WO2003025135A2

WO2003025135A2 - Genes associated with malignant neoplasms

Info

Publication number: WO2003025135A2
Application number: PCT/US2002/029371
Authority: WO
Inventors: Michele Nation; James C. Diggans; Mark Porter; Sun Lu; Michael Orr
Original assignee: Gene Logic, Inc.
Priority date: 2001-09-14
Filing date: 2002-09-16
Publication date: 2003-03-27
Also published as: WO2003025135A3; AU2002362309A1

Abstract

The invention relates generally to the changes in gene expression in malignant neoplasms. The invention relates specifically to human genes which correspond to mRNA species that are differentially expressed in malignant neoplasms compared to normal tissues, such as neoplasms of the cervix, endometrium, uterus, breast, colon, rectum, intestine, kidney, liver, lung, stomach, ovary, pancreas, thyroid gland, lymph node, omentum, skin, esophagus, larynx, adrenal gland, prostate, vulva, connective tissue or soft tissue.

Description

GENES ASSOCIATED WITH MALIGNANT NEOPLASMS

INVENTORS: Michele Nation, James C. Diggans, Mark Porter, Sun Lu and Michael Orr

RELATED APPLICATIONS

This application is related to U.S. Provisional Applications 60/322,468, 60/324,050,

60/322,733, 60/322,790, 60/323,078, 60/373,595, 60/318,891, 60/322,732, 60/318,905, 60/324,246, 60/324,910 and 60/324,621, all of which are herein incorporated by reference in their entirety.

FIELD OF THE INVENTION

The invention relates generally to the changes in gene expression in malignant neoplastic tissue from cancer patients compared to normal human tissue. The invention relates specifically to human genes which are differentially expressed in cervical, endometrial, uterine, breast, colon, rectal, intestinal, kidney, liver, lung, stomach, ovarian, pancreatic, thyroid, lymph node, omentum, skin, esophageal, laryngeal, adrenal, prostate, vulvar, connective tissue and soft tissue from patients with malignant neoplasms (tumors) compared to normal tissues.

BACKGROUND OF THE INVENTION Cancer is group of many related diseases that begin in cells following the proliferation of cells not needed by the body. This abnormal growth results in tissue masses known as tumors, which may be benign or malignant. Benign tumors, which are not cancer, can usually be removed and do not return or spread to other parts of the body. They are rarely fatal. In contrast, malignant, or cancerous, tumors are composed of cells that divide without control or order. They can invade or damage surrounding tissues and/or spread to other parts of the body via the lymph system. Most cancers are named for the type of organ or tissue in which they arise, i.e., cancer that begins in the lung is lung cancer. As a cancer spreads, cancer cells are found in nearby lymph nodes, an indication that the cancer has spread to other organs as well. A later tumor found another part of the body has the same kind of abnormal cells as the first tumor. For example, if lung cancer spreads to the brain, the cancer cells in the brain are lung cancer cells, not brain cancer cells.

Risk factors for developing cancer include tobacco smoking, eating a high-fat diet, obesity, ultraviolet radiation, heavy alcohol consumption, and exposure to x-rays, pesticides, metals, certain pharmaceuticals and environmental toxins. Decreasing the risk of fatal cancer can be achieved by regular screening for common types of cancer, such as breast, cervical, prostate, and colon and rectal cancers.

Because cancer patients are largely asymptomatic in the early stages, most cancers are not detected at an early stage. Any symptoms that do appear (e.g., lumps, changes in moles, sores that do not heal, coughing or hoarseness, changes in bowel or bladder habits, difficulty in swallowing or indigestion, unexpected weight loss, or unusual bleeding or discharge), are not specific and may have a variety of causes. Cancers are typically diagnosed by imaging techniques, such as x-ray, CT scan (computed tomography), radionuclide scanning, ultrasound or MRI. Additional diagnostic techniques include endoscopy, needle biopsy and surgical biopsy. Treatment options for cancer, depending on the extent of the disease, are removal of the affected area, removal of the entire affected organ (or, for tissues such as skin or soft tissues, removal of more than the affected area), radiation therapy and chemotherapy. Usually, surgical removal is combined with radiation and/or chemotherapy.

The most common type of cancer is breast cancer in women and prostate cancer in men. Lung cancer is the second most common type of cancer in men and in women and the leading cause of cancer deaths for either gender (over 85% of lung cancer cases are caused by smoking). The fourth most common type of cancer for either gender is colo-rectal cancer, and the fifth is non-Hodgkins lymphomas. Although liver and pancreatic cancers are low in incidence (less than 2% each), they have among the lowest survival rates. Among women alone, cancers of the uterus and ovaries rank fourth and fifth in incidence (http://www.cancer.gov/).

Molecular Changes in Malignant Neoplasms

Little is known about the molecular changes in neoplastic cells associated with the development and progression of malignant tumors. It has been demonstrated that the expression levels of a number of individual genes are changed compared to normal cells, (e.g., GenBank Accession No. BC012980, differentially expressed in melanomas of the skin, Strausberg R., direct submission to the NTH Mammalian Gene Collection, Cancer Genomics Office, NCI; in lung cancer, neural cell adhesion molecule (NCAM) (Miyahara et al., (2001) J Surg Oncol 77(l):49-54; in cervical cancer, cyclooxygenase 2, Kulkarni et a/.(2001), Clin Cancer Res, l(2):429-434).

There exists a need for the identification of new molecular markers associated with the development and progression of malignant neoplasms. Furthermore, if intervention is expected to be successful in halting or slowing down the malignant tumors, means of accurately assessing the early manifestations of the neoplasms need to be established. One way to accurately assess the early manifestations of malignant neoplasms is to identify markers which are uniquely associated with disease progression. Likewise, the development of therapeutics to prevent or stop the progression of malignant neoplasms relies on the identification of genes that play a significant role in these diseases.

SUMMARY OF THE INVENTION

The present invention is based on the discovery of new gene families that are differentially expressed in malignant neoplastic tissues compared to normal tissues. The invention includes isolated nucleic acid molecules selected from the group consisting of an isolated nucleic acid molecule that encodes all or a portion of one or more of the amino acid sequences of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48, an isolated nucleic acid molecule that encodes a fragment of at least 10 contiguous amino acids of one or more of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48, an isolated nucleic acid molecule that hybridizes to the complement of a nucleic acid molecule comprising SEQ ED NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45 or 47 and an isolated nucleic acid molecule which hybridizes to the complement of a nucleic acid molecule encoding the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48. Nucleic acid molecules of the invention may encode a protein having at least about

50%, 60%, or 65% amino acid sequence identity to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48, preferably at least about 70% or 75% sequence identity, more preferably at least about 80% or 85% sequence identity, and even more preferably at least about 90% or 95% sequence identity to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48.

The present invention further includes the nucleic acid molecules operably linked to one or more expression control elements, including vectors comprising the isolated nucleic acid molecules. The invention further includes host cells transformed to contain the nucleic acid molecules of the invention and methods for producing a protein comprising the step of culturing a host cell transformed with a nucleic acid molecule of the invention under conditions in which the protein is expressed.

The invention further provides an isolated polypeptide selected from the group consisting of an isolated polypeptide comprising the amino acid sequence of all or a portion of one or more of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48, an isolated polypeptide comprising a fragment of at least 6 contiguous amino acids of one or more of SEQ ED NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20,

22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48, an isolated polypeptide comprising one or more conservative amino acid substitutions of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48 and an isolated polypeptide comprising naturally occurring amino acid sequence variants of SEQ ID NOS:

2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48.

Polypeptides of the invention also include polypeptides with an amino acid sequence having at least about 50%, 60%, 70% or 75% amino acid sequence identity with the sequences set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48, preferably at least about 80% or 85%, more preferably at least about 90%, and most preferably at least about 95% sequence identity with the sequence set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48. The invention further provides an isolated antibody or antigen-binding antibody fragment that specifically binds to a polypeptide of the invention, including monoclonal and polyclonal antibodies.

The invention further provides methods of identifying an agent which modulates the expression of a nucleic acid molecule encoding a protein of the invention, comprising exposing cells which express the nucleic acid molecule to the agent and determining whether the agent modulates expression of said nucleic acid molecule, thereby identifying an agent which modulates the expression of a nucleic acid molecule encoding the protein.

