.

Alternatively, allele-specific amplification methods that rely on selective PCR amplification may be used with the present invention. Oligonucleotides used as primers for specific amplification may be located at the center of the molecule (so that the amplification depends on differential hybridization) (Gibbs et al. (1989) Nucleic Acid Res. 17: 2437-1448), or one primer. Contains the mutation of interest at the 3 'end of this, at which position under appropriate conditions a mismatch can prevent or inhibit polymerase extension (
Prossner (1993) Tibtech 11: 238). Furthermore, it is desirable to perform cleavage-based detection by introducing a new restriction site in the region of the mutation (Gasparini et al. (1).
992) Mol. Cell Probes 6: 1). In some embodiments, amplification may be performed using Taq ligase for amplification (Barany (1991) Proc. Natl. Acad. Sci. USA 88: 189). In such a case, only when there is a perfect partner at the 3 'end of the 5' sequence, a ligation reaction occurs, and by searching whether amplification occurs, the presence of a known mutation at a specific site is detected. It becomes possible.

The methods described herein can be performed, for example, by using a pre-packed diagnostic kit that includes at least one probe nucleic acid or antibody reagent described herein. This diagnostic kit can be suitably used in a clinical setting, for example, for diagnosing a patient presenting with symptoms or a family history of a disease or disease associated with the gene encoding the polypeptide of the present invention. Furthermore, any cell type or tissue in which the polypeptide of the invention is expressed, preferably peripheral blood leukocytes,
It may be used in the prognostic assays described in the present invention.

[0229] 3. Pharmacological Genetics By administering to a subject an agent having a stimulatory or inhibitory effect on the activity or expression of the polypeptide of the present invention identified by the screening assay described herein, or a modulator, the abnormalities of the polypeptide can be increased. A disorder associated with activity can be treated (for prophylactic or therapeutic purposes). With such treatment, one may consider the pharmacogenetics of an individual (ie, a study of the relationship between an individual's genotype and the individual's response to a foreign compound or drug). Differences in the metabolism of the therapeutic agent can lead to severe toxicity or treatment failure by altering the relationship between pharmacologically active drug dose and blood concentration. Thus, the pharmacogenetics of an individual allows for the selection of an effective agent (eg, a drug) for prevention or treatment, taking into account the genotype of the individual. Such pharmacogenetics can also be used to determine the appropriate dosage and treatment. Thus, by determining the activity of a polypeptide of the invention, expression of a nucleic acid of the invention, or the content of a mutation in a gene of the invention in an individual, selection of an appropriate agent for treatment or prophylaxis of the individual. Can be.

Pharmacogenetics treats severe hereditary variations in response to drugs with altered drug propensity and abnormal effects in patients clinically. Linder (1997) Clin. Chem
43 (2): 254-266. In general, pharmacogenetic conditions can be distinguished into two types. Genetic conditions that are transmitted as one factor that alters the way drugs act on the body are termed "altered drug effects." Genetic conditions that are transmitted as a factor that alters the way the body acts on drugs are referred to as "altered drug metabolism." These pharmacogenetic conditions can occur as either rare defects or polymorphisms. For example, glucose-6-phosphate dehydrogenase deficiency (G6PD)
It is a common inherited enzyme disorder and its major clinical complications are oxidant drugs (antimalarial drugs, sulfonamides, central nervous system stimulants, nitrofuran), and hemolysis after consumption of fava beans. It is.

As an exemplary embodiment, the activity of a drug metabolizing enzyme is a major determinant of the intensity and duration of drug action. The discovery of genetic polymorphisms for drug metabolizing enzymes (eg, N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymes CYP2D6 and CYP2C19) has led to the expected drug effect after a standard and safe drug dose. It was possible to explain why some patients did not obtain or exhibited excessive drug response and severe toxicity. These polymorphisms are expressed as two phenotypes in the population: extensive (EM) and poor (EM). Within the different populations, PM is dominant. For example, the gene encoding CYP2D6 is highly polymorphic and several mutations have been identified in PM, all of which lead to the absence of functional CYP2D6. Poorly metabolized forms of CYP2D6 and CYP2D19 quite frequently suffer from excessive drug responses and side effects when they receive standard doses. When the metabolite is the active therapeutic component, PM shows no therapeutic response, as demonstrated for the analgesic effect of codeine mediated by its CYP2D6-forming metabolite morphine. The other extreme is the so-called ultra-rapid metabolism, which does not respond to standard doses. Recently, it has been confirmed that the molecular principle of ultra-rapid metabolism is due to CYP2D6 gene amplification.

Thus, by detecting the activity of a polypeptide of the present invention in an individual, the expression of a nucleic acid encoding the polypeptide, or the mutation content of a gene encoding the polypeptide, the treatment or treatment of the individual is detected. An appropriate drug for prophylactic treatment can be selected. In addition, by using pharmacogenetic studies, genotyping of polymorphic alleles encoding drug metabolizing enzymes can be applied to the identification of an individual's drug responsive phenotype. Applying this finding to dosage determination or drug selection can avoid adverse reactions or treatment failures, and thus include modulators identified by one of the exemplary screening assays described herein. The therapeutic or prophylactic efficiency in treating patients with modulators of the activity or expression of the polypeptide of the invention can be increased.

[0233] 4. Monitoring effects during clinical trials Monitoring the effect of an agent (eg, drug, compound) on the expression or activity of a polypeptide of the invention (eg, aberrant cell proliferation chemotaxis and / or ability to modulate differentiation) It can be applied not only to basic drug screening but also to clinical trials. For example, the effectiveness of an agent that enhances gene expression, protein level or protein activity, as measured by the screening assays described herein, is indicative of a subject that exhibits reduced gene expression, protein level or protein activity. Can be monitored in clinical trials. Alternatively, the effectiveness of an agent that suppresses gene expression, protein level or protein activity, as measured by a screening assay, can be monitored in clinical trials of subjects that show an increase in gene expression, protein level or protein activity. it can. In such clinical trials, the expression or activity of the polypeptide of the present invention, and preferably, for example, the expression or activity of other polypeptides that have been implicated in cell proliferation disorders, may be determined by measuring the immunoreactivity of specific cells. Can be used as

For example, but not limited to, the activity or expression of a polypeptide of the present invention (eg,
For example, to identify genes, including those of the present invention, that are regulated in cells by treatment with an agent (eg, a compound, drug or small molecule) that modulates (eg, identified by the screening assays described herein). Can be. So, for example,
In clinical trials, to determine the effect of a drug on a cell proliferative disorder, cells are isolated, RNA is prepared, and analyzed for expression levels of the gene of the invention or other genes involved in the disorder. Gene expression (ie, gene expression pattern) is determined by Northern blot analysis or RT-PCR, as described herein, or the amount of protein produced using one of the methods described herein. Or, alternatively, it can be quantified by measuring the activity level of the gene of the present invention or other genes. Thus, the gene expression pattern is
Acts as a marker and indicates the physiological response of the cell to the drug. Thus, this response state can be measured before treatment of the individual with the drug, as well as at various points during treatment.

In a preferred embodiment, the invention relates to an agent (eg, an agonist, antagonist, mimetic peptide, protein, peptide, nucleic acid, small molecule, or other candidate drug identified by a screening assay described herein). C) monitoring the efficacy of the treatment of the patient according to (i) taking a pre-dose sample from the patient prior to administration of the drug; and (ii) determining the level of the polypeptide or nucleic acid of the invention in the pre-dose sample. (Iii) obtaining one or more post-dose samples from the patient; (iv) detecting the level of a polypeptide or nucleic acid of the invention in the post-dose sample; (v) detecting the level of the polypeptide of the invention in the pre-dose sample. Comparing the level of the peptide or nucleic acid with the level of the polypeptide or nucleic acid of the invention in the sample after administration;
(vi) providing a method that includes varying the administration of the drug to the patient accordingly. For example, to increase the expression or activity of the polypeptide to a level higher than that detected, ie, to increase the effectiveness of the agent, it is desirable to increase the dosage of the agent. In contrast, to reduce the expression or activity of the polypeptide to a level lower than that detected, ie, reduce the efficacy of the drug, it is desirable to reduce the dosage of the drug.

C. Therapeutic Methods The present invention provides both prophylactic and therapeutic methods of treating subjects at risk of (or susceptible to) or having a disorder associated with abnormal expression or activity of a polypeptide of the present invention. For example, disorders characterized by abnormal expression or activity of a polypeptide of the present invention include proliferative disorders such as psoriasis and cancer. In addition, the polypeptides of the present invention can be used for promoting hair growth, promoting wound healing, and other uses described herein.

[0237] 1. In one embodiment of the method of prevention , the invention provides for administering to a patient an agent that modulates expression or at least one activity of a polypeptide of the invention in the patient.
Methods are provided for preventing a disorder or condition associated with abnormal expression or activity of the polypeptide. Due to abnormal expression or activity of the polypeptide of the present invention,
Or a subject at risk of developing a disease to which it contributes, can be identified, for example, by any of the diagnostic or prognostic assays described herein, or a combination thereof. The administration of a prophylactic agent can be performed before the onset of a specific abnormal symptom to prevent or delay the progress of the disease or disorder. Depending on the type of abnormality, for example, an agonist or antagonist can be used to treat the patient. For example, an antagonist of the ELVIS protein can be used to treat a proliferative disorder associated with abnormal ELVIS expression or activity, eg, psoriasis. Suitable agents can be determined based on the screening assays described herein.

[0238] 2. Another embodiment of the therapeutic method of the invention, for therapeutic purposes, to methods of modulating the expression or activity of a polypeptide of the present invention. The modulation method of the present invention comprises contacting a cell with an agent that modulates at least one activity of the polypeptide. The agent that modulates the activity can be an agent described herein, such as a nucleic acid or protein, a cognate ligand of the naturally occurring polypeptide, a peptide, a pseudopeptide, or other small molecule. In one embodiment, the agent stimulates one or more biological activities of the polypeptide. Examples of such stimulants include active polypeptides of the invention, as well as nucleic acids encoding polypeptides of the invention introduced into cells. In another embodiment, the agent inhibits one or more biological activities of the polypeptide of the invention. Examples of such inhibitors include antisense nucleic acid molecules and antibodies. These modulating methods can be performed in vitro (eg, by culturing cells with the drug) or alternatively in vivo (eg, by administering the drug to a subject). Thus, the present invention provides a method of treating an individual suffering from a disorder or condition characterized by abnormal expression or activity of a polypeptide of the present invention. In one embodiment, the method comprises an agent that modulates expression or activity (eg, up-regulates or down-regulates) (an agent identified based on the screening assays described herein), or an agent of these agents. Including administration of the combination. In another embodiment, the method comprises compensating for reduced or abnormal expression of the polypeptide by administering a polypeptide of the invention or a nucleic acid of the invention as a therapy.

In situations where the activity or expression is abnormally low or down-regulated and / or where increased activity would have a beneficial effect, such as wound healing, stimulation of activity is desirable. Conversely, abnormally high activity or expression,
Alternatively, inhibition of activity is desirable in situations where the activity is up-regulated and / or where reduced activity would have a beneficial effect, such as in the treatment of growth disorders such as seafood.

When a modulator of the expression or activity of a protein of the invention is required in treating contact dermatitis, treating burns, regulating epithelial cell proliferation, and treating alopecia or unwanted hair growth. There is also.

The invention will be described in more detail with reference to the following examples, which are not intended to be limiting. References, patents and published patent applications cited throughout this specification are hereby incorporated by reference.

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

[Brief description of the drawings]

FIG. 1 shows the cDNA sequence of human ELVIS-1 (SEQ ID NO: 1) and the deduced amino acid sequence of ELVIS-1 (SEQ ID NO: 2). The open reading frame of SEQ ID NO: 1
From nucleotide 31 to nucleotide 492 (SEQ ID NO: 3).

FIG. 2 shows a hydropathy plot of human ELVIS-1. Relatively hydrophobic residues are shown above the horizontal dashed line, and relatively hydrophilic residues are shown below the horizontal dashed line. Cysteine residues (cys) and potential N-glycosylation sites (Ngly) are indicated by a short vertical line immediately below the hydropathy race. Below the hydropathy plot, ELVIS-1
Shows the amino acid sequence of The amino acid sequence of the signal sequence of ELVIS-1 is underlined and "S
The amino acid sequence of the transmembrane domain of ELVIS-1 is underlined and "TM"
It is indicated by adding.

FIG. 3 shows the cDNA sequence of ELVIS-2 (SEQ ID NO: 4), which is a mutant form of ELVIS-1, and the deduced amino acid sequence of ELVIS-2 (SEQ ID NO: 5). The open reading frame of SEQ ID NO: 4 extends from nucleotide 15 to nucleotide 323 of SEQ ID NO: 4 (SEQ ID NO: 6).

FIG. 4 shows a hydropathy plot of human ELVIS-2. Relatively hydrophobic residues are shown above the horizontal dashed line, and relatively hydrophilic residues are shown below the horizontal dashed line. Cysteine residues (cys) and potential N-glycosylation sites (Ngly) are indicated by a short vertical line immediately below the hydropathy race. Below the hydropathy plot, ELVIS-2
And the signal sequence of ELVIS-2 is indicated by underlining and "S".

FIG. 5. cDNA sequence of another variant of ELVIS-1, ELVIS-3 (SEQ ID NO: 7), and
The deduced amino acid sequence of ELVIS-3 (SEQ ID NO: 8) is shown. The open reading frame of SEQ ID NO: 7 extends from nucleotide 12 to nucleotide 294 of SEQ ID NO: 7 (SEQ ID NO: 9).

FIG. 6 shows a hydropathy plot of human ELVIS-3, another variant of ELVIS-1. Relatively hydrophobic residues are shown above the horizontal dashed line, and relatively hydrophilic residues are shown below the horizontal dashed line. Cysteine residues (cys) and potential N-glycosylation sites (Ngly) are indicated by a short vertical line immediately below the hydropathy race. The amino acid sequence of ELVIS-3 is shown below the hydropathy plot, and the signal sequence of ELVIS-3 is indicated by underlining and "S".

FIG. 7: Amino acid sequences of human ELVIS-1 (SEQ ID NO: 2), human ELVIS-2 (SEQ ID NO: 5), human ELVIS-3 (SEQ ID NO: 8), and amino acid sequence of human TGF-α (SEQ ID NO: 13)
3 shows the alignment. The amino acid sequence of human ELVIS-1 and human TGF-α is 24.4%
Has the sequence identity of Multiple alignments were performed using the ClustalW alignment program with a PAM250 scoring matrix, gap penalty of 10, and gap extension penalty of 0.05.

FIG. 8 shows an alignment of the nucleotide sequences encoding human ELVIS-1 (SEQ ID NO: 1), ELVIS-2 (SEQ ID NO: 4) and ELVIS-3 (SEQ ID NO: 7).

FIG. 9 shows the more complete sequence of the ELVIS-3 cDNA shown in FIG. 5 (SEQ ID NO: 21).

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61K 39/395 A61P 17/00 4C085 45/00 17/06 4C086 48/00 25/18 4H045 A61P 17/00 35/00 17/06 43/00 111 25/18 C07K 14/485 35/00 16/22 43/00 111 19/00 C07K 14/485 C12P 21/02 H 16/22 C12Q 1/02 19/00 1 / 68 A C12N 5/10 G01N 33/53 D C12P 21/02 C12N 15/00 ZNAA C12Q 1/02 5/00 B 1/68 A61K 37/02 G01N 33/53 37/24 (81) Designated country EP ( AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, L, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ) , MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM , EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR , TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Gearin , David, copy. United States 02181 Welshley, Massachusetts Welshley, Standesh Road 23 F-term (reference) 4B024 AA01 AA11 BA21 CA04 DA02 EA04 GA11 HA01 HA12 HA15 4B063 QA01 QA18 QQ20 QQ42 QQ52 QR56 QR59 QR80 QS24 QS25 QS34 4B064 AG13 CA10 AACA19 CC AB01 AC14 BA02 CA24 CA44 CA46 4C084 AA01 AA02 AA06 AA07 AA13 AA17 BA01 BA18 BA19 BA20 BA21 BA22 CA18 CA53 DB53 NA14 ZA18 ZA89 ZB26 ZC02 4C085 AA13 AA14 AA19 BB11 CC21 DD62 EE01 4C02 AAA ZAA A14A04 ZA18 AA11 AA20 AA30 BA10 BA41 CA40 DA20 DA76 EA22 EA28 EA50 FA74

Claims (22)

[Claims] 1. The following nucleic acid molecules: a) SEQ ID NO: 1, 3, 4, 6, 7, 9, or the nucleotide sequence of the cDNA insert of the plasmid deposited at the ATCC under accession number 98981, 98982 or 98983, or its complement. A nucleic acid molecule comprising a nucleotide sequence having at least 55% identity to the body; b) SEQ ID NO: 1, 3, 4, 6, 7, 9, or the plasmid deposited at the ATCC under accession numbers 98981, 98982 or 98983. A nucleic acid molecule comprising a nucleotide sequence of at least 300 nucleotides of the cDNA sequence of the cDNA insert or its complement; c) the amino acid sequence of SEQ ID NO: 2, 5 or 8, or accession number 98981,
A nucleic acid molecule encoding a polypeptide comprising the amino acid sequence encoded by the cDNA insert of the plasmid deposited as 98982 or 98983; d) SEQ ID NO: 2, 5, 8, or deposited with the ATCC as accession number 98981, 98982 or 98983. A nucleic acid molecule encoding a polypeptide fragment comprising the amino acid sequence of the polypeptide encoded by the cDNA insert of the plasmid, wherein the fragment comprises the amino acid sequence of SEQ ID NO: 2, 5 or 8, or the ATCC accession number 98981. , 9
Comprising at least 15 contiguous amino acids of the amino acid sequence of the polypeptide encoded by the cDNA insert of the plasmid deposited as 8982 or 98983.
Said nucleic acid molecule; and e) the amino acid sequence of SEQ ID NO: 2, 5 or 8, or accession number 98981,
A nucleic acid molecule encoding a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence encoded by the cDNA insert of the plasmid deposited as 98982 or 98983, wherein the nucleic acid molecule comprises SEQ ID NO: 3, 6, or 9 or a complement thereof. An isolated nucleic acid molecule selected from the group consisting of: a nucleic acid molecule that hybridizes under stringent conditions with a nucleic acid molecule containing the nucleic acid molecule. 2. The following nucleic acid molecules: a) the nucleotide sequence of the cDNA insert of SEQ ID NO: 1, 3, 4, 6, 7, 9, or the plasmid deposited with the ATCC as accession number 98981, 98982 or 98983, or its complement. And b) the amino acid sequence of SEQ ID NO: 2, 5 or 8, or accession number 98981,
2. The isolated nucleic acid molecule of claim 1, wherein the nucleic acid molecule is selected from the group consisting of: a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence encoded by the cDNA insert of the plasmid deposited as 98982 or 98983. 3. The nucleic acid molecule of claim 1, further comprising a vector nucleic acid sequence. 4. The nucleic acid molecule of claim 1, further comprising a nucleic acid sequence encoding a heterologous polypeptide. 5. A host cell containing the nucleic acid molecule according to claim 1. 6. The host cell according to claim 5, which is a mammalian host cell. 7. A non-human mammalian host cell containing the nucleic acid molecule according to claim 1. 8. The following polypeptide: a) a fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, 5 or 8, comprising at least 15 contiguous amino acids of SEQ ID NO: 2, 5 or 8. A fragment; b) the amino acid sequence of SEQ ID NO: 2, 5 or 8, or the ATCC under accession number 98981,
A naturally occurring allelic variant of a polypeptide comprising the amino acid sequence encoded by the cDNA insert of the plasmid deposited as 98982 or 98983, wherein the nucleic acid molecule comprises SEQ ID NO: 1, 4 or 7 or the complement thereof. Said variant encoded by a nucleic acid molecule which hybridizes under stringent conditions; and c) at least 55% identity to a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 1, 4 or 7 or its complement An isolated polypeptide selected from the group consisting of: a polypeptide encoded by a nucleic acid molecule comprising a nucleotide sequence having: 9. The isolated polypeptide according to claim 8, comprising the amino acid sequence of SEQ ID NO: 2, 5 or 8. 10. The polypeptide of claim 8, further comprising a heterologous amino acid sequence. 11. An antibody which selectively binds to the polypeptide according to claim 8. 12. The following polypeptides: a) the amino acid sequence of SEQ ID NO: 2, 5 or 8, or the ATCC accession number 98981.
A) a polypeptide comprising the amino acid sequence encoded by the cDNA insert of the plasmid deposited as No. 98982 or 98983; b) the amino acid sequence of SEQ ID NOs: 2, 5 or 8, or accession number 98981 to the ATCC.
, 98982 or 98983, comprising a fragment of the amino acid sequence encoded by the cDNA insert of the plasmid, wherein the fragment is SEQ ID NO: 2, 5 or 8 or the ATCC as accession number 98981, 98982 or 98983. The polypeptide comprising at least 15 contiguous amino acids of the amino acid sequence encoded by the cDNA insert of the deposited plasmid; and c) the amino acid sequence of SEQ ID NO: 2, 5, or 8, or accession number 98981 to the ATCC.
, A naturally occurring allelic variant of a polypeptide comprising the amino acid sequence encoded by the cDNA insert of the plasmid deposited as 98982 or 98983, wherein the nucleic acid molecule comprises SEQ ID NO: 1, 4 or 7 or a complement thereof. And a variant encoded by a nucleic acid molecule that hybridizes under stringent conditions to a polypeptide selected from the group consisting of: The above method, comprising culturing under the conditions under which expression is performed. 13. A method for detecting the presence of a polypeptide according to claim 8 in a sample, comprising: a) contacting the sample with a compound that selectively binds to the polypeptide according to claim 8; ) Determining whether the compound binds to the polypeptide in the sample;
The above method, comprising: 14. The method of claim 13, wherein the compound that binds to the polypeptide is an antibody. 15. A kit comprising a compound that selectively binds to the polypeptide according to claim 8 and an instruction manual. 16. A method for detecting the presence of a nucleic acid molecule according to claim 1 in a sample, comprising the steps of: a) contacting the sample with a nucleic acid probe or primer that selectively hybridizes with said nucleic acid molecule. Allowing the nucleic acid probe or primer to bind to the nucleic acid molecule in a sample. 17. The method of claim 16, wherein the sample comprises an mRNA molecule and the sample is contacted with a nucleic acid probe. 18. A kit comprising a compound that selectively hybridizes with the nucleic acid molecule according to claim 1 and an instruction manual. 19. A method for identifying a compound that binds to the polypeptide according to claim 8, comprising the following steps: a) contacting the polypeptide according to claim 8 or a cell expressing the polypeptide with a test compound; Allowing the polypeptide to bind to a test compound. 20. A method for detecting binding between a polypeptide and a test compound by the following detection methods: a) a method for detecting binding by direct detection of a test compound / polypeptide bond; b) a method for detecting binding using a competitive binding assay; 20. The method of claim 19, wherein the method comprises the step of: c) detecting binding using an assay for ELVIS-mediated signaling. 21. A method for modulating the activity of the polypeptide according to claim 8, wherein the compound that binds the polypeptide according to claim 8 or a cell expressing the polypeptide to the polypeptide, Such a method, comprising contacting at a concentration sufficient to modulate the activity of the polypeptide. 22. A method for identifying a compound that modulates the activity of the polypeptide according to claim 8, comprising: a) contacting the polypeptide according to claim 8 with a test compound; and b) contacting the test compound with the test compound. Determining the effect on the activity of the polypeptide, thereby identifying a compound that modulates the activity of the polypeptide.