EP1280922A2 - 21953, a human prolyl oligopeptidase family member and uses thereof - Google Patents

Abstract

The invention provides isolated nucleic acids molecules, designated 21953 nucleic acid molecules, which encode novel prolyl oligopeptidase members. The invention also provides antisense nucleic acid molecules, recombinant expression vectors containing 21953 nucleic acid molecules, host cells into which the expression vectors have been introduced, and nonhuman transgenic animals in which a 21953 gene has been introduced or disrupted. The invention still further provides isolated 21953 proteins, fusion proteins, antigenic pepides and anti-21953 antibodies. Diagnostic methods utilizing compositions of the invention are also provided.

Description

21953, A NOVEL HUMAN PROLYL OLIGOPEPTIDASE FAMILY MEMBER

AND USES THEREOF

Related Applications [01] This application claims priority to U.S. provisional application number

60/197,508 filed on April 18, 2000, the contents of which are incorporated herein by reference.

Background ofthe Invention [02] Proline residues confer unique structural constraints on peptide chains and markedly influence the susceptibility of proximal peptide bonds to protease activity. For example, proline residues are sterically constrained by the imino group. Prolyl oligopeptidases are a distinct sub-group of endopeptidases that degrade a variety of proline- containing peptides by cleaving the peptide bond at the carboxyl side of proline residues. Some prolyl oligopeptidases prefer smaller polypeptides or oligopeptides as substrates. [03] The natural substrates of prolyl oligopeptidases include many biologically active peptides such as peptide messenger molecules. For example, they are involved in the metabolism of peptide hormones and neuropeptides. Prolyl oligopeptidases have few naturally occurring inhibitors and their distinctive specificity prevents them from interacting with β-macroglobulin, unlike the great majority of endopeptidases. The specificity of an oligopeptidase depends on the three dimensional stracture of its active site, which includes a putative catalytic triad, which contains aspartate, serine and histidine residues. [04] Examples of known prolyl oligopeptidases include human prolyl oligopeptidase

(Yoshimoto et al. Genebank AB020018), mouse prolyl oligopeptidase (Ishino et al., J. Biochem. 123 (3), 540-545 (1998)), pig prolyl oligopeptidase (Rennix et al, Biochemistry, 30:2195-2203, 1991), rat dipeptidyl-peptidase IV (Ognata et al., J. Biol. Chem, 264:3596- 3601, 1989), F. meningosepticum prolyl oligopeptidase (Yoshimoto et al., J. Biochem. 110:873-878, 1991), andE. coli protease II (Kanatani et al., J. Biochemistry (Tokyo), 110: 315-320, 1991).

[05] Prolyl oligopeptidases also control the activity of other peptides present in body fluids such as bradykinin and angiotensin. Bradykinin is a very potent vasodilator that increases the permeability of post capillary venules and acts on endothelial cells to activate phospholipase A2. Angiotensin causes contraction of vascular smooth muscle, raising blood pressure and stimulating aldosterone release from the adrenal glands. Other members ofthe prolyl oligopeptidase family mediate the degradation of neuropeptides such as substance P, thyrotropin releasing hormone, hippocampal cholinergic neurostimulating peptide (HCNP), neuropeptide Y (NPY), and neuropeptides derived from pro-opiomelanocortin (POMC) and neurohypophyseal hormones.

Summary ofthe Invention

[06] The present invention is based, in part, on the discovery of a novel prolyl oligopeptidase family member, referred to herein as "21953". The nucleotide sequence of a cDNA encoding 21953 is shown in SEQ ID NO:l, and the amino acid sequence of a 21953 polypeptide is shown in SEQ ID NO:2. In addition, the nucleotide sequences ofthe coding region are depicted in SEQ ID NO:3.

[07] Accordingly, in one aspect, the invention features a nucleic acid molecule which encodes a 21953 protein or polypeptide, e.g., a biologically active portion ofthe 21953 protein. In a preferred embodiment the isolated nucleic acid molecule encodes a polypeptide having the amino acid sequence of SEQ ID NO:2. In other embodiments, the invention provides isolated 21953 nucleic acid molecules having the nucleotide sequence shown in SEQ ID NO:l, SEQ ID NO:3, or the sequence ofthe DNA insert ofthe plasmid deposited with ATCC Accession Number . In still other embodiments, the invention provides nucleic acid molecules that are substantially identical (e.g., naturally occurring allelic variants) to the nucleotide sequence shown in SEQ ID NO:l, SEQ ID NO:3. or the sequence ofthe DNA insert ofthe plasmid deposited with ATCC Accession Number .

In other embodiments, the invention provides a nucleic acid molecule which hybridizes under a stringency condition described herein to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:l, 3, or the sequence ofthe DNA insert ofthe plasmid deposited with ATCC Accession Number , wherein the nucleic acid encodes a full length 21953 protein or an active fragment thereof.

[08] In a related aspect, the invention further provides nucleic acid constructs wliich include a 21953 nucleic acid molecule described herein. In certain embodiments, the nucleic acid molecules ofthe invention are operatively linked to native or heterologous regulatory sequences. Also included, are vectors and host cells containing the 21953 nucleic acid molecules ofthe invention e.g., vectors and host cells suitable for producing 21953 nucleic acid molecules and polypeptides. [09] In another related aspect, the invention provides nucleic acid fragments suitable as primers or hybridization probes for the detection of 21953-encoding nucleic acids. [10] In still another related aspect, isolated nucleic acid molecules that are antisense to a 21953 encoding nucleic acid molecule are provided.

[11] In another aspect, the invention features, 21953 polypeptides, and biologically active or antigenic fragments thereof that are useful, e.g., as reagents or targets in assays applicable to treatment and diagnosis of 21953-mediated or -related disorders. In another embodiment, the invention provides 21953 polypeptides having a 21953 activity. Preferred polypeptides are 21953 proteins including at least one prolyl oligopeptidase domain, and, preferably, having a 21953 activity, e.g., a 21953 activity as described herein. [12] In other embodiments, the invention provides 21953 polypeptides, e.g., a 21953 polypeptide having the amino acid sequence shown in SEQ ID NO:2 or the amino acid sequence encoded by the cDNA insert ofthe plasmid deposited with ATCC Accession

Number ; an amino acid sequence that is substantially identical to the a ino acid sequence shown in SEQ ID NO:2 or the amino acid sequence encoded by the cDNA insert ofthe plasmid deposited with ATCC Accession Number ; or an amino acid sequence encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under a stringency condition described herein to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:l, SEQ ID NO: 3, or the sequence ofthe DNA insert ofthe plasmid deposited with ATCC Accession Number , wherein the nucleic acid encodes a full length 21953 protein or an active fragment thereof.

[13] In a related aspect, the invention further provides nucleic acid constructs which include a 21953 nucleic acid molecule described herein.

[14] In a related aspect, the invention provides 21953 polypeptides or fragments operatively linked to non-21953 polypeptides to form fusion proteins.

[15] In another aspect, the invention features antibodies and antigen-binding fragments thereof, that react with, or more preferably specifically bind 21953 polypeptides.

[16] In another aspect, the invention provides a method of evaluating a sample. The method includes: providing a sample; detecting a 21953 polypeptide or nucleic acid in the sample; and, optionally, comparing the level of expressed 21953 molecules to a reference sample. For example, an increased level of 21953 molecules can be an indication that the sample includes cells transiting from the Gl cell cycle phase to S phase. In other examples, the level of 21953 molecules can be an indication that a sample includes a proliferating cell, e.g., a proliferating lung, breast, ovary, or colon cell; or a heart cell, a prostate cell, a vascular cell (e.g., a smooth muscle or an endothelial cell), or a brain cell. [17] In another aspect, the invention provides methods of screening for compounds that modulate the expression or activity ofthe 21953 polypeptides or nucleic acids. The invention also provides assays for deteπnining the activity of or the presence or absence of 21953 polypeptides or nucleic acid molecules in a biological sample, including for disease diagnosis.

[18] In still another aspect, the invention provides a process for modulating 21953 polypeptide or nucleic acid expression or activity, e.g. using the screened compounds. In certain embodiments, the methods involve treatment of conditions or disorders related to aberrant activity or expression ofthe 21953 polypeptides or nucleic acids, such as conditions or disorders involving aberrant or deficient cell proliferation or differentiation, e.g., a cancer (e.g. a cancer ofthe lung, breast, ovary, prostate, or colon), or conditions or disorders ofthe cardiovascular (including vascular, e.g., a smooth muscle or an endothelial cell), neuronal, or reproductive (e.g., prostatic) systems.

[19] In yet another aspect, the invention provides methods for modulating the activity of a 21953-expressing cell, e.g., a hyper-proliferative 21953 -expressing cell. In one embodiment, the activity is modulated by one of more of: inhibiting the proliferation or migration, or inducing the differentiation or killing ofthe 21953-expressing cell. The method includes contacting the cell with a compound (e.g., a compound identified using the methods described herein) that modulates the activity, or expression, ofthe 21953 polypeptide or nucleic acid, such that the activity ofthe 21953-expressing cell is modulated. [20] In a preferred embodiment, the contacting step is effective in vitro or ex vivo. In other embodiments, the contacting step is effected in vivo, e.g., in a subject (e.g., a mammal, e.g., a human), as part of a therapeutic or prophylactic protocol.

[21] In a preferred embodiment, the cell is a hyperproliferative cell, e.g., a cell found in a solid tumor, a soft tissue tumor, or a metastatic lesion. For example, the cell is a lung, breast, ovary, prostate, or colon cell. In a preferred embodiment, the cell is lung cell. [22] In other embodiments, the cell is a neural cell (e.g., a neuronal or a glial cell), a vascular cell (e.g., smooth muscle or an endothelial cell), a heart cell, a prostatic cell, or an immune cell.

[23] In a preferred embodiment, the compound is an inhibitor of a 21953 polypeptide. Preferably, the inhibitor is chosen from a peptide, a phosphopeptide, a small organic molecule, a small inorganic molecule and an antibody (e.g., an antibody conjugated to a therapeutic moiety selected from a cytotoxin, a cytotoxic agent and a radioactive metal ion). The inhibitor can also be a protease inhibitor or a derivative thereof, or a peptidomimetic, e.g., a phosphonate analog of a peptide substrate such as a prolyl peptide substrate. In another preferred embodiment, the compound is an inhibitor of a 21953 nucleic acid, e.g., an antisense, a ribozyme, or a triple helix molecule. [24] In a preferred embodiment, the compound is administered in combination with a cytotoxic agent. Examples of cytotoxic agents include anti-microtubule agent, a topoisomerase I inhibitor, atopoisomerase II inhibitor, an anti-metabolite, a mitotic inhibitor, an alkylating agent, an intercalating agent, an agent capable of interfering with a signal transduction pathway, an agent that promotes apoptosis or necrosis, and radiation. [25] In another aspect, the invention features methods for treating or preventing a disorder characterized by aberrant activity, e.g., cellular proliferation or differentiation, of a 21953-expressing cell, in a subject. Preferably, the method includes comprising administering to the subject (e.g., a mammal, e.g., a human) an effective amount of a compound (e.g., a compound identified using the methods described herein) that modulates the activity, or expression, ofthe 21953 polypeptide or nucleic acid. [26] In a preferred embodiment, the disorder is a cancerous or pre-cancerous condition, e.g., relating to proliferation of a lung, breast, ovary, prostate, or colon cell. In another preferred embodiment, the disorder is an immune, a neuronal, cardiovascular, reproductive disorder, e.g., a disorder relating to aberrant processing of a polypeptide hormone. [27] In a further aspect, the invention provides methods for evaluating the efficacy of a treatment of a disorder, e.g., proliferative disorder (e.g., lung cancer), or a neuronal disorder. The method includes: treating a subject, e.g., a patient or an animal, with a protocol under evaluation (e.g., treating a subject with one or more of: chemotherapy, radiation, and/or a compound identified using the methods described herein); and evaluating the expression of a 21953 nucleic acid or polypeptide before and after treatment. A change, e.g., a decrease or increase, in the level of a 21953 nucleic acid (e.g., mRNA) or polypeptide after treatment, relative to the level of expression before treatment, is indicative ofthe efficacy ofthe treatment ofthe disorder. The level of 21953 nucleic acid or polypeptide expression can be detected, e.g., by a method described herein.

[28] In a preferred embodiment, the evaluating step includes obtaining a sample (e.g., a tissue sample, e.g., a biopsy, or a fluid sample) from the subject, before and after treatment and comparing the level of expressing of a 21953 nucleic acid (e.g., mRNA) or polypeptide before and after treatment.

[29] In another aspect, the invention provides methods for evaluating the efficacy of a therapeutic or prophylactic agent (e.g., an anti-neoplastic agent). The method includes: contacting a sample with an agent (e.g., a compound identified using the methods described herein, a cytotoxic agent) and, evaluating the expression of 21953 nucleic acid or polypeptide in the sample before and after the contacting step. A change, e.g., a decrease or increase, in the level of 21953 nucleic acid (e.g., mRNA) or polypeptide in the sample obtained after the contacting step, relative to the level of expression in the sample before the contacting step, is indicative ofthe efficacy ofthe agent. The level of 21953 nucleic acid or polypeptide expression can be detected by any method described herein. In a preferred embodiment, the sample includes cells obtained from a cancerous, a neuronal, immune, a cardiovascular, or prostatic tissue. The cancerous tissue can include, for example, cells of lung, breast, ovary, prostate, or colon.

[30] In further aspect, the invention provides assays for determining the presence or absence of a genetic alteration in a 21953 polypeptide or nucleic acid molecule, including for disease diagnosis. In a still further aspect, the invention features a method of processing a polypeptide hormone precursor, e.g., in vitro.

[31] In another aspect, the invention features a two dimensional array having a plurality of addresses, each address ofthe plurality being positionally distinguishable from each other address ofthe plurality, and each address ofthe plurality having a unique capture probe, e.g., a nucleic acid or peptide sequence. At least one address ofthe plurality has a capture probe that recognizes a 21953 molecule. In one embodiment, the capture probe is a nucleic acid, e.g., a probe complementary to a 21953 nucleic acid sequence. In another embodiment, the capture probe is a polypeptide, e.g., an antibody specific for 21953 polypeptides. Also featured is a method of analyzing a sample by contacting the sample to the aforementioned array and detecting binding ofthe sample to the array. [32] Other features and advantages ofthe invention will be apparent from the following detailed description, and from the claims.

Brief Description ofthe Drawings

[33] Figure 1 depicts a hydropathy plot of human 21953. Relative hydrophobic residues are shown above the dashed horizontal line, and relative hydrophilic residues are below the dashed horizontal line. Numbers corresponding to positions in the amino acid sequence of human 21953 are indicated.

[34] Figure 2 depicts an alignment ofthe prolyl oligopeptidase domain of human

21953 with a consensus amino acid sequence derived from a hidden Markov model for prolyl oligopeptidase domains. The upper sequence is the consensus amino acid sequence

(SEQ ID NO:4), while the lower amino acid sequence corresponds to amino acids 672 to

744 ofSEQ ID O:2.

[35] Figures 3 A and 3B depict an alignment of human dipeptidyl peptidase IV

(Accession Number P48147) (upper line, SEQ ID NO:5), to the 21953 amino acid sequence.

The * symbol indicates identities, and the : or . symbols indicate similarities. The alignment was generated by ClustalW (Thompson et al. (1994) Nucleic Acids Res. 22:4673-4680).

Detailed Description

[36] The human 21953 sequence (see SEQ ID NO: 1 as recited in Example 1), which is approximately 3143 nucleotides long, including untranslated regions, contains a predicted metliionine-initiated coding sequence of about 2649 nucleotides, including the termination codon. The coding sequence encodes a 882 amino acid protein (see SEQ ID NO:2 as recited in Example 1).

[37] Human 21953 contains the following regions or other structural features: a predicted prolyl oligopeptidase domain (PFAM Accession PF00326) located at about amino acids 672-744 of SEQ ID NO:2; two predicted cAMP phosphorylation sites and cGMP- dependent protein kinase phosphorylation domains (Prosite Accession PS00004) located at about amino acid residues 231 to 234 of SEQ ID NO:2 and about amino acid residues 476- 479 of SEQ ID NO:2; ten predicted Protein Kinase C sites (PS00005) at about amino acids 52 to 54, 80 to 82, 115 to 117, 307 to 309, 312 to 314, 326 to 328, 551 to 553, 594 to 596, 776 to 778, and 850 to 852 of SEQ ID NO:2; 11 predicted Casein Kinase II sites (PS00006) located at about amino 133 tol36, 227 to 230, 293 to 296, 412 to 415, 443 to 446, 499 to 502, 530 to 533, 587 to 590, 603 to 606, 615 to 618, and 723 to 726 of SEQ ID NO:2; five predicted tyrosine phosphorylation sites (PS00007) at about amino acids 29 to 36, 47 to 55, 308 to 315, 549 to 555, and 837 to 844 of SEQ ID NO:2; four predicted N-myristylation sites (PS00008) from about amino 176 to 181, 741 to 746, 762 to 767 and 873 to 878 of SEQ ID NO:2 and one predicted amidation site (PS00009) from about amino acid 642 to 645 ofSEQ IDNO:2. [38] For general information regarding PFAM identifiers, PS prefix and PF prefix domain identification numbers, refer to Sonnhammer et al. (1997) Protein 28:405-420 and http://www.psc.edu general/ software/packages/pfairi/pfam.html.

[39] A plasmid containing the nucleotide sequence encoding human 21953 was deposited with American Type Culture Collection (ATCC), 10801 University Boulevard,

Manassas. VA 20110-2209, on and assigned Accession Number . This deposit will be maintained under the terms ofthe Budapest Treaty on the International Recognition ofthe Deposit of Microorganisms for the Purposes of Patent Procedure. This deposit was made merely as a convenience for those of skill in the art and is not an admission that a deposit is required under 35 U.S.C. §112. [40] The 21953 polypeptide contains a significant number of structural characteristics in common with members ofthe human prolyl oligopeptidase family. The term Υamily" when referring to the protein and nucleic acid molecules ofthe invention means two or more proteins or nucleic acid molecules having a common structural domain or motif and having sufficient amino acid or nucleotide sequence homology as defined herein. Such family members can be naturally or non-naturally occurring and can be from either the same or different species. For example, a family can contain a first protein of human origin as well as other distinct proteins of human origin, or alternatively, can contain homologues of non-human origin, e.g., rat or mouse proteins. Members of a family can also have common functional characteristics.

[41] Polypeptide ofthe prolyl oligopeptidase family such as a 21953 polypeptide typically include an N-terminal seven-blade β-propeller domain and a C-terminal α/β hydrolase domain. The N-teπninal seven-blade β-propeller domain can include a "DPP IV N-terminal domain" or regions homologous with a "DPP IV N-terminal domain." The C- terminal α/β hydrolase domain, e.g., the C-terminal region of a 21953 polypeptide, can include a "prolyl oligopeptidase domain" or regions homologous with a "prolyl oligopeptidase domain". The "prolyl oligopeptidase domain" can include a catalytic active site, which generally occurs at the C-terminal region ofthe polypeptide chain, which is involved in the hydrolysis of proline-containing peptide bonds. A prolyl oligopeptidase can be soluble. An alignment of human dipeptidyl peptidase IV (Accession Number P48147) to the 21953 amino acid sequence is depicted in Figures 3A-3B.

[42] As used herein, the term "prolyl oligopeptidase domain" includes an amino acid sequence of at least about 60 amino acid residues in length and having a bit score for the alignment ofthe sequence to the Pfam Hidden Markov Model (HMM) PF00326 of at least 10. Preferably, a prolyl oligopeptidase domain includes at least about 30 to 180 amino acids, more preferably about 50 to 140 amino acid residues, or about 60 to 80 amino acids and has a bit score for the alignment ofthe sequence to the prolyl oligopeptidase domain (HMM) of at least 10, 20, 30 or greater. An alignment ofthe prolyl oligopeptidase domain (amino acids 672 to 744 of SEQ ID NO:2) of human 21953 with a consensus amino acid sequence derived from a hidden Markov model is depicted in Figure 3. In a preferred embodiment, a human 21953 polypeptide has a serine peptidase active site, e.g., an active site that is nearly identical to the Prosite signature PDOC00587. The active site can have a conserved catalytic triad with a conserved serine, e.g., a serine residue located at about amino acid 739 of SEQ ID NO:2, a conserved aspartic acid, e.g., an aspartic acid residue located at about amino acid 817 of SEQ ID NO:2, and a conserved histidine, e.g., a histidine residue located at about amino acid 849 of SEQ ID NO:2. [43] In a preferred embodiment 21953 polypeptide or protein has a "prolyl oligopeptidase domain" or a region which includes at least about 30-300, more preferably about 50-150, or 60-80 amino acid residues and has at least about 50%, 60%, 70% 80% 90% 95%, 99%, or 100% homology with a "prolyl oligopeptidase domain," e.g., the prolyl oligopeptidase domain of human 21953 (e.g., residues 672-744 of SEQ ID NO:2). [44] To identify the presence of a "prolyl oligopeptidase" domain in a 21953 protein sequence, and make the determination that a polypeptide or protein of interest has a particular profile, the amino acid sequence ofthe protein can be searched against a database of HMMs (e.g., the Pfam database, release 2.1) using the default parameters (http://www. sanger. ac. ulc/Sof vare/Pfam HMM_search). For example, the hmmsf program, which is available as part ofthe HMMER package of search programs, is a family specific default program for MILPAT0063 and a score of 15 is the default threshold score for determining a hit. Alternatively, the threshold score for determining a hit can be lowered (e.g., to 8 bits). A description ofthe Pfam database can be found in Sonhammer et al. (1997) Proteins 28(3):405-420 and a detailed description of HMMs can be found, for example, in Gribskov et α/. (1990) Meth. Enzymol. 183:146-159; Gribskov et α . (1987) Proc. Natl. Acad Sci. USA 84:4355-4358; Krogh etα/.(1994)J. Mol. Biol. 235:1501-1531; and Stultz et .(1993) Protein Sci. 2:305-314, the contents of which are incorporated herein by reference. A search was performed against the HMM database resulting in the identification of a "prolyl oligopeptidase domain" domain in the amino acid sequence of human 21953 at about residues 672-744 of SEQ ID NO:2 (see Figure 2).

[45] In a preferred embodiment, a 21953 polypeptide includes an N-terminal seven- blade β-propeller domain, e.g., residues about 88 to 663 of SEQ ID NO:2. The amino acid sequence of this region can be aligned to the HMM profile for DPP IV N-terminal domain or the human DPP IV amino acid sequence (P27487). As used herein, the term "DPP IV N- terrninal domain" refers to an amino acid sequence at least 60% identical to residues about 88 to 663 of SEQ ID NO:2.

[46] A 21953 family member can include a prolyl oligopeptidase domain and may also include a cAMP phosphorylation site and cGMP-dependent protein kinase phosphorylation domain, a predicted Protein Kinase C site, a predicted Casein Kinase II site, a predicted tyrosine phosphorylation site, a predicted N-myristylation site, and an amidation site.

[47] As the 21953 polypeptides ofthe invention may modulate 21953-mediated activities, e.g., a dipeptidyl peptidase activity such as a prolyl oligopeptidase activity, they may be useful for developing novel diagnostic and therapeutic agents for 21953-mediated or related disorders, as described below. The 21953 polypeptide ofthe invention are highly expressed in tumors, for example in breast and lung tumors. Further, 21953 polypeptide expression is increased at the Gl-S phase transition ofthe mammalian cell cycle. Additional expression data for 21953 polypeptides are described below and in the Figures. Generally, increased prolyl oligopeptidase activity has been detected in human prostate, lung, and sigmoid tumors relative to healthy normal tissue. Such increased activity can result from 21953 increased expression.

[48] As used herein, a "21953 activity", "biological activity of 21953" or "functional activity of 21953", refers to an activity exerted by a 21953 protein, polypeptide or nucleic acid molecule on, e.g., a 21953-responsive cell or on a 21953 substrate, e.g., a oligopeptide substrate, as determined in vivo or in vitro. In one embodiment, a 21953 activity is a direct activity, such as an association with a 21953 target molecule. A "target molecule" or "binding partner" is a molecule with which a 21953 protein binds or interacts in nature. For example, the 21953 proteins ofthe present invention can have one or more ofthe following activities: (1) hydrolyzing peptide bonds at the carboxyl side of proline residues; (2) mediating degradation of proline-containing peptides, e.g., a prolyl endopeptidases activity; (3) processing of peptide factors (e.g., peptide hormones, chemokines, cytokines, neuropeptides, and vasoactive peptides); (4) processing N-terminal dipeptides of unmodified N-termini wherein the penultimate residue is proline; (5) modulating cell proliferation and/or modulating cell differentiation (e.g., of a lung, breast, lymphoid, or colon cell); (6) modulating the regulation of transmission of intracellular signals, e.g., during immunological processes; (7) modulating metabolism of neurotransmitters or neuropeptides; (8) modulating neurodegeneration; or (9) modulating follicular development. [49] As used herein, a "dipetidyl peptidase activity" refers to a catalytic activity that accelerates the scission of a peptide bond between an amino acid sequence of less than four amino acids and the remainder ofthe polypeptide. Preferably, the cleaved peptide is a dipeptide having two amino acids. The catalytic activity can be mediated by the side chain of a serine amino acid and surrounding residues in the active site. [50] As used herein, a "prolyl endopeptidases activity" refers to a catalytic activity that accelerates the scission of a peptide bond adjacent to a proline amino acid in a peptide or polypeptide chain. This catalytic activity has been detected, for example, in primary human lung tumors, squamous cell lung carcinomas, and lung adenocarcinomas. For example, squamous cell lung carcinomas and lung adenocarcinomas showed significantly higher levels of prolyl endopeptidases activity relative to normal lung parenchyma. [51] In accordance with the above-described sequence similarities and observed polypeptide expression pattern, the 21953 molecules ofthe present invention can have similar biological activities as related prolyl oligopeptidase family members. Members of the prolyl oligopeptidase family can play an important role in the metabolism of a variety of proline containing peptides by cleaving prolyl bonds. These peptides can be less than about 200, 150, 100, or 50 residues in length. Prolyl oligopeptidases are involved, e.g., alone or together with other factors, in the regulation, e.g., processing, activation, or degradation of biological factors, e.g., peptide hormones (such as growth hormone, insulin, prolactin, adrenocorticotropic hormone, placental lactogen, calcitonin, parathyroid hormone, and thyroid stimulating hormone); chemokines; cytokines; neuropeptides; and vasoactive peptides.

[52] As the 21953 mRNA is highly expressed, for example, in cancerous tissues (e.g., lung and breast tumors), as well as normal cardiovascular, neural, and prostatic tissues, the molecules ofthe invention can be used to treat, prevent and/or diagnose disorders involving aberrant activity of 21953-expressing cells. Accordingly, the 21953 molecules can act as novel diagnostic targets and therapeutic agents for controlling disorders associated with the aberrant activity or degradation of peptide hormones, e.g., disorders associated with cell differentiation and proliferation (e.g., a cancer ofthe lung, breast, ovary, and colon tissues), immune function (e.g., T cell activities, e.g., lymphomas, leukemias, and immune disorders), reproductive, neurological and cardiovascular functioa

[53] As used herein, the terms "cancer", "hyperproliferative" and "neoplastic" refer to cells having the capacity for autonomous growth, i.e., an abnormal state or condition characterized by rapidly proliferating cell growth. Hyperproliferative and neoplastic disease states may be categorized as pathologic, i.e., characterizing or constituting a disease state, or may be categorized as non-pathologic, i.e., a deviation from normal but not associated with a disease state. The term is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness. "Pathologic hyperproliferative" cells occur in disease states characterized by malignant tumor growth Examples of non-pathologic hyperproliferative cells include proliferation of cells associated with wound repair. [54] Examples of cellular proliferative and/or differentiative disorders include cancer, e.g., carcinoma, sarcoma, or metastatic disorders. The 21953 molecules can act as novel diagnostic targets and therapeutic agents for controlling lung cancer, breast cancer, ovarian cancer, colon cancer, metastasis of such cancers and the like. A metastatic tumor can arise from a multitude of primary tumor types, including but not Umited to those of lung, breast, liver, colon and ovarian origin.

[55] Examples of cellular proliferative and/or differentiative disorders ofthe lung include, but are not limited to, squamous cell lung carcinomas, small cell lung carcinoma, lung adenocarcinomas, bronchogenic carcinoma, including paraneoplastic syndromes, bronchioloalveolar carcinoma, neuroendocrine tumors, such as bronchial carcinoid, miscellaneous tumors, and metastatic tumors; pathologies ofthe pleura, including inflammatory pleural effusions, noninflammatory pleural effusions, pneumothorax, and pleural tumors, including solitary fibrous tumors (pleural fibroma) and malignant mesothelioma

[56] Examples of cellular proliferative and/or differentiative disorders ofthe breast include, but are not limited to, proliferative breast disease including, e.g., epithelial hyperplasia, sclerosing adenosis, and small duct papillomas; tumors, e.g., stromal tumors such as fibroadenoma, phyllodes tumor, and sarcomas, and epithelial tumors such as large duct papilloma; carcinoma ofthe breast including in situ (noninvasive) carcinoma that includes ductal carcinoma in situ (including Paget 's disease) and lobular carcinoma in situ, and invasive (infiltrating) carcinoma including, but not limited to, invasive ductal carcinoma, invasive lobular carcinoma, medullary carcinoma, colloid (mutinous) carcinoma, tubular carcinoma, and invasive papillary carcinoma, and miscellaneous malignant neoplasms. Disorders in the male breast include, but are not limited to, gynecomastia and carcinoma.

[57] Examples of cellular proliferative and/or differentiative disorders ofthe colon include, but are not limited to, non-neoplastic polyps, adenomas, familial syndromes, colorectal carcinogenesis, colorectal carcinoma, and carcinoid tumors. [58] Examples of cellular proliferative and or differentiative disorders ofthe Uver include, but are not limited to, nodular hyperplasias, adenomas, and malignant tumors, including primary carcinoma ofthe liver and metastatic tumors.

[59] Examples of cellular proliferative and/or differentiative disorders ofthe ovary include, but are not limited to, ovarian tumors such as, tumors of coelomic epithelium, serous tumors, mutinous tumors, endometeriod tumors, clear cell adenocarcinoma, cystadenofibroma, brenner tumor, surface epithelial tumors; germ cell tumors such as mature (benign) teratomas, monodermal teratomas, immature malignant teratomas, dysgerminoma, endodermal sinus tumor, choriocarcinoma; sex cord-stomal tumors such as, granulosa-theca cell tumors, thecoma-fibromas, androblastomas, hill cell tumors, and gonadoblastoma; and metastatic tumors such as Krukenberg tumors. [60] Additional examples of proliferative disorders include hematopoietic neoplastic disorders. As used herein, the term "hematopoietic neoplastic disorders" includes diseases involving hyperplastic/neoplastic cells of hematopoietic origin, e.g., arising from myeloid, lymphoid or erythroid lineages, or precursor cells thereof. Preferably, the diseases arise from poorly differentiated acute leukemias, e.g., erythroblastic leukemia and acute megakaryoblastic leukemia Additional exemplary myeloid disorders include, but are not limited to, acute promyeloid leukemia (APML), acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML) (reviewed in Vaickus, L. (1991) CritRev. in Oncol/Hemotol. 11:267-97); lymphoid malignancies include, but are not limited to acute lymphoblastic leukemia (ALL) which includes B-lineage ALL and T-lineage ALL, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) and Waldenstrom's macroglobulinemia (WM). Additional forms of mahgnant lymphomas include, but are not Umited to non-Hodgkin lymphoma and variants thereof, peripheral T cell lymphomas, adult T cell leukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), large granular lymphocytic leukemia (LGF), Hodgkin's disease and Reed-Sternberg disease.

[61] The 21953 nucleic acid and protein ofthe invention can be used to treat and/or diagnose a variety of immune disorders, e.g., as a result of aberrant 21953 activity in T cells. Examples of immune disorders or diseases include, but are not Umited to, autoimmune diseases (including, for example, diabetes mellitus, arthritis (including rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis), multiple sclerosis, encephalomyeUtis, myasthenia gravis, systemic lupus erythematosis, autoimmune thyroiditis, dermatitis (including atopic dermatitis and eczematous dermatitis), psoriasis, Sjόgren's Syndrome, Crohn's disease, aphthous ulcer, iritis, conjunctivitis, keratoconjunctivitis, ulcerative colitis, asthma, allergic asthma, cutaneous lupus erythematosus, scleroderma, vaginitis, proctitis, drug eruptions, leprosy reversal reactions, erythema nodosum leprosum, autoimmune uveitis, allergic encephalomyeUtis, acute necrotizing hemorrhagic encephalopathy, idiopathic bilateral progressive sensorineural hearing loss, aplastic anemia, pure red cell anemia, idiopathic thrombocytopenia, polychondritis, Wegener's granulomatosis, chronic active hepatitis, Stevens- Johnson syndrome, idiopathic sprue, Uchen planus, Graves' disease, sarcoidosis, primary biliary cirrhosis, uveitis posterior, and interstitial lung fibrosis), graft-versus-host disease, cases of transplantation, and allergy such as, atopic aUergy.

[62] Examples of neuronal disorders include, but are not limited to disorders involving neurons, and disorders involving glia, such as astrocytes, oUgodendrocytes, ependymal cells, and microglia; cerebral edema, raised intracranial pressure and herniation, and hydrocephalus; malformations and developmental diseases, such as neural tube defects, forebrain anomalies, posterior fossa anomalies, and syringomyelia and hydromyelia; perinatal brain injury; transmissible spongiform encephalopathies (prion diseases); demyelinating diseases, including multiple sclerosis, multiple sclerosis variants, acute disseminated encephalomyeUtis and acute necrotizing hemorrhagic encephalomyeUtis, and other diseases with demyelination; degenerative diseases, such as degenerative diseases affecting the cerebral cortex, including Alzheimer disease and Pick disease, degenerative diseases of basal ganglia and brain stem, including Parkinsonism, idiopathic Parkinson disease (paralysis agitans), progressive supranuclear palsy, corticobasal degeneration, multiple system atrophy, including striatonigral degeneration, Shy-Drager syndrome, and olivopontocerebeUar atrophy, and Huntington disease; spinocerebellar degenerations, including spinocerebellar ataxias, including Friedreich ataxia, and ataxia-telanglectasia, degenerative diseases affecting motor neurons, including amyotrophic lateral sclerosis (motor neuron disease), bulbospinal atrophy (Kennedy syndrome), and spinal muscular atrophy; tumors, such as gliomas, including astrocytoma, including fibrillary (diffuse) astrocytoma and glioblastoma multiforme, pilocytic astrocytoma, pleomorphic xanthoasttocvtoma, and brain stem glioma, oligodendroglioma, and ependymoma and related paraventricular mass lesions, neuronal tumors, poorly differentiated neoplasms, including medulloblastoma, other parenchymal tumors, including primary brain lymphoma, germ cell tumors, and pineal parenchymal tumors, meningiomas, metastatic tumors, paraneoplastic syndromes, peripheral nerve sheath tumors, including schwannoma, neurofibroma, and malignant peripheral nerve sheath tumor (malignant schwannoma), and neurocutaneous syndromes (phakomatoses), including neurofibromotosis, including Type 1 neurofibromatosis (NFl) and TYPE 2 neurofibromatosis (NF2), tuberous sclerosis, and Von Hippel-Lindau disease.

[63] The term "vascular disorder" includes disorders involving aberrant activity (e.g., proliferation, metaboUsm, angiogenesis, vascularization) of blood vessel-associated ceUs, e.g., smooth muscle or endothelial cells. Examples of such disorders include but are not limited to hypertension (e.g., arterial hypertension), vascular restenosis, ischemic disease (e.g., atherosclerosis), tumorigenesis, tumor metastasis, diabetic retinopathy, endometriosis, Grave's disease. Aberrant vascular activity may also affect cardiovascular function, and thus the molecules ofthe invention can be used to treat, prevent and/or diagnose cardiovascular disorders. Examples of cardiovascular disorders, include but are not Umited to, heart failure, cardiac hypertrophy, left-sided heart failure, and right-sided heart failure; ischemic heart disease, including but not limited to angina pectoris, myocardial infarction, chronic ischemic heart disease, and sudden cardiac death; hypertensive heart disease, including but not limited to, systemic (left-sided) hypertensive heart disease and pulmonary (right-sided) hypertensive heart disease; valvular heart disease, including but not Umited to, valvular degeneration caused by calcification, such as calcific aortic stenosis, calcification of a congenitally bicuspid aortic valve, and mitral annular calcification, and myxomatous degeneration ofthe mitral valve (mitral valve prolapse), rheumatic fever and rheumatic heart disease, infective endocarditis, and noninfected vegetations, such as nonbacterial thrombotic endocarditis and endocarditis of systemic lupus erythematosus (Libman-Sacks disease), carcinoid heart disease, and complications of artificial valves; myocardial disease, including but not limited to dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, and myocarditis; pericardia! disease, including but not Umited to, pericardial effusion and hemopericardium and pericarditis, including acute pericarditis and healed pericarditis, and rheumatoid heart disease; neoplastic heart disease, including but not limited to, primary cardiac tumors, such as myxoma, Upoma, papillary fibroelastoma, rhabdomyoma, and sarcoma, and cardiac effects of noncardiac neoplasms; congenital heart disease, including but not Umited to, left-to-right shunts—late cyanosis, such as atrial septal defect, ventricular septal defect, patent ductus arteriosus, and atrioventricular septal defect, right-to-left shunts—early cyanosis, such as tetralogy of fallot, transposition of great arteries, truncus arteriosus, tricuspid atresia, and total anomalous pulmonary venous connection, obstructive congenital anomalies, such as coarctation of aorta, pulmonary stenosis and atresia, and aortic stenosis and atresia, and disorders involving cardiac transplantation. [64] As used herein, "a prostate disorder" refers to an abnormal condition occurring in the male pelvic region characterized by, e.g., male sexual dysfunction and/or urinary symptoms. This disorder may be manifested in the form of genitourinary inflammation (e.g., inflammation of smooth muscle cells) as in several common diseases ofthe prostate including prostatitis, benign prostatic hyperplasia and cancer, e.g., adenocarcinoma or carcinoma, ofthe prostate.

[65] The 21953 nucleic acid and protein ofthe invention can be used to treat and/or diagnose a variety of conditions, in addition to the ones described above (see "Methods of Treatment" for additional examples).

[66] The presence of 21953 RNA or protein can also be used to identify a cell or tissue, or other biological sample, as being derived from breast, T-cell, kidney, liver, and aorta, or being of human origin. Expression can also be used to diagnose or stage a disorder, e.g., a cancer (e.g., a cancer ofthe lung or breast), or a breast, lymphoid, lung, ovarian, or liver disorder. Expression can be determined by evaluating RNA, e.g., by hybridization of a 21953 specific probe, or with a 21953 specific antibody.

[67] The 21953 protein, fragments thereof and derivatives and other variants ofthe sequence in SEQ ID NO:2 thereof are collectively referred to as "polypeptides or proteins of the invention" or "21953 polypeptides or proteins". Nucleic acid molecules encoding such polypeptides or proteins are coUectively referred to as "nucleic acids ofthe invention" or "21953 nucleic acids." 21953 molecules refer to 21953 nucleic acids, polypeptides, and antibodies.

[68] As used herein, the term "nucleic acid molecule" includes DNA molecules (e.g., a cDNA or genomic DNA), RNA molecules (e.g., an mRNA) and analogs ofthe DNA or RNA A DNA or RNA analog can be synthesized from nucleotide analogs. The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA

[69] The term "isolated nucleic acid molecule" or "purified nucleic acid molecule" includes nucleic acid molecules that are separated from other nucleic acid molecules present in the natural source ofthe nucleic acid. For example, with regards to genomic DNA, the term "isolated" includes nucleic acid molecules which are separated from the chromosome with which the genomic DNA is naturally associated. Preferably, an "isolated" nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5' and/or 3* ends ofthe nucleic acid) in the genomic DNA ofthe organism from which the nucleic acid is derived. For example, in various embodiments, the isolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of 5' and/or 3' nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA ofthe cell from which the nucleic acid is derived. Moreover, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. [70] As used herein, the term "hybridizes under low stringency, medium stringency, high stringency, or very high stringency conditions" describes conditions for hybridization and washing. Guidance for performing hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6, which is incorporated by reference. Aqueous and nonaqueous methods are described in that reference and either can be used. Specific hybridization conditions referred to herein are as follows: 1) low stringency hybridization conditions in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by two washes in 0.2X SSC, 0.1% SDS at least at 50°C (the temperature ofthe washes can be increased to 55°C for low stringency conditions); 2) medium stringency hybridization conditions in 6X SSC at about 45°C, foUowed by one or more washes in 0.2X SSC, 0.1% SDS at 60°C; 3) high stringency hybridization conditions in 6X SSC at about 45°C, foUowed by one or more washes in 0.2X SSC, 0.1% SDS at 65°C; and preferably 4) very high stringency hybridization conditions are 0.5M sodium phosphate, 7% SDS at 65°C, followed by one or more washes at 0.2X SSC, 1% SDS at 65°C. Very high stringency conditions (4) are the preferred conditions and the ones that should be used unless otherwise specified.

[71] Preferably, an isolated nucleic acid molecule ofthe invention that hybridizes under a stringency condition described herein to the sequence of SEQ ID NO:l or SEQ ID NO:3, corresponds to anaturaUy-occurring nucleic acid molecule.

[72] As used herein, a "naturaUy-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature. For example a naturally occurring nucleic acid molecule can encode a natural protein. [73] As used herein, the terms "gene" and "recombinant gene" refer to nucleic acid molecules which include at least an open reading frame encoding a 21953 protein. The gene can optionally further include non-coding sequences, e.g., regulatory sequences and introns. Preferably, a gene encodes a mammalian 21953 protein or derivative thereof. [74] An "isolated" or "purified" polypeptide or protein is substantially free of cellular material or other contaminating proteins from the ceU or tissue source from which the protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. "Substantially free" means that a preparation of 21953 protein is at least 10% pure. In a preferred embodiment, the preparation of 21953 protein has less than about 30%, 20%, 10% and more preferably 5% (by dry weight), of non-21953 protein (also referred to herein as a "contaminating protein"), or of chemical precursors or non-21953 chemicals. When the 21953 protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% ofthe volume ofthe protein preparation. The invention includes isolated or purified preparations of at least 0.01, 0.1, 1.0, and 10 milligrams in dry weight. [75] A "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence of 21953 without abolishing or substantially altering a 21953 activity. Preferably the alteration does not substantially alter the 21953 activity, e.g., the activity is at least 20%, 40%, 60%, 70% or 80% of wild-type. An "essential" amino acid residue is a residue that, when altered from the wUd-type sequence of 21953, results in abolishing a 21953 activity such that less than 20% ofthe wild-type activity is present. For example, conserved amino acid residues in 21953 are predicted to be particularly unamenable to alteration.

[76] A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., aianine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in a 21953 protein is preferably replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of a 21953 coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for 21953 biological activity to identify mutants that retain activity. FoUowing mutagenesis of SEQ ID NO: 1 or SEQ ID NO:3, the encoded protein can be expressed recombinantly and the activity of he protein can be determined. [77] As used herein, a "biologically active portion" of a 21953 protein includes a fragment of a 21953 protein which participates in an interaction, e.g., an intramolecular or an inter-molecular interaction. An inter-molecular interaction can be a specific binding interaction or an enzymatic interaction (e.g., the interaction can be transient and a covalent bond is formed or broken). An inter-molecular interaction can be between a 21953 molecule and a non-21953 molecule or between a first 21953 molecule and a second 21953 molecule (e.g., a dimerization interaction). Biologically active portions of a 21953 protein include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequence ofthe 21953 protein, e.g., the amino acid sequence shown in SEQ ID NO:2, which include less amino acids than the full length 21953 proteins, and exhibit at least one activity of a 21953 protein. Typically, biologically active portions comprise a domain or motif with at least one activity ofthe 21953 protein, e.g., prolyl oligopeptidase activity. A biologically active portion of a 21953 protein can be a polypeptide which is, for example, 10, 25, 50, 100, 200 or more amino acids in length. BiologicaUy active portions of a 21953 protein can be used as targets for developing agents which modulate a 21953 mediated activity, e.g., prolyl oligopeptidase activity. [78] Calculations of homology or sequence identity between sequences (the terms are used interchangeably herein) are performed as foUows.

[79] To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aUgned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% ofthe length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology"). [80] The percent identity between the two sequences is a function ofthe number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment ofthe two sequences. [81] The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com), using aNWSgapdnaCMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and the one that should be used unless otherwise specified) are a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5. [82] The percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of E. Meyers and W. Miller ((1989) CABIOS, 4:11-17) which has been incorporated into the ALIGN program (version 2.0), using a P AMI 20 weight residue table, a gap length penalty of 12 and a gap penalty of 4. [83] The nucleic acid and protein sequences described herein can be used as a "query sequence" to perform a search against pubUc databases to, for example, identify other farnily members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to 21953 nucleic acid molecules ofthe invention. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to 21953 protein molecules ofthe invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al, (1997) Nucleic Acids Res. 25:3389-3402. When utiUzing BLAST and Gapped BLAST programs, the default parameters ofthe respective programs (e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlmnih.gov.

[84] Particularly preferred 21953 polypeptides ofthe present invention have an amino acid sequence substantially identical to the amino acid sequence of SEQ ID NO:2. In the context of an amino acid sequence, the term "substantially identical" is used herein to refer to a first amino acid that contains a sufficient or minimum number of amino acid residues that are i) identical to, or ii) conservative substitutions of aligned amino acid residues in a second amino acid sequence such that the first and second amino acid sequences can have a common structural domain and/or common functional activity. For example, amino acid sequences that contain a common structural domain having at least about 60%, or 65% identity, likely 75% identity, more likely 85%, 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:2 are termed substantially identical.

[85] In the context of nucleotide sequence, the term "substantially identical" is used herein to refer to a first nucleic acid sequence that contains a sufficient or minimum number of nucleotides that are identical to aUgned nucleotides in a second nucleic acid sequence such that the first and second nucleotide sequences encode a polypeptide having common functional activity, or encode a common structural polypeptide domain or a common functional polypeptide activity. For example, nucleotide sequences having at least about 60%, or 65% identity, likely 75% identity, more likely 85%, 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 1 or 3 are termed substantially identical.

[86] "Misexpression or aberrant expression", as used herein, refers to a non-wildtype pattern of gene expression at the RNA or protein level. It includes: expression at non- wild type levels, i.e., over- or under-expression; a pattern of expression that differs from wild type in terms ofthe time or stage at which the gene is expressed, e.g., increased or decreased expression (as compared with wild type) at a predetermined developmental period or stage; a pattern of expression that differs from wild type in terms of altered, e.g., increased or decreased, expression (as compared with wild type) in a predetermined cell type or tissue type; a pattern of expression that differs from wild type in terms ofthe splicing size, translated amino acid sequence, post-transitional modification, or biological activity ofthe expressed polypeptide; a pattern of expression that differs from wild type in terms ofthe effect of an environmental stimulus or extracellular stimulus on expression ofthe gene, e.g., a pattern of increased or decreased expression (as compared with wild type) in the presence of an increase or decrease in the strength ofthe stimulus.

[87] "Subject," as used herein, refers to human and non-human animals. The term

"non-human animals" ofthe invention includes all vertebrates, e.g., mammals, such as non- human primates (particularly higher primates), sheep, dog, rodent (e.g., mouse or rat), guinea pig, goat, pig, cat, rabbits, cow, and non-mammals, such as chickens, amphibians, reptiles, etc. In a preferred embodiment, the subject is a human. In another embodiment, the subject is an experimental animal or animal suitable as a disease model [88] A "purified preparation of cells", as used herein, refers to an in vitro preparation of ceUs. In the case cells from multicellular organisms (e.g., plants and animals), a purified preparation of ceUs is a subset of cells obtained from the organism, not the entire intact organism. In the case of unicellular microorganisms (e.g., cultured ceUs and microbial cells), it consists of a preparation of at least 10% and more preferably 50% ofthe subject cells. [89] Various aspects ofthe invention are described in further detail below.

Isolated Nucleic Acid Molecules

[90] In one aspect, the invention provides, an isolated or purified, nucleic acid molecule that encodes a 21953 polypeptide described herein, e.g., a full-length 21953 protein or a fragment thereof e.g., a biologically active portion of 21953 protein. Also included is a nucleic acid fragment suitable for use as a hybridization probe, which can be used, e.g., to identify a nucleic acid molecule encoding a polypeptide ofthe invention, 21953 mRNA, and fragments suitable for use as primers, e.g., PCR primers for the amplification or mutation of nucleic acid molecules.

[91] In one embodiment, an isolated nucleic acid molecule ofthe invention includes the nucleotide sequence shown in SEQ ID NO:l, or a portion of any of these nucleotide sequences. In one embodiment, the nucleic acid molecule includes sequences encoding the human 21953 protein (i.e., "the coding region" of SEQ ID NO: 1, as shown in SEQ ID NO: 3), as well as 5' untranslated sequences. Alternatively, the nucleic acid molecule can include only the coding region of SEQ ID NO:l (e.g., SEQ ID NO:3) and, e.g., no flanking sequences which normally accompany the subject sequence. In another embodiment, the nucleic acid molecule encodes a sequence corresponding to a fragment ofthe protein that includes amino acid 672 to 744, 88 to 663, or 88 to 744 of SEQ ID NO:2. [92] In another embodiment, an isolated nucleic acid molecule ofthe invention includes a nucleic acid molecule which is a complement ofthe nucleotide sequence shown in SEQ ID NO: l_or SEQ ID NO:3, or a portion of any of these nucleotide sequences. In other embodiments, the nucleic acid molecule ofthe invention is sufficiently complementary to the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO:3, such that it can hybridize (e.g., under a stringency condition described herein) to the nucleotide sequence shown in SEQ ID NO: 1 or 3, thereby forming a stable duplex.

[93] In one embodiment, an isolated nucleic acid molecule ofthe present invention includes a nucleotide sequence which is at least about 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more homologous to the entire length ofthe nucleotide sequence shown in SEQ ID NO:l or SEQ ID NO:3, or a portion, preferably ofthe same length, of any of these nucleotide sequences.

21953 Nucleic Acid Fragments

[94] A nucleic acid molecule ofthe invention can include only a portion ofthe nucleic acid sequence of SEQ ID NO: 1 or 3. For example, such a nucleic acid molecule can include a fragment which can be used as a probe or primer or a fragment encoding a portion of a 21953 protein, e.g., an immunogenic or biologically active portion of a 21953 protein. A fragment can comprise those nucleotides of SEQ ID NO: 1, which encode a prolyl oligopeptidase domain of human 21953. The nucleotide sequence determined from the cloning ofthe 21953 gene aUows for the generation of probes and primers designed for use in identifying and/or cloning other 21953 family members, or fragments thereof as weU as 21953 homologues, or fragments thereof from other species. [95] In another embodiment, a nucleic acid includes a nucleotide sequence that includes part, or all ofthe coding region and extends into either (or both) the 5' or 3' noncoding regioα Other embodiments include a fragment which includes a nucleotide sequence encoding an amino acid fragment described herein. Nucleic acid fragments can encode a specific domain or site described herein or fragments thereof, particularly fragments thereof which are at least 100, 150, 200, 300, 360, 400, 600, 650, or 700 amino acids in length Fragments also include nucleic acid sequences corresponding to specific amino acid sequences described above or fragments thereof Nucleic acid fragments should not to be construed as encompassing those fragments that may have been disclosed prior to the invention

[96] A nucleic acid fragment can include a sequence corresponding to a domain, region, or functional site described herein. A nucleic acid fragment can also include one or more domain, region, or functional site described herein. Thus, for example, a 21953 nucleic acid fragment can include a sequence corresponding to a prolyl oligopeptidase domain. [97] 21953 probes and primers are provided. Typically a probe/primer is an isolated or purified oUgonucleotide. The oligonucleotide typically includes a region of nucleotide sequence that hybridizes under a stringency condition described herein to at least about 7, 12 or 15, preferably about 20 or 25, more preferably about 30, 35, 40, 45, 50, 55, 60, 65, or 75 consecutive nucleotides of a sense or antisense sequence of SEQ ID NO: 1 or SEQ ID NO: 3, or of a naturaUy occurring allelic variant or mutant of SEQ ID NO: 1 or SEQ ID NO:3. [98] In a preferred embodiment the nucleic acid is a probe which is at least 5 or 10, and less than 200, more preferably less than 100, or less than 50, base pairs in length. It should be identical, or differ by 1, or less than in 5 or 10 bases, from a sequence disclosed herein. If alignment is needed for this comparison the sequences should be aligned for maximum homology. "Looped" out sequences from deletions or insertions, or mismatches, are considered differences.

[99] A probe or primer can be derived from the sense or anti-sense strand of a nucleic acid which encodes: a fragment ofthe protein that includes amino acid 672 to 744, 88 to 663, or 88 to 744 of SEQ ID NO:2.

[100] In another embodiment a set of primers is provided, e.g., primers suitable for use in a PCR, which can be used to amplify a selected region of a 21953 sequence, e.g., a domain, region, site or other sequence described herein. The primers should be at least 5, 10, or 50 base pairs in length and less than 100, or less than 200, base pairs in length. The primers should be identical, or differs by one base from a sequence disclosed herein or from a naturally occurring variant. For example, primers suitable for amplifying all or a portion of any ofthe following regions are provided: a prolyl oligopeptidase domain from about amino acid 672 to 744, 88 to 663, or 88 to 744 of SEQ ID NO:2.

[101] A nucleic acid fragment can encode an epitope bearing region of a polypeptide described herein.

[102] A nucleic acid fragment encoding a "biologically active portion of a 21953 polypeptide" can be prepared by isolating a portion ofthe nucleotide sequence of SEQ ID NO:l or 3, which encodes a polypeptide having a 21953 biological activity (e.g., the biological activities ofthe 21953 proteins are described herein), expressing the encoded portion ofthe 21953 protein (e.g., by recombinant expression in vitro) and assessing the activity ofthe encoded portion ofthe 21953 proteia For example, a nucleic acid fragment encoding a biologically active portion of 21953 includes a prolyl oUgopeptidase domain, e.g., amino acid residues about 672 to 744, 88 to 663, or 88 to 744 of SEQ ID NO:2. A nucleic acid fragment encoding a biologically active portion of a 21953 polypeptide, may comprise a nucleotide sequence which is greater than 361, 470, 800, 1000, 1600, or more nucleotides in length.

[103] In preferred embodiments, a nucleic acid includes a nucleotide sequence which is about 300, 400, 500, 700, 800, 1000, 1100, 1200, 1500, 1600, 2000, 2400 or more nucleotides in length and hybridizes under a stringency condition described herein to a nucleic acid molecule of SEQ ID NO: 1, or SEQ ID NO:3. In a preferred embodiment, the nucleic acid includes a contiguous sequence that includes approximately nucleotide 1640, or 1642 of SEQ ID NO:l, e.g., the region from nucleotide 1635 to 1645 of SEQ ID NO:l. In other embodiment the nucleic acid includes a contiguous sequence that includes about nucleotides 1 to 25, 1 to 66, 100 to 300, 300 to 700, 500 to 800, 800 to 1200, 1000 to 1400, or 1200 to 1600 of SEQ ID NO:l.

21953 Nucleic Acid Variants

[104] The invention further encompasses nucleic acid molecules that differ from the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO:3. Such differences can be due to degeneracy ofthe genetic code (and result in a nucleic acid which encodes the same 21953 proteins as those encoded by the nucleotide sequence disclosed herein. In another embodiment, an isolated nucleic acid molecule ofthe invention has a nucleotide sequence encoding a protein having an amino acid sequence which differs, by at least 1, but less than 5, 10, 20, 50, or 100 amino acid residues that shown in SEQ ID NO:2. If aUgnment is needed for this comparison the sequences should be aligned for maximum homology. "Looped" out sequences from deletions or insertions, or mismatches, are considered differences.

[105] Nucleic acids ofthe inventor can be chosen for having codons, which are preferred, or non-preferred, for a particular expression system. E.g., the nucleic acid can be one in which at least one codon, at preferably at least 10%, or 20% ofthe codons has been altered such that the sequence is optimized for expression in E. coli, yeast, human, insect, or CHO cells.

[106] Nucleic acid variants can be naturaUy occurring, such as aUeUc variants (same locus), homologs (different locus), and orthologs (different organism) or can be non naturally occurring. Non-naturally occurring variants can be made by mutagenesis techniques, including those applied to polynucleotides, cells, or organisms. The variants can contain nucleotide substitutions, deletions, inversions and insertions. Variation can occur in either or both the coding and non-coding regions. The variations can produce both conservative and non- conservative amino acid substitutions (as compared in the encoded product). [107] In a preferred embodiment, the nucleic acid differs from that of SEQ ID NO: 1 or 3, e.g., as follows: by at least one but less than 10, 20, 30, or 40 nucleotides; at least one but less than 1%, 5%, 10% or 20% ofthe nucleotides in the subject nucleic acid. If necessary for this analysis the sequences should be aligned for maximum homology. "Looped" out sequences from deletions or insertions, or mismatches, are considered differences. [108] Orthologs, homologs, and aUelic variants can be identified using methods known in the art. These variants comprise a nucleotide sequence encoding a polypeptide that is 50%, at least about 55%, typically at least about 70-75%, more typically at least about 80-85%, and most typicaUy at least about 90-95% or more identical to the nucleotide sequence shown in SEQ ID NO:2 or a fragment of this sequence. Such nucleic acid molecules can readUy be identified as being able to hybridize under a stringency condition described herein, to the nucleotide sequence shown in SEQ ID NO 2 or a fragment ofthe sequence. Nucleic acid molecules corresponding to orthologs, homologs, and allelic variants ofthe 21953 cDNAs of the invention can further be isolated by mapping to the same chromosome or locus as the 21953 gene.

[109] Preferred variants include those that are correlated with dipeptidyl peptidase or prolyl endopeptidases activity.

[110] Allelic variants of 21953, e.g., human 21953, include both functional and nonfunctional proteins. Functional allelic variants are naturally occurring amino acid sequence variants ofthe 21953 protein within a population that maintain the abiUty to bind and/or cleave polypeptide substrates, e.g., a polypeptide having a proline residue. Functional alleUc variants will typically contain only conservative substitution of one or more amino acids of SEQ ID NO:2, or substitution, deletion or insertion of non-critical residues in non-critical regions ofthe protein. Non-functional allelic variants are naturally-occurring amino acid sequence variants ofthe 21953, e.g., human 21953, protein within a population that do not have the ability to bind and/or cleave polypeptide substrates, e.g., a polypeptide having a proline residue. Non-functional allelic variants will typically contain anon-conservative substitution, a deletion, or insertion, or premature truncation ofthe amino acid sequence of SEQ ID NO:2, or a substitution, insertion, or deletion in critical residues or critical regions ofthe protein.

[Ill] Moreover, nucleic acid molecules encoding other 21953 family members and, thus, which have a nucleotide sequence which differs from the 21953 sequences of SEQ ID NO:l or SEQ ID NO:3 are intended to be within the scope ofthe invention.

Antisense Nucleic Acid Molecules. Ribozvmes and Modified 21953 Nucleic Acid Molecules

[112] In another aspect, the invention features, an isolated nucleic acid molecule which is antisense to 21953. An "antisense" nucleic acid can include a nucleotide sequence which is complementary to a "sense" nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence. The antisense nucleic acid can be complementary to an entire 21953 coding strand, or to only a portion thereof (e.g., the coding region of human 21953 corresponding to SEQ ID NO:3). In another embodiment, the antisense nucleic acid molecule is antisense to a "noncoding region" ofthe coding strand of a nucleotide sequence encoding 21953 (e.g., the 5' and 3' untranslated regions). [113] An antisense nucleic acid can be designed such that it is complementary to the entire coding region of 21953 mRNA, but more preferably is an oUgonucleotide which is antisense to only a portion ofthe coding or noncoding region of 21953 mRNA For example, the antisense oUgonucleotide can be complementary to the region surrounding the translation start site of 21953 mRNA, e.g., between the -10 and +10 regions ofthe target gene nucleotide sequence of interest. An antisense oUgonucleotide can be, for example, about 7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or more nucleotides in length.

[114] An antisense nucleic acid ofthe invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability ofthe molecules or to increase the physical stability ofthe duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used. The antisense nucleic acid also can be produced biologicaUy using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).

[115] The antisense nucleic acid molecules ofthe invention are typically administered to a subject (e.g., by direct injection at atissue site), or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a 21953 protein to thereby inhibit expression ofthe protein, e.g., by inhibiting transcription and/or translation. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies which bind to cett surface receptors or antigens. The antisense nucleic acid molecules can also be deUvered to ceUs using the vectors described herein. To achieve sufficient intracellular concentrations ofthe antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred. [116] In yet another embodiment, the antisense nucleic acid molecule ofthe invention is an α-anomeric nucleic acid molecule. An α-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual β -units, the strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641). The antisense nucleic acid molecule can also comprise a 2'-o- methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res. 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBSLett. 215:327-330). [117] In still another embodiment, an antisense nucleic acid ofthe invention is a ribozyme. A ribozyme having specificity for a 21953-encoding nucleic acid can include one or more sequences complementary to the nucleotide sequence of a 21953 cDNA disclosed herein (i.e., SEQ ID NO:l or SEQ ID NO:3), and a sequence having known catalytic sequence responsible for mRNA cleavage (see U.S. Pat. No. 5,093,246 or Haselhoff and Geriach (1988) Nature 334:585-591). For example, a derivative of a Tetrahymena L-19 JNS RΝA can be constructed in which the nucleotide sequence ofthe active site is complementary to the nucleotide sequence to be cleaved in a 21953-encoding mRΝA See, e.g., Cechetα/. U.S. Patent No. 4,987,071; and Cech etal. U.S. Patent No. 5,116,742. Alternatively, 21953 mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel, D. and Szostak, J.W. (1993) Science 261:1411-1418.

[118] 21953 gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region ofthe 21953 (e.g., the 21953 promoter and/or enhancers) to form triple helical structures that prevent transcription ofthe 21953 gene in target ceUs. See generally, Helene, C. (1991) Anticancer Drug Des. 6:569-84; Helene, C. i (1992) Ann. N.Y. Acad. Sci. 660:27-36; and Maher, L.J. (1992) Bioassays 14:807-15. The potential sequences that can be targeted for triple helix formation can be increased by creating a so-called "switchback" nucleic acid molecule. Switchback molecules are synthesized in an alternating 5'-3', 3 -5' manner, such that they base pair with first one strand of a duplex and then the other, eliminating the necessity for a sizeable stretch of either purines or pyrimidines to be present on one strand of a duplex.

[119] The invention also provides detectably labeled oUgonucleotide primer and probe molecules. Typically, such labels are chemiluminescent, fluorescent, radioactive, or colorimetric. [120] A 21953 nucleic acid molecule can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility ofthe molecule. For non-Umiting examples of synthetic oligonucleotides with modifications see Toulme (2001) Nature Biotech. 19:17 and Faria etα . (2001) Nature Biotech. 19:40-44. Such phosphoramidite oUgonucleotides can be effective antisense agents. [121] For example, the deoxyribose phosphate backbone ofthe nucleic acid molecules can be modified to generate peptide nucleic acids (see Hyrup B. et al. (1996) Bioorganic & Medicinal Chemistry 4: 5-23). As used herein, the terms "peptide nucleic acid" or "PNA" refers to a nucleic acid mimic, e.g., a DNA mimic, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained. The neutral backbone of a PNA can allow for specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oUgomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup B. et al. (1996) supra and Perry-O'Keefe etal. Proc. Natl. Acad. Sci. 93: 14670-675. [122] PNAs of 21953 nucleic acid molecules can be used in therapeutic and diagnostic appUcations. For example, PNAs can be used as antisense or antigene agents for sequence- specific modulation of gene expression by, for example, inducing transcription or translation arrest or inhibiting repUcatioa PNAs of 21953 nucleic acid molecules can also be used in the analysis of single base pair mutations in a gene, (e.g., by PNA-directed PCR clamping); as 'artificial restriction enzymes' when used in combination with other enzymes, (e.g., Sl nucleases (Hyrup B. et al. (1996) supra)); or as probes or primers for DNA sequencing or hybridization (Hyrup B. etal. (1996) supra; Perry-O'Keefe supra). [123] In other embodiments, the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the ceU membrane (see, e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA 86:6553-6556; Lemaitre etal. (1987) Proc. Natl. Acad. Sci. USA 84:648-652; PCT Publication No. W088/09810) or the blood-brain barrier (see, e.g., PCT Publication No. W089/10134). In addition, oUgonucleotides can be modified with hybridization-triggered cleavage agents (see, e.g., Krol etal. (1988) Bio-Techniques 6:958-976) or intercalating agents (see, e.g., Zon (1988) Pharm. Res. 5:539-549). To this end, the oligonucleotide may be conjugated to another molecule, (e.g., a peptide, hybridization triggered cross-linking agent, transport agent, or hybridization-triggered cleavage agent). [124] The invention also includes molecular beacon oligonucleotide primer and probe molecules having at least one region which is complementary to a 21953 nucleic acid ofthe invention, two complementary regions one having a fluorophore and one a quencher such that the molecular beacon is useful for quantitating the presence ofthe 21953 nucleic acid of the invention in a sample. Molecular beacon nucleic acids are described, for example, in Lizardi etal, U.S. Patent No. 5,854,033; Nazarenko etal, U.S. Patent No. 5,866,336, and Livak et al, U.S. Patent 5,876,930.

Isolated 21953 Polypeptides

[125] In another aspect, the invention features, an isolated 21953 protein, or fragment, e.g., a biologically active portion, for use as immunogens or antigens to raise or test (or more generaUy to bind) anti-21953 antibodies. 21953 protein can be isolated from cells or tissue sources using standard protein purification techniques. 21953 protein or fragments thereof can be produced by recombinant DNA techniques or synthesized chemically. [126] Polypeptides ofthe invention include those which arise as a result ofthe existence of multiple genes, alternative transcription events, alternative RNA splicing events, and alternative translational and post-translational events. The polypeptide can be expressed in systems, e.g., cultured cells, which result in substantially the same post- translational modifications present when expressed the polypeptide is expressed in a native cell, or in systems which result in the alteration or omission of post-translational modifications, e.g., glycosylation or cleavage, present when expressed in a native cell. [127] In a preferred embodiment, a 21953 polypeptide has one or more ofthe following characteristics:

[128] (i) it has the ability to promote the degradation of proUne-containing peptides by cleaving the peptide bond at the carboxyl side of proUne residues; [129] (ii) it has a molecular weight, (e.g., about 97 KDa), amino acid composition, or other physical characteristic, of a 21953 polypeptide, e.g., a polypeptide of SEQ ID NO:2; [130] (iii) it has an overaU sequence similarity ofat least 60%, more preferably at least 70, 80, 90, or 95%, with a polypeptide of SEQ ID NO:2;

[131] (iv) it has a prolyl oligopeptidase domain which has preferably about 70%,

80%, 90% or 95% sequence similarity with amino acid residues 672-744 of SEQ ID NO:2; or [132] (v) it has at least 70%, preferably 80%, and most preferably 90% ofthe cysteines found in the amino acid sequence ofthe native protein (SEQ ID NO:2). [133] In a preferred embodiment the 21953 protein, or fragment thereof, differs from the corresponding sequence in SEQ ID:2. In one embodiment it differs by at least one but by less than 15, 10 or 5 amino acid residues. In another it differs from the corresponding sequence in SEQ ID NO:2 by at least one residue but less than 20%, 15%, 10% or 5% ofthe residues in it differ from the corresponding sequence in SEQ ID NO:2. (If this comparison requires alignment the sequences should be aligned for maximum homology. "Looped" out sequences from deletions or insertions, or mismatches, are considered differences.) The differences are, preferably, differences or changes at anon-essential residue or a conservative substitution. In a preferred embodiment the differences are not in the prolyl oligopeptidase domain and/or the DPP IV N-terminal domain. In another preferred embodiment one or more differences are in the prolyl oUgopeptidase domain and/or the DPP IN N-terminal domain.

[134] Other embodiments include a protein that contain one or more changes in amino acid sequence, e.g., a change in an amino acid residue which is not essential for activity. Such 21953 proteins differ in amino acid sequence from SEQ ID NO:2, yet retain biological activity.

[135] In some embodiments, the protein includes an amino acid sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more homologous to SEQ ID NO:2. In some embodiments, the protein includes at least one contiguous amino acid from the region of about amino acid 1 to 200, 100 to 300, 200 to 400, 300 to 500, 400 to 600, 500 to 700, or 600 to 800 of SEQ ID NO:2.

[136] A 21953 protein or fragment is provided which varies from the sequence of SEQ

ID NO:2 in regions defined by amino acids about 672 to 744 by at least one but by less than 15, 10 or 5 amino acid residues in the protein or fragment but which does not differ from SEQ ID NO:2 in regions defined by amino acids about 672 to 744. (If this comparison requires aUgnment the sequences should be aligned for maximum homology. "Looped" out sequences from deletions or insertions, or mismatches, are considered differences.) In some embodiments the difference is at a non-essential residue or is a conservative substitution, while in others the difference is at an essential residue or is a non-conservative substitution. [137] In one embodiment, a biologically active portion of a 21953 protein includes a prolyl oUgopeptidase domain. Moreover, other biologically active portions, in wliich other regions ofthe protein are deleted, can be prepared by recombinant techniques and evaluated for one or more ofthe functional activities of a native 21953 protein. [138] In a preferred embodiment, the 21953 protein has an amino acid sequence shown in SEQ ID NO:2. In other embodiments, the 21953 protein is substantially identical to SEQ ID NO:2. In yet another embodiment, the 21953 protein is substantially identical to SEQ ID NO:2 and retains the functional activity ofthe protein of SEQ ID NO: 2, as described in detail in the subsections above.

[139] In another preferred embodiment, the 21953 protein has a K_m for the substrate

H-Gly-Pro- -nitroanilide (NA)/HC1 (Sigma Corp, MO, USA) (H-Gly-Pro-pNA) of less than about 10 mM, 5 mM, 1 mM, 0.5 mM, 0.2 mM, or 0.1 mM, and/or a V_max for H-Gly-Pro-pNA of about at least 100, 500, 1000, 3000, 5000, or 10000 absorbance umts-min^"1. Such parameters can be determined using a prolyl oUgopeptidase assay described herein, e.g., as described in "Screening Assays," below.

21953 Chimeric or Fusion Proteins

[140] In another aspect, the invention provides 21953 chimeric or fusion proteins. As used herein, a 21953 "chimeric protein" or "fusion protein" includes a 21953 polypeptide linked to a non-21953 polypeptide. A "non-21953 polypeptide" refers to a polypeptide having an amino acid sequence corresponding to a protein which is not substantially homologous to the 21953 protein, e.g., a protein which is different from the 21953 protein and which is derived from the same or a different organism. The 21953 polypeptide ofthe fusion protein can correspond to all or a portion e.g., a fragment described herein of a 21953 amino acid sequence. In a preferred embodiment, a 21953 fusion protein includes at least one (or two) biologically active portion of a 21953 protein. The non-21953 polypeptide can be fused to the N-terminus or C-terminus ofthe 21953 polypeptide. [141] The fusion protein can include a moiety which has a high affinity for a Ugand.

For example, the fusion protein can be a GST-21953 fusion protein in which the 21953 sequences are fused to the C-terminus ofthe GST sequences. Such fusion proteins can facilitate the purification of recombinant 21953. Alternatively, the fusion protein can be a 21953 protein containing a heterologous signal sequence at its N-terminus. In certain host cells (e.g., mammalian host cells), expression and/or secretion of 21953 can be increased through use of a heterologous signal sequence. [142] Fusion proteins can include all or a part of a serum protein, e.g., an IgG constant region, or human serum albumin.

[143] The 21953 fusion proteins ofthe invention can be incorporated into pharmaceutical compositions and administered to a subject in vivo. The 21953 fusion proteins can be used to affect the bioavailability of a 21953 substrate. 21953 fusion proteins may be useful therapeutically for the treatment of disorders caused by, for example, (i) aberrant modification or mutation of a gene encoding a 21953 protein; (ii) mis-regulation of the 21953 gene; and (Ui) aberrant post-translational modification of a 21953 proteiα [144] Moreover, the 21953-fusion proteins ofthe invention can be used as immunogens to produce anti-21953 antibodies in a subject, to purify 21953 Ugands and in screening assays to identify molecules which inhibit the interaction of 21953 with a 21953 substrate.

[145] Expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide). A 21953-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the 21953 protein.

Variants of 21953 Proteins

[146] In another aspect, the invention also features a variant of a 21953 polypeptide, e.g., which functions as an agonist (mimetics) or as an antagonist. Variants ofthe 21953 proteins can be generated by mutagenesis, e.g., discrete point mutation, the insertion or deletion of sequences or the truncation of a 21953 protein. An agonist ofthe 21953 proteins can retain substantially the same, or a subset, ofthe biological activities ofthe naturaUy occurring form of a 21953 protein. An antagonist of a 21953 protein can inhibit one or more ofthe activities ofthe naturally occurring form ofthe 21953 protein by, for example, competitively modulating a 21953-mediated activity of a 21953 protein. Thus, specific biological effects can be elicited by treatment with a variant of limited functioa Preferably, treatment of a subject with a variant having a subset ofthe biological activities ofthe naturally occurring form ofthe protein has fewer side effects in a subject relative to treatment with the naturally occurring form ofthe 21953 protein. [147] Variants of a 21953 protein can be identified by screening combinatorial libraries of mutants, e.g., truncation mutants, of a 21953 protein for agonist or antagonist activity. [148] Libraries of fragments e.g., N terminal, C terminal, or internal fragments, of a

21953 protein coding sequence can be used to generate a variegated population of fragments for screening and subsequent selection of variants of a 21953 proteia Variants in which a cysteine residues is added or deleted or in which a residue which is glycosylated is added or deleted are particularly preferred.

[149] Methods for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property are known in the art. Such methods are adaptable for rapid screening of the gene Ubraries generated by combinatorial mutagenesis of 21953 proteins. Recursive ensemble mutagenesis (REM), a new technique which enhances the frequency of functional mutants in the Ubraries, can be used in combination with the screening assays to identify 21953 variants (Arkin and Yourvan (1992) Proc. Natl. Acad. Sci. USA 59:7811-7815; Delgrave et al. (1993) Protein Engineering 6:327-331).

[150] Cell based assays can be exploited to analyze a variegated 21953 library. For example, a tibrary of expression vectors can be transfected into a cell line, e.g., a ceU line, which ordinarily responds to 21953 in a substrate-dependent manner. The transfected ceUs are then contacted with 21953 and the effect ofthe expression ofthe mutant on signaling by the 21953 substrate can be detected, e.g., by measuring prolyl oligopeptidase as described below. Plasmid DNA can then be recovered from the ceUs which score for inhibition, or alternatively, potentiation of signaling by the 21953 substrate, and the individual clones further characterized.

[151] In another aspect, the invention features a method of making a 21953 polypeptide, e.g., a peptide having a non- wild type activity, e.g., an antagonist, agonist, or super agonist of a naturally occurring 21953 polypeptide, e.g., a naturally occurring 21953 polypeptide. The method includes: altering the sequence of a 21953 polypeptide, e.g., altering the sequence , e.g., by substitution or deletion of one or more residues of a non- conserved region, a domain or residue disclosed herein, and testing the altered polypeptide for the desired activity.

[152] In another aspect, the invention features a method of making a fragment or analog of a 21953 polypeptide a biological activity of a naturaUy occurring 21953 polypeptide. The method includes: altering the sequence, e.g., by substitution or deletion of one or more residues, of a 21953 polypeptide, e.g., altering the sequence of a non-conserved region, or a domain or residue described herein, and testing the altered polypeptide for the desired activity.

Anti-21953 Antibodies

[153] In another aspect, the invention provides an anti-21953 antibody, or a fragment thereof (e.g., an antigen-binding fragment thereof). The term "antibody" as used herein refers to an immunoglobulin molecule or immunologically active portion thereof) i.e., an antigen-binding portioa As used herein, the term "antibody" refers to a protein comprising at least one, and preferably two, heavy (H) chain variable regions (abbreviated herein as VH), and at least one and preferably two light (L) chain variable regions (abbreviated herein as VL). The VH and VL regions can be further subdivided into regions of hypervariability, termed "complementarity determining regions" ("CDR"), interspersed with regions that are more conserved, termed "framework regions" (FR). The extent ofthe framework region and CDR's has been precisely defined (see, Kabat, E. A, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NTH Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917, which are incorporated herein by reference). Each VH and VL is composed of three CDR's and four FRs, arranged from arnino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[154] The anti-21953 antibody can further include a heavy and Ught chain constant region, to thereby form a heavy and Ught immunoglobulin chain, respectively. In one embodiment, the antibody is a tetramer of two heavy immunoglobulin chains and two light immunoglobulin chains, wherein the heavy and light immunoglobulin chains are interconnected by, e.g., disulfide bonds. The heavy chain constant region is comprised of three domains, CHI, CH2 and CH3. The light chain constant region is comprised of one domain, CL. The variable region ofthe heavy and light chains contains a binding domain that interacts with an antigea The constant regions ofthe antibodies typically mediate the binding ofthe antibody to host tissues or factors, including various cells ofthe immune system (e.g., effector cells) and the first component (Clq) ofthe classical complement system.

[155] As used herein, the term "immunoglobulin" refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes. The recognized human immunoglobulin genes include the kappa, lambda, alpha (IgAl and IgA2), gamma (IgGl, IgG2, IgG3, IgG4), delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Full-length immunoglobulin "light chains" (about 25 KDa or 214 amino acids) are encoded by a variable region gene at the NH2- terrninus (about 110 amino acids) and a kappa or lambda constant region gene at the COOH— terminus. FuU-length immunoglobulin "heavy chains" (about 50 KDa or 446 amino acids), are similarly encoded by a variable region gene (about 116 amino acids) and one ofthe other aforementioned constant region genes, e.g., gamma (encoding about 330 amino acids).

[156] The term "antigen-binding fragment" of an antibody (or simply "antibody portion," or "fragment"), as used herein, refers to one or more fragments of a full-length antibody that retain the ability to specifically bind to the antigen, e.g., 21953 polypeptide or fragment thereof. Examples of antigen-binding fragments ofthe anti-21953 antibody include, but are not Umited to: (i) a Fab fragment, a monovalent fragment consisting ofthe VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two

Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting ofthe VH and CHI domains; (iv) a Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al, (1989) Nature 341:544-546). which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains ofthe Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird etal (1988) Science 242:423-426; and Huston etal. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also encompassed within the term "antigen-binding fragment" of an antibody. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

[157] The anti-21953 antibody can be a polyclonal or a monoclonal antibody. In other embodiments, the antibody can be recombinantly produced, e.g., produced by phage display or by combinatorial methods.

[158] Phage display and combinatorial methods for generating anti-21953 antibodies are known in the art (as described in, e.g., Ladner et al. U.S. Patent No. 5,223,409; Kang et al. International Publication No. WO 92/18619; Dower et al. International Publication No. WO 91/17271; Winter et al. International Publication WO 92/20791; Markland et al. International PubUcationNo. WO 92/15679; Breitling et al. International Publication WO 93/01288; McCafferty et al. International Publication No. WO 92/01047; Garrard et al. International PubUcationNo. WO 92/09690; Ladner et al. International PubUcationNo. WO 90/02809; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum Antibod Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffths et al. (1993) EMBO J 12:725-734; Hawkins et al. (1992) JMolBiol 226:889-896; Clackson et al. (1991) Nature 352:624-628: Gram et al. (1992) PNAS 89:3576-3580; Garrad et al. (1991) Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137; and Barbas et al. (1991) PNAS 88:7978-7982, the contents of all of wliich are incorporated by reference herein).

[159] In one embodiment, the anti-21953 antibody is a fully human antibody (e.g., an antibody made in a mouse wliich has been genetically engineered to produce an antibody from a human immunoglobulin sequence), or a non-human antibody, e.g., a rodent (mouse or rat), goat, primate (e.g., monkey), camel antibody. Preferably, the non-human antibody is a rodent (mouse or rat antibody). Method of producing rodent antibodies are known in the art.

[160] Human monoclonal antibodies can be generated using transgenic mice carrying the human immunoglobulin genes rather than the mouse system Splenocytes from these transgenic mice immunized with the antigen of interest are used to produce hybridomas that secrete human mAbs with specific affinities for epitopes from a human protein (see, e.g., Wood et al. International Application WO 91/00906, Kucherlapati et al. PCT pubUcation WO 91/10741; Lonberg et al. International AppUcation WO 92/03918; Kay et al. International Application 92/03917; Lonberg, N. et al. 1994 Nature 368:856-859; Green, L.L. et al. 1994 Nature Genet. 7:13-21; Morrison, S.L. et al. 1994 Proc. Natl. Acad. Sci. USA 81:6851-6855; Bruggeman et al. 1993 Year Immunol 7:33-40; Tuaillon et al. 1993 PNAS 90:3720-3724; Bruggeman et al. 1991 Eur J Immunol 21:1323-1326). [161] An anti-21953 antibody can be one in which the variable region, or a portion thereof, e.g., the CDR's, are generated in a non-human organism, e.g., a rat or mouse. Chimeric, CDR-grafted, and humanized antibodies are within the inventioa Antibodies generated in anon-human organism, e.g., a rat or mouse, and then modified, e.g., in the variable framework or constant region, to decrease antigenicity in a human are within the invention. [162] Chimeric antibodies can be produced by recombinant DNA techniques known in the art. For example, a gene encoding the Fc constant region of a rnurine (or other species) monoclonal antibody molecule is digested with restriction enzymes to remove the region encoding the rnurine Fc, and the equivalent portion of a gene encoding a human Fc constant region is substituted (see Robinson et al, International Patent Publication PCT/US86/02269; Akira, et al., European Patent AppUcation 184,187; Taniguchi, M., European Patent Application 171,496; Morrison et al., European Patent Application 173,494; Neuberger et al., International Application WO 86/01533; Cabilly et al. U.S. Patent No. 4,816,567; CabUly et al., European Patent Application 125,023; Better et al. (1988 Science 240:1041- 1043); Liu et al. (19S1)PNAS 84:3439-3443; Liu et al., 1987, J. Immunol. 139:3521-3526; Sun et al. (1987) PNAS 84:214-218; Nishimura et al., 1987, Cane. Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; and Shaw et al., 1988, J. Natl Cancer Inst. 80:1553-1559). [163] A humanized or CDR-grafted antibody wul have at least one or two but generally all three recipient CDR's (of heavy and or light immuoglobulin chains) replaced with a donor CDR. The antibody may be replaced with at least a portion of a non-human CDR or only some ofthe CDR's may be replaced with non-human CDR's. It is only necessary to replace the number of CDR's required for binding ofthe humanized antibody to a 21953 or a fragment thereof. Preferably, the donor will be a rodent antibody, e.g., a rat or mouse antibody, and the recipient will be a human framework or a human consensus framework. TypicaUy, the immunoglobulin providing the CDR's is called the "donor" and the immunoglobulin providing the framework is called the "acceptor." In one embodiment, the donor immunoglobulin is a non-human (e.g., rodent). The acceptor framework is a naturally-occurring (e.g., a human) framework or a consensus framework, or a sequence about 85% or higher, preferably 90%, 95%, 99% or higher identical thereto. [164] As used herein, the term "consensus sequence" refers to the sequence formed from the most frequently occurring amino acids (or nucleotides) in a family of related sequences (See e.g., Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987). In a family of proteins, each position in the consensus sequence is occupied by the amino acid occurring most frequently at that position in the family. If two amino acids occur equaUy frequently, either can be included in the consensus sequence. A "consensus framework" refers to the framework region in the consensus immunoglobulin sequence. [165] An antibody can be humanized by methods known in the art. Humanized antibodies can be generated by replacing sequences ofthe Fv variable region which are not directly involved in antigen binding with equivalent sequences from human Fv variable regions. General methods for generating humanized antibodies are provided by Morrison, S. L., 1985, Science 229:1202-1207, by Oi et al., 1986, BioTechniques 4:214, and by Queen et al. US 5,585,089, US 5,693,761 and US 5,693,762, the contents of aU of which are hereby incorporated by reference. Those methods include isolating, manipulating, and expressing the nucleic acid sequences that encode all or part of immunoglobulin Fv variable regions from at least one of a heavy or light chain. Sources of such nucleic acid are weU known to those skUled in the art and, for example, may be obtained from a hybridoma producing an antibody against a 21953 polypeptide or fragment thereof. The recombinant DNA encoding the humanized antibody, or fragment thereof, can then be cloned into an appropriate expression vector.

[166] Humanized or CDR-grafted antibodies can be produced by CDR-grafting or

CDR substitution, wherein one, two, or all CDR's of an immunoglobulin chain can be replaced. See e.g., U.S. Patent 5,225,539; Jones et al. (1986) Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534; Beidler et al. (1988) J. Immunol. 141:4053-4060; Winter US 5,225,539, the contents of aU of which are hereby expressly incorporated by reference. Winter describes a CDR-grafting method which may be used to prepare the humanized antibodies ofthe present invention (UK Patent Application GB 2188638 A, filed on March 26, 1987; Winter US 5,225,539), the contents of which is expressly incorporated by reference.

[167] Also within the scope ofthe invention are humanized antibodies in which specific amino acids have been substituted, deleted or added. Preferred humanized antibodies have amino acid substitutions in the framework region, such as to improve binding to the antigen. For example, a humanized antibody will have framework residues identical to the donor framework residue or to another amino acid other than the recipient framework residue. To generate such antibodies, a selected, small number of acceptor framework residues ofthe humanized immunoglobulin chain can be replaced by the corresponding donor amino acids. Preferred locations ofthe substitutions include amino acid residues adjacent to the CDR, or which are capable of interacting with a CDR (see e.g., US 5,585,089). Criteria for selecting amino acids from the donor are described in US 5,585,089, e.g., columns 12-16 of US 5,585,089, the e.g., columns 12-16 of US 5,585,089, the contents of which are hereby incorporated by reference. Other techniques for humanizing antibodies are described in Padlan et al. EP 519596 Al, pubUshed on December 23, 1992.

[168] In preferred embodiments an antibody can be made by immunizing with purified

21953 antigen, or a fragment thereof) e.g., a fragment described herein, tissue, e.g., crude tissue preparations, whole cells, preferably Uving cells, lysed cells, or ceU fractions. [169] A full-length 21953 protein or, antigenic peptide fragment of 21953 can be used as an immunogen or can be used to identify anti-21953 antibodies made with other immunogens, e.g., cells, membrane preparations, and the Uke. The antigenic peptide of 21953 should include at least 8 amino acid residues ofthe amino acid sequence shown in SEQ ID NO:2 and encompasses an epitope of 21953. Preferably, the antigenic peptide includes at least 10 amino acid residues, more preferably at least 15 amino acid residues, even more preferably at least 20 amino acid residues, and most preferably at least 30 amino acid residues.

[170] Hydrophilic fragments of 21953, e.g., those which include residues 20 to 40, 65 to 80, or 780 to 790, can be used to make, e.g., used as immunogens or used to characterize the specificity of an antibody, antibodies against hydrophUic regions ofthe 21953 protein. Similarly, a hydrophobic fragment of 21953, e.g. which include residues 250 to 270, 370 to 390, or 681 to 695, can be used to make an antibody against a hydrophobic region ofthe 21953 protein; a fragment of 21953 which include residues about 672 to 744, 672 to 690, 690 to 710, or 710 to 744 can be used to make an antibody against the prolyl oUgopeptidase domain ofthe 21953 protein.

[171] Antibodies reactive with, or specific for, any of these regions, or other regions or domains described herein are provided.

[172] Antibodies which bind only native 21953 protein, only denatured or otherwise non-native 21953 protein, or which bind both, are with in the invention. Antibodies with linear or conformational epitopes are within the invention. Conformational epitopes can sometimes be identified by identifying antibodies which bind to native but not denatured 21953 proteia

[173] Preferred epitopes encompassed by the antigenic peptide are regions of 21953 are located on the surface ofthe protein, e.g., hydrophilic regions, as weU as regions with high antigenicity. For example, an Emini surface probability analysis ofthe human 21953 protein sequence can be used to indicate the regions that have a particularly high probability of being localized to the surface ofthe 21953 protein and are thus likely to constitute surface residues useful for targeting antibody production.

[174] The anti-21953 antibody can be a single chain antibody. A single-chain antibody (scFV) may be engineered (see, for example, Colcher, D. et al. (1999) Ann N Y

Acad Sci 880:263-80; and Reiter, Y. (1996) Clin Cancer Res 2:245-52). The single chain antibody can be dimerized or multimerized to generate multivalent antibodies having specificities for different epitopes ofthe same target 21953 protein.

[175] In a preferred embodiment the antibody has: effector function; and can fix complement. In other embodiments the antibody does not; recruit effector ceUs; or fix complement.

[176] In a preferred embodiment, the antibody has reduced or no ability to bind an Fc receptor. For example., it is a isotype or subtype, fragment or other mutant, which does not support binding to an Fc receptor, e.g., it has a mutagenized or deleted Fc receptor binding region.

[177] In a preferred embodiment, an anti-21953 antibody alters (e.g., increases or decreases) the prolyl oligopeptidase activity of a 21953 polypeptide. For example, the antibody can specifically bind a residue ofthe active site of 21953 polypeptide, e.g., a residue located between about 730 to 750, 805 to 830, 835 to 860 of SEQ ID NO:2. The antibody can block the binding of substrate to the 21953 polypeptide.

[178] In another preferred embodiment, the antibody specifically binds a residue in the prolyl oUgopeptidase domain, e.g., from about amino acid 672 to 744, or 610 to 883 of SEQ

ID NO:2, or in the DPP IN Ν-terminal residue, e.g., a residue between about amino acids 88 to 663 of SEQ ID ΝO:2.

[179] The antibody can be coupled to a toxin, e.g., a polypeptide toxin, e,g, ricin or diphtheria toxin or active fragment hereof, or a radioactive nucleus, or imaging agent, e.g. a radioactive, enzymatic, or other, e.g., imaging agent, e.g., a NMR contrast agent. Labels which produce detectable radioactive emissions or fluorescence are preferred.

[180] An anti-21953 antibody (e.g., monoclonal antibody) can be used to isolate

21953 by standard techniques, such as affinity chromatography or immunoprecipitation.

Moreover, an anti-21953 antibody can be used to detect 21953 protein (e.g., in a ceUular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression ofthe protein. Anti-21953 antibodies can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (i.e., physically Unking) the antibody to a detectable substance (i.e., antibody labelling). Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dicWorotriazmylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include ¹²⁵I, ^B11, ³⁵S or ³H.

[181] The invention also includes a nucleic acid which encodes an anti-21953 antibody, e.g., an anti-21953 antibody described herein. Also included are vectors which include the nucleic acid and cells transformed with the nucleic acid, particularly cells which are useful for producing an antibody, e.g., mammalian cells, e.g. CHO or lymphatic cells. [182] The invention also includes cell lines, e.g., hybridomas, which make an anti-

21953 antibody, e.g., and antibody described herein, and method of using said ceUs to make a 21953 antibody.

Recombinant Expression Vectors. Host Cells and Genetically Engineered Cells

[183] In another aspect, the invention includes, vectors, preferably expression vectors, containing a nucleic acid encoding a polypeptide described herein. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked and can include a plasmid, cosmid or viral vector. The vector can be capable of autonomous replication or it can integrate into a host DNA Viral vectors include, e.g., repUcation defective retroviruses, adeno viruses and adeno-associated viruses. [184] A vector can include a 21953 nucleic acid in a form suitable for expression of the nucleic acid in a host cell. Preferably the recombinant expression vector includes one or more regulatory sequences operatively linked to the nucleic acid sequence to be expressed. The term "regulatory sequence" includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence, as weU as tissue-specific regulatory and/or inducible sequences. The design ofthe expression vector can depend on such factors as the choice ofthe host cell to be transformed, the level of expression of protein desired, and the like. The expression vectors ofthe invention can be introduced into host ceUs to thereby produce proteins or polypeptides, including fusion proteins or polypeptides, encoded by nucleic acids as described herein (e.g., 21953 proteins, mutant forms of 21953 proteins, fusion proteins, and the like).

[185] The recombinant expression vectors ofthe invention can be designed for expression of 21953 proteins in prokaryotic or eukaryotic cells. For example, polypeptides ofthe invention can be expressed inE. coli, insect ceUs (e.g., using baculovirus expression vectors), yeast cells or mammalian cells. Suitable host ceUs are discussed further in Goeddel, (1990) Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.

[186] Expression of proteins in prokaryotes is most often carried out in E. coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus ofthe recombinant proteia Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility ofthe recombinant protein; and 3) to aid in the purification ofthe recombinant protein by acting as a ligand in affinity purification. Often, a proteolytic cleavage site is introduced at the junction ofthe fusion moiety and the recombinant protein to enable separation ofthe recombinant protein from the fusion moiety subsequent to purification ofthe fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith, D.B. and Johnson, KS. (1988) Gene 67:31-40), pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant proteia Additional convenient fusion moieties include the hexa- histidine tag which can be inserted in frame at either terminus of coding region, or in loop regions or inter-domain linkers. A polypeptide that includes a hexa-histidine tag can be purified by immobilized metal chelate chromatography, e.g., using Ni²⁺-NTA resin (Qiagen, Inc.). [187] Purified fusion proteins can be used in 21953 activity assays, (e.g., direct assays or competitive assays described in detail below), or to generate antibodies specific for 21953 proteins. In a preferred embodiment, a fusion protein expressed in a retroviral expression vector ofthe present invention can be used to infect bone marrow ceUs which are subsequently transplanted into irradiated recipients. The pathology ofthe subject recipient is then examined after sufficient time has passed (e.g., six weeks). [188] To maximize recombinant protein expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman, S., (1990) Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California 119-128). Another strategy is to alter the nucleic acid sequence ofthe nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (Wada et al. , (1992) Nucleic Acids Res. 20:2111 -2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques. [189] The 21953 expression vector can be a yeast expression vector, a vector for expression in insect ceUs, e.g., a baculovirus expression vector or a vector suitable for expression in mammalian ceUs.

[190] When used in mammalian cells, the expression vector's control functions can be provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40. [191] In another embodiment, the promoter is an inducible promoter, e.g., a promoter regulated by a steroid hormone, by a polypeptide hormone (e.g., by means of a signal transduction pathway), or by a heterologous polypeptide (e.g., the tetracycline-inducible systems, 'Tet-On" and 'Tet-Off '; see, e.g., Clontech Inc., CA, Gossen and Bujard (1992) Proc. Natl. Acad. Sci. USA 89:5547, and Paillard (1989) Human Gene Therapy 9:983). [192] In another embodiment, the recombinant mammalian expression vector is capable of directing expression ofthe nucleic acid preferentially in a particular ceU type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Non-Umiting examples of suitable tissue-specific promoters include the albumin promoter (Uver-specific; Pinkert etal. (1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton (1988)_4αv. Immunol. 43:235-275), in particular promoters of T ceU receptors (Winoto and Baltimore (1989) EMBOJ. 8:729-733) and immunoglobulins (Banerji etal. (1983) Cell 33:729-740; Queen and Baltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477), pancreas-specific promoters (Edlund etal. (1985) Science 230:912-916), and naammary gland-specific promoters (e.g., milk whey promoter; U.S. Patent No. 4,873,316 and European Application Publication No. 264,166). Developmentally-regulated promoters are also encompassed, for example, the rnurine hox promoters (Kessel and Grass (1990) Science 249:374-379) and the α-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3:537-546).

[193] The invention further provides a recombinant expression vector comprising a

DNA molecule ofthe invention cloned into the expression vector in an antisense orientation. Regulatory sequences (e.g., viral promoters and/or enhancers) operatively linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the constitutive, tissue specific or ceU type specific expression of antisense RNA in a variety of cell types. The antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus.

[194] Another aspect the invention provides a host cell which includes a nucleic acid molecule described herein, e.g., a 21953 nucleic acid molecule within a recombinant expression vector or a 21953 nucleic acid molecule containing sequences which allow it to homologously recombine into a specific site ofthe host cell's genome. The terms "host cell" and "recombinant host cell" are used interchangeably herein. Such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent ceU, but are still included within the scope ofthe term as used hereia

[195] A host cell can be any prokaryotic or eukaryotic cell. For example, a 21953 protein can be expressed in bacterial cells (such as E. coli), insect ceUs, yeast or n-ammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells). Other suitable host ceUs are known to those skilled in the art.

[196] Vector DNA can be introduced into host cells via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co- precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. [197] A host ceU ofthe invention can be used to produce (i.e., express) a 21953 protein. Accordingly, the invention further provides methods for producing a 21953 protein using the host ceUs ofthe inventioa In one embodiment, the method includes culturing the host ceU ofthe invention (into which a recombinant expression vector encoding a 21953 protein has been introduced) in a suitable medium such that a 21953 protein is produced. In another embodiment, the method further includes isolating a 21953 protein from the medium or the host cell.

[198] In another aspect, the invention features, a ceU or purified preparation of ceUs which include a 21953 transgene, or which otherwise misexpress 21953. The ceU preparation can consist of human or non-human cells, e.g., rodent cells, e.g., mouse or rat cells, rabbit cells, or pig cells. In preferred embodiments, the cell or ceUs include a 21953 transgene, e.g., a heterologous form of a 21953, e.g., a gene derived from humans (in the case of anon-human cell). The 21953 transgene can be misexpressed, e.g., overexpressed or underexpressed. In other preferred embodiments, the cell or cells include a gene that mis- expresses an endogenous 21953, e.g., a gene the expression of which is disrupted, e.g., a knockout. Such ceUs can serve as a model for studying disorders that are related to mutated or mis-expressed 21953 aUeles or for use in drug screening.

[199] In another aspect, the invention features, a human cell, e.g., a hematopoietic stem cell, transformed with nucleic acid which encodes a subject 21953 polypeptide. [200] Also provided are ceUs, preferably human cells, e.g., human hematopoietic or fibroblast cells, in which an endogenous 21953 is under the control of a regulatory sequence that does not normally control the expression ofthe endogenous 21953 gene. The expression characteristics of an endogenous gene within a cell, e.g., a ceU line or microorganism, can be modified by inserting a heterologous DNA regulatory element into the genome of he cell such that the inserted regulatory element is operably linked to the endogenous 21953 gene. For example, an endogenous 21953 gene which is "transcriptionally silent," e.g., not normally expressed, or expressed only at very low levels, may be activated by inserting a regulatory element which is capable of promoting the expression of a normally expressed gene product in that ceU. Techniques such as targeted homologous recombinations, can be used to insert the heterologous DNA as described in, e.g., Chappel, US 5,272,071; WO 91/06667, published in May 16, 1991. [201] In a preferred embodiment, recombinant ceUs described herein can be used for replacement therapy in a subject. For example, a nucleic acid encoding a 21953 polypeptide operably linked to an inducible promoter (e.g., a steroid hormone receptor-regulated promoter) is introduced into a human or nonhuman, e.g., mammalian, e.g., porcine recombinant ceU. The ceU is cultivated and encapsulated in a biocompatible material, such as poly-lysine alginate, and subsequently implanted into the subject. See, e.g., Lanza (1996) Nat. Biotechnol. 14:1107; Joki etal. (2001) Nat. Biotechnol 19:35; and U.S. Patent No. 5,876,742. Production of 21953 polypeptide can be regulated in the subject by administering an agent (e.g., a steroid hormone) to the subject. In another preferred embodiment, the implanted recombinant cells express and secrete an antibody specific for a 21953 polypeptide. The antibody can be any antibody or any antibody derivative described herein.

Transgenic Animals

[202] The invention provides non-human transgenic animals. Such animals are useful for studying the function and/or activity of a 21953 protein and for identifying and/or evaluating modulators of 21953 activity. As used herein, a "transgenic animal" is a non- human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more ofthe cells ofthe animal includes a transgene. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, and the like. A transgene is exogenous DNA or a rearrangement, e.g., a deletion of endogenous chromosomal DNA, which preferably is integrated into or occurs in the genome ofthe ceUs of a transgenic animal. A transgene can direct the expression of an encoded gene product in one or more cell types or tissues ofthe transgemc animal, other transgenes, e.g., a knockout, reduce expression. Thus, a transgenic animal can be one in which an endogenous 21953 gene has been altered by, e.g., by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell ofthe animal, e.g., an embryonic ceU ofthe animal, prior to development ofthe animal. [203] Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression ofthe transgene. A tissue-specific regulatory sequence(s) can be operably linked to a transgene ofthe invention to direct expression of a 21953 protein to particular cells. A transgenic founder animal can be identified based upon the presence of a 21953 transgene in its genome and/or expression of 21953 mRNA in tissues or cells ofthe animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene encoding a 21953 protein can further be bred to other transgenic animals carrying other transgenes.

[204] 21953 proteins or polypeptides can be expressed in transgenic animals or plants, e.g., a nucleic acid encoding the protein or polypeptide can be introduced into the genome of an animal. In preferred embodiments the nucleic acid is placed under the control of a tissue specific promoter, e.g., a milk or egg specific promoter, and recovered from the milk or eggs produced by the animal. Suitable animals are mice, pigs, cows, goats, and sheep.

[205] The invention also includes a population of cells from a transgenic animal, as discussed, e.g., below.

Uses

[206] The nucleic acid molecules, proteins, protein homologues, and antibodies described herein can be used in one or more ofthe following methods: a) screening assays; b) predictive medicine (e.g., diagnostic assays, prognostic assays, monitoring clinical trials, and pharmacogenetics); c) methods of treatment (e.g., therapeutic and prophylactic); and d) in vitro modification of polypeptide hormones.

[207] The isolated nucleic acid molecules ofthe invention can be used, for example, to express a 21953 protein (e.g., via a recombinant expression vector in a host ceU in gene therapy applications), to detect a 21953 mRNA (e.g., in a biological sample) or a genetic alteration in a 21953 gene, and to modulate 21953 activity, as described further below. The 21953 proteins can be used to treat disorders characterized by insufficient or excessive production of a 21953 substrate or production of 21953 inhibitors. In addition, the 21953 proteins can be used to screen for naturally occurring 21953 substrates, to screen for drugs or compounds which modulate 21953 activity, as well as to treat disorders characterized by insufficient or excessive production of 21953 protein or production of 21953 protein forms which have decreased, aberrant or unwanted activity compared to 21953 wild type protein (e.g., a cell proliferative or cell differentiative disorder, e.g., a cancer, e.g., a cancer ofthe lung, prostate, breast, ovary, or colon). Moreover, the anti-21953 antibodies ofthe invention can be used to detect and isolate 21953 proteins, regulate the bioavailability of 21953 proteins, and modulate 21953 activity.

[208] A method of evaluating a compound for the ability to interact with, e.g., bind, a subject 21953 polypeptide is provided. The method includes: contacting the compound with the subject 21953 polypeptide; and evaluating ability ofthe compound to interact with, e.g., to bind or form a complex with the subject 21953 polypeptide. This method can be performed in vitro, e.g., in a ceU free system, or in vivo, e.g., in a two-hybrid interaction trap assay. This method can be used to identify naturally occurring molecules that interact with subject 21953 polypeptide. It can also be used to find natural or synthetic inhibitors of subject 21953 polypeptide. Screening methods are discussed in more detail below. [209] The 21953 polypeptide is also an enzyme useful for processing polypeptide hormone precursors. For example, the 21953 polypeptide can be used in a method that includes a) providing a polypeptide hormone precursor; b) combining the polypeptide hormone polypeptide with a 21953 polypeptide or active fragment thereof (e.g., in an effective amount) to provide a reaction mixture; and c) maintaining the mixture under conditions such that the polypeptide hormone precursor is modified to yield the processed polypeptide hormone, e.g., an active form thereof. The method can further include d) separating the processed polypeptide hormone from the 21953 polypeptide. The polypeptide hormone precursor can be obtained from a synthetic process or from a producing cell. The method can be used in the preparation of a pharmaceutical composition that includes the processed hormone.

Screening Assays

[210] The invention provides methods (also referred to herein as "screening assays") for identifying modulators, i.e., candidate or test compounds or agents (e.g., proteins, peptides, peptidomimetics, peptoids, small molecules or other drugs) which bind to 21953 proteins, have a stimulatory or inhibitory effect on, for example, 21953 expression or 21953 activity, or have a stimulatory or inhibitory effect on, for example, the expression or activity of a 21953 substrate. Compounds thus identified can be used to modulate the activity of target gene products (e.g., 21953 genes) in a therapeutic protocol, to elaborate the biological function ofthe target gene product, or to identify compounds that disrupt normal target gene interactions.

[211] The prolyl oligopeptidase activity of a 21953 polypeptide can be assayed in vitro using an enzymatic assay such as described in Abbott etal. (199) FEBSLett. 458:278-284 and Abbott et al. (2000) Eur. J. Biochem 267:6140-4150. A sample to be assayed is combined with substrate in phosphate buffer pH 7.4. Substrates include H-Gly-Pro- - nitroanilide (NA)/HC1 (Sigma Corp, MO, USA), and Gly-Pro-7-amino-4- trifluoromethylcoumarin (Calbiochem, San Diego, CA, USA) and other peptidyl substrates. The reaction is incubated for 30 minutes at 37°C. For example, hydrolysis of H-Gly-Pro- p A is monitored spectroscopically at 405 nm. The sample to be assayed can be a purified 21953 polypeptide, e.g., a 21953 polypeptide or a 21953 fusion protein purified by a method described hereia Routine Michaelis-Menten analysis of kinetic parameters can be used to quantify the enzymatic activity. Alternatively, the reaction can be quenched and total substrate hydrolyzed can be measured as indication ofthe activity.

[212] In one embodiment, the invention provides assays for screening candidate or test compounds which are substrates of a 21953 protein or polypeptide or a biologically active portion thereof. In another embodiment, the invention provides assays for screening candidate or test compounds that bind to or modulate the activity of a 21953 protein or polypeptide or a biologically active portion thereof. The afore-mentioned assay can be used by adding a candidate or test compound to the reaction mixture, either before or together with addition ofthe substrate.

[213] The test compounds ofthe present invention can be obtained using any ofthe numerous approaches in combinatorial library methods known in the art, including: biological libraries; peptoid Ubraries (libraries of molecules having the functionalities of peptides, but with a novel, non-peptide backbone which are resistant to enzymatic degradation but which nevertheless remain bioactive; see, e.g., Zuckermaiin, RN. et al. (1994) J. Med. Chem. 37:2678-85); spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the 'one-bead one- compound' Ubrary method; and synthetic library methods using affinity chromatography selectioa The biological library and peptoid library approaches are limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oUgomer or smatt molecule libraries of compounds (Lam (1997) Anticancer Drug Des. 12:145). [214] Examples of methods for the synthesis of molecular Ubraries can be found in the art, for example in: DeWitt etal. (1993) Proc. Natl. Acad. Sci. USA. 90:6909; Erb etal. (1994) Proc. Natl. Acad. Sci. USA 91:11422; Zuckermann et α . (1994). J. Med. Chem. 37:2678; Cho etal. (1993) Science 261:1303; Carrell etal. (1994) Angew. Chem. Int. Ed. Engl 33:2059; Carell etal. (1994) Angew. Chem. Int. Ed. Engl 33:2061; and GaUop etal (1994) J Med. Chem. 37:1233. Combinatorial chemical Ubraries can be designed based on known substrates of oligopeptidases. For example, compounds can be designed that are peptidomimetics, e.g., phosphonate analogs of a peptide substrate, such as a proline- containing peptide. [215] Libraries of compounds may be presented in solution (e.g., Houghten (1992)

Biotechniques 13:412-421), or on beads (Lam (1991) Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (Ladner, U.S. Patent No. 5,223,409), spores (Ladner U.S. Patent No. 5,223,409), plasmids (Cull etal (1992) Proc Natl Acad Sci USA 89:1865- 1869) or on phage (Scott and Smith (1990) Science 249:386-390; Devlin (1990) Science 249:404-406; Cwirla et al. (1990) Proc. Natl. Acad. Sci. 87:6378-6382; Felici (1991) J. Mol. Biol. 222:301-310; Ladner supra.).

[216] In one embodiment, an assay is a cell-based assay in which a cell which expresses a 21953 protein or biologically active portion thereof is contacted with a test compound, and the ability ofthe test compound to modulate 21953 activity is determined. Determining the ability ofthe test compound to modulate 21953 activity can be accomplished by monitoring, for example, prolyl oligopeptidase activity. The cell, for example, can be of mammalian origin, e.g., human.

[217] The ability ofthe test compound to modulate 21953 binding to a compound, e.g., a 21953 substrate, orto bind to 21953 can also be evaluated. This can be accomplished, for example, by coupling the compound, e.g., the substrate, with a radioisotope or enzymatic label such that binding ofthe compound, e.g., the substrate, to 21953 can be determined by detecting the labeled compound, e.g., substrate, in a complex. Alternatively, 21953 could be coupled with a radioisotope or enzymatic label to monitor the ability of a test compound to modulate 21953 binding to a 21953 substrate in a complex. For example, compounds (e.g., 21953 substrates) can be labeled with ¹²⁵1, ³⁵S, ¹⁴C, or ³H, either directly or indirectly, and the radioisotope detected by direct counting of radioemmission or by scintillation counting. Alternatively, compounds can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.

[218] The ability of a compound (e.g., a 21953 substrate) to interact with 21953 with or without the labeling of any ofthe interactants can be evaluated. For example, a microphysiometer can be used to detect the interaction of a compound with 21953 without the labeling of either the compound or the 21953. McConnell, H. M. et al. (1992) Science 257:1906-1912. As used herein, a "microphysiometer" (e.g., Cytosensor) is an analytical instrument that measures the rate at which a cell acidifies its environment using a light- addressable potentiometric sensor (LAPS). Changes in this acidification rate can be used as an indicator ofthe interaction between a compound and 21953. [219] In yet another embodiment, a cell-free assay is provided in which a 21953 protein or biologically active portion thereof is contacted with a test compound and the ability ofthe test compound to bind to the 21953 protein or biologically active portion thereof is evaluated. Preferred biologically active portions ofthe 21953 proteins to be used in assays ofthe present invention include fragments which participate in interactions with non-21953 molecules, e.g., fragments with high surface probability scores. [220] Soluble and/or membrane-bound forms of isolated proteins (e.g., 21953 proteins or biologically active portions thereof) can be used in the cell-free assays ofthe inventioa When membrane-bound forms ofthe protein are used, it may be desirable to utilize a solubilizing agent. Examples of such solubilizing agents include non-ionic detergents such as n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton® X-100, Triton® X-114, Thesit®, Isotridecypoly(ethylene glycol ether^, 3-[(3-cholarmdopropyl)dimethylamminio]-l-propane sulfonate (CHAPS), 3-[(3 -cholamidopropyl)dimethylamminio] -2-hydroxy- 1 -propane sulfonate (CHAPSO), or N-dodecyl=N,N-dimethyl-3-ammonio-l-propane sulfonate. [221] Cell-free assays involve preparing a reaction mixture ofthe target gene protein and the test compound under conditions and for a time sufficient to allow the two components to interact and bind, thus forming a complex that can be removed and/or detected.

[222] The interaction between two molecules can also be detected, e.g., using fluorescence energy transfer (FET) (see, for example, Lakowicz et al, U.S. Patent No. 5,631,169; Stavrianopoulos, etal, U.S. Patent No. 4,868,103). Afluorophore label on the first, 'donor' molecule is selected such that its emitted fluorescent energy will be absorbed by a fluorescent label on a second, 'acceptor' molecule, which in turn is able to fluoresce due to the absorbed energy. Alternately, the 'donor' protein molecule may simply utiUze the natural fluorescent energy of tryptophan residues. Labels are chosen that emit different wavelengths of Ught, such that the 'acceptor' molecule label may be differentiated from that ofthe 'donor'. Since the efficiency of energy transfer between the labels is related to the distance separating the molecules, the spatial relationship between the molecules can be assessed. la a situation in which binding occurs between the molecules, the fluorescent emission ofthe 'acceptor' molecule label in the assay should be maximal An FET binding event can be conveniently measured through standard fluorometric detection means well known in the art (e.g., using a fluorimeter).

[223] In another embodiment, determining the ability ofthe 21953 protein to bind to a target molecule can be accomplished using real-time Biomolecular Interaction Analysis (BIA) (see, e.g., Sjolander, S. and Urbaniczky, C. (1991) Anal. Chem. 63:2338-2345 and Szabo etal. (1995) Curr. Opin. Struct. Biol. 5:699-705). "Surface plasmon resonance" or "BIA" detects biospecific interactions in real time, without labeling any ofthe interactants (e.g., BIAcore). Changes in the mass at the binding surface (indicative of a binding event) result in alterations ofthe refractive index of Ught near the surface (the optical phenomenon of surface plasmon resonance (SPR)), resulting in a detectable signal which can be used as an indication of real-time reactions between biological molecules. [224] In one embodiment, the target gene product or the test substance is anchored onto a soUd phase. The target gene product/test compound complexes anchored on the soUd phase can be detected at the end ofthe reaction. Preferably, the target gene product can be anchored onto a solid surface, and the test compound, (which is not anchored), can be labeled, either directly or indirectly, with detectable labels discussed herein. [225] It may be desirable to immobilize either 21953, an anti-21953 antibody or its target molecule to facilitate separation of complexed from uncomplexed forms of one or both ofthe proteins, as well as to accommodate automation ofthe assay. Binding of a test compound to a 21953 protein, or interaction of a 21953 protein with atarget molecule in the presence and absence of a candidate compound, can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided which adds a domain that allows one or both ofthe proteins to be bound to a matrix. For example, glutathione-S-transferase/21953 fusion proteins or glutathione-S-transferase/target fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microtiter plates, which are then combined with the test compound or the test compound and either the non-adsorbed target protein or 21953 protein, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate weUs are washed to remove any unbound components, the matrix immobUized in the case of beads, complex determined either directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of 21953 binding or activity determined using standard techniques.

[226] Other techniques for immobilizing either a 21953 protein or a target molecule on matrices include using conjugation of biotin and streptavidin Biotinylated 21953 protein or target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, IL), and immobilized in the weUs of streptavidin-coated 96 well plates (Pierce Chemical). [227] In order to conduct the assay, the non-immobilized component is added to the coated surface containing the anchored component. After the reaction is complete, unreacted components are removed (e.g., by washing) under conditions such that any complexes formed will remain immobilized on the solid surface. The detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the previously non-immobilized component is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the previously non-immobilized component is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the immobilized component (the antibody, in turn, can be directly labeled or indirectly labeled with, e.g., a labeled anti-Ig antibody).

[228] In one embodiment, this assay is performed utiUzing antibodies reactive with

21953 protein or target molecules but which do not interfere with binding ofthe 21953 protein to its target molecule. Such antibodies can be derivatized to the weUs ofthe plate, and unbound target or 21953 protein trapped in the weUs by antibody conjugation Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the 21953 protein or target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the 21953 protein or target molecule. [229] Alternatively, cell free assays can be conducted in a liquid phase. In such an assay, the reaction products are separated from unreacted components, by any of a number of standard techniques, including but not limited to: differential centrifugation (see, for example, Rivas, G, and Minton, AP., (1993) Trends Biochem Sci 18:284-7); chromatography (gel filtration chromatography, ion-exchange chromatography); electrophoresis (see, e.g., Ausubel, F. et al, eds. Current Protocols in Molecular Biology 1999, J. Wiley: New York.); and immunoprecipitation (see, for example, Ausubel F. etal, eds. (1999) Current Protocols in Molecular Biology, J. Wiley: New York). Such resins and chromatographic techniques are known to one skilled in the art (see, e.g., Heegaard, N.H, (1998)J o/ Recognit 11:141-8; Hage, D.S., and Tweed, S.A (1997) J Chromatogr B Biomed Sci Appl. 699:499-525). Further, fluorescence energy transfer may also be conveniently utilized, as described herein, to detect binding without further purification of the complex from solution

[230] In a preferred embodiment, the assay includes contacting the 21953 protein or biologically active portion thereof with a known compound which binds 21953 to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of he test compound to interact with a 21953 protein, wherein determining the ability ofthe test compound to interact with a 21953 protein includes determining the ability ofthe test compound to preferentially bind to 21953 or biologically active portion thereof) or to modulate the activity of a target molecule, as compared to the known compound. [231] The target gene products ofthe invention can, in vivo, interact with one or more cellular or extracellular macromolecules, such as proteins. For the purposes of this discussion, such cellular and extracellular macromolecules are referred to herein as "binding partners." Compounds that disrupt such interactions can be useful in regulating the activity ofthe target gene product. Such compounds can include, but are not limited to molecules such as antibodies, peptides, and small molecules. The preferred target genes/products for use in this embodiment are the 21953 genes herein identified. In an alternative embodiment, the invention provides methods for determining the ability ofthe test compound to modulate the activity of a 21953 protein through modulation ofthe activity of a downstream effector of a 21953 target molecule. For example, the activity ofthe effector molecule on an appropriate target can be determined, or the binding ofthe effector to an appropriate target can be determined, as previously described.

[232] To identify compounds that interfere with the interaction between the target gene product and its cellular or extracellular binding partner(s), a reaction mixture containing the target gene product and the binding partner is prepared, under conditions and for a time sufficient, to allow the two products to form complex. In order to test an inhibitory agent, the reaction mixture is provided in the presence and absence ofthe test compound. The test compound can be initiaUy included in the reaction mixture, or can be added at a time subsequent to the addition ofthe target gene and its cellular or extraceUular binding partner. Control reaction mixtures are incubated without the test compound or with a placebo. The formation of any complexes between the target gene product and the ceUular or extraceUular binding partner is then detected. The formation of a complex in the control reaction, but not in the reaction mixture containing the test compound, indicates that the compound interferes with the interaction ofthe target gene product and the interactive binding partner. Additionally, complex formation within reaction mixtures containing the test compound and normal target gene product can also be compared to complex formation within reaction mixtures containing the test compound and mutant target gene product. This comparison can be important in those cases wherein it is desirable to identify compounds that disrupt interactions of mutant but not normal target gene products. [233] These assays can be conducted in a heterogeneous or homogeneous format.

Heterogeneous assays involve anchoring either the target gene product or the binding partner onto a solid phase, and detecting complexes anchored on the solid phase at the end ofthe reaction. In homogeneous assays, the entire reaction is carried out in a Uquid phase. In either approach, the order of addition of reactants can be varied to obtain different information about the compounds being tested. For example, test compounds that interfere with the interaction between the target gene products and the binding partners, e.g., by competition, can be identified by conducting the reaction in the presence ofthe test substance. Alternatively, test compounds that disrupt preformed complexes, e.g., compounds with higher binding constants that displace one ofthe components from the complex, can be tested by adding the test compound to the reaction mixture after complexes have been formed. The various formats are briefly described below. [234] In a heterogeneous assay system, either the target gene product or the interactive cellular or extracellular binding partner, is anchored onto a soUd surface (e.g., a microtiter plate), while the non-anchored species is labeled, either directly or indirectly. The anchored species can be immobilized by non-covalent or covalent attachments. Alternatively, an immobilized antibody specific for the species to be anchored can be used to anchor the species to the solid surface.

[235] In order to conduct the assay, the partner ofthe immobilized species is exposed to the coated surface with or without the test compound. After the reaction is complete, unreacted components are removed (e.g., by washing) and any complexes formed will remain immobilized on the soUd surface. Where the non-immobilized species is pre- labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the non-immobilized species is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the initially non-immobilized species (the antibody, in turn, can be directly labeled or indirectly labeled with, e.g., a labeled anti-Ig antibody). Depending upon the order of addition of reaction components, test compounds that inhibit complex formation or that disrupt preformed complexes can be detected.

[236] Alternatively, the reaction can be conducted in a Uquid phase in the presence or absence ofthe test compound, the reaction products separated from unreacted components, and complexes detected; e.g., using an immobilized antibody specific for one ofthe binding components to anchor any complexes formed in solution, and a labeled antibody specific for the other partner to detect anchored complexes. Again, depending upon the order of addition of reactants to the liquid phase, test compounds that inhibit complex or that disrupt preformed complexes can be identified.

[237] In an alternate embodiment ofthe invention, a homogeneous assay can be used.

For example, a preformed complex ofthe target gene product and the interactive cellular or extracellular binding partner product is prepared in that either the target gene products or their binding partners are labeled, but the signal generated by the label is quenched due to complex formation (see, e.g., U.S. Patent No. 4,109,496 that utilizes this approach for immunoassays). The addition of a test substance that competes with and displaces one of the species from the preformed complex will result in the generation of a signal above background. In this way, test substances that disrupt target gene product-binding partner interaction can be identified.

[238] In yet another aspect, the 21953 proteins can be used as "bait proteins" in a two- hybrid assay or three-hybrid assay (see, e.g., U.S. Patent No. 5,283,317; Zervos etal. (1993) Cell 72:223-232; Madura etal. (1993) J. Biol. Chem. 268:12046-12054; Bartel et /. (1993) Biotechniques 14:920-924; Iwabuchi etal. (1993) Oncogene 8:1693-1696; and Brent WO94/10300), to identify other proteins, which bind to or interact with 21953 ("21953- binding proteins" or "21953-bp") and are involved in 21953 activity. Such 21953-bps can be activators or inhibitors of signals by the 21953 proteins or 21953 targets as, for example, downstream elements of a 21953-mediated signaling pathway. [239] The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. In one construct, the gene that codes for a 21953 protein is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct, a DNA sequence, from a Ubrary of DNA sequences, that encodes an unidentified protein ("prey" or "sample") is fused to a gene that codes for the activation domain ofthe known transcription factor. (Alternatively the: 21953 protein can be the fused to the activator domain.) If the "bait" and the "prey" proteins are able to interact, in vivo, forming a 21953-dependent complex, the DNA-binding and activation domains ofthe transcription factor are brought into close proximity. This proximity aUows transcription of a reporter gene (e.g., lacZ) which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression ofthe reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene which encodes the protein which interacts with the 21953 protein.

[240] In another embodiment, modulators of 21953 expression are identified. For example, a ceU or ceU free mixture is contacted with a candidate compound and the expression of 21953 mRNA or protein evaluated relative to the level of expression of 21953 mRNA or protein in the absence ofthe candidate compound. When expression of 21953 mRNA or protein is greater in the presence ofthe candidate compound than in its absence, the candidate compound is identified as a stimulator of 21953 mRNA or protein expression. Alternatively, when expression of 21953 mRNA or protein is less (statistically significantly less) in the presence ofthe candidate compound than in its absence, the candidate compound is identified as an inhibitor of 21953 mRNA or protein expression. The level of 21953 mRNA or protein expression can be determined by methods described herein for detecting 21953 mRNA or protein.

[241] In another aspect, the invention pertains to a combination of two or more ofthe assays described herein. For example, a modulating agent can be identified using a cell- based or a cell free assay, and the abUity ofthe agent to modulate the activity of a 21953 protein can be confirmed in vivo, e.g., in an animal such as an animal model for a ceU proUferative or cell differentiative disorder, e.g., a cancer, e.g., a cancer ofthe lung, prostate, breast, or colon; an animal model for an immunological disorder; or an animal model for a neurological disorder.

[242] This invention further pertains to novel agents identified by the above-described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein (e.g., a 21953 modulating agent, an antisense 21953 nucleic acid molecule, a 21953-specific antibody, or a 21953-binding partner) in an appropriate animal model to determine the efficacy, toxicity, side effects, or mechanism of action, of treatment with such an agent. Furthermore, novel agents identified by the above-described screening assays can be used for treatments as described herein

Detection Assays

[243] Portions or fragments ofthe nucleic acid sequences identified herein can be used as polynucleotide reagents. For example, these sequences can be used to: (i) map their respective genes on a chromosome e.g., to locate gene regions associated with genetic disease or to associate 21953 with a disease; (u) identify an individual from a minute biological sample (tissue typing); and (in) aid in forensic identification of a biological sample. These appUcations are described in the subsections below.

Chromosome Mapping

[244] The 21953 nucleotide sequences or portions thereof can be used to map the location ofthe 21953 genes on a chromosome. This process is called chromosome mapping.

Chromosome mapping is useful in correlating the 21953 sequences with genes associated with disease.

[245] Briefly, 21953 genes can be mapped to chromosomes by preparing PCR primers

(preferably 15-25 bp in length) from the 21953 nucleotide sequences. These primers can then be used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the 21953 sequences will yield an ampUfied fragment.

[246] A panel of somatic cell hybrids in which each cell line contains either a single human chromosome or a smaU number of human chromosomes, and a full set of mouse chromosomes, can allow easy mapping of individual genes to specific human chromosomes.

(DΕustachio P. etal. (1983) Science 220:919-924).

[247] Other mapping strategies e.g., in situ hybridization (described in Fan, Y. et al.

(1990) Proc. Natl. Acad. Sci. USA, 87:6223-27), pre-screening with labeled flow-sorted chromosomes, and pre-selection by hybridization to chromosome specific cDNA Ubraries can be used to map 21953 to a chromosomal location

[248] Fluorescence in situ hybridization (FISH) of a DNA sequence to a metaphase chromosomal spread can further be used to provide a precise chromosomal location in one step. The FISH technique can be used with a DNA sequence as short as 500 or 600 bases. However, clones larger than 1,000 bases have a higher Ukelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection Preferably 1,000 bases, and more preferably 2,000 bases will suffice to get good results at a reasonable amount of time. For a review of this technique, see Verma et al, Human Chromosomes: A Manual of Basic Techniques ((1988) Pergamon Press, New York). [249] Reagents for chromosome mapping can be used individually to mark a single chromosome or a single site on that chromosome, or panels of reagents can be used for marking multiple sites and/or multiple chromosomes. Reagents corresponding to noncoding regions ofthe genes actuaUy are preferred for mapping purposes. Coding sequences are more hkely to be conserved within gene families, thus increasing the chance of cross hybridizations during chromosomal mapping.

[250] Once a sequence has been mapped to a precise chromosomal location, the physical position ofthe sequence on the chromosome can be correlated with genetic map data. (Such data are found, for example, in V. McKusick, Mendelian Inheritance in Man, available on-Une through Johns Hopkins University Welch Medical Library). The relationship between a gene and a disease, mapped to the same chromosomal region, can then be identified through linkage analysis (co-inheritance of physically adjacent genes), described in, for example, Egeland, J. etal. (1987) Nature, 325:783-787. [251] Moreover, differences in the DNA sequences between individuals affected and unaffected with a disease associated with the 21953 gene, can be determined. If a mutation is observed in some or all ofthe affected individuals but not in any unaffected individuals, then the mutation is likely to be the causative agent ofthe particular disease. Comparison of affected and unaffected individuals generaUy involves first looking for structural alterations in the chromosomes, such as deletions or translocations that are visible from chromosome spreads or detectable using PCR based on that DNA sequence. Ultimately, complete sequencing of genes from several individuals can be performed to confirm the presence of a mutation and to distinguish mutations from polymorphisms.

Tissue Typing

[252] 21953 sequences can be used to identify individuals from biological samples using, e.g., restriction fragment length polymorphism (RFLP). In this technique, an individual's genomic DNA is digested with one or more restriction enzymes, the fragments separated, e.g., in a Southern blot, and probed to yield bands for identification. The sequences ofthe present invention are useful as additional DNA markers for RFLP (described in U.S. Patent 5,272,057).

[253] Furthermore, the sequences ofthe present invention can also be used to determine the actual base-by-base DNA sequence of selected portions of an individual's genome. Thus, the 21953 nucleotide sequences described herein can be used to prepare two PCR primers from the 5' and 3' ends ofthe sequences. These primers can then be used to amplify an individual's DNA and subsequently sequence it. Panels of corresponding DNA sequences from individuals, prepared in this manner, can provide unique individual identifications, as each individual wUl have a unique set of such DNA sequences due to aUelic differences.

[254] Allelic variation occurs to some degree in the coding regions of these sequences, and to a greater degree in the noncoding regions. Each ofthe sequences described herein can, to some degree, be used as a standard against which DNA from an individual can be compared for identification purposes. Because greater numbers of polymorphisms occur in the noncoding regions, fewer sequences are necessary to differentiate individuals. The noncoding sequences of SEQ ID NO.l can provide positive individual identification with a panel of perhaps 10 to 1,000 primers which each yield a noncoding amplified sequence of 100 bases. If predicted coding sequences, such as those in SEQ ID NO:3 are used, a more appropriate number of primers for positive individual identification would be 500-2,000. [255] If a panel of reagents from 21953 nucleotide sequences described herein is used to generate a unique identification database for an individual those same reagents can later be used to identify tissue from that individual. Using the unique identification database, positive identification ofthe individual, living or dead, can be made from extremely small tissue samples.

Use of Partial 21953 Sequences in Forensic Biology

[256] DNA-based identification techniques can also be used in forensic biology. To make such an identification, PCR technology can be used to amplify DNA sequences taken from very small biological samples such as tissues, e.g., hair or skin, or body fluids, e.g., blood, saliva, or semen found at a crime scene. The amplified sequence can then be compared to a standard, thereby aUowing identification ofthe origin ofthe biological sample. [257] The sequences ofthe present invention can be used to provide polynucleotide reagents, e.g., PCR primers, targeted to specific loci in the human genome, which can enhance the reUability of DNA-based forensic identifications by, for example, providing another "identification marker" (i.e. another DNA sequence that is unique to a particular individual). As mentioned above, actual base sequence information can be used for identification as an accurate alternative to patterns formed by restriction enzyme generated fragments. Sequences targeted to noncoding regions of SEQ ID NO:l (e.g., fragments derived from the noncoding regions of SEQ ID NO: 1 having a length of at least 20 bases, preferably at least 30 bases) are particularly appropriate for this use. [258] The 21953 nucleotide sequences described herein can further be used to provide polynucleotide reagents, e.g., labeled or labelable probes which can be used in, for example, an in situ hybridization technique, to identify a specific tissue. This can be very useful in cases where a forensic pathologist is presented with a tissue of unknown origin Panels of such 21953 probes can be used to identify tissue by species and/or by organ type. [259] In a similar fashion, these reagents, e.g., 21953 primers or probes can be used to screen tissue culture for contamination (i.e. screen for the presence of a mixture of different types of cells in a culture).

Predictive Medicine

[260] The present invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, and monitoring clinical trials are used for prognostic

(predictive) purposes to thereby treat an individual.

[261] Generally, the invention provides, a method of determining if a subject is at risk for a disorder related to a lesion in or the misexpression of a gene which encodes 21953.

[262] Such disorders include, e.g., a disorder associated with the misexpression of

21953 gene; a disorder of cell proliferation (such as lung, breast, colon, prostate, or ovarian cancer) or ofthe nervous system

[263] The method includes one or more ofthe following:

[264] detecting, in a tissue ofthe subject, the presence or absence of a mutation which affects the expression ofthe 21953 gene, or detecting the presence or absence of a mutation in a region which controls the expression ofthe gene, e.g., a mutation in the 5' control region; [265] detecting, in a tissue ofthe subject, the presence or absence of a mutation which alters the structure ofthe 21953 gene;

[266] detecting, in a tissue ofthe subject, the misexpression ofthe 21953 gene, at the mRNA level, e.g., detecting a non-wild type level of a mRNA ;

[267] detecting, in a tissue ofthe subject, the misexpression ofthe gene, at the protein level, e.g., detecting anon-wUd type level of a 21953 polypeptide.

[268] In preferred embodiments the method includes: ascertaining the existence of at least one of: a deletion of one or more nucleotides from the 21953 gene; an insertion of one or more nucleotides into the gene, a point mutation, e.g., a substitution of one or more nucleotides ofthe gene, a gross chromosomal rearrangement ofthe gene, e.g., a translocation, inversion, or deletion.

[269] For example, detecting the genetic lesion can include: (i) providing a probe/primer including an oligonucleotide containing a region of nucleotide sequence which hybridizes to a sense or antisense sequence from SEQ ID NO: 1, or naturaUy occurring mutants thereof or 5' or 3' flanking sequences naturally associated with the 21953 gene; (n) exposing the probe/primer to nucleic acid ofthe tissue; and detecting, by hybridization, e.g., in situ hybridization, ofthe probe primer to the nucleic acid, the presence or absence ofthe genetic lesion.

[270] In preferred embodiments detecting the misexpression includes ascertaining the existence of at least one of: an alteration in the level of a messenger RNA transcript ofthe

21953 gene; the presence of a non- wild type splicing pattern of a messenger RNA transcript ofthe gene; or a non- wild type level of 21953.

[271] Methods ofthe invention can be used prenatally or to determine if a subject's offspring will be at risk for a disorder.

[272] In preferred embodiments the method includes determining the stracture of a

21953 gene, an abnormal structure being indicative of risk for the disorder.

[273] In preferred embodiments the method includes contacting a sample from the subject with an antibody to the 21953 protein or a nucleic acid, which hybridizes specifically with the gene. These and other embodiments are discussed below. Diagnostic and Prognostic Assays

[274] Diagnostic and prognostic assays ofthe invention include method for assessing the expression level of 21953 molecules and for identifying variations and mutations in the sequence of 21953 molecules.

[275] Expression Monitoring and Profiling. The presence, level, or absence of 21953 protein or nucleic acid in a biological sample can be evaluated by obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting 21953 protein or nucleic acid (e.g., mRNA, genomic DNA) that encodes 21953 protein such that the presence of 21953 protein or nucleic acid is detected in the biological sample. The term "biological sample" includes tissues, ceUs and biological fluids isolated from a subject, as weU as tissues, cells and fluids present within a subject. A preferred biological sample is serum The level of expression ofthe 21953 gene can be measured in a number of ways, including, but not Umited to: measuring the mRNA encoded by the 21953 genes; measuring the amount of protein encoded by the 21953 genes; or measuring the activity ofthe protein encoded by the 21953 genes.

[276] The level of mRNA corresponding to the 21953 gene in a cell can be determined both by in situ and by in vitro formats.

[277] The isolated mRNA can be used in hybridization or amplification assays that include, but are not limited to, Southern or Northern analyses, polymerase chain reaction analyses and probe arrays. One preferred diagnostic method for the detection of mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to the mRNA encoded by the gene being detected. The nucleic acid probe can be, for example, a full-length 21953 nucleic acid, such as the nucleic acid of SEQ ID NO:l, or a portion thereof) such as an oUgonucleotide of at least 7, 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to 21953 mRNA or genomic DNA. The probe can be disposed on an address of an array, e.g., an array described below. Other suitable probes for use in the diagnostic assays are described herein.

[278] In one format, mRNA (or cDNA) is immobilized on a surface and contacted with the probes, for example by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane, such as nitroceUulose. In an alternative format, the probes are immobiUzed on a surface and the mRNA (or cDNA) is contacted with the probes, for example, in a two-dimensional gene chip array described below. A skilled artisan can adapt known mRNA detection methods for use in detecting the level of mRNA encoded by the 21953 genes.

[279] The level of mRNA in a sample that is encoded by one of 21953 can be evaluated with nucleic acid amplification, e.g., by rtPCR (MulUs (1987) U.S. Patent No. 4,683,202), ligase chain reaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189-193), self sustained sequence replication (Guatelli etal, (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwohetα/., (1989), Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-BetaReplicase (Lizardi tα/., (1988) Bio/Technology 6:1197), rolling circle repUcation (Lizardi et al, U.S. Patent No. 5,854,033) or any other nucleic acid amplification method, followed by the detection ofthe amplified molecules using techniques known in the art. As used herein, amplification primers are defined as being a pair of nucleic acid molecules that can anneal to 5' or 3' regions of a gene (plus and minus strands, respectively, or vice- versa) and contain a short region in between. In general, amplification primers are from about 10 to 30 nucleotides in length and flank a region from about 50 to 200 nucleotides in length. Under appropriate conditions and with appropriate reagents, such primers permit the amplification of a nucleic acid molecule comprising the nucleotide sequence flanked by the primers.

[280] For in situ methods, a cell or tissue sample can be prepared/processed and immobilized on a support, typically a glass slide, and then contacted with a probe that can hybridize to mRNA that encodes the 21953 gene being analyzed. [281] In another embodiment, the methods further contacting a control sample with a compound or agent capable of detecting 21953 mRNA, or genomic DNA, and comparing the presence of 21953 mRNA or genomic DNA in the control sample with the presence of 21953 mRNA or genomic DNA in the test sample. In still another embodiment, serial analysis of gene expression, as described in U.S. Patent No. 5,695,937, is used to detect 21953 transcript levels.

[282] A variety of methods can be used to determine the level of protein encoded by

21953. In general these methods include contacting an agent that selectively binds to the protein, such as an antibody with a sample, to evaluate the level of protein in the sample. In a preferred embodiment, the antibody bears a detectable label. Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab')2) can be used. The term "labeled", with regard to the probe or antibody, is intended to encompass direct labeling ofthe probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as weU as indirect labeling ofthe probe or antibody by reactivity with a detectable substance. Examples of detectable substances are provided herein.

[283] The detection methods can be used to detect 21953 protein in a biological sample in vitro as well as in vivo. In vitro techniques for detection of 21953 protein include enzyme linked immunosorbent assays (ELISAs), immunoprecipitations, immunofluorescence, enzyme immunoassay (EIA), radioimmunoassay (RIA), and Western blot analysis. In vivo techniques for detection of 21953 protein include introducing into a subject a labeled anti-21953 antibody. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques. In another embodiment, the sample is labeled, e.g., biotinylated and then contacted to the antibody, e.g., an anti-21953 antibody positioned on an antibody array (as described below). The sample can be detected, e.g., with avidin coupled to a fluorescent label.

[284] In another embodiment, the methods further include contacting the control sample with a compound or agent capable of detecting 21953 protein, and comparing the presence of 21953 protein in the control sample with the presence of 21953 protein in the test sample.

[285] The invention also includes kits for detecting the presence of 21953 in a biological sample. For example, the kit can include a compound or agent capable of detecting 21953 protein or mRNA in a biological sample; and a standard. The compound or agent can be packaged in a suitable container. The kit can further comprise instructions for using the kit to detect 21953 protein or nucleic acid.

[286] For antibody-based kits, the kit can include: (1) a first antibody (e.g., attached to a solid support) which binds to a polypeptide corresponding to a marker ofthe invention; and, optionally, (2) a second, different antibody which binds to either the polypeptide or the first antibody and is conjugated to a detectable agent.

[287] For oligonucleotide-based kits, the kit can include: (1) an oligonucleotide, e.g., a detectably labeled oUgonucleotide, which hybridizes to a nucleic acid sequence encoding a polypeptide corresponding to a marker ofthe invention or (2) a pair of primers useful for amplifying a nucleic acid molecule corresponding to a marker ofthe iaventioa The kit can also includes a buffering agent, a preservative, or a protein stabilizing agent. The kit can also includes components necessary for detecting the detectable agent (e.g., an enzyme or a substrate). The kit can also contain a control sample or a series of control samples which can be assayed and compared to the test sample contained. Each component ofthe kit can be enclosed within an individual container and aU ofthe various containers can be within a single package, along with instructions for interpreting the results ofthe assays performed using the kit.

[288] The diagnostic methods described herein can identify subjects having, or at risk of developing, a disease or disorder associated with misexpressed or aberrant or unwanted 21953 expression or activity. As used herein, the term "unwanted" includes an unwanted phenomenon involved in a biological response such as a cell proUferative or ceU differentiative disorder, e.g., a cancer, e.g., a cancer ofthe lung, prostate, breast, or colon or deregulated cell proliferation

[289] In one embodiment, a disease or disorder associated with aberrant or unwanted

21953 expression or activity is identified. A test sample is obtained from a subject and 21953 protein or nucleic acid (e.g., mRNA or genomic DNA) is evaluated, wherein the level, e.g., the presence or absence, of 21953 protein or nucleic acid is diagnostic for a subject having or at risk of developing a disease or disorder associated with aberrant or unwanted 21953 expression or activity. As used herein, a "test sample" refers to a biological sample obtained from a subject of interest, including a biological fluid (e.g., serum), cell sample, or tissue.

[290] The prognostic assays described herein can be used to determine whether a subject can be administered an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate) to treat a disease or disorder associated with aberrant or unwanted 21953 expression or activity. For example, such methods can be used to determine whether a subject can be effectively treated with an agent for a ceU a cell proliferative or cell differentiative disorder, e.g., a cancer, e.g., a cancer of the lung, prostate, breast, or colon disorder.

[291] In another aspect, the invention features a computer medium having a plurality of digitally encoded data records. Each data record includes a value representing the level of expression of 21953 in a sample, and a descriptor ofthe sample. The descriptor ofthe sample can be an identifier ofthe sample, a subject from which the sample was derived (e.g., a patient), a diagnosis, or a treatment (e.g., a preferred treatment). In a preferred embodiment, the data record further includes values representing the level of expression of genes other than 21953 (e.g., other genes associated with a 21953-disorder, or other genes on an array). The data record can be structured as a table, e.g., a table that is part of a database such as a relational database (e.g., a SQL database ofthe Oracle or Sybase database environments).

[292] Also featured is a method of evaluating a sample. The method includes providing a sample, e.g., from the subject, and determining a gene expression profile ofthe sample, wherein the profile includes a value representing the level of 21953 expressioa The method can further include comparing the value or the profile (i.e., multiple values) to a reference value or reference profile. The gene expression profile ofthe sample can be obtained by any ofthe methods described herein (e.g., by providing a nucleic acid from the sample and contacting the nucleic acid to an array). The method can be used to diagnose a a cell proUferative or cell differentiative disorder, e.g., a cancer, e.g., a cancer ofthe lung, in a subject wherein an increase in 21953 expression is an indication that the subject has or is disposed to having a cell proliferative or cell differentiative disorder, e.g., a cancer, e.g., a cancer ofthe lung. The method can be used to monitor a treatment for a cell proUferative or cell differentiative disorder, e.g., a cancer, e.g., a cancer ofthe lung, prostate, breast, or colon in a subject. For example, the gene expression profile can be determined for a sample from a subject undergoing treatment. The profile can be compared to a reference profile or to a profile obtained from the subject prior to treatment or prior to onset ofthe disorder (see, e.g., Golub etal. (1999) Science 286:531).

[293] In yet another aspect, the invention features a method of evaluating a test compound (see also, "Screening Assays", above). The method includes providing a cell and a test compound; contacting the test compound to the cell; obtaining a subject expression profile for the contacted ceU; and comparing the subject expression profile to one or more reference profiles. The profiles include a value representing the level of 21953 expression. In a preferred embodiment, the subject expression profile is compared to a target profile, e.g., a profile for a normal cell or for desired condition of a ceU. The test compound is evaluated favorably if the subject expression profile is more similar to the target profile than an expression profile obtained from an uncontacted ceU.

[294] In another aspect, the invention features, a method of evaluating a subject. The method includes: a) obtaining a sample from a subject, e.g., from a caregiver, e.g., a caregiver who obtains the sample from the subject; b) determining a subject expression profile for the sample. Optionally, the method further includes either or both of steps: c) comparing the subject expression profile to one or more reference expression profiles; and d) selecting the reference profile most similar to the subject reference profile. The subject expression profile and the reference profiles include a value representing the level of 21953 expressioa A variety of routine statistical measures can be used to compare two reference profiles. One possible metric is the length ofthe distance vector that is the difference between the two profiles. Each ofthe subject and reference profile is represented as a multidimensional vector, wherein each dimension is a value in the profile. [295] The method can further include transmitting a result to a caregiver. The result can be the subject expression profile, a result of a comparison ofthe subject expression profile with another profile, a most similar reference profile, or a descriptor of any ofthe aforementioned. The result can be transmitted across a computer network, e.g., the result can be in the form of a computer transmission, e.g., a computer data signal embedded in a carrier wave.

[296] Also featured is a computer medium having executable code for effecting the following steps: receive a subject expression profile; access a database of reference expression profiles; and either i) select a matching reference profile most similar to the subject expression profile or ii) determine at least one comparison score for the similarity of the subject expression profile to at least one reference profile. The subject expression profile, and the reference expression profiles each include a value representing the level of 21953 expression.

Arrays and Uses Thereof

[297] In another aspect, the invention features an array that includes a substrate having a plurality of addresses. At least one address ofthe plurality includes a capture probe that binds specifically to a 21953 molecule (e.g., a 21953 nucleic acid or a 21953 polypeptide). The array can have a density of at least than 10, 50, 100, 200, 500, 1,000, 2,000, or 10,000 or more addresses/cm², and ranges between. In a preferred embodiment, the plurality of addresses includes at least 10, 100, 500, 1,000, 5,000, 10,000, 50,000 addresses. In a preferred embodiment, the plurality of addresses includes equal to or less than 10, 100, 500, 1,000, 5,000, 10,000, or 50,000 addresses. The substrate can be a two-dimensional substrate such as a glass slide, a wafer (e.g., silica or plastic), a mass spectroscopy plate, or a three- dimensional substrate such as a gel pad. Addresses in addition to address ofthe plurality can be disposed on the array. [298] In a preferred embodiment, at least one address ofthe plurality includes a nucleic acid capture probe that hybridizes specifically to a 21953 nucleic acid, e.g., the sense or anti-sense strand. In one preferred embodiment, a subset of addresses ofthe plurality of addresses has a nucleic acid capture probe for 21953. Each address ofthe subset can include a capture probe that hybridizes to a different region of a 21953 nucleic acid. In another preferred embodiment, addresses ofthe subset include a capture probe for a 21953 nucleic acid. Each address ofthe subset is unique, overlapping, and complementary to a different variant of 21953 (e.g., an aUelic variant, or all possible hypothetical variants). The array can be used to sequence 21953 by hybridization (see, e.g., U.S. Patent No. 5,695,940). [299] An array can be generated by various methods, e.g., by photolithographic methods (see, e.g., U.S. Patent Nos. 5,143,854; 5,510,270; and 5,527,681), mechanical methods (e.g., directed-flow methods as described in U.S. Patent No. 5,384,261), pin-based methods (e.g., as described in U.S. Pat. No. 5,288,514), and bead-based techniques (e.g., as described in PCT US/93/04145).

[300] In another preferred embodiment, at least one address ofthe plurality includes a polypeptide capture probe that binds specifically to a 21953 polypeptide or fragment thereof. The polypeptide can be a naturally-occurring interaction partner of 21953 polypeptide. Preferably, the polypeptide is an antibody, e.g., an antibody described herein (see "Anti- 21953 Antibodies," above), such as a monoclonal antibody or a single-chain antibody. [301] In another aspect, the invention features a method of analyzing the expression of

21953. The method includes providing an array as described above; contacting the array with a sample and detecting binding of a 21953-molecule (e.g., nucleic acid or polypeptide) to the array. In a preferred embodiment, the array is a nucleic acid array. Optionally the method further includes amplifying nucleic acid from the sample prior or during contact with the array.

[302] In another embodiment, the array can be used to assay gene expression in a tissue to ascertain tissue specificity of genes in the array, particularly the expression of 21953. If a sufficient number of diverse samples is analyzed, clustering (e.g., hierarchical clustering, k-means clustering, Bayesian clustering and the like) can be used to identify other genes which are co-regulated with 21953. For example, the array can be used for the quantitation ofthe expression of multiple genes. Thus, not only tissue specificity, but also the level of expression of a battery of genes in the tissue is ascertained. Quantitative data can be used to group (e.g., cluster) genes on the basis of their tissue expression per se and level of expression in that tissue.

[303] For example, array analysis of gene expression can be used to assess the effect of ceU-cell interactions on 21953 expression. A first tissue can be perturbed and nucleic acid from a second tissue that interacts with the first tissue can be analyzed. In this context, the effect of one ceU type on another cell type in response to a biological stimulus can be determined, e.g., to monitor the effect of cell-cell interaction at the level of gene expression. [304] In another embodiment, cells are contacted with a therapeutic agent. The expression profile ofthe cells is determined using the array, and the expression profile is compared to the profile of Uke cells not contacted with the agent. For example, the assay can be used to determine or analyze the molecular basis of an undesirable effect ofthe therapeutic agent. If an agent is administered therapeutically to treat one cell type but has an undesirable effect on another ceU type, the invention provides an assay to determine the molecular basis ofthe undesirable effect and thus provides the opportunity to co-administer a counteracting agent or otherwise treat the undesired effect. Similarly, even within a single cell type, undesirable biological effects can be determined at the molecular level. Thus, the effects of an agent on expression of other than the target gene can be ascertained and counteracted.

[305] In another embodiment, the array can be used to monitor expression of one or more genes in the array with respect to time. For example, samples obtained from different time points can be probed with the array. Such analysis can identify and/or characterize the development of a 21953-associated disease or disorder; and processes, such as a cellular transformation associated with a 21953-associated disease or disorder. The method can also evaluate the treatment and/or progression of a 21953-associated disease or disorder [306] The array is also useful for ascertaining differential expression patterns of one or more genes in normal and abnormal cells. This provides a battery of genes (e.g. , including 21953) that could serve as a molecular target for diagnosis or therapeutic intervention. [307] In another aspect, the invention features an array having a plurality of addresses.

Each address ofthe plurality includes a unique polypeptide. At least one address ofthe pluraUty has disposed thereon a 21953 polypeptide or fragment thereof. Methods of producing polypeptide arrays are described in the art, e.g., in De Wildt et al. (2000). Nature Biotech. 18, 989-994; Lueking et α/. (1999). Anal. Biochem. 270, 103-111; Ge, H. (2000). Nucleic Acids Res. 28, e3, 1-VII; MacBeaih, G, and Schreiber, S.L. (2000). Science 289, 1760-1763; and WO 99/51773A1. In a preferred embodiment, each addresses ofthe pluraUty has disposed thereon a polypeptide at least 60, 70, 80,85, 90, 95 or 99 % identical to a 21953 polypeptide or fragment thereof. For example, multiple variants of a 21953 polypeptide (e.g., encoded by allelic variants, site-directed mutants, random mutants, or combinatorial mutants) can be disposed at individual addresses ofthe plurality. Addresses in addition to the address ofthe plurality can be disposed on the array. [308] The polypeptide array can be used to detect a 21953 binding compound, e.g., an antibody in a sample from a subject with specificity for a 21953 polypeptide or the presence of a 21953-binding protein or Ugand.

[309] The array is also useful for ascertaining the effect ofthe expression of a gene on the expression of other genes in the same cell or in different ceUs (e.g., ascertaining the effect of 21953 expression on the expression of other genes). This provides, for example, for a selection of alternate molecular targets for therapeutic intervention if the ultimate or downstream target cannot be regulated.

[310] In another aspect, the invention features a method of analyzing a plurality of probes. The method is useful, e.g., for analyzing gene expression The method includes: providing a two dimensional array having a plurality of addresses, each address ofthe pluraUty being positionally distinguishable from each other address ofthe plurality having a unique capture probe, e.g., wherein the capture probes are from a cell or subject which express 21953 or from a cell or subject in which a 21953 mediated response has been eUcited, e.g., by contact ofthe cell with 21953 nucleic acid or protein, or administration to the cell or subject 21953 nucleic acid or protein; providing a two dimensional array having a plurality of addresses, each address ofthe plurality being positionally distinguishable from each other address ofthe plurality, and each address ofthe pluraUty having a unique capture probe, e.g., wherein the capture probes are from a cell or subject which does not express 21953 (or does not express as highly as in the case ofthe 21953 positive pluraUty of capture probes) or from a cell or subject which in which a 21953 mediated response has not been eUcited (or has been eUcited to a lesser extent than in the first sample); contacting the array with one or more inquiry probes (which is preferably other than a 21953 nucleic acid, polypeptide, or antibody), and thereby evaluating the plurality of capture probes. Binding, e.g., in the case of anucleic acid, hybridization with a capture probe at an address ofthe pluraUty, is detected, e.g., by signal generated from a label attached to the nucleic acid, polypeptide, or antibody. [311] In another aspect, the invention features a method of analyzing a pluraUty of probes or a sample. The method is useful e g-, for analyzing gene expression. The method includes: providing a two dimensional array having a plurality of addresses, each address of the plurality being positionally distinguishable from each other address ofthe plurality having a unique capture probe, contacting the array with a first sample from a cell or subject which express or mis-express 21953 or from a cell or subject in which a 21953-mediated response has been eUcited, e.g., by contact ofthe ceU with 21953 nucleic acid or protein, or administration to the ceU or subject 21953 nucleic acid or protein; providing a two dimensional array having a plurality of addresses, each address ofthe plurality being positionally distinguishable from each other address ofthe plurality, and each address ofthe pluraUty having a unique capture probe, and contacting the array with a second sample from a cell or subject which does not express 21953 (or does not express as highly as in the case ofthe 21953 positive pluraUty of capture probes) or from a cell or subject which in which a 21953 mediated response has not been elicited (or has been elicited to a lesser extent than in the first sample); and comparing the binding ofthe first sample with the binding ofthe second sample. Binding, e.g., in the case of anucleic acid, hybridization with a capture probe at an address ofthe plurality, is detected, e.g., by signal generated from a label attached to the nucleic acid, polypeptide, or antibody. The same array can be used for both samples or different arrays can be used. If different arrays are used the plurality of addresses with capture probes should be present on both arrays. [312] In another aspect, the invention features a method of analyzing 21953, e.g., analyzing structure, function, or relatedness to other nucleic acid or amino acid sequences. The method includes: providing a 21953 nucleic acid or amino acid sequence; comparing the 21953 sequence with one or more preferably a plurality of sequences from a collection of sequences, e.g., anucleic acid or protein sequence database; to thereby analyze 21953.

Detection of Variations or Mutations

[313] The methods ofthe invention can also be used to detect genetic alterations in a

21953 gene, thereby determining if a subject with the altered gene is at risk for a disorder characterized by misregulation in 21953 protein activity or nucleic acid expression, such as a ceU proliferative or cell differentiative disorder, e.g., a cancer, e.g., a cancer ofthe lung, prostate, breast, or colon disorder. In preferred embodiments, the methods include detecting, in a sample from the subject, the presence or absence of a genetic alteration characterized by at least one of an alteration affecting the integrity of a gene encoding a 21953-protein, or the mis-expression ofthe 21953 gene. For example, such genetic alterations can be detected by ascertaining the existence of at least one of 1) a deletion of one or more nucleotides from a 21953 gene; 2) an addition of one or more nucleotides to a 21953 gene; 3) a substitution of one or more nucleotides of a 21953 gene, 4) a chromosomal rearrangement of a 21953 gene; 5) an alteration in the level of a messenger RNA transcript of a 21953 gene, 6) aberrant modification of a 21953 gene, such as ofthe methylation pattern ofthe genomic DNA, 7) the presence of a non- wild type splicing pattern of a messenger RNA transcript of a 21953 gene, 8) anon-wild type level of a 21953-protein, 9) aUelic loss of a 21953 gene, and 10) inappropriate post-translational modification of a 21953-protein.

[314] An alteration can be detected without a probe/primer in a polymerase chain reaction, such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR), the latter of which can be particularly useful for detecting point mutations in the 21953-gene. This method can include the steps of collecting a sample of cells from a subject, isolating nucleic acid (e.g., genomic, mRNA or both) from the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to a 21953 gene under conditions such that hybridization and amplification ofthe 21953-gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size ofthe amplification product and comparing the length to a control sample. It is anticipated that PCR and/or LCR may be desirable to use as a preliminary amplification step in conjunction with any ofthe techniques used for detecting mutations described herein. Alternatively, other amplification methods described herein or known in the art can be used. [315] In another embodiment, mutations in a 21953 gene from a sample cell can be identified by detecting alterations in restriction enzyme cleavage patterns. For example,

e.g., chip based arrays. Such arrays include a plurality of addresses, each of which is positionally distinguishable from the other. A different probe is located at each address of the plurality. A probe can be complementary to a region of a 21953 nucleic acid or a putative variant (e.g., allelic variant) thereof. A probe can have one or more mismatches to a region of a 21953 nucleic acid (e.g., a destabUizing mismatch). The arrays can have a high density of addresses, e.g., can contain hundreds or thousands of oligonucleotides probes (Cronin, M.T. et al. (1996) HumanMutation 7: 244-255; Koza MJ. et al. (1996) Nature Medicine 2: 753-759). For example, genetic mutations in 21953 can be identified in two- dimensional arrays containing Ught-generated DNA probes as described in Cronin, M.T. et al. supra. Briefly, a first hybridization array of probes can be used to scan through long stretches of DNA in a sample and control to identify base changes between the sequences by making linear arrays of sequential overlapping probes. This step aUows the identification of point mutations. This step is followed by a second hybridization array that aUows the characterization of specific mutations by using smaller, specialized probe arrays complementary to all variants or mutations detected. Each mutation array is composed of paraUel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene.

[317] In yet another embodiment, any of a variety of sequencing reactions known in the art can be used to directly sequence the 21953 gene and detect mutations by comparing the sequence ofthe sample 21953 with the corresponding wild-type (control) sequence. Automated sequencing procedures can be utilized when performing the diagnostic assays ((1995) Biotechniques 19:448), including sequencing by mass spectrometry. [318] Other methods for detecting mutations in the 21953 gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNAheteroduplexes (Myers etal. (1985) Science 230:1242; Cotton etal. (1988) Proc. NatlAcadSci USA 85:4397; Saleebaetα/. (1992) Methods Enzymol 217:286-295). [319] In still another embodiment, the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so caUed "DNA mismatch repair" enzymes) in defined systems for detecting and mapping point mutations in 21953 cDNAs obtained from samples of cells. For example, the mutY enzyme of E. coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T at G/T mismatches (Hsu etal. (1994) Carcinogenesis 15:1657-1662; U.S. Patent No. 5,459,039). [320] In other embodiments, alterations in electrophoretic mobility will be used to identify mutations in 21953 genes. For example, single strand conformation polymorphism (SSCP) may be used to detect differences in electrophoretic mobility between mutant and wild type nucleic acids (Orita et al. (1989) Proc Natl. Acad. Sci USA: 86:2766, see also Cotton (1993)Mutat. Res. 285:125-144; and Hayashi (1992) Genet. Anal. Tech. Appl. 9:73- 79). Single-stranded DNA fragments of sample and control 21953 nucleic acids will be denatured and aUowed to renature. The secondary structure of single-stranded nucleic acids varies according to sequence, the resulting alteration in electrophoretic mobility enables the detection of even a single base change. The DNA fragments may be labeled or detected with labeled probes. The sensitivity ofthe assay may be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a change in sequence. In a preferred embodiment, the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility (Keen etal. (1991) Trends Genet 7:5).

[321] In yet another embodiment, the movement of mutant or wild-type fragments in poly aery lamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE) (Myers et al. (1985) Nature 313:495). When DGGE is used as the method of analysis, DNA wUl be modified to insure that it does not completely denature, for example by adding a GC clamp of approximately 40 bp of high-melting GC-rich DNA by PCR. In a further embodiment, a temperature gradient is used in place of a denaturing gradient to identify differences in the mobility of control and sample DNA (Rosenbaum and Reissner (1987) Biophys Chem 265:12753).

[322] Examples of other techniques for detecting point mutations include, but are not limited to, selective oUgonucleotide hybridization, selective amplification, or selective primer extension (Saiki et al. (1986) Nature 324: 163); Saiki et al. (1989) Proc. Natl Acad. Sci USA 86:6230). A further method of detecting point mutations is the chemical ligation of oligonucleotides as described in Xu et al. ((2001) Nature Biotechnol. 19:148). Adjacent oligonucleotides, one of which selectively anneals to the query site, are ligated together if the nucleotide at the query site ofthe sample nucleic acid is complementary to the query oligonucleotide; ligation can be monitored, e.g., by fluorescent dyes coupled to the oligonucleotides.

[323] Alternatively, allele specific amplification technology that depends on selective

PCR amplification may be used in conjunction with the instant invention. OUgonucleotides used as primers for specific amplification may carry the mutation of interest in the center of the molecule (so that amplification depends on differential hybridization) (Gibbs et al. (1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3' end of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (Prossner (1993) Tibtech 11:238). In addition it may be desirable to introduce a novel restriction site in the region ofthe mutation to create cleavage-based detection (Gasparini et al (1992) Mol. Cell Probes 6:1). It is anticipated that in certain embodiments amplification may also be performed using Taq Ugase for amplification (Barany (1991) Proc. Natl. Acad. Sci USA 88: 189). In such cases, ligation will occur only if there is a perfect match at the 3' end ofthe 5' sequence making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification. [324] In another aspect, the invention features a set of oligonucleotides. The set includes a plurality of oligonucleotides, each of which is at least partially complementary (e.g., at least 50%, 60%, 70%, 80%, 90%, 92%, 95%, 97%, 98%, or 99% complementary) to a 21953 nucleic acid.

[325] In a preferred embodiment the set includes a first and a second oligonucleotide.

The first and second oligonucleotide can hybridize to the same or to different locations of SEQ ID NO: 1 or the complement of SEQ ID NO: 1. Different locations can be different but overlapping or non-overlapping on the same strand. The first and second oUgonucleotide can hybridize to sites on the same or on different strands.

[326] The set can be useful, e.g., for identifying SNP's, or identifying specific alleles of 21953. In a preferred embodiment, each oligonucleotide ofthe set has a different nucleotide at an interrogation position In one embodiment, the set includes two oligonucleotides, each complementary to a different allele at a locus, e.g., a biallelic or polymorphic locus.

[327] In another embodiment, the set includes four oUgonucleotides, each having a different nucleotide (e.g., adenine, guanine, cytosine, or thymidine) at the interrogation position The interrogation position can be a SNP or the site of a mutation In another preferred embodiment, the oligonucleotides ofthe plurality are identical in sequence to one another (except for differences in length). The oligonucleotides can be provided with differential labels, such that an oUgonucleotide that hybridizes to one allele provides a signal that is distinguishable from an oligonucleotide that hybridizes to a second aUele. In still another embodiment, at least one ofthe oligonucleotides ofthe set has a nucleotide change at a position in addition to a query position, e.g., a destabilizing mutation to decrease the T_m ofthe oUgonucleotide. In another embodiment, at least one oligonucleotide ofthe set has a non-natural nucleotide, e.g., inosine. In a preferred embodiment, the oUgonucleotides are attached to a solid support, e.g., to different addresses of an array or to different beads or nanoparticles.

[328] I-n a preferred embodiment the set of oligo nucleotides can be used to specifically amplify, e.g., by PCR, or detect, a 21953 nucleic acid.

[329] The methods described herein may be performed, for example, by utUizing prepackaged diagnostic kits comprising at least one probe nucleic acid or antibody reagent described herein, which may be conveniently used, e.g., in clinical settings to diagnose patients exhibiting symptoms or family history of a disease or illness involving a 21953 gene.

Use of 21953 Molecules as Surrogate Markers

[330] The 21953 molecules ofthe invention are also useful as markers of disorders or disease states, as markers for precursors of disease states, as markers for predisposition of disease states, as markers of drag activity, or as markers ofthe pharmacogenomic profile of a subject. Using the methods described herein, the presence, absence and/or quantity ofthe 21953 molecules ofthe invention may be detected, and may be correlated with one or more biological states in vivo. For example, the 21953 molecules ofthe invention may serve as surrogate markers for one or more disorders or disease states or for conditions leading up to disease states. As used herein, a "surrogate marker" is an objective biochemical marker which correlates with the absence or presence of a disease or disorder, or with the progression of a disease or disorder (e.g., with the presence or absence of a tumor). The presence or quantity of such markers is independent ofthe disease. Therefore, these markers may serve to indicate whether a particular course of treatment is effective in lessening a disease state or disorder. Surrogate markers are of particular use when the presence or extent of a disease state or disorder is difficult to assess through standard methodologies (e.g., early stage tumors), or when an assessment of disease progression is desired before a potentially dangerous clinical endpoint is reached (e.g., an assessment of cardiovascular disease may be made using cholesterol levels as a surrogate marker, and an analysis of FUN infection may be made using HTV RΝA levels as a surrogate marker, weU in advance ofthe undesirable clinical outcomes of myocardial infarction or fully-developed AIDS). Examples ofthe use of surrogate markers in the art include: Koomen et al (2000) J. Mass. Spectrom. 35: 258-264; and James (1994) AIDS Treatment News Archive 209. [331] The 21953 molecules ofthe invention are also useful as pharmacodynamic markers. As used herein, a "pharmacodynamic marker" is an objective biochemical marker which correlates specifically with drug effects. The presence or quantity of a pharmacodynamic marker is not related to the disease state or disorder for which the drug is being administered; therefore, the presence or quantity ofthe marker is indicative ofthe presence or activity ofthe drug in a subject. For example, a pharmacodynamic marker may be indicative ofthe concentration ofthe drag in a biological tissue, in that the marker is either expressed or transcribed or not expressed or transcribed in that tissue in relationship to the level ofthe drug. In this fashion, the distribution or uptake ofthe drag may be monitored by the pharmacodynamic marker. Similarly, the presence or quantity ofthe pharmacodynamic marker may be related to the presence or quantity ofthe metaboUc product of a drug, such that the presence or quantity ofthe marker is indicative ofthe relative breakdown rate ofthe drag in vivo. Pharmacodynamic markers are of particular use in increasing the sensitivity of detection of drug effects, particularly when the drug is administered in low doses. Since even a small amount of a drug may be sufficient to activate multiple rounds of marker (e.g., a 21953 marker) transcription or expression, the amplified marker may be in a quantity which is more readily detectable than the drug itself. Also, the marker may be more easily detected due to the nature ofthe marker itself; for example, using the methods described herein, anti-21953 antibodies may be employed in an immune-based detection system for a 21953 protein marker, or 21953-specific radiolabeled probes may be used to detect a 21953 mRNA marker. Furthermore, the use of a pharmacodynamic marker may offer mechanism-based pYrediction of risk due to drug treatment beyond the range of possible direct observations. Examples ofthe use of pharmacodynamic markers in the art include: Matsuda et al. US 6,033,862; Hattis et al. (1991) Env. Health Perspect. 90: 229-238; Schentag (1999).4m. J. Health-Syst. Pharm. 56 Suppl. 3: S21-S24; and Nicolau (1999).4m, J. Health-Syst. Pharm. 56 Suppl 3: S16-S20. [332] The 21953 molecules ofthe invention are also useful as pharmacogenomic markers. As used herein, a "pharmacogenomic marker" is an objective biochemical marker which correlates with a specific clinical drag response or susceptibility in a subject (see, e.g., McLeod etal. (1999) Eur. J. Cancer 35:1650-1652). The presence or quantity ofthe pharmacogenomic marker is related to the predicted response ofthe subject to a specific drug or class of drugs prior to administration ofthe drag. By assessing the presence or quantity of one or more pharmacogenomic markers in a subject, a drug therapy which is most appropriate for the subject, or which is predicted to have a greater degree of success, may be selected. For example, based on the presence or quantity of RNA, or protein (e.g., 21953 protein or RNA) for specific tumor markers in a subject, a drug or course of treatment may be selected that is optimized for the treatment ofthe specific tumor likely to be present in the subject. Similarly, the presence or absence of a specific sequence mutation in 21953 DNA may correlate 21953 drag response. The use of pharmacogenomic markers therefore permits the application ofthe most appropriate treatment for each subject without having to administer the therapy.

Pharmaceutical Compositions

[333] The nucleic acid and polypeptides, fragments thereof, as weU as anti-21953 antibodies (also referred to herein as "active compounds") ofthe invention can be incorporated into pharmaceutical compositions. Such compositions typically include the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier. As used herein the language "pharmaceutically acceptable carrier" includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions.

[334] A pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal aclministration Solutions or suspensions used for parenteral, intradermal or subcutaneous application can include the following components: a sterile diluent such as water for injection, saUne solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. [335] 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, Cremophor EL™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringabUity exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol propylene glycol, and liquid polyetheylene glycol and the like), and suitable mixtures thereof The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use of surfactants. Prevention ofthe 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 manitol, sorbitol, sodium chloride in the composition Prolonged absoφtion ofthe injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin. [336] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder ofthe active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[337] Oral compositions generally include an inert diluent or an edible carrier. 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, e.g., gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part ofthe composition The tablets, pills, capsules, troches and the like can contain any of the foUowing ingredients, or compounds of a similar nature: a binder such as microcrystaUine ceUulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[338] For administration by inhalation, the compounds are deUvered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propeUant, e.g., a gas such as carbon dioxide, or a nebulizer.

[339] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generaUy known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art. [340] The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[341] In one embodiment, the active compounds are prepared with carriers that wiU protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated deUvery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skiUed in the art, for example, as described in U.S. Patent No. 4,522,811.

[342] It is advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. [343] Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% ofthe population) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds which exhibit high therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cetts and, thereby, reduce side effects.

[344] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with Uttle or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography. [345] As defined herein, a therapeutically effective amount of protein or polypeptide

(i.e., an effective dosage) ranges from about 0.001 to 30 mg kg body weight, preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.1 to 20 mg kg body weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight. The protein or polypeptide can be administered one time per week for between about 1 to 10 weeks, preferably between 2 to 8 weeks, more preferably between about 3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not Umited to the severity ofthe disease or disorder, previous treatments, the general health and/or age ofthe subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a protein, polypeptide, or antibody can include a single treatment or, preferably, can include a series of treatments.

[346] For antibodies, the preferred dosage is 0.1 mg/kg of body weight (generally 10 mg/kg to 20 mg/kg). If the antibody is to act in the brain, a dosage of 50 mg kg to 100 mg/kg is usually appropriate. Generally, partially human antibodies and fully human antibodies have a longer half-life within the human body than other antibodies. Accordingly, lower dosages and less frequent administration is often possible. Modifications such as Upidation can be used to stabilize antibodies and to enhance uptake and tissue penetration (e.g., into the brain). A method for Upidation of antibodies is described by Cruikshank et al. ((1997) J. Acquired Immune Deficiency Syndromes and Human Retrovirology 14:193).

[347] The present invention encompasses agents which modulate expression or activity. An agent may, for example, be a small molecule. For example, such small molecules include, but are not limited to, peptides, peptidomimetics (e.g., peptoids), amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, organic or inorganic compounds (i.e.,. including heteroorganic and organometaUic compounds) having a molecular weight less than about 10,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, and other pharmaceuticaUy acceptable forms of such compounds. [348] Exemplary doses include milligram or microgram amounts ofthe small molecule per kilogram of subject or sample weight (e.g., about 1 microgram per kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 5 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram It is furthermore understood that appropriate doses of a small molecule depend upon the potency ofthe small molecule with respect to the expression or activity to be modulated. When one or more of these small molecules is to be administered to an animal (e.g., a human) in order to modulate expression or activity of a polypeptide or nucleic acid ofthe invention, a physician, veterinarian, or researcher may, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained. In addition, it is understood that the specific dose level for any particular animal subject wUl depend upon a variety of factors including the activity ofthe specific compound employed, the age, body weight, general health, gender, and diet ofthe subject, the time of administration, the route of administration, the rate of excretion, any drag combination, and the degree of expression or activity to be modulated.

[349] An antibody (or fragment thereof) may be conjugated to a therapeutic moiety such as a cytotoxin, a therapeutic agent or a radioactive metal ion. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include taxol cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Therapeutic agents include, but are not Umited to, antimetaboUtes (e.g., methotrexate, 6-mercaptopurine, 6- thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambutil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine).

[350] The conjugates ofthe invention can be used for modifying a given biological response, the drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, α- interferon, β-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator; or, biological response modifiers such as, for example, lymphokines, interleukin-1 ('TL-1"), interleukin-2 ('TL-2"), interleukin-6 ('TL-6"), granulocyte macrophase colony stimulating factor ("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or other growth factors.

[351] Alternatively, an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Patent No. 4,676,980. [352] The nucleic acid molecules ofthe invention can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be deUvered to a subject by, for example, intravenous injection, local administration (see U.S. Patent 5,328,470) or by stereotactic injection (see e.g., Chen etal. (1994) Proc. Natl. Acad. Sci. USA 91:3054-3057). The pharmaceutical preparation ofthe gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene deUvery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant ceUs, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system. [353] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

Methods of Treatment

[354] The present invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with aberrant or unwanted 21953 expression or activity. As used herein, the term "treatment" is defined as the appUcation or administration of a therapeutic agent to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient, who has a disease, a symptom of disease or a predisposition toward a disease, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease, the symptoms of disease or the predisposition toward disease. A therapeutic agent includes, but is not limited to, small molecules, peptides, antibodies, ribozymes and antisense oligonucleotides.

[355] With regards to both prophylactic and therapeutic methods of treatment, such treatments may be specificaUy tailored or modified, based on knowledge obtained from the field of pharmacogenomics. "Pharmacogenomics", as used herein, refers to the application of genomics technologies such as gene sequencing, statistical genetics, and gene expression analysis to drugs in clinical development and on the market. More specifically, the term refers the study of how a patient's genes determine his or her response to a drug (e.g., a patient's "drug response phenotype", or "drag response genotype".) Thus, another aspect of the invention provides methods for tailoring an individual's prophylactic or therapeutic treatment with either the 21953 molecules ofthe present invention or 21953 modulators according to that individual's drug response genotype. Pharmacogenomics allows a clinician or physician to target prophylactic or therapeutic treatments to patients who will most benefit from the treatment and to avoid treatment of patients who will experience toxic drug-related side effects.

[356] In one aspect, the invention provides a method for preventing in a subject, a disease or condition associated with an aberrant or unwanted 21953 expression or activity, by administering to the subject a 21953 or an agent which modulates 21953 expression or at least one 21953 activity. Subjects at risk for a disease which is caused or contributed to by aberrant or unwanted 21953 expression or activity can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein. Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic ofthe 21953 aberrance, such that a disease or disorder is prevented or, alternatively, delayed in its progression Depending on the type of 21953 aberrance, for example, a 21953, 21953 agonist or 21953 antagonist agent can be used for treating the subject. The appropriate agent can be determined based on screening assays described herein. [357] It is possible that some 21953 disorders can be caused, at least in part, by an abnormal level of gene product, or by the presence of a gene product exhibiting abnormal activity. As such, the reduction in the level and/or activity of such gene products would bring about the amelioration of disorder symptoms.

[358] The 21953 molecules can act as novel diagnostic targets and therapeutic agents for controlUng one or more of cellular proliferative and/or differentiative disorders, disorders associated with bone metabolism, immune disorders, cardiovascular disorders, liver disorders, viral diseases, pain or metaboUc disorders.

[359] Examples of cellular proliferative and/or differentiative disorders include cancers and proliferative disorders mentioned above. Further examples of cancers or neoplastic conditions, in addition to the ones described above include, but are not Umited to, a fibrosarcoma, myosarcoma, Uposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, gastric cancer, esophageal cancer, rectal cancer, pancreatic cancer, ovarian cancer, prostate cancer, uterine cancer, cancer ofthe head and neck, skin cancer, brain cancer, squamous ceU carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal ceU carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testicular cancer, small cell lung carcinoma, non-small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, meduUoblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendrogUoma, meningioma, melanoma, neuroblastoma, retinoblastoma, leukemia, lymphoma, or Kaposi sarcoma

[360] Disorders involving T-cells include, but are not Umited to, ceU-mediated hypersensitivity, such as delayed type hypersensitivity and T-cell-mediated cytotoxicity, and transplant rejection; autoimmune diseases, such as systemic lupus erythematosus, Sjόgren syndrome, systemic sclerosis, inflammatory myopathies, mixed connective tissue disease, and polyarteritis nodosa and other vasculitides; immunologic deficiency syndromes, including but not Umited to, primary immunodeficiencies, such as thymic hypoplasia, severe combined immunodeficiency diseases, and AIDS; leukopenia; reactive (inflammatory) proliferations of white cells, including but not Umited to, leukocytosis, acute nonspecific lymphadenitis, and chronic nonspecific lymphadenitis; neoplastic proUferations of white cells, including but not limited to lymphoid neoplasms, such as precursor T-cell neoplasms, such as acute lymphoblastic leukemia/lymphoma, peripheral T-cell and natural killer cell neoplasms that include peripheral T-cell lymphoma, unspecified, adult T-cell leukemia/lymphoma, mycosis fungoides and Sezary syndrome, and Hodgkin disease.

[361] Additionally, 21953 may play an important role in the regulation of metabolism or pain disorders, e.g., by processing neuropeptides and metabolic peptide hormones. Diseases of metabolic imbalance include, but are not Umited to, obesity, anorexia nervosa, cachexia, Upid disorders, and diabetes. Examples of pain disorders include, but are not limited to, pain response elicited during various forms of tissue injury, e.g., inflammation, infection, and ischemia, usuaUy referred to as hyperalgesia (described in, for example, Fields, H.L. (1987) Pain, New York:McGraw-Hill); pain associated with musculoskeletal disorders, e.g., joint pain; tooth pain; headaches; pain associated with surgery; pain related to irritable bowel syndrome; or chest pain.

[362] As discussed, successful treatment of 21953 disorders can be brought about by techniques that serve to inhibit the expression or activity of target gene products. For example, compounds, e.g., an agent identified using an assays described above, that proves to exhibit negative modulatory activity, can be used in accordance with the invention to prevent and/or ameliorate symptoms of 21953 disorders. Such molecules can include, but are not Umited to peptides, phosphopeptides, smaU organic or inorganic molecules, or antibodies (including, for example, polyclonal, monoclonal, humanized, anti-idiotypic, chimeric or single chain antibodies, and Fab, F(ab')₂ and Fab expression library fragments, scFV molecules, and epitope-binding fragments thereof).

[363] Further, antisense and ribozyme molecules that inhibit expression ofthe target gene can also be used in accordance with the invention to reduce the level of target gene expression, thus effectively reducing the level of target gene activity. StiU further, triple helix molecules can be utilized in reducing the level of target gene activity. Antisense, ribozyme and triple helix molecules are discussed above.

[364] It is possible that the use of antisense, ribozyme, and/or triple helix molecules to reduce or inhibit mutant gene expression can also reduce or inhibit the transcription (triple helix) and/or translation (antisense, ribozyme) of mRNA produced by normal target gene alleles, such that the concentration of normal target gene product present can be lower than is necessary for a normal phenotype. In such cases, nucleic acid molecules that encode and express target gene polypeptides exhibiting normal target gene activity can be introduced into cells via gene therapy method. Alternatively, in instances in that the target gene encodes an extracellular protein, it can be preferable to co-administer normal target gene protein into the cell or tissue in order to maintain the requisite level of cellular or tissue target gene activity.

[365] Another method by which nucleic acid molecules may be utilized in treating or preventing a disease characterized by 21953 expression is through the use of aptamer molecules specific for 21953 protein. Aptamers are nucleic acid molecules having a tertiary structure which permits them to specifically bind to protein ligands (see, e.g., Osborne, etal. (1997) Curr. Opin. Chem Biol. 1: 5-9; and Patel, D.J. (1997) Curr Opin Chem Biol 1:32- 46). Since nucleic acid molecules may in many cases be more conveniently introduced into target ceUs than therapeutic protein molecules may be, aptamers offer a method by which 21953 protein activity may be specifically decreased without the introduction of drugs or other molecules which may have pluripotent effects.

[366] Antibodies can be generated that are both specific for target gene product and that reduce target gene product activity. Such antibodies may, therefore, by administered in instances whereby negative modulatory techniques are appropriate for the treatment of 21953 disorders. For a description of antibodies, see the Antibody section above. [367] In circumstances wherein injection of an animal or a human subject with a

21953 protein or epitope for stimulating antibody production is harmful to the subject, it is possible to generate an immune response against 21953 through the use of anti-idiotypic antibodies (see, for example, Herlyn, D. (1999) Ann Med 31:66-78; and Bhattacharya- Chatterjee, M., and Foon, K.A (1998) Cancer Treat Res. 94:51-68). If an anti-idiotypic antibody is introduced into a mammal or human subject, it should stimulate the production of anti-anti-idiotypic antibodies, which should be specific to the 21953 proteia Vaccines directed to a disease characterized by 21953 expression may also be generated in this fashioa

[368] In instances where the target antigen is intracellular and whole antibodies are used, internalizing antibodies may be preferred. Lipofectin or Uposomes can be used to deUver the antibody or a fragment ofthe Fab region that binds to the target antigen into cells. Where fragments ofthe antibody are used, the smallest inhibitory fragment that binds to the target antigen is preferred. For example, peptides having an amino acid sequence corresponding to the Fv region ofthe antibody can be used. Alternatively, single chain neutralizing antibodies that bind to intracellular target antigens can also be administered. Such single chain antibodies can be administered, for example, by expressing nucleotide sequences encoding single-chain antibodies within the target cell population (see e.g., Marasco etal. (1993) Proc. Natl. Acad. Sci. USA 90:7889-7893). [369] The identified compounds that inhibit target gene expression, synthesis and/or activity can be administered to a patient at therapeutically effective doses to prevent, treat or ameliorate 21953 disorders. A therapeutically effective dose refers to that amount ofthe compound sufficient to result in amelioration of symptoms ofthe disorders. Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures as described above.

[370] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED₅₀ with Uttle or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method ofthe invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC₅₀ (le., the concentration ofthe test compound that achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance Uquid chromatography. [371] Another example of determination of effective dose for an individual is the ability to directly assay levels of "free" and "bound" compound in the seram ofthe test subject. Such assays may utiUze antibody mimics and/or "biosensors" that have been created through molecular imprinting techniques. The compound which is able to modulate 21953 activity is used as a template, or "imprinting molecule", to spatially organize polymerizable monomers prior to their polymerization with catalytic reagents. The subsequent removal of the imprinted molecule leaves a polymer matrix which contains a repeated "negative image" ofthe compound and is able to selectively rebind the molecule under biological assay conditions. A detailed review of this technique can be seen in Ansell, R. J. et al (1996) Current Opinion in Biotechnology 7:89-94 and in Shea, K.J. (1994) Trends in Polymer Science 2:166-173. Such "imprinted" affinity matrixes are amenable to ligand-binding assays, whereby the immobilized monoclonal antibody component is replaced by an appropriately imprinted matrix. An example ofthe use of such matrixes in this way can be seen in Vlatakis, G. et al (1993) Nature 361:645-647. Through the use of isotope-labeling, the "free" concentration of compound which modulates the expression or activity of 21953 can be readily monitored and used in calculations of IC₅₀.

[372] Such "imprinted" affinity matrixes can also be designed to include fluorescent groups whose photon-emitting properties measurably change upon local and selective binding of target compound. These changes can be readily assayed in real time using appropriate fiberoptic devices, in turn allowing the dose in a test subject to be quickly optimized based on its individual IC5₀. An rudimentary example of such a "biosensor" is discussed in Kriz, D. etal (1995) Analytical Chemistry 67:2142-2144. [373] Another aspect ofthe invention pertains to methods of modulating 21953 expression or activity for therapeutic purposes. Accordingly, in an exemplary embodiment, the modulatory method ofthe invention involves contacting a cell with a 21953 or agent that modulates one or more ofthe activities of 21953 protein activity associated with the cell. An agent that modulates 21953 protein activity can be an agent as described herein, such as anucleic acid or a protein, a naturally-occurring target molecule of a 21953 protein (e.g., a 21953 substrate or receptor), a 21953 antibody, a 21953 agonist or antagonist, a peptidomimetic of a 21953 agonist or antagonist, or other small molecule. [374] In one embodiment, the agent stimulates one or 21953 activities. Examples of such stimulatory agents include active 21953 protein and a nucleic acid molecule encoding 21953. In another embodiment, the agent inhibits one or more 21953 activities. Examples of such inhibitory agents include antisense 21953 nucleic acid molecules, anti-21953 antibodies, and 21953 inhibitors. These modulatory methods can be performed in vitro (e.g., by culturing the ceU with the agent) or, alternatively, in vivo (e.g., by administering the agent to a subject). As such, the present invention provides methods of treating an individual afflicted with a disease or disorder characterized by aberrant or unwanted expression or activity of a 21953 protein or nucleic acid molecule. In one embodiment, the method involves administering an agent (e.g., an agent identified by a screening assay described herein), or combination of agents that modulates (e.g., up regulates or down regulates) 21953 expression or activity. In another embodiment, the method involves administering a 21953 protein or nucleic acid molecule as therapy to compensate for reduced, aberrant, or unwanted 21953 expression or activity. [375] Stimulation of 21953 activity is desirable in situations in which 21953 is abnormally downregulated and/or in which increased 21953 activity is likely to have a beneficial effect. For example, stimulation of 21953 activity is desirable in situations in which a 21953 is downregulated and/or in which increased 21953 activity is likely to have a beneficial effect. Likewise, inhibition of 21953 activity is desirable in situations in which 21953 is abnormally upregulated and/or in which decreased 21953 activity is likely to have a beneficial effect.

Pharmacogenomics

[376] The 21953 molecules ofthe present invention, as well as agents, or modulators which have a stimulatory or inhibitory effect on 21953 activity (e.g., 21953 gene expression) as identified by a screening assay described herein can be administered to individuals to treat (prophylactically or therapeutically) 21953 associated disorders (e.g., a cell proliferative or cell differentiative disorder, e.g., a cancer, e.g., a cancer ofthe lung, prostate, breast, or colon) associated with aberrant or unwanted 21953 activity. In conjunction with such treatment, pharmacogenomics (i.e., the study ofthe relationship between an individual's genotype and that individual's response to a foreign compound or drag) may be considered. Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration ofthe pharmacologically active drag. Thus, a physician or clinician may consider applying knowledge obtained in relevant pharmacogenomics studies in deterrnining whether to administer a 21953 molecule or 21953 modulator as weU as tailoring the dosage and/or therapeutic regimen of treatment with a 21953 molecule or 21953 modulator. [377] Pharmacogenomics deals with clinically significant hereditary variations in the response to drugs due to altered drag disposition and abnormal action in affected persons. See, for example, Eichelbaum, M. etal. (1996) Clin. Exp. Pharmacol. Physiol 23:983-985 and Linder, M.W. et al. (1997) Clin. Chem. 43:254-266. In general, two types of pharmacogenetic conditions can be differentiated. Genetic conditions transmitted as a single factor altering the way drags act on the body (altered drug action) or genetic conditions transmitted as single factors altering the way the body acts on drugs (altered drug metaboUsm). These pharmacogenetic conditions can occur either as rare genetic defects or as naturaUy-occurring polymorphisms. For example, glucose-6-phosphate dehydrogenase deficiency (G6PD) is a common inherited enzymopathy in which the main clinical complication is haemolysis after ingestion of oxidant drags (anti-malarials, sulfbnamides, analgesics, nitrofurans) and consumption of fava beans.

[378] One pharmacogenomics approach to identifying genes that predict drug response, known as "a genome-wide association", relies primarily on a high-resolution map ofthe human genome consisting of already known gene-related markers (e.g., a "bi-allelic" gene marker map which consists of 60,000-100,000 polymoφhic or variable sites on the human genome, each of which has two variants.) Such a high-resolution genetic map can be compared to a map ofthe genome of each of a statistically significant number of patients taking part in a Phase II/JJI drug trial to identify markers associated with a particular observed drag response or side effect. Alternatively, such a high resolution map can be generated from a combination of some ten-million known single nucleotide polymoφhisms (SNPs) in the human genome. As used herein, a "SNP" is a common alteration that occurs in a single nucleotide base in a stretch of DNA For example, a SNP may occur once per every 1000 bases of DNA A SNP may be involved in a disease process, however, the vast majority may not be disease-associated. Given a genetic map based on the occurrence of such SNPs, individuals can be grouped into genetic categories depending on a particular pattern of SNPs in their individual genome. In such a manner, treatment regimens can be taUored to groups of genetically similar individuals, taking into account traits that may be common among such geneticaUy similar individuals. [379] Alternatively, a method termed the "candidate gene approach," can be utilized to identify genes that predict drag response. According to this method, if a gene that encodes a drug's target is known (e.g., a 21953 protein ofthe present invention), aU common variants of that gene can be fairly easily identified in the population and it can be determined if having one version ofthe gene versus another is associated with a particular drag response. [380] Alternatively, a method termed the "gene expression profiling," can be utilized to identify genes that predict drug response. For example, the gene expression of an animal dosed with a drug (e.g., a 21953 molecule or 21953 modulator ofthe present invention) can give an indication whether gene pathways related to toxicity have been turned on. [381] Information generated from more than one ofthe above pharmacogenomics approaches can be used to determine appropriate dosage and treatment regimens for prophylactic or therapeutic treatment of an individual. This knowledge, when applied to dosing or drug selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic or prophylactic efficiency when treating a subject with a 21953 molecule or 21953 modulator, such as a modulator identified by one ofthe exemplary screening assays described hereia

[382] The present invention further provides methods for identifying new agents, or combinations, that are based on identifying agents that modulate the activity of one or more ofthe gene products encoded by one or more ofthe 21953 genes ofthe present invention, wherein these products may be associated with resistance ofthe cells to a therapeutic agent. Specifically, the activity ofthe proteins encoded by the 21953 genes ofthe present invention can be used as a basis for identifying agents for overcoming agent resistance. By blocking the activity of one or more ofthe resistance proteins, target cells, e.g., human cells, will become sensitive to treatment with an agent that the unmodified target ceUs were resistant to.

[383] Monitoring the influence of agents (e.g., drags) on the expression or activity of a

21953 protein can be applied in clinical trials. For example, the effectiveness of an agent determined by a screening assay as described herein to increase 21953 gene expression, protein levels, or upregulate 21953 activity, can be monitored in clinical trials of subjects exhibiting decreased 21953 gene expression, protein levels, or downregulated 21953 activity. Alternatively, the effectiveness of an agent determined by a screening assay to decrease 21953 gene expression, protein levels, or downregulate 21953 activity, can be monitored in clinical trials of subjects exhibiting increased 21953 gene expression, protein levels, or upregulated 21953 activity. In such clinical trials, the expression or activity of a 21953 gene, and preferably, other genes that have been implicated in, for example, a 21953- associated disorder can be used as a 'read out" or markers ofthe phenotype of a particular cell.

21953 Informatics

[384] The sequence of a 21953 molecule is provided in a variety of media to facilitate use thereof. A sequence can be provided as a manufacture, other than an isolated nucleic acid or amino acid molecule, which contains a 21953. Such a manufacture can provide a nucleotide or amino acid sequence, e.g., an open reading frame, in a form which allows examination ofthe manufacture using means not directly applicable to examining the nucleotide or amino acid sequences, or a subset thereof, as they exists in nature or in purified for The sequence information can include, but is not limited to, 21953 full-length nucleotide and/or amino acid sequences, partial nucleotide and/or amino acid sequences, polymoφhic sequences including single nucleotide polymoφhisms (SNPs), epitope sequence, and the like. In a preferred embodiment, the manufacture is a machine-readable medium, e.g., a magnetic, optical chemical or mechanical information storage device. [385] As used herein, "machine-readable media" refers to any medium that can be read and accessed directly by a machine, e.g., a digital computer or analogue computer. Non-Umiting examples of a computer include a desktop PC, laptop, mainframe, server (e.g., a web server, network server, or server farm), handheld digital assistant, pager, mobile telephone, and the like. The computer can be stand-alone or connected to a communications network, e.g., a local area network (such as a VPN or intranet), a wide area network (e.g., an Extranet or the Internet), or a telephone network (e.g., a wireless, DSL, or ISDN network). Machine-readable media include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as CD-ROM; electrical storage media such as RAM, ROM, EPROM, EEPROM, flash memory, and the like; and hybrids of these categories such as magnetic/optical storage media.

[386] A variety of data storage structures are avaUable to a skiUed artisan for creating a machine-readable medium having recorded thereon a nucleotide or amino acid sequence of the present invention. The choice ofthe data storage stracture will generaUy be based on the means chosen to access the stored information In addition, a variety of data processor programs and formats can be used to store the nucleotide sequence information ofthe present invention on computer readable medium The sequence information can be represented in a word processing text file, formatted in commercially-available software such as WordPerfect and Microsoft Word, or represented in the form of an ASCII file, stored in a database application, such as DB2, Sybase, Oracle, or the like. The skilled artisan can reacUly adapt any number of data processor stracturing formats (e.g. , text file or database) in order to obtain computer readable medium having recorded thereon the nucleotide sequence information ofthe present invention.

[387] In a preferred embodiment, the sequence information is stored in a relational database (such as Sybase or Oracle). The database can have a first table for storing sequence (nucleic acid and/or amino acid sequence) information. The sequence information can be stored in one field (e.g., a first column) of a table row and an identifier for the sequence can be store in another field (e.g., a second column) ofthe table row. The database can have a second table, e.g., storing annotations. The second table can have a field for the sequence identifier, a field for a descriptor or annotation text (e.g., the descriptor can refer to a functionality ofthe sequence, a field for the initial position in the sequence to which the annotation refers, and a field for the ultimate position in the sequence to which the annotation refers. Non-limiting examples for annotation to nucleic acid sequences include polymoφhisms (e.g., SNP's) translational regulatory sites and splice junctions. Non- limiting examples for annotations to amino acid sequence include polypeptide domains, e.g., a domain described herein; active sites and other functional amino acids; and modification sites.

[388] By providing the nucleotide or amino acid sequences ofthe invention in computer readable form, the skilled artisan can routinely access the sequence information for a variety of puφoses. For example, one skilled in the art can use the nucleotide or amino acid sequences ofthe invention in computer readable form to compare a target sequence or target structural motif with the sequence information stored within the data storage means. A search is used to identify fragments or regions of he sequences ofthe invention which match a particular target sequence or target motif. The search can be a BLAST search or other routine sequence comparison, e.g., a search described herein. [389] Thus, in one aspect, the invention features a method of analyzing 21953, e.g., analyzing stracture, function, or relatedness to one or more other nucleic acid or amino acid sequences. The method includes: providing a 21953 nucleic acid or amino acid sequence; comparing the 21953 sequence with a second sequence, e.g., one or more preferably a pluraUty of sequences from a collection of sequences, e.g., a nucleic acid or protein sequence database to thereby analyze 21953. The method can be performed in a machine, e.g., a computer, or manually by a skilled artisan.

[390] The method can include evaluating the sequence identity between a 21953 sequence and a database sequence. The method can be performed by accessing the database at a second site, e.g., over the Internet.

[391] As used herein, a "target sequence" can be any DNA or amino acid sequence of six or more nucleotides or two or more amino acids. A skilled artisan can readily recognize that the longer a target sequence is, the less likely a target sequence will be present as a random occurrence in the database. Typical sequence lengths of a target sequence are from about 10 to 100 amino acids or from about 30 to 300 nucleotide residues. However, it is well recognized that commercially important fragments, such as sequence fragments involved in gene expression and protein processing, may be of shorter length. [392] Computer software is publicly available which allows a skilled artisan to access sequence information provided in a computer readable medium for analysis and comparison to other sequences. A variety of known algorithms are disclosed publicly and a variety of commercially available software for conducting search means are and can be used in the computer-based systems ofthe present invention. Examples of such software include, but are not limited to, MacPattern (EMBL), BLASTN and BLASTX (NCBI). [393] Thus, the invention features a method of making a computer readable record of a sequence of a 21953 sequence which includes recording the sequence on a computer readable matrix. In a preferred embodiment the record includes one or more ofthe following: identification of an ORF; identification of a domain, region, or site; identification ofthe start of transcription; identification ofthe transcription terminator; the full length amino acid sequence ofthe protein, or a mature form thereof; the 5' end ofthe translated region

[394] In another aspect, the invention features, a method of analyzing a sequence. The method includes: providing a 21953 sequence, or record, in machine-readable form; comparing a second sequence to the 21953 sequence; thereby analyzing a sequence. Comparison can include comparing to sequences for sequence identity or determining if one sequence is included within the other, e.g., deterrnining if the 21953 sequence includes a sequence being compared. In a preferred embodiment the 21953 or second sequence is stored on a first computer, e.g., at a first site and the comparison is performed, read, or recorded on a second computer, e.g., at a second site. For example, the 21953 or second sequence can be stored in a public or proprietary database in one computer, and the results ofthe comparison performed, read, or recorded on a second computer. In a preferred embodiment the record includes one or more ofthe following: identification of an ORF; identification of a domain, region, or site; identification ofthe start of transcription; identification ofthe transcription terminator; the full length amino acid sequence ofthe protein, or a mature form thereof; the 5' end ofthe translated region. [395] In another aspect, the invention provides a machine-readable medium for holding instructions for performing a method for determining whether a subject has a 21953- associated disease or disorder or a pre-disposition to a 21953-associated disease or disorder, wherein the method comprises the steps of determining 21953 sequence information associated with the subject and based on the 21953 sequence information, determining whether the subject has a 21953-associated disease or disorder or a pre-disposition to a 21953-associated disease or disorder and/or recommending a particular treatment for the disease, disorder or pre-disease condition.

[396] The invention further provides in an electronic system and/or in a network, a method for deterniining whether a subject has a 21953-associated disease or disorder or a pre-disposition to a disease associated with a 21953 wherein the method comprises the steps of determining 21953 sequence information associated with the subject, and based on the 21953 sequence information, determining whether the subject has a 21953-associated disease or disorder or a pre-disposition to a 21953-associated disease or disorder, and/or recommending a particular treatment for the disease, disorder or pre-disease condition. In a preferred embodiment, the method further includes the step of receiving information, e.g., phenotypic or genotypic information, associated with the subject and/or acquiring from a network phenotypic information associated with the subject. The information can be stored in a database, e.g., a relational database. In another embodiment, the method further includes accessing the database, e.g., for records relating to other subjects, comparing the 21953 sequence ofthe subject to the 21953 sequences in the database to thereby determine whether the subject as a 21953-associated disease or disorder, or a pre-disposition for such [397] The present invention also provides in a network, a method for deterrnining whether a subject has a 21953 associated disease or disorder or a pre-disposition to a 21953- associated disease or disorder associated with 21953, said method comprising the steps of receiving 21953 sequence information from the subject and/or information related thereto, receiving phenotypic information associated with the subject, acquiring information from the network corresponding to 21953 and/or corresponding to a 21953-associated disease or disorder (e.g., a cell proliferative or ceU differentiative disorder, e.g., a cancer, e.g., a cancer ofthe lung, prostate, breast, or colon), and based on one or more ofthe phenotypic information, the 21953 information (e.g., sequence information and/or information related thereto), and the acquired information, determining whether the subject has a 21953- associated disease or disorder or a pre-disposition to a 21953-associated disease or disorder. The method may further comprise the step of recommending a particular treatment for the disease, disorder or pre-disease condition.

[398] The present invention also provides a method for determining whether a subject has a 21953 -associated disease or disorder or a pre-disposition to a 21953-associated disease or disorder, said method comprising the steps of receiving information related to 21953 (e.g., sequence information and or information related thereto), receiving phenotypic information associated with the subject, acquiring information from the network related to 21953 and/or related to a 21953-associated disease or disorder, and based on one or more of the phenotypic information, the 21953 information, and the acquired information, determining whether the subject has a 21953-associated disease or disorder or a predisposition to a 21953-associated disease or disorder. The method may further comprise the step of recommending a particular treatment for the disease, disorder or pre-disease condition.

[399] This invention is further illustrated by the following examples that should not be construed as limiting. The contents of aU references, patents and pubUshed patent appUcations cited throughout this application are incoφorated herein by reference.

EXAMPLES

Example 1: Identification and Characterization of Human 21953 cDNA

[400] The human 21953 nucleic acid sequence is recited as foUows:

[401] CTATAGGGAGTCGCCCACGCGTCCGGCCTCCGAGGCCAAGGCCGCTG

CTACTC CGCCGCTGCTTCTTAGTGCCGCGTTCGCCGCCTGGGTTGTCACCGGCG

CCGCCGCCGAGGAAGCCACTACAACCAGGACCGGAGTGGAGGCGGCGCAGCAT GAAGCGGCCTCAGGCCCGCTCCATAGCCCACGTCGGGACGGTCCGGGCGGGGCC GGGGGGAAGGAAAATGCAACATGGCAGCAGCAATGGAAACAGAACAGCTGGG TGTTGAGATATTTGAAACTGCGGACTGTGAGGAGAATATTGAATCACAGGATCG

GCCTAAAT GGAGCCT TTΓATGTTGAGCGGTATTCCTGGAGTCAGCTTAAAAA GCTGCTTGCCGATACCAGAAAATATCATGGCTACATGATGGCTAAGGCACCACA

TGATTTCATGTTTGTGAAGAGGAATGATCCAGATGGACCTCATTCAGACAGAAT

CTAT ACCTTGCCATGTCTGGTGAGAACAGAGAAAATACACTGTTTTATTCTGAA

ATTCCCAAAACTATCAATAGAGCAGCAGTCTTAATGCTCTCTTGGAAGCCTCTTT

TGGATCTTTTTCAGGCAACACTGGACTATGGAATGTATTCTCGAGAAGAAGAAC

TATTAAGAGAAAGAAAACGCATTGGAACAGTCGGAATTGCTTCTTACGATTATC

ACCAAGGAAGTGGAACATTTCTGTTΓCAAG CGGTAGTGGAATTTATCACGTAA

AAGATGGAGGGCCACAAGGATTTACGCAACAACCTTTAAGGCCCAATCTAGTG

GAAACTAGTTGTCCCAACATACGGATGGATCCAAAATTATGCCCTGCTGATCCA

GACTCGATTGCTTTΓATACATAC AACGATATTTGGATATCTAACATCGTAACCA

GAGAAGAAAGGAGACTCACTTATGTGCACAATGAGCTAGCCAACATGGAAGAA

GATGCCAGATCAGCTGGAGTCGCTACCTTTGTTCTCCAAGAAGAATTTGATAGA

TATTCTGGCTATTGGTGGTGTCCAAAAGCTGAAACAACTCCCAGTGGTGGTAAA

AT CTTAGAATTCTATATGAAGAAAATGATGAATCTGAGGTGGAAATTATTCAT

GTTACATCCCCTATGTTGGAAACAAC 3AGGGCAGATTCATTCCGTTATCCTAAA

ACAGGTACAGCAAATCCTAAAGTCACTTTTAAGATGTCAGAAATAATGATTGAT

GCTGAAGGAAGGATCATAGATGTCATAGATAAGGAACTAATTCAACCTTTTGAG

ATTCTATTTGAAGGAGTTGAATATATTGCCAGAGCTGGATGGACTCCTGAGGGA

AAATATGCTTGGTCCATCCTACTAGATCGCTCCCAGACTCGCCTGCAGATAGTGT

TGATCTCACCTGAATTATΓTATCCCAGTAGAAGATGATGTTATGGAAAGGCAGA

GACTCATTGAGTCAGTGCCTGATTCTGTGACGCCACTAATTATCTATGAAGAAA

CAACAGACATCTGGATAAATATCCATGACATCTTTCATGTTΓTTCCCCAAAGTCA

CGAAGAGGAAATTGAGTTTATTT1 GCCTCTGAATGCAAAACAC<}TTTCCGTCA

TTTATACAAAAT ACATCTATTTTAAAGGAAAGCAAATATAAACGATCCAGTGG

TGGGCTGCCTGCTCCAAGTGATTTCAAGTGTCCTATCAAAGAGGAGATAGCAAT

TACCAGTCTGTGAATGGGAAGTTCTTGGCCGGCATGGATCTAATATCCAAGTTGA

TGAAGTCAGAAGGCTGGTATATTI GAAGGCACCAAAGACTCCCCTTTAGAGCA

TCACCTGTACGTAGTCAGTTACGTAAATCCTGGAGAGGTGACAAGGCTGACTGA

CCGTGGCTACTCACATTCTTGCTGCATCAGTCAGCACTGTGACTTCTTTATAAGT AAGTATAGTAACCAGAAGAATCCACACTGTGTGTCCCTTTACAAGCTATCAAGT

CCTGAAGATGACCCAACTTGCAAAACAAAGGAATTTTGGGCCACCATTTTGGAT

TCAGCAGGTCCTCTTCCTGACTATACTCCTCCAGAAATTTTCTCTTTTGAAAGTA

CTACTGGATTTACATTGTATGGGATGCTCTACAAGCCTCATGATCTACAGCCTGG

AAAGAAATATCCTACTGTGCTGTTCATATATGGTGGTCCTCAGGTGCAGTTGGT

GAATAATCGGTTTAAAGGAGTCAAGTATTTCCGCTTGAATACCCTAGCCTCTCTA

GGTTATGTGGTTGTAGTGATAGACAACAGGGGATCCTGTCACCGAGGGCTTAAA

TTTGAAGGCGCCTTTAAATATAAAATGGGTCAAATAGAAATTGACGATCAGGTG

GAAGGACTCCAATATCTAGCTTCTCGATATGATTTCATTGACTTAGATCGTGTGG

GCATCCACGGCTGGTCCTATGGAGGATACCTCTCCCTGATGGCATTAATGCAGA

C^JTCAGATATCTTCAGGGTTGCTATTGCTGGGGCCCCAGTCACTCTGTGGATCTT

CTATGATACAGGATACACGGAACGTTATATGGGTCACCCTGACCAGAATGAACA

GGGCTATTACTTAGGATCTGTGGCCATGCAAGCAGAAAAGTTCCCCTCTGAACC

AAATCGTTTACTGCTCTTACATC<ΪTTTCCTGGATGAGAATGTCCATTTTGCACAT

ACCAGTATAT ACTGAGTTTTTTAGTGAGGGCTGGAAAGCCATATGATTTACAG

ATCTATCCTCAGGAGAGACACAGCATAAGAGTTCCTGAATCGGGAGAACATTAT

GAACTGCATCT TTGCACTACCTTCAAGAAAACCTTGGATCACGTATTGCTGCTC

TAAAAGTGATATAATTTTGACCTGTGTAGAACTCTCTGGTATACACTGGCTATTT

AACCAAATGAGGAGGTTTAATCAACAGAAAACACAGAATTGATCATCACATTTT

GATACCTGCCATGTAACATCTACTCCTGAAAATAAATGTGGTGCCATGCAGGGG

TCTACGGTTTGTGGTAGTAATCTAATACCTTAACCCCACATGCTCAAAATCAAAT

GATACATATTCCTGAGAGACCCAGCAATACCATAAGAATTACTAAAAAAAAAA

AAAAAAAAA ( SEQ ID NO:l).

[402] The human 21953 nucleic acid sequence (SEQ ID NO: 1) is approximately 3143 nucleotides long. The nucleic acid sequence includes an initiation codon (ATG) and a termination codon (TAA) which are underlined above. The region between and inclusive of the initiation codon and the teπnination codon is a methionine-initiated coding sequence of about 2646 nucleotides (nucleotides 229-2874 of SEQ ID NO: 1, designated as SEQ ID

NO:3). The coding sequence encodes an 882 amino acid protein, the sequence of which is recited as follows:

[403] MAAAMETEQLGVEIFETADCEENIESQDRPKLEPFYVERYSWSQLKKLL ADTΈKYHGYMMAKAPHDFMFVKRNDPDGPHSDRIYYLAMSGENRENTLFYSEIPK TINRAAVLMLSWKPLLDLFQATLDYGMYSREEELLRERKRIGTNGIASYDYHQGSG TTLFQAGSGRYL^IADGGPQGFTQQPLRPNLVETSCPMRMDPKLCPADPDWIAFTHS

NDIWISNIVTREERRLTYVHNELANFMEEDARSAGVATFVLQEEFDRYSGYWWCPK

AETTPSGGKILMLYEENDESEVEI1I VTSPMLETRRADSFRYPKTGTANPKVTFKMS

EIMIDAEGRIIDVIDKELIQPFEILFEGVEYIARAGWΓPEGKYAWSILLDRSQTRLQΓVL

ISPELΠPVEDDVIVIERQRLIESVPDSVTPLIIYEETTDIWΓNΓHDIIΉVIΦQSHEEEIEFIF

ASECKTGFRHLYKITSILKESKYKRSSGGLPAPSDFKCPIKEEIAITSGEWEVLGRHGS

MQVDEVRRLVYFEGTIA->SPLEHHLYVVSYVNPGEVTRLTDRGYSHSCCISQHCDFF

ISKYSNQKNPHCVSLYKLSSPEDDPTCKTKEFWAΉLDSAGPLPDYTPPEIFSFESTTG

F LYGMLYKPHDLQPGKKYPTNLFIYGGPQVQLVNNRFKGVKYFRLNTLASLGYV

VVVIDNRGSCHRGLKFEGAFKYKMGQIEIDDQVEGLQYLASRYDFIDLDRVGIHGW SYGGYLSLMALMQRSDIFRVAIAGAPVTLWIFYDTGYTERYMGHPDQNEQGYYLG SVAMQAEKFPSEPNRLLLLHGFLDENVHFAHTSILLSFLVRAGKPYDLQiYPQERHSI RVPESGEHYELHLLHYLQENLGSRIAALKVI (SEQ ID NO:2).

Example 2: 21953 mRNA Expression

[404] Endogenous human 21953 gene expression was determined using the Perkin-

Elmer/ABI 7700 Sequence Detection System which employs TaqMan technology. Briefly, TaqMan technology relies on standard RT-PCR with the addition of a third gene-specific oligonucleotide (referred to as a probe) which has a fluorescent dye coupled to its 5' end (typically 6-FAM) and a quenching dye at the 3' end (typically TAMRA). When the fluorescently tagged oUgonucleotide is intact, the fluorescent signal from the 5' dye is quenched. As PCR proceeds, the 5' to 3' nucleolytic activity of Taq polymerase digests the labeled primer, producing a free nucleotide labeled with 6-FAM, which is now detected as a fluorescent signal. The PCR cycle where fluorescence is first released and detected is directly proportional to the starting amount ofthe gene of interest in the test sample, thus providing a quantitative measure ofthe initial template concentration. Samples were internally controlled by the addition of a second set of primers/probe specific for a reference gene such as β2-macroglobulin, GAPDH which has been labeled with a different fluorophore on the 5' end (typically VIC).

[405] To determine the level of 21953 in various human tissues a primer/probe set was designed. Total RNA was prepared from a series of human tissues using an RNeasy kit from Qiagen. First strand cDNA was prepared from 1 μg total RNA using an oligo-dT primer and Superscript II reverse transcriptase (Gibco/BRL). cDNA obtained from approximately 50 ng total RNA was used per TaqMan reaction. Tissues tested include the human tissues and several cell lines shown in the left column ofthe tables below. [406] 21953 mRNA expression was elevated in 85% of clinical lung tumor samples tested, and is similarly elevated in a number of breast tumor and colon tumor samples (see, e.g., Table 1 below).

Table 1.

Sample Relative Expression

Breast Normal 0.02

Breast Normal 0.07

Breast Tumor 0.08

Breast Tumor 0.07

Breast Tumor 0.19

Breast Tumor 0.21

Breast Tumor 0.07

Breast Tumor 0.30

Ovary Normal 0.37

Ovary Normal 0.26

Ovary Normal 0.33

Ovary Tumor 0.16

Ovary Tumor 0.13

Ovary Tumor 0.17

Ovary Tumor 0.10

Ovary Tumor 0.12

Ovary Tumor 0.08

Ovary Tumor 0.52

Ovary Tumor 0.06

Lung Normal 0.02

Lung Normal 0.01

Lung Normal 0.10

Lung Normal 0.01

Lung Tumor 0.59

Lung Tumor 0.18

Lung Tumor 0.24

Lung Tumor 0.04

Lung Tumor 0.78

Lung Tumor 0.37

Lung Tumor 0.16

[407] Many tested lung tumor samples in Table 1 (6 of 7) expressed 21953 mRNA at higher levels than did normal lung tumor samples. Similarly, a number of breast tumor samples expressed 21953 mRNA to a greater extent that did normal breast tumor samples. Table 2.

Sample Relative Expression

Colon Normal 0.00

Colon Normal 0.02

Colon Normal 0.05

Colon Normal 0.01

Colon Tumor 0.03

Colon Tumor 0.24

Colon Tumor 0.07

Colon Tumor 0.03

Colon Tumor 0.03

Colon Tumor 0.04

Liver Metastatic 0.07

Liver Metastatic 0.16

Liver Metastatic 0.23

Liver Normal 0.05

Liver Normal 0.19

Brain Normal 1.50

Brain Normal 0.98

Astrocyte 0.37

Brain Tumor 0.04

Brain Tumor 0.10

Brain Tumor 0.04

Brain Tumor 0.13

HMVEC-Arr 0.22

HMVEC-Prol 0.26

Placenta 0.11

Fetal Adrenal 0.15

Fetal Adrenal 0.18

Fetal Liver 0.71

Fetal Liver 0.18

[408] The mRNA expression data for 21953 mRNA tabulated in Table 2 indicated that

(1) 21953 mRNA can be overexpressed in some colon tumor samples relative to normal colon tissue samples; (2) 21953 mRNA is weU expressed in metastatic liver samples; (3) 21953 mRNA is highly expressed in normal brain tissue (e.g., increased expression relative to brain tumors), astrocytes, and fetal liver; and (4) 21953 mRNA is also expressed in HMVEC (human microvascular endothelial cells), and fetal adrenal cells. Table 3.

Sample Relative Expression

Aorta / normal 0.00

Fetal heart/ normal 2.42

Heart normal 0.66

Heart/ CHF 0.72

Vein/ Normal 0.13

SMC (Aortic) 0.89

Spinal cord Normal 0.66

Brain cortex/ Normal 5.94

Brain hypothalamus/ Normal 4.13

Glial cells (Astrocytes) 1.35

Brain/ Glioblastoma 1.12

Breast /Normal 0.18

Breast tumor/ IDC 0.38

Ovary/ Normal 0.39

Ovary/ Tumor 0.16

Pancreas 0.25

Prostate/ Normal 0.18

Prostate/ Tumor 0.15

Colon normal 0.07

Colon/tumor 0.56

Colon/IBD 0.10

Kidney/ normal 0.71

Liver/ normal 0.10

Liver fibrosis 0.22

Fetal Liver/normal 2.21

Lung / normal 0.16

Lung/ tumor 0.39

Lung/ COPD 0.22

Spleen/ normal 0.14

Tonsil normal 0.11

Lymph node/ normal 0.27

Thymus/ normal 1.16

Epithelial Cells (prostate) 2.04

Endothelial Cells (aortic) 0.27

Skeletal Muscle/ Normal 1.22

Fibroblasts (Dermal) 0.18

Skin/ normal 0.35

Adipose/ Normal 0.06

Osteoblasts (primary) 0.44

Osteoblasts (Undiff) 0.32

Osteoblasts(Diff) 0.29

Osteoclasts 0.08

Aortic SMC Early 1.27

Aortic SMC Late 2.61 shear HUVEC 3.39 static HUVEC 2.14 [409] The mRNA expression data for 21953 mRNA tabulated in Table 3 indicated that

21953 mRNA is highly expressed, for example, in fetal heart, brain cortex, brain hypothalamus, fetal liver, epitheUal cells from prostate, aortic smooth muscle ceUs, and human umbilical vein endotheUal cells under both shear and static conditions.

Table 4

Sample Relative Expression

MCF-7 Breast Tumor 15.15

ZR75 Breast Tumor 6.11

T47D Breast Tumor 1.50

MD A 231 Breast Tumor 0.01

MDA 435 Breast Tumor 0.00

DLD 1 ColonT (stageC) 22.33

SW480 ColonT (stageB) 0.06

SW620 ColonT (stageC) 5.23

HCT116 0.63

HT29 0.01

Colo 205 0.00

NCIH125 0.75

NCIH69 23.28

NCIH322 20.91

NCIH460 1.25

A549 7.11

NHBE 0.83

SKOV-3 ovary 0.22

OVCAR-3 ovary 17.28

293 ovary 44.97

293T ovary 59.75

A549 t6 0.83

A549 t9 1.27

A549 tl8 14.63

A549124 1.99

[410] Tumor cell lines were xenografted into nude mice. Expression of human 21953 mRNA in tumors harvested from the mice was analyzed using TaqMan. Results are tabulated in Table 4 (excepting the final four rows, see below). The results indicated that, for example, 21953 mRNA is highly expressed in some xenografted colon tumor samples (colonT), some xenografted breast tumor samples, and xenografted ovarian ceU lines. [411] The final four rows of Table 4 tabulate relative 21953 mRNA expression in samples of A549 human lung cancer ceUs at various hourly time points (time in hours being indicated with the prefix t) after release from aphidocolin treatment. 21953 mRNA expression peaked at the Gl to S phase transition

Table 5

Sample Relative Expression

PIT 337 Colon Normal 0.28 CHT 410 Colon Normal 0.03 CHT 425 Colon Normal 0.13 CHT 371 Colon Normal 0.03

CHT 414 Colonic ACA-B 0.16 CHT 841 Colonic ACA-B 0.07 CHT 807 Colonic ACA-B 0.21 CHT 382 Colonic ACA-B 0.32 CHT 596 Colonic ACA-C 0.00 CHT 907 Colonic ACA-C 0.13 CHT 372 Colonic ACA-C 0.49 NDR 210 Colonic ACA-C 0.13 CHT 1365 Colonic ACA-C 0.03

CLN 741 Liver Normal 0.00

NDR 165 Liver Normal 0.00

NDR 150 Liver Normal 0.06

PIT 236 Liver Normal 0.00

CHT 077 Col Liver Metastatis 0.06 CHT 119 Col Liver Metastatis 4.79 CHT 131 Col Liver Metastatis 0.76 CHT 218 Col Liver Metastatis 1.12 CHT 739 Col Liver Metastatis 0.18

CHT 215 Col Abdominal Metastatis 0.01

[412] 21953 mRNA is cell cycle regulated in the lung carcinoma cell line A549.

A549 cells were synchronized with aphidocholin, and then released. mRNA was prepared from the cells at regular intervals after release. 21953 expression peaked during the Gl to S phase transition

[413] In situ hybridization experiments which provided additional confirmatory results are tabulated in Table 6. 21953 mRNA was observed by in situ hybridization in lung small cell carcinoma and differentiated tumors, but not in normal lung tissue. Similarly, by this analysis, 21953 mRNA expression was elevated in colon tumor samples (2 of 2), metastatic colon tumor samples (2 of 2), and in a differentiated papillary ovarian tumor sample. 21953 mRNA was also detected in normal breast tissue (1 of 1), normal ovarian tissue (1 of 1), and ovarian tumors (2 of 2). Table 6.

Tissue Diagnosis Results

Breast Normal

Breast Intraductal Carcinoma

Colon Normal

Colon Normal

Colon Tumor +

Colon Tumor +

Colon Metastasis +

Colon Metastasis

Liver Normal

Lung Normal

Lung Small Cell Carcinoma

Lung Differentiated

Lung Differentiated +/-

Lung Differentiated

Ovary Normal +

Ovary Tumor (well differentiated carcinoma) +

Ovary Tumor (moderately differentiated papillary) -H

Example 3: Recombinant Expression of 21953 in Bacterial Cells

[414] In this example, 21953 is expressed as a recombinant glutathione-S-transferase

(GST) fusion polypeptide in E. coli and the fusion polypeptide is isolated and characterized. Specifically, 21953 is fused to GST and this fusion polypeptide is expressed in E. coli, e.g., strain PΕB199. Expression ofthe GST-21953 fusion protein in PEB199 is induced with IPTG. The recombinant fusion polypeptide is purified from crude bacterial lysates ofthe induced PEB 199 strain by affinity chromatography on glutathione beads. Using polyacry lamide gel electrophoretic analysis ofthe polypeptide purified from the bacterial lysates, the molecular weight ofthe resultant fusion polypeptide is determined.

Example 4: Expression of Recombinant 21953 Protein in COS Cells

[415] To express the 21953 gene in COS cells, the pcDNA Amp vector by Invitrogen

Coφoration (San Diego, CA) is used. This vector contains an SV40 origin of repUcation, an ampicillin resistance gene, an E. coli replication origin, a CMV promoter foUowed by a polylinker region, and an SV40 intron and polyadenylation site. A DNA fragment encoding the entire 21953 protein and an HA tag (Wilson et al. (1984) Cell 37:767) or a FLAG tag fused in-frame to its 3' end ofthe fragment is cloned into the polylinker region ofthe vector, thereby placing the expression ofthe recombinant protein under the control ofthe CMV promoter.

[416] To constract the plasmid, the 21953 DNA sequence is ampUfied by PCR using two primers. The 5' primer contains the restriction site of interest followed by approximately twenty nucleotides ofthe 21953 coding sequence starting from the initiation codon; the 3' end sequence contains complementary sequences to the other restriction site of interest, a translation stop codon, the HA tag or FLAG tag and the last 20 nucleotides ofthe 21953 coding sequence. The PCR amplified fragment and the pCDNA/Amp vector are digested with the appropriate restriction enzymes and the vector is dephosphorylated using the CIAP enzyme (New England Biolabs, Beverly, MA). Preferably the two restriction sites chosen are different so that the 21953_gene is inserted in the correct orientation The ligation mixture is transformed into E. coli cells (strains HB101, DH5α, SURE, avaUable from Stratagene Cloning Systems, La Jolla, C A, can be used), the transformed culture is plated on ampicillin media plates, and resistant colonies are selected. Plasmid DNA is isolated from transformants and examined by restriction analysis for the presence ofthe correct fragment.

[417] COS cells are subsequently transfected with the 21953-pcDNA/Amp plasmid

DNA using the calcium phosphate or calcium chloride co-precipitation methods, DEAE- dextran-mediated transfection, lipofection, or electroporation. Other suitable methods for transfecting host ceUs can be found in Sambrook, J., Fritsh, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. The expression ofthe 21953 polypeptide is detected by radiolabelling (³⁵S-methionine or ³⁵S-cysteine available from NEN, Boston, MA, can be used) and immunoprecipitation (Harlow, E. and Lane, D. (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) using an HA specific monoclonal antibody. Briefly, the cells are labeled for 8 hours with ³5s-methionine (or 35s-cysteine). The culture media are then collected and the cells are lysed using detergents (RIPA buffer, 150 mM NaCI, 1% NP-40, 0.1% SDS, 0.5% DOC, 50 mM Tris, pH 7.5). Both the cell lysate and the culture media are precipitated with an HA specific monoclonal antibody. Precipitated polypeptides are then analyzed by SDS- PAGE. [418] Alternatively, DNA containing the 21953 coding sequence is cloned directly into the polylinker o the pCDNA/Amp vector using the appropriate restriction sites. The resulting plasmid is transfected into COS ceUs in the manner described above, and the expression ofthe 21953 polypeptide is detected by radiolabelling and immunoprecipitation using a 21953 specific monoclonal antibody.

Equivalents

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

Ill -

Claims

What is claimed is:

1. An isolated nucleic acid molecule selected from the group consisting of: a) a nucleic acid molecule comprising a nucleotide sequence which is at least 80% identical to the nucleotide sequence of SEQ ID NO: 1, or SEQ ID NO:3; b) a nucleic acid molecule comprising a fragment of at least 800 nucleotides of the nucleotide sequence of SEQ ID NO: 1, or SEQ ID NO:3; c) a nucleic acid molecule which encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:2; d) a nucleic acid molecule which encodes a fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO:2, wherein the fragment comprises at least 15 contiguous amino acids of SEQ ID NO:2; and e) a nucleic acid molecule which encodes a naturaUy occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, wherein the nucleic acid molecule hybridizes to anucleic acid molecule comprising SEQ ID NO:l, 3, or a complement thereof) under stringent conditions.

2. The isolated nucleic acid molecule of claim 1 , which is selected from the group consisting of: a) a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO:3; and b) a nucleic acid molecule which encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:2.

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 ceU which contains the nucleic acid molecule of claim 1.

6. An isolated polypeptide selected from the group consisting of: a) a polypeptide which is encoded by a nucleic acid molecule comprising a nucleotide sequence which is at least 85% identical to a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO:3; b) a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO:2, wherein the polypeptide is encoded by a nucleic acid molecule which hybridizes to a nucleic acid molecule comprising SEQ ID NO: 1 , SEQ ID NO:3, or a complement thereof under stringent conditions; and c) a fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO:2, wherein the fragment comprises at least 15 contiguous amino acids of SEQ ID NO:2.

7. The isolated polypeptide of claim 6 comprising the amino acid sequence of SEQ IDNO.2.

8. The polypeptide of claim 6 further comprising a heterologous amino acid sequence.

9. An antibody which selectively binds to a polypeptide of claim 6.

10. A method for producing a polypeptide selected from the group consisting of: a) a polypeptide comprising the amino acid sequence of SEQ ID NO:2; b) a polypeptide comprising a fragment ofthe a ino acid sequence of SEQ ID NO:2, wherein the fragment comprises at least 15 contiguous amino acids of SEQ ID NO:2; and c) a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO:2, wherein the polypeptide is encoded by a nucleic acid molecule which hybridizes to a nucleic acid molecule comprising SEQ ID NO : 1 , SEQ ID NO:3, or a complement thereof under stringent conditions; the method, comprising culturing the host cell of claim 5 under conditions in which the nucleic acid molecule is expressed.

11. A method for detecting the presence of a polypeptide of claim 6 in a sample, comprising: a) contacting the sample with a compound which selectively binds to a polypeptide of claim 6; and b) determining whether the compound binds to the polypeptide in the sample.

12. The method of claim 11, wherein the compound is an antibody.

13. A kit comprising a compound which selectively binds to a polypeptide of claim 6 and instructions for use.

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

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

16. A method for identifying a compound which binds to a polypeptide of claim 6 comprising the steps of: a) contacting a polypeptide, or a ceU expressing a polypeptide of claim 6 with a test compound; and b) determining whether the polypeptide binds to the test compound.

17. The method of claim 16, wherein the binding of the test compound to the polypeptide is detected by a method selected from the group consisting of: a) detection of binding by direct detecting of test compound/polypeptide binding; b) detection of binding using a competition binding assay; c) detection of binding using an assay for peptide cleavage.

18. A method for modulating the activity or expression of a polypeptide of claim 6 comprising contacting a polypeptide or a ceU expressing a polypeptide of claim 6 with a compound which binds to the polypeptide in a sufficient concentration to modulate the activity or expression ofthe polypeptide.

19. A method for identifying a compound which modulates the activity of a polypeptide of claim 6, comprising: a) contacting a polypeptide of claim 6 with a test compound; and b) determining the effect ofthe test compound on the proteolytic activity ofthe polypeptide to thereby identify a compound which modulates the proteolytic activity ofthe polypeptide.

20. A method of treating or preventing a disorder characterized by aberrant activity or expression of a 21953 nucleic acid or polypeptide in a subject, the method comprising administering to the subject an effective amount of an agent that modulates the activity or expression of a 21953 nucleic acid or polypeptide such that the disorder is ameliorated or prevented.

21. A method of modulating the activity of a 21953 -expressing ceU, comprising contacting the cell with an amount of an agent that modulates the activity or expression of a 21953 nucleic acid or polypeptide such that the activity ofthe cell is modulated.

22. The method of claim 21, wherein the disorder is a cellular proliferative or cUfferentiative disorder.

23. The method of claim 22, wherein the disorder is lung cancer, colon cancer, or breast cancer.

24. The method of claim 21, wherein the agent is a peptide, a phosphopeptide, a smaU molecule, an antibody, or any combination thereof.

25. The method of claim 21, wherein the agent is an antisense, a ribozyme, a triple helix molecule, a 21953 nucleic acid, or any combination thereof.

26. A method of evaluating a subject for the presence of a ceU proliferative disorder, the method comprising: a) providing a sample from the subject; b) detecting an expression level ofthe nucleic acid of claim 1 in the sample; c) comparing the expression level to a reference expression level, wherein an increased expression level relative to the reference expression level is an indication of a ceU proliferative disorder.

27. The method of claim 26 wherein the sample comprises cells from lung, breast, ovarian, prostate, or colonic tissue.

28. A method of evaluating a subject for the presence of a ceU proliferative disorder, the method comprising: a) providing a sample from the subject; b) detecting an expression level ofthe polypeptide of claim 6 in the sample; c) comparing the abundance ofthe polypeptide to a reference expression level, wherein an increased abundance relative to the reference expression level is an indication of a cell proliferative disorder.

29. The method of claim 28 wherein the sample comprises cells from lung, breast, ovarian, prostate, or colonic tissue.