The invention further provides methods of identifying an agent which modulates the level of or at least one activity of a protein of the invention, comprising exposing cells which express the protein to the agent and determining whether the agent modulates the level of or at least one activity of said protein, thereby identifying an agent which modulates the level of or at least one activity of the protein.

The invention further provides methods of identifying binding partners for a protein of the invention, comprising exposing said protein to a potential binding partner and determining if the potential binding partner binds to said protein, thereby identifying binding partners for the protein.

The present invention further provides methods of modulating the expression of a nucleic acid molecule encoding a protein of the invention, comprising administering an effective amount of an agent which modulates the expression of a nucleic acid molecule encoding the protein. The invention also provides methods of modulating at least one activity of a protein of the invention, comprising administering an effective amount of an agent which modulates at least one activity of the protein. Preferably, the modulation is effective for the treatment of a malignant neoplasm, and the expression and/or activity is regulated in the direction of restoring expression and/or activity to levels in normal tissue.

The invention includes a method of diagnosing a disease state in a subject, comprising determining the level of expression of a nucleic acid molecule or protein of the invention. Preferably, the disease state is a malignant neoplasm.

The present invention further includes non-human transgenic animals modified to contain the nucleic acid molecules of the invention, or non-human transgenic animals modified to contain mutated nucleic acid molecules such that expression of the encoded polypeptides of the invention is altered or prevented.

The present invention also includes non-human transgenic animals in which all or a portion of one or more genes comprising all or a portion of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45 or 47 has been knocked out or deleted from the genome of the animal.

The invention further provides methods of diagnosing a malignant neoplasm, comprising determining the level of expression of a nucleic acid molecule of the invention or polypeptide of the invention. The invention further includes compositions comprising a diluent and a polypeptide or protein selected from the group consisting of an isolated polypeptide comprising all or a portion of one or more of the amino acid sequences of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 , 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48, an isolated polypeptide comprising a fragment of at least 10 contiguous amino acids of one or more of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or

48, an isolated polypeptide comprising one or more conservative amino acid substitutions of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48, naturally occurring amino acid sequence variants of SEQ ED NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48, and an isolated polypeptide with an amino acid sequence having at least about 50%, 60%, 70% or 75% amino acid sequence identity with the sequences set forth in SEQ ED NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48, preferably at least about 80%), or 85% more preferably at least about 90%, and most preferably at least about 95%> sequence identity with the sequences set forth in SEQ ED NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

I. General Description The present invention is based in part on the identification of new families of genes that are differentially expressed in human, malignant neoplastic tissue compared to normal human tissue. These genes correspond to the human cDNAs of SEQ ID NOS: 1, 3, 5, 7, 9,

II, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45 or 47. Genes corresponding to those that encode the human protein of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48 may also be found in other animal species, particularly mammalian species.

The genes and proteins of the invention may be used as diagnostic agents or markers to detect malignant neoplasms or the progression of malignant neoplasms in a subject or sample. They can also serve as a target for agents that modulate gene expression or activity. For example, agents may be identified that modulate biological processes associated with malignant tumor growth, including the processes associated with malignant hyperplasia.

The present invention is further based on the development of methods for isolating binding partners that bind to the proteins. Additionally, the proteins provide novel targets for the screening of synthetic small molecules and combinatorial or naturally occurring compound libraries to discover novel therapeutics to regulate cell growth and division.

II. Specific Embodiments

A. The Proteins Associated with Malignant Neoplasms

The present invention provides isolated proteins, allelic variants of the proteins, and conservative amino acid substitutions of the proteins associated with malignant neoplasms. As used herein, the "protein" or "polypeptide" refers, in part, to a protein that has the human amino acid sequence depicted in SEQ ED NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48. The terms also refer to naturally occurring allelic variants and proteins that have a slightly different amino acid sequence than that specifically recited above. Allelic variants, though possessing a slightly different amino acid sequence than those recited above, will still have the same or similar biological functions associated with these proteins.

The present invention also encompasses proteins translated from alternative splice variants of the genes encoding the identified proteins. As used herein, the family of proteins related to the human amino acid sequence of

SEQ ED NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44,

46, or 48 also includes the corresponding proteins that have been isolated from organisms other than humans. The methods used to identify and isolate other members of the families of proteins related to these proteins are described below.

The proteins of the present invention are preferably in isolated form. As used herein, a protein is said to be isolated when physical, mechanical or chemical methods are employed to remove the protein from cellular constituents that are normally associated with the protein. A skilled artisan can readily employ standard purification methods to obtain an isolated protein.

Some embodiments of the present invention include compositions containing all or a portion of at least one protein corresponding to SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48, each of these protein components having a purity of at least about 50%, 60%, or 65%, preferably at least about 70%> or 75%, more preferably at least about 80 or 85%, and even more preferably at least about 90% or 95%.

The proteins of the present invention further include insertion, deletion or conservative amino acid substitution variants of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48. As used herein, a conservative variant refers to alterations in the amino acid sequence that do not adversely affect the biological functions of the protein. A substitution, insertion or deletion is said to adversely affect the protein when the altered sequence prevents or disrupts a biological function associated with the protein. For example, the overall charge, structure or hydrophobic/hydrophilic properties of the protein can be altered without adversely affecting a biological activity. Accordingly, the amino acid sequence can be altered, for example to render the peptide more hydrophobic or hydrophilic, without adversely affecting the biological activities of the protein.

Ordinarily, the allelic variants, the conservative substitution variants, and the members of the protein families, will have an amino acid sequence having at least about 50%, 60%, 10% or 75% amino acid sequence identity with the sequences set forth in SEQ ED NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48, more preferably at least about 80% or 85%, even more preferably at least about 90%, and most preferably at least about 95%, 97%, 98% or 99% sequence identity. Identity or homology with respect to such sequences is defined herein as the percentage of amino acid residues in the candidate sequence that are identical with the known peptides, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent homology, and not considering any conservative substitutions as part of the sequence identity (see section B for the relevant parameters). Fusion proteins, or N-terminal, C-terminal or internal extensions, deletions, or insertions into the peptide sequence shall not be construed as affecting homology.

Thus, the proteins of the present invention include molecules having the amino acid sequence disclosed in SEQ TD NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,

34, 36, 38, 40, 42, 44, 46, or 48; fragments thereof having a consecutive sequence of at least about 3, 4, 5, 6, 10, 15, 20, 25, 30, 35 or more amino acid residues of these proteins; amino acid sequence variants wherein one or more amino acid residues has been inserted N- or C-terminal to, or within, the disclosed coding sequence; and amino acid sequence variants of the disclosed sequence, or their fragments as defined above, that have been substituted by at least one residue. Such fragments, also referred to as peptides or polypeptides, may contain antigenic regions, functional regions of the protein identified as regions of the amino acid sequence which correspond to known protein domains, as well as regions of pronounced hydrophilicity. The regions are all easily identifiable by using commonly available protein sequence analysis software such as MacVector (Oxford Molecular).

Contemplated variants further include those containing predetermined mutations by, e.g. , homologous recombination, site-directed or PCR mutagenesis, and the corresponding proteins of other animal species, including but not limited to rabbit, mouse, rat, porcine, bovine, ovine, equine and non-human primate species, and the alleles or other naturally occurring variants of the family of proteins; and derivatives wherein the protein has been covalently modified by substitution, chemical, enzymatic, or other appropriate means with a moiety other than a naturally occurring amino acid (for example a detectable moiety such as an enzyme or radioisotope).

The present invention further provides compositions comprising a protein or polypeptide of the invention and a diluent. Suitable diluents can be aqueous or non-aqueous solvents or a combination thereof, and can comprise additional components, for example water-soluble salts or glycerol, that contribute to the stability, solubility, activity, and/or storage of the protein or polypeptide.

As described below, members of a family of proteins can be used: (1) to identify agents which modulate the level of or at least one activity of the protein, (2) to identify binding partners for the protein, (3) as an antigen to raise polyclonal or monoclonal antibodies, (4) as a therapeutic agent or target and (5) as a diagnostic agent or marker of malignant neoplastic diseases.

B. Nucleic Acid Molecules The present invention further provides nucleic acid molecules that encode the proteins having SEQ ED NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48 and the related proteins herein described, preferably in isolated form. As used herein, "nucleic acid" is defined as RNA or DNA that encodes a protein or peptide as defined above, is complementary to a nucleic acid sequence encoding such proteins or peptides, hybridizes to the nucleic acid of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45 or 47 and remains stably bound to it under appropriate stringency conditions, encodes a polypeptide sharing at least about 50%, 60%, 70% or 75%, preferably at least about 80%, more preferably at least about 85%, and most preferably at least about 90%, 95%, 97%, 98%, 99% or more identity with the peptide sequence of SEQ ED NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48, or exhibits at least about 50%, 60%, 70%> or 75%, preferably at least about 80%, more preferably at least about 85%, and even more preferably at least about 90%, 95%, 99% or more nucleotide sequence identity over the open reading frames of SEQ ED NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45 or 47.

Specifically contemplated are genomic DNA, cDNA, mRNA and antisense molecules, as well as nucleic acids based on alternative backbones or including alternative bases, whether derived from natural sources or synthesized. Such hybridizing or complementary nucleic acids, however, are defined further as being novel and unobvious over any prior art nucleic acid including that which encodes, hybridizes under appropriate stringency conditions, or is complementary to nucleic acid encoding a protein according to the present invention.

Homology or identity at the nucleotide or amino acid sequence level is determined by BLAST (Basic Local Alignment Search Tool) analysis using the algorithm employed by the programs blastp, blastn, blastx, tblastn and tblastx (Altschul S.F. et al. (1997) Nucleic Acids Res 25:3389-3402, and Karlin et al, (1990) Proc Natl Acad Sci USA 87:2264-2268, both fully incorporated by reference) which are tailored for sequence similarity searching. The approach used by the BLAST program is to first consider similar segments, with and without gaps, between a query sequence and a database sequence, then to evaluate the statistical significance of all matches that are identified and finally to summarize only those matches which satisfy a preselected threshold of significance. For a discussion of basic issues in similarity searching of sequence databases, see Altschul et al, (1994) Nature

Genetics 6:119-129 which is fully incorporated by reference. The search parameters for histogram, descriptions, alignments, expect (i.e., the statistical significance threshold for reporting matches against database sequences), cutoff, matrix and filter (low complexity) are at the default settings. The default scoring matrix used by blastp, blastx, tblastn, and tblastx is the BLOSUM62 matrix (Henikoff et al, (1992) Proc Natl Acad Sci USA 89:

10915-10919, fully incorporated by reference), recommended for query sequences over 85 nucleotides or amino acids in length.

For blastn, the scoring matrix is set by the ratios of M (i.e., the reward score for a pair of matching residues) to N (i.e., the penalty score for mismatching residues), wherein the default values for M and N are 5 and -4, respectively. Four blastn parameters were adjusted as follows: Q=10 (gap creation penalty); R=10 (gap extension penalty); wink^l (generates word hits at every wink^th position along the query); and gapw=16 (sets the window width within which gapped alignments are generated). The equivalent Blastp parameter settings were Q=9; R=2; wink=l; and gapw=32. A Bestfit comparison between sequences, available in the GCG package version 10.0, uses DNA parameters GAP=50 (gap creation penalty) and LEN=3 (gap extension penalty) and the equivalent settings in protein comparisons are GAP=8 and LEN=2.

"Stringent conditions" are those that (1) employ low ionic strength and high temperature for washing, for example, 0.015 M NaCl/0.0015 M sodium citrate/0.1% SDS at 50°C, or (2) employ during hybridization a denaturing agent such as formamide, for example, 50% (vol/vol) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42°C. Another example is hybridization in 50% formamide, 5x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5x Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42°C, with washes at 42°C in 0.2χ SSC and 0.1% SDS. A skilled artisan can readily determine and vary the stringency conditions appropriately to obtain a clear and detectable hybridization signal. Preferred molecules are those that hybridize under the above conditions to the complement of SEQ TD NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45 or 47 and which encode a functional protein. Even more preferred hybridizing molecules are those that hybridize under the above conditions to the complement strand of the open reading frame of SEQ ED

NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45 or 47.

As used herein, a nucleic acid molecule is said to be "isolated" when the nucleic acid molecule is substantially separated from contaminant nucleic acid molecules encoding other polypeptides.

The present invention further provides fragments of the encoding nucleic acid molecule. As used herein, a fragment of an encoding nucleic acid molecule refers to a small portion of the entire protein coding sequence. The size of the fragment will be determined by the intended use. For example, if the fragment is chosen so as to encode an active portion of the protein, the fragment will need to be large enough to encode the functional region(s) of the protein. For instance, fragments which encode peptides corresponding to predicted antigenic regions may be prepared. If the fragment is to be used as a nucleic acid probe or PCR primer, then the fragment length is chosen so as to obtain a relatively small number of false positives during probing priming (see the discussion in Section H).

Fragments of the encoding nucleic acid molecules of the present invention (i.e., synthetic oligonucleotides) that are used as probes or specific primers for the polymerase chain reaction (PCR), or to synthesize gene sequences encoding proteins of the invention, can easily be synthesized by chemical techniques, for example, the phosphoramidite method of Matteucci et al, (1981) (J Am Chem Soc 103:3185-3191) or using automated synthesis methods. In addition, larger DNA segments can readily be prepared by well known methods, such as synthesis of a group of oligonucleotides that define various modular segments of the gene, followed by ligation of oligonucleotides to build the complete modified gene. The encoding nucleic acid molecules of the present invention may further be modified so as to contain a detectable label for diagnostic and probe purposes. A variety of such labels are known in the art and can readily be employed with the encoding molecules herein described. Suitable labels include, but are not limited to, biotin, radiolabeled nucleotides and the like. A skilled artisan can readily employ any such label to obtain labeled variants of the nucleic acid molecules of the invention.

Modifications to the primary structure itself by deletion, addition, or alteration of the amino acids incorporated into the protein sequence during translation can be made without destroying the activity of the protein. Such substitutions or other alterations result in proteins having an amino acid sequence encoded by a nucleic acid falling within the contemplated scope of the present invention.

C. Isolation of Other Related Nucleic Acid Molecules

As described above, the identification and characterization of the nucleic acid molecule having SEQ ED NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45 or 47 allows a skilled artisan to isolate nucleic acid molecules that encode other members of the protein families in addition to the sequences herein described.

For instance, a skilled artisan can readily use the amino acid sequence of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48 to generate antibody probes to screen expression libraries prepared from appropriate cells. Typically, polyclonal antiserum from mammals such as rabbits immunized with the purified protein (as described below) or monoclonal antibodies can be used to probe a mammalian cDNA or genomic expression library, such as lambda gtll library, to obtain the appropriate coding sequence for other members of the protein family. The cloned cDNA sequence can be expressed as a fusion protein, expressed directly using its own control sequences, or expressed by constructions using control sequences appropriate to the particular host used for expression of the enzyme.

Alternatively, a portion of the coding sequence herein described can be synthesized and used as a probe to retrieve DNA encoding a member of the protein family from any mammalian organism. Oligomers containing approximately 18-20 nucleotides (encoding about a 6-7 amino acid stretch) are prepared and used to screen genomic DNA or cDNA libraries to obtain hybridization under stringent conditions or conditions of sufficient stringency to eliminate an undue level of false positives.

Additionally, pairs of oligonucleotide primers can be prepared for use in a polymerase chain reaction (PCR) to selectively clone an encoding nucleic acid molecule. A PCR denature-anneal-extend cycle for using such PCR primers is well known in the art and can readily be adapted for use in isolating other encoding nucleic acid molecules.

Nucleic acid molecules encoding other members of the protein family may also be identified in existing genomic or other sequence information using any available computational method, including but not limited to: PSI-BLAST (Altschul, et al. (1997) Nucleic Acids Res 25:3389-3402); PHI-BLAST (Zhang, et al. (1998) Nucleic Acids Res 26:3986-3990), 3D-PSSM (Kelly et al. (2000) JMol Biol 299(2):499-520); and other computational analysis methods (Shi et al. (1999) Biochem Biophys Res Commun 262(1): 132-8 and Matsunami et. al. (2000) Nature 404(6778):601-4. D. rDNA molecules Containing a Nucleic Acid Molecule

The present invention further provides recombinant DNA molecules (rDNAs) that contain a coding sequence. As used herein, a rDNA molecule is a DNA molecule that has been subjected to molecular manipulation in situ. Methods for generating rDNA molecules are well known in the art, for example, see Sambrook et al, Molecular Cloning- A Laboratory Manual, 3d Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001. In the preferred rDNA molecules, a coding DNA sequence is operably linked to expression control sequences and/or vector sequences. The choice of vector and/or expression control sequences to which one of the protein family encoding sequences of the present invention is operably linked depends directly, as is well known in the art, on the functional properties desired, e.g., protein expression, and the host cell to be transformed. A vector contemplated by the present invention is at least capable of directing the replication or insertion into the host chromosome, and preferably also expression, of the structural gene included in the rDNA molecule.

Expression control elements that are used for regulating the expression of an operably linked protein encoding sequence are known in the art and include, but are not limited to, inducible promoters, constitutive promoters, secretion signals, and other regulatory elements. Preferably, the inducible promoter is readily controlled, such as being responsive to a nutrient in the host cell's medium.

In one embodiment, the vector containing a coding nucleic acid molecule will include a prokaryotic replicon, i.e., a DNA sequence having the ability to direct autonomous replication and maintenance of the recombinant DNA molecule extrachromosomally in a prokaryotic host cell, such as a bacterial host cell, transformed therewith. Such replicons are well known in the art. In addition, vectors that include a prokaryotic replicon may also include a gene whose expression confers a detectable marker such as a drug resistance. Typical bacterial drug resistance genes are those that confer resistance to ampicillin, kanamycin, chloramphenicol or tetracycline.

Vectors that include a prokaryotic replicon can further include a prokaryotic or bacteriophage promoter capable of directing the expression (transcription and translation) of the coding gene sequences in a bacterial host cell, such as E. coli. A promoter is an expression control element formed by a DNA sequence that permits binding of RNA polymerase and transcription to occur. Promoter sequences compatible with bacterial hosts are typically provided in plasmid vectors containing convenient restriction sites for insertion of a DNA segment of the present invention. Typical of such vector plasmids are pUC8, pUC9, pBR322 and pBR329 available from BioRad Laboratories, (Richmond, CA), pPL and pKK223 available from Pharmacia (Piscataway, NJ).

Expression vectors compatible with eukaryotic cells, preferably those compatible with mammalian cells, such as cervical, endometrial, uterine, breast, colon, rectal, intestinal, kidney, liver, lung, stomach, ovarian, pancreatic, thyroid, lymph node, omentum, skin, esophageal, laryngeal, adrenal, prostate, vulvar, connective tissue or soft tissue cells, can also be used to form rDNA molecules that contain a coding sequence. Eukaryotic cell expression vectors, including viral vectors, are well known in the art and are available from several commercial sources. Typically, such vectors are provided containing convenient restriction sites for insertion of the desired DNA segment. Typical of such vectors are pSVL and pKSV-10 (Pharmacia), pBPV-l/pML2d (International Biotechnologies, Inc.), pTDTl (ATCC, #31255), the vector pCDM8 described herein, and the like eukaryotic expression vectors. Vectors may be modified to include cervical, endometrial, uterine, breast, colon, rectal, intestinal, kidney, liver, lung, stomach, ovarian, pancreatic, thyroid, lymph node, omentum, skin, esophageal, laryngeal, adrenal, prostate, vulvar, connective tissue or soft tissue cell-specific promoters if needed.

Eukaryotic cell expression vectors used to construct the rDNA molecules of the present invention may further include a selectable marker that is effective in an eukaryotic cell, preferably a drug resistance selection marker. A preferred drug resistance marker is the gene whose expression results in neomycin resistance, i.e., the neomycin phosphotransferase (neo) gene. (Southern et al, (1982) J Mol Anal Genet 1:327-341) Alternatively, the selectable marker can be present on a separate plasmid, and the two vectors are introduced by co-transfection of the host cell, and selected by culturing in the appropriate drug for the selectable marker.

E. Host Cells Containing an Exogenously Supplied Coding Nucleic Acid Molecule

The present invention further provides host cells transformed with a nucleic acid molecule that encodes a protein of the present invention. The host cell can be either prokaryotic or eukaryotic. Eukaryotic cells useful for expression of a protein of the invention are not limited, so long as the cell line is compatible with cell culture methods and compatible with the propagation of the expression vector and expression of the gene product. Preferred eukaryotic host cells include, but are not limited to, yeast, insect and mammalian cells, preferably vertebrate cells such as those from a mouse, rat, monkey or human cell line. Preferred eukaryotic host cells include Chinese hamster ovary (CHO) cells available from the ATCC as CCL61, NEH Swiss mouse embryo cells (NEH 3T3) available from the ATCC as CRL 1658, baby hamster kidney cells (BHK), and the like eukaryotic tissue culture cell lines. Any prokaryotic host can be used to express a rDNA molecule encoding a protein of the invention. The preferred prokaryotic host is E. coli.

Transformation of appropriate cell hosts with a rDNA molecule of the present invention is accomplished by well known methods that typically depend on the type of vector used and host system employed. With regard to transformation of prokaryotic host cells, electroporation and salt treatment methods are typically employed, see, for example, Cohen et al, (1912) Proc Natl Acad Sci USA 69:2110; and Sambrook et al. Molecular Cloning- A Laboratory Manual, 3d Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001. With regard to transformation of vertebrate cells with vectors containing rDNAs, electroporation, cationic lipid or salt treatment methods are typically employed, see, for example, Graham et al, (1973) Virol 52, 456; Wigler et al, (1979) Proc Natl Acad Sci USA 76, 1373-1376.

Successfully transformed cells, i.e., cells that contain a rDNA molecule of the present invention, can be identified by well known techniques including the selection for a selectable marker. For example, cells resulting from the introduction of an rDNA of the present invention can be cloned to produce single colonies. Cells from those colonies can be harvested, lysed and their DNA content examined for the presence of the rDNA using a method such as that described by Southern (1975) JMol Biol 98:503 or Berent et al, (1985) Biotech 3:208, or the proteins produced from the cell assayed via an immunological method.

F. Production of Recombinant Proteins using a rDNA Molecule

The present invention further provides methods for producing a protein of the invention using nucleic acid molecules herein described. In general terms, the production of a recombinant form of a protein typically involves the following steps: First, a nucleic acid molecule is obtained that encodes all or a portion of a protein of the invention, such as a nucleic acid molecule comprising, consisting essentially of or consisting of SEQ TD NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45 or 47, or the portions corresponding to the open reading frames of these nucleic acid molecules, with or without the stop codons. The positions of the open reading frames and lengths of the protein molecules encoded are given in Example 2, below. If the encoding sequence is uninterrupted by introns, as are these open-reading-frames, it is directly suitable for expression in any host.

The nucleic acid molecule is then preferably placed in operable linkage with suitable control sequences, as described above, to form an expression unit containing the protein open reading frame. The expression unit is used to transform a suitable host and the transformed host is cultured under conditions that allow the production of the recombinant protein. Optionally the recombinant protein is isolated from the medium or from the cells; recovery and purification of the protein may not be necessary in some instances where some impurities may be tolerated.

Each of the foregoing steps can be done in a variety of ways. For example, the desired coding sequences may be obtained from genomic fragments and used directly in appropriate hosts. The construction of expression vectors that are operable in a variety of hosts is accomplished using appropriate replicons and control sequences, as set forth above. The control sequences, expression vectors, and transformation methods are dependent on the type of host cell used to express the gene and were discussed in detail earlier. Suitable restriction sites can, if not normally available, be added to the ends of the coding sequence so as to provide an excisable gene to insert into these vectors. A skilled artisan can readily adapt any host/expression system known in the art for use with the nucleic acid molecules of the invention to produce recombinant protein.

G. Methods to Identify Binding Partners

Another embodiment of the present invention provides methods for isolating and identifying binding partners of proteins of the invention. In general, a protein of the invention is mixed with a potential binding partner or an extract or fraction of a cell under conditions that allow the association of potential binding partners with the protein of the invention. After mixing, peptides, polypeptides, proteins or other molecules that have become associated with a protein of the invention are separated from the mixture. The binding partner that bound to the protein of the invention can then be removed and further analyzed. To identify and isolate a binding partner, the entire protein, for instance a protein comprising the entire amino acid sequence of SEQ TD NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48 can be used. Alternatively, a fragment of the protein can be used.

As used herein, a cellular extract refers to a preparation or fraction which is made from a lysed or disrupted cell. The preferred source of cellular extracts will be cells derived from neoplastic, human tissue or cells, for instance, biopsy tissue or tissue culture cells from malignant tumors. Alternatively, cellular extracts may be prepared from normal tissue or available cell lines, particularly cell lines derived from cervical, endometrial, uterine, breast, colon, rectal, intestinal, kidney, liver, lung, stomach, ovarian, pancreatic, thyroid, lymph node, omentum, skin, esophageal, laryngeal, adrenal, prostate, vulvar, connective tissue or soft tissue.

A variety of methods can be used to obtain an extract of a cell. Cells can be disrupted using either physical or chemical disruption methods. Examples of physical disruption methods include, but are not limited to, sonication and mechanical shearing. Examples of chemical lysis methods include, but are not limited to, detergent lysis and enzyme lysis. A skilled artisan can readily adapt methods for preparing cellular extracts in order to obtain extracts for use in the present methods.

Once an extract of a cell is prepared, the extract is mixed with the protein of the invention under conditions in which association of the protein with the binding partner can occur. A variety of conditions can be used, the most preferred being conditions that closely resemble conditions found in the cytoplasm of a human cell. Features such as osmolarity, pH, temperature, and the concentration of cellular extract used, can be varied to optimize the association of the protein with the binding partner. After mixing under appropriate conditions, the bound complex is separated from the mixture. A variety of techniques can be utilized to separate the mixture. For example, antibodies specific to a protein of the invention can be used to immunoprecipitate the binding partner complex. Alternatively, standard chemical separation techniques such as chromatography and density/sediment centrifugation can be used. After removal of non-associated cellular constituents found in the extract, the binding partner can be dissociated from the complex using conventional methods. For example, dissociation can be accomplished by altering the salt concentration or pH of the mixture.

To aid in separating associated binding partner pairs from the mixed extract, the protein of the invention can be immobilized on a solid support. For example, the protein can be attached to a nitrocellulose matrix or acrylic beads. Attachment of the protein to a solid support aids in separating peptide/binding partner pairs from other constituents found in the extract. The identified binding partners can be either a single protein or a complex made up of two or more proteins. Alternatively, binding partners may be identified using a Far- Western assay according to the procedures of Takayama et al, (1997) Methods Mol

Biol 69:171-184 or Sauder et al, (1996) J Gen Virol 77:991-996 or identified through the use of epitope tagged proteins or GST fusion proteins.

Alternatively, the nucleic acid molecules of the invention can be used in a yeast two- hybrid system or other in vivo protein-protein detection system. The yeast two-hybrid system has been used to identify other protein partner pairs and can readily be adapted to employ the nucleic acid molecules herein described.

H. Methods to Identify Agents that Modulate the Expression a Nucleic Acid Encoding the Genes Associated With Malignant Neoplasms

Another embodiment of the present invention provides methods for identifying agents that modulate the expression of a nucleic acid encoding a protein of the invention such as a protein having the amino acid sequence of SEQ TD NOS: 2, 4, 6, 8, 10, 12, 14, 16,

18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48. Such assays may utilize any available means of monitoring for changes in the expression level of the nucleic acids of the invention. As used herein, an agent is said to modulate the expression of a nucleic acid of the invention if it is capable of up- or down-regulating expression of the nucleic acid in a cell.

In one assay format, cell lines that contain reporter gene fusions between nucleotides encoding all or a portion of the open reading frame defined by nucleotides nucleotides

1049-1549 of SEQ ID NO: 1, or nucleotides 81-1580 of SEQ ED NO: 3, or nucleotides 78- 1181 of SEQ ED NO: 5, or nucleotides 14-1057 of SEQ TD NO: 7, or nucleotides 13-1056 of SEQ ED NO: 9, or nucleotides 136-1977 of SEQ ID NO: 11, or nucleotides 37-654 of SEQ TD NO: 13, or nucleotides 303-1097 of SEQ TD NO: 15, or nucleotides 186-1352 of SEQ TD NO: 17, or nucleotides 739-2205 of SEQ TD NO: 19, or nucleotides 301-795 of SEQ ED NO: 21, or nucleotides 133-807 of SEQ ED NO: 23, or nucleotides 131-1723 of SEQ ED NO: 25, or nucleotides 271-603 of SEQ TD NO: 27, or nucleotides 12-638 of SEQ ED NO: 29, or nucleotides 94-909 of SEQ ED NO: 31, or nucleotides 14-1057 of SEQ TD NO: 33, or nucleotides 246-1811 of SEQ ED NO: 35, or nucleotides 300-1865 of SEQ ED NO: 37, or nucleotides 31-507 of SEQ ED NO: 39, or nucleotides 40-1677 of SEQ TD NO: 41, or nucleotides 52-1335 of SEQ TD NO: 43, or nucleotides 6-2018 of SEQ TD NO: 45, or nucleotides 10-786 of SEQ TD NO: 47, and/or the 5 'and/or 3' regulatory elements and any assayable fusion partner may be prepared. Numerous assayable fusion partners are known and readily available including the firefly luciferase gene and the gene encoding chloramphenicol acetyltransferase (Alam et al, (1990) Anal Biochem 188:245-254). Cell lines containing the reporter gene fusions are then exposed to the agent to be tested under appropriate conditions and time. Differential expression of the reporter gene between samples exposed to the agent and control samples identifies agents which modulate the expression of a nucleic acid of the invention.

Additional assay formats may be used to monitor the ability of the agent to modulate the expression of one or more of the nucleic acids encoding proteins of the invention, such as the proteins having SEQ TD NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,

34, 36, 38, 40, 42, 44, 46, or 48. For instance, mRNA expression may be monitored directly by hybridization to the nucleic acids of the invention. Cell lines are exposed to the agent to be tested under appropriate conditions and time and total RNA or mRNA is isolated by standard procedures such those disclosed in Sambrook et al, Molecular Cloning- A Laboratory Manual, 3d Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001. The preferred cells will be those derived from human, malignant tumorous tissue, for instance, cervical, endometrial, uterine, breast, colon, rectal, intestinal, kidney, liver, lung, stomach, ovarian, pancreatic, thyroid, lymph node, omentum, skin, esophageal, laryngeal, adrenal, prostate, vulvar, connective tissue or soft tissue, or cultured cells from normal or cancer patients, for example, the human malignant melanoma cell line A-375, ATCC Catalog No. CRL-1619 or the non-small cell lung carcinoma cell line containing genomic DNA from NCI-H2126, ATCC Catalog No. 45512. Alternatively, other available cells or cell lines may be used.

Probes to detect differences in RNA expression levels between cells exposed to the agent and control cells may be prepared from the nucleic acids of the invention. It is preferable, but not necessary, to design probes which hybridize only with target nucleic acids under conditions of high stringency. Only highly complementary nucleic acid hybrids form under conditions of high stringency. Accordingly, the stringency of the assay conditions determines the amount of complementarity which should exist between two nucleic acid strands in order to form a hybrid. Stringency should be chosen to maximize the difference in stability between the probe:target hybrid and probe:non-target hybrids.

Probes may be designed from the nucleic acids of the invention through methods known in the art. For instance, the G+C content of the probe and the probe length can affect probe binding to its target sequence. Methods to optimize probe specificity are commonly available in Sambrook et al. (supra) or Ausubel et al. , Short Protocols in Molecular Biology. 4th Ed., John Wiley & Sons, Inc., New York, 1999.

Hybridization conditions are modified using known methods, such as those described by Sambrook et al. and Ausubel et al as required for each probe. Hybridization of total cellular RNA or RNA enriched for polyA RNA can be accomplished in any available format. For instance, total cellular RNA or RNA enriched for polyA RNA can be affixed to a solid support and the solid support exposed to at least one probe comprising at least one, or part of one of the sequences of the invention under conditions in which the probe will specifically hybridize. Alternatively, nucleic acid fragments comprising at least one, or part of one of the sequences of the invention can be affixed to a solid support, such as a silicon chip, porous glass wafer or membrane. The solid support can then be exposed to total cellular RNA or polyA RNA from a sample under conditions in which the affixed sequences will specifically hybridize. Such solid supports and hybridization methods are widely available, for example, those disclosed by Beattie, (1995) WO 95/11755. By examining for the ability of a given probe to specifically hybridize to an RNA sample from an untreated cell population and from a cell population exposed to the agent, agents which up- or down-regulate the expression of a nucleic acid encoding the protein having the sequence of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48 are identified.

Hybridization for qualitative and quantitative analysis of mRNAs may also be carried out by using a RNase Protection Assay (i.e., RPA, see Ma et al (1996) Methods 10: 273-238). Briefly, an expression vehicle comprising cDNA encoding the gene product and a phage specific DNA dependent RNA polymerase promoter (e.g., TI, T3 or SP6 RNA polymerase) is linearized at the 3' end of the cDNA molecule, downstream from the phage promoter, wherein such a linearized molecule is subsequently used as a template for synthesis of a labeled antisense transcript of the cDNA by in vitro transcription. The labeled transcript is then hybridized to a mixture of isolated RNA (i.e., total or fractionated mRNA) by incubation at 45 °C overnight in a buffer comprising 80% formamide, 40 mM Pipes, pH 6.4, 0.4 M NaCl and 1 mM EDTA. The resulting hybrids are then digested in a buffer comprising 40 μg/ml ribonuclease A and 2 μg/ml ribonuclease. After deactivation and extraction of extraneous proteins, the samples are loaded onto urea/polyacrylamide gels for analysis.

In another assay format, to identify agents which affect the expression of the instant gene products, cells or cell lines are first identified which express the gene products of the invention physiologically. Cell and/or cell lines so identified would be expected to comprise the necessary cellular machinery such that the fidelity of modulation of the transcriptional apparatus is maintained with regard to exogenous contact of agent with appropriate surface transduction mechanisms and/or the cytosolic cascades. Further, such cells or cell lines would be transduced or transfected with an expression vehicle (e.g., a plasmid or viral vector) construct comprising an operable non-translated 5 '-promoter containing end of the structural gene encoding the instant gene products fused to one or more antigenic fragments, which are peculiar to the instant gene products, wherein said fragments are under the transcriptional control of said promoter and are expressed as polypeptides whose molecular weight can be distinguished from the naturally occurring polypeptides or may further comprise an immuno logically distinct tag or other detectable marker. Such a process is well known in the art (see Sambrook et al, supra).

Cells or cell lines transduced or transfected as outlined above are then contacted with agents under appropriate conditions. For example, the agent in a pharmaceutically acceptable excipient is contacted with cells in an aqueous physiological buffer such as phosphate buffered saline (PBS) at physiological pH, Eagles balanced salt solution (BSS) at physiological pH, PBS or BSS comprising serum or conditioned media comprising PBS or BSS and/or serum incubated at 37°C. Said conditions may be modulated as deemed necessary by one of skill in the art. Subsequent to contacting the cells with the agent, said cells will be disrupted and the polypeptides of the lysate are fractionated such that a polypeptide fraction is pooled and contacted with an antibody to be further processed by immunological assay (e.g., ELISA, immunoprecipitation or Western blot). The pool of proteins isolated from the "agent-contacted" sample will be compared with a control sample where only the excipient is contacted with the cells and an increase or decrease in the immunologically generated signal from the "agent-contacted" sample compared to the control will be used to distinguish the effectiveness of the agent.

I. Methods to Identify Agents that Modulate the Level or at Least One Activity of the Malignant Neoplasm Associated Proteins

Another embodiment of the present invention provides methods for identifying agents that modulate the level or at least one activity of a protein of the invention such as the protein having the amino acid sequence of SEQ ED NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18,

20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48. Such methods or assays may utilize any means of monitoring or detecting the desired activity.

In one format, the relative amounts of a protein of the invention between a cell population that has been exposed to the agent to be tested compared to an un-exposed control cell population may be assayed. In this format, probes such as specific antibodies are used to monitor the differential expression of the protein in the different cell populations. Cell lines or populations are exposed to the agent to be tested under appropriate conditions and time. Cellular lysates may be prepared from the exposed cell line or population and a control, unexposed cell line or population. The cellular lysates are then analyzed with the probe.

Antibody probes are prepared by immunizing suitable mammalian hosts in appropriate immunization protocols using the peptides, polypeptides or proteins of the invention if they are of sufficient length, or, if desired, or if required to enhance immunogenicity, conjugated to suitable carriers. Methods for preparing immunogenic conjugates with carriers such as BSA, KLH, or other carrier proteins are well known in the art. In some circumstances, direct conjugation using, for example, carbodiimide reagents may be effective; in other instances linking reagents such as those supplied by Pierce Chemical Co. (Rockford, IL), may be desirable to provide accessibility to the hapten. The hapten peptides can be extended at either the amino or carboxy terminus with a cysteine residue or interspersed with cysteine residues, for example, to facilitate linking to a carrier. Administration of the immunogens is conducted generally by injection over a suitable time period and with use of suitable adjuvants, as is generally understood in the art. During the immunization schedule, titers of antibodies are taken to determine adequacy of antibody formation.

While the polyclonal antisera produced in this way may be satisfactory for some applications, for pharmaceutical compositions, use of monoclonal preparations is preferred. Immortalized cell lines which secrete the desired monoclonal antibodies may be prepared using the standard method of Kohler and Milstein (Nature (1975) 256:495-497) or modifications which effect immortalization of lymphocytes or spleen cells, as is generally known. The immortalized cell lines secreting the desired antibodies are screened by immunoassay in which the antigen is the peptide hapten, polypeptide or protein. When the appropriate immortalized cell culture secreting the desired antibody is identified, the cells can be cultured either in vitro or by production in ascites fluid.

The desired monoclonal antibodies are then recovered from the culture supernatant or from the ascites supernatant. Fragments of the monoclonal antibodies or the polyclonal antisera which contain the immunologically significant (antigen-binding) portion can be used as antagonists, as well as the intact antibodies. Use of immunologically reactive (antigen-binding) antibody fragments, such as the Fab, Fab', or F(ab')₂ fragments is often preferable, especially in a therapeutic context, as these fragments are generally less immunogenic than the whole immunoglobulin.

The antibodies or antigen-binding fragments may also be produced, using current technology, by recombinant means. Antibody regions that bind specifically to the desired regions of the protein can also be produced in the context of chimeras with multiple species origin, such as humanized antibodies.

Agents that are assayed in the above method can be randomly selected or rationally selected or designed. As used herein, an agent is said to be randomly selected when the agent is chosen randomly without considering the specific sequences involved in the association of a protein of the invention alone or with its associated substrates, binding partners, etc. An example of randomly selected agents is the use a chemical library or a peptide combinatorial library, or a growth broth of an organism.

As used herein, an agent is said to be rationally selected or designed when the agent is chosen on a nonrandom basis which takes into account the sequence of the target site and/or its conformation in connection with the agent's action. Agents can be rationally selected or rationally designed by utilizing the peptide sequences that make up these sites. For example, a rationally selected peptide agent can be a peptide whose amino acid sequence is identical to or a derivative of any functional consensus site. The agents of the present invention can be, as examples, peptides, small molecules, vitamin derivatives, as well as carbohydrates. Probes based on the proteins can be used as capture probes to isolate potential binding partners, such as other proteins. Dominant negative proteins, DNAs encoding these proteins, antibodies to these proteins, peptide fragments of these proteins or mimics of these proteins may be introduced into cells to affect function. "Mimic" used herein refers to the modification of a region or several regions of a peptide molecule to provide a structure chemically different from the parent peptide but topographically and functionally similar to the parent peptide (see G.A. Grant in: Molecular Biology and Biotechnology, Meyers, ed., pp. 659-664, VCH Publishers, New York, 1995). A skilled artisan can readily recognize that there is no limit as to the structural nature of the agents of the present invention.

The peptide agents of the invention can be prepared using standard solid phase (or solution phase) peptide synthesis methods, as is known in the art. In addition, the DNA encoding these peptides may be synthesized using commercially available oligonucleotide synthesis instrumentation and produced recombinantly using standard recombinant production systems. The production using solid phase peptide synthesis is necessitated if non-gene-encoded amino acids are to be included.

Another class of agents of the present invention are antibodies immunoreactive with critical positions of proteins of the invention. Antibody agents are obtained by immunization of suitable mammalian subjects with peptides, containing as antigenic regions, those portions of the protein intended to be targeted by the antibodies.

J. Uses for Agents that Modulate the Expression or at least one Activity of the Proteins of the Invention As provided in the Examples, the proteins and nucleic acids of the invention, such as one or more of the proteins having the amino acid sequence of SEQ TD NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48 are differentially expressed in malignant tumorous tissue. Agents that up- or down- regulate or modulate the expression of the protein or at least one activity of the protein, such as agonists or antagonists, may be used to modulate biological and pathologic processes associated with the protein's function and activity.

As used herein, a subject can be any mammal, so long as the mammal is in need of modulation of a pathological or biological process mediated by a protein of the invention. The term "mammal" is defined as an individual belonging to the class Mammalia. The invention is particularly useful in the treatment of human subjects.

Pathological processes refer to a category of biological processes which produce a deleterious effect. For example, expression of a protein of the invention may be associated with malignant cell growth or regulation. As used herein, an agent is said to modulate a pathological process when the agent reduces the degree or severity of the process. For instance, malignant tumors may be prevented or disease progression modulated by the administration of agents which up- or down-regulate or modulate in some way the expression or at least one activity of a protein of the invention.

The agents of the present invention can be provided alone, or in combination with other agents that modulate a particular pathological process. For example, an agent of the present invention can be administered in combination with other known drugs. As used herein, two agents are said to be administered in combination when the two agents are administered simultaneously or are administered independently in a fashion such that the agents will act at the same time.

The agents of the present invention can be administered via parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, or buccal routes.

Alternatively, or concurrently, administration may be by the oral route. The dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired. The present invention further provides compositions containing one or more agents which modulate expression or at least one activity of a protein of the invention. While individual needs vary, determination of optimal ranges of effective amounts of each component is within the skill of the art. Typical dosages comprise 0.1 to 100 μg/kg body wt. The preferred dosages comprise 0.1 to 10 μg/kg body wt. The most preferred dosages comprise 0.1 to 1 μg/kg body wt.

In addition to the pharmacologically active agent, the compositions of the present invention may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically for delivery to the site of action. Suitable formulations for parenteral administration include aqueous solutions of the active compounds in water- soluble form, for example, water-soluble salts. In addition, suspensions of the active compounds as appropriate oily injection suspensions may be administered. Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension include, for example, sodium carboxymethyl cellulose, sorbitol, and/or dextran. Optionally, the suspension may also contain stabilizers. Liposomes can also be used to encapsulate the agent for delivery into the cell.

The pharmaceutical formulation for systemic administration according to the invention may be formulated for enteral, parenteral or topical administration. Indeed, all three types of formulations may be used simultaneously to achieve systemic administration of the active ingredient.

Suitable formulations for oral administration include hard or soft gelatin capsules, pills, tablets, including coated tablets, elixirs, suspensions, syrups or inhalations and controlled release forms thereof.

In practicing the methods of this invention, the compounds of this invention may be used alone or in combination, or in combination with other therapeutic or diagnostic agents. In certain preferred embodiments, the compounds of this invention may be coadministered along with other compounds typically prescribed for these conditions according to generally accepted medical practice. The compounds of this invention can be utilized in vivo, ordinarily in mammals, such as humans, sheep, horses, cattle, pigs, dogs, cats, rats and mice, or in vitro.

K. Transgenic Animals

Transgenic animals containing mutant, knock-out or modified genes corresponding to the cDNA sequence of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45 or 47, or the open reading frame encoding the polypeptide sequence of SEQ TD NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48 or fragments thereof having a consecutive sequence of at least about 3, 4, 5, 6, 10, 15, 20, 25, 30, 35 or more amino acid residues, are also included in the invention. Transgenic animals are genetically modified animals into which recombinant, exogenous or cloned genetic material has been experimentally transferred. Such genetic material is often referred to as a "transgene." The nucleic acid sequence of the transgene, in some embodiments, all or a portion of one or more of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45 or 47, may be integrated either at a locus of a genome where that particular nucleic acid sequence is not otherwise normally found or at the normal locus for the transgene. The transgene may consist of nucleic acid sequences derived from the genome of the same species or of a different species than the species of the target animal.

In some embodiments, transgenic animals in which all or a portion of one or more genes comprising SEQ ED NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45 or 47 is deleted may be constructed. In those cases where the gene corresponding to SEQ ED NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45 or 47 contains one or more introns, the entire gene- all exons, introns and the regulatory sequences- may be deleted. Alternatively, less than the entire gene may be deleted. For example, a single exon and/or intron may be deleted, so as to create an animal expressing a modified version of a protein of the invention.

The term "germ cell line transgenic animal" refers to a transgenic animal in which the genetic alteration or genetic information was introduced into a germ line cell, thereby conferring the ability of the transgenic animal to transfer the genetic information to offspring. If such offspring in fact possess some or all of that alteration or genetic information, then they too are transgenic animals.

The alteration or genetic information may be foreign to the species of animal to which the recipient belongs, foreign only to the particular individual recipient, or may be genetic information already possessed by the recipient. In the last case, the altered or introduced gene may be expressed differently than the native gene.

Transgenic animals can be produced by a variety of different methods including transfection, electroporation, microinjection, gene targeting in embryonic stem cells and recombinant viral and retroviral infection (see, e.g., U.S. Patent No. 4,736,866; U.S. Patent

No. 5,602,307; Mullins et al, (1993) Hypertension 22:630-633; Brenin et al, (1997) Surg

Oncol 6:99-110; Tuan (1997) Recombinant Gene Expression Protocols, Methods in

Molecular Biology, Humana Press). A number of recombinant or transgenic mice have been produced, including those which express an activated oncogene sequence (U.S. Patent No. 4,736,866); express simian SV40 T-antigen (U.S. Patent No. 5,728,915); lack the expression of interferon regulatory factor 1 (ERF-1) (U.S. Patent No. 5,731,490); exhibit dopaminergic dysfunction (U.S. Patent No. 5,723,719); express at least one human gene which participates in blood pressure control (U.S. Patent No. 5,731,489); display greater similarity to the conditions existing in naturally occurring Alzheimer's disease (U.S. Patent No. 5,720,936); have a reduced capacity to mediate cellular adhesion (U.S. Patent No. 5,602,307); possess a bovine growth hormone gene (Clutter et al, (1996) Genetics 143:1753-1760); or, are capable of generating a fully human antibody response (McCarthy (1997) Lancet 349, 405). While mice and rats remain the animals of choice for most transgenic experimentation, in some instances it is preferable or even necessary to use alternative animal species. Transgenic procedures have been successfully utilized in a variety of non- murine animals, including sheep, goats, pigs, dogs, cats, monkeys, chimpanzees, hamsters, rabbits, cows and guinea pigs (see, e.g., Kim et al, (1997) Mol Reprod Dev 46:515-526; Houdebine (1995) Reprod Nutr Dev 35:609-617; Petters (1994) Reprod Fertil Dev 6:643- 645; Schnieke et al, (1997) Science 278:2130-2133; and Amoah (1997) J Animal Science 75:578-585).

The method of introduction of nucleic acid fragments into recombination competent mammalian cells can be by any method which favors co-transformation of multiple nucleic acid molecules. Detailed procedures for producing transgenic animals are readily available to one skilled in the art, including the disclosures in U.S. Patent No. 5,489,743 and U.S. Patent No. 5,602,307. L. Diagnostic Methods

As the genes and proteins of the invention are differentially expressed in malignant neoplastic tissue compared to normal tissue, the genes and proteins of the invention may be used to diagnose or monitor malignant tumors, to monitor regions of the body experiencing unusual cell growth, or to track disease progression. One means of diagnosing malignant neoplasms using the nucleic acid molecules or proteins of the invention involves obtaining cervical, endometrial, uterine, breast, colon, rectal, intestinal, kidney, liver, lung, stomach, ovarian, pancreatic, thyroid, lymph node, omentum, skin, esophageal, laryngeal, adrenal, prostate, vulvar, connective tissue, soft tissue or other tissues from living subjects. Such tissue samples may be obtained by any conventional means, for example, by biopsy. When possible, urine, blood or peripheral lymphocyte samples may be used as the tissue sample in the assay. Commonly, in hyperplastic diseases, genes which are up- or down-regulated in the affected tissue are also up- or down-regulated in lymphocytes, which may be isolated from whole blood. The use of molecular biological tools has become routine in forensic technology.

For example, nucleic acid probes comprising all or at least part of the sequence of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45 or 47 may be used to determine the expression of a nucleic acid molecule in forensic/pathology specimens. Further, nucleic acid assays may be carried out by any means of conducting a transcriptional profiling analysis. In addition to nucleic acid analysis, forensic methods of the invention may target the proteins of the invention, particularly a protein comprising SEQ TD NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48, to determine up- or down-regulation of the genes (Shiverick et al, (1975) Biochim Biophys Acta 393:124-133). Methods of the invention may involve treatment of tissues with collagenases or other proteases to make the tissue amenable to cell lysis (Semenov et al, (1987) Biull Eksp Biol Med 104:113-116). Further, it is possible to obtain biopsy samples from different regions of the malignant tumor for analysis.

Assays to detect nucleic acid or protein molecules of the invention may be in any available format. Typical assays for nucleic acid molecules include hybridization or PCR based formats. Typical assays for the detection of proteins, polypeptides or peptides of the invention include the use of antibody probes in any available format such as in situ binding assays, etc. (see Harlow & Lane, Antibodies- A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988). In preferred embodiments, assays are carried-out with appropriate controls.

The above methods may also be used in other diagnostic protocols, including protocols and methods to detect disease states in other tissues or organs, for example in tissues in which the gene is detected.

Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present invention and practice the claimed methods. The following working examples therefore, specifically point out preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.

EXAMPLES Example 1 Identification of Differentially Expressed mRNA in Malignant Neoplasms

Human tissue samples were obtained from cervical, endometrial, uterine, breast, colon, rectal, intestinal, kidney, liver, lung, stomach, ovarian, pancreatic, thyroid, lymph node, omentum, skin, esophageal, laryngeal, adrenal, prostate, vulvar, connective tissue or soft tissue from normal individuals and from patients with malignant neoplastic disease as listed in Table 1. These normal and disease state samples were used to identify differentially expressed genes.

Genes differentially expressed in diseased versus normal tissue were identified using the standard techniques of gene chip analysis (see, for example, the Affymetrix GeneChip® Expression Analysis Manual), and full length cDNAs were obtained and sequenced. The extent to which each gene is up- or down-regulated, expressed as a fold change, in each tissue type where there is differential expression, is also indicated in Table 1. A box with a (-) sign indicates down-regulation compared to control tissues, while a box with no sign before the fold-change values indicates up-regulation compared to control tissues. For example, down-regulation of expression (1.9 to 2.9-fold) is observed for SEQ TD NO: 13, Clone No. P5F10, in adenocarcinomas of the lung. Down-regulation is also observed for SEQ ED NO: 13 in cases of pulmonary squamous cell carcinoma (1.6 to 4.2-fold), large cell carcinoma (5-fold), neuroendocrine carcinoma (5.1 -fold) and mixed cell type carcinoma (3.3-fold).

Table 1 further provides an overview of the organs used as sample sources, the diseases associated with each organ, and the types (based on morphology) of cancerous tumors seen in each disease used to evaluate the differential expression of the mRNAs corresponding to SEQ TD NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45 or 47.

Example 2

Cloning of a Full Length Human cDNAs Corresponding to the differentially expressed mRNA species

The full length human cDNAs (SEQ ED NOS: 1, 3, 5, 7, 9,11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45 or 47) corresponding to the differentially expressed mRNA species identified were obtained by using the GeneTrapper® Kit. Briefly, a pair of gene specific primers were synthesized and human cDNA libraries from brain, liver, ovarian, lung, testicular, pancreatic and spleen tissues were screened. The cDNA libraries were prepared by Life Technologies, Inc. using Superscript® technology. Of a number of positive clones obtained, the longest one that corresponded well to the predicted size of the

RNA transcript, as determined by Northern blotting, was selected for DNA sequencing. The nucleotide sequences of the full-length human cDNA corresponding to the differentially regulated mRNAs detected above are set forth in SEQ TD NOS: 1, 3, 5, 7, 9,

11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45 and 47. The corresponding amino acid sequences for the encoded protein are set forth in SEQ TD NOS:

2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48, respectively.

The nucleotide positions for the open reading frame (ORF), including the stop codon, within the cDNA nucleotide sequences corresponding to each of SEQ ED NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45 and 47 is shown below. The number of amino acids encoded by each open reading frame is also indicated below.

Example 3

Detection of mRNA for Malignant Neoplasm Screening

The expression level of mRNA corresponding to SEQ TD NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45 or 47 may be determined in tissue biopsy samples from cervical, endometrial, uterine, breast, colon, rectal, intestinal, kidney, liver, lung, stomach, ovarian, pancreatic, thyroid, lymph node, omentum, skin, esophageal, laryngeal, adrenal, prostate, vulvar, connective tissue or soft tissue, or, where appropriate, from other subject-derived samples including, but not limited to, lymphocytes from blood samples, as described above. In some preferred examples, expression levels may be determined by screening mRNA samples on a GeneChip® or by semi-quantitative PCR analysis using a fluorescent detection system (e.g., the GADPH system from Perkin Elmer, used in conjunction with SYBR green primers from Molecular Probes). Tissue samples from patients with malignant tumors and from normal subjects may be used as positive and negative controls. A level of expression different from that of the normal control is indicative of malignant neoplastic disease or a likelihood of developing malignant neoplastic disease.

Although the present invention has been described in detail with reference to examples above, it is understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims. All cited patents, patent applications and publications referred to in this application are herein incorporated by reference in their entirety.

Claims

What is claimed is:

1. An isolated nucleic acid molecule selected from the group consisting of: (a) an isolated nucleic acid molecule that encodes all or a portion of the amino acid sequence of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48; (b) an isolated nucleic acid molecule that encodes a fragment of at least 10 contiguous amino acids of one or more of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48; (c) an isolated nucleic acid molecule which hybridizes to the complement of a nucleic acid molecule comprising SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45 or 47; (d) an isolated nucleic acid molecule which hybridizes to the complement of a nucleic acid molecule that encodes the amino acid sequence of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48; (e) an isolated nucleic acid molecule that encodes a protein that is differentially expressed in malignant neoplasms and that exhibits at least about 60% nucleotide sequence identity over the open reading frame of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45 or 47; and (f) an isolated nucleic acid molecule comprising the complement of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45 or 47.

2. The isolated nucleic acid molecule of claim 1, wherein the nucleic acid molecule comprises an open reading frame encoding a protein selected from the group consisting of SEQ TD NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48.

3. The isolated nucleic acid molecule of claim 1, wherein the nucleic acid molecule consists of an open reading frame encoding a protein selected from the group consisting of SEQ TD NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48.

4. The isolated nucleic acid molecule of claim 1, wherein the nucleic acid molecule comprises a naturally occurring variant of a protein selected from the group consisting of SEQ TD NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48.

5. The isolated nucleic acid molecule of any one of claims 1-4, wherein said nucleic acid molecule is operably linked to one or more expression control elements.

6. A vector comprising an isolated nucleic acid molecule of any one of claims 1-4.

7. A host cell transformed to contain the nucleic acid molecule of any one of claims 1-4.

8. A host cell comprising a vector of claim 6.

9. A host cell of claim 8, wherein said host cell is selected from the group consisting of prokaryotic host cells and eukaryotic host cells.

10. A method for producing a polypeptide comprising culturing a host cell transformed with the nucleic acid molecule of any one of claims 1 -4 under conditions in which the protein encoded by said nucleic acid molecule is expressed.

11. The method of claim 10, wherein said host cell is selected from the group consisting of prokaryotic host cells and eukaryotic host cells.

12. An isolated polypeptide produced by the method of claim 10.

13. An isolated polypeptide or protein selected from the group consisting of: (a) an isolated polypeptide comprising the amino acid sequence of any one of SEQ TD NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48; (b) an isolated polypeptide comprising a fragment of at least 10 contiguous amino acids of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48; (c) an isolated polypeptide comprising one or more conservative amino acid substitutions of any one of SEQ TD NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48; (d) an isolated polypeptide comprising naturally occurring amino acid sequence variants of any one of SEQ TD NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48; and (e) an isolated polypeptide exhibiting at least about 60% amino acid sequence identity with any one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48.

14. An isolated antibody or antigen-binding antibody fragment that binds to a polypeptide of claim 13.

15. An antibody of claim 14 wherein said antibody is a monoclonal or a polyclonal antibody.

16. A method of identifying an agent which modulates the expression of a nucleic acid encoding a protein of claim 13 , comprising:

(a) exposing cells which express the nucleic acid to the agent; and

(b) determining whether the agent modulates expression of said nucleic acid, thereby identifying an agent which modulates the expression of a nucleic acid encoding the protein.

17. A method of identifying an agent which modulates the level of or at least one activity of a protein of claim 13, comprising:

(a) exposing cells which express the protein to the agent; and

(b) determining whether the agent modulates the level of or at least one activity of said protein, thereby identifying an agent which modulates the level of or at least one activity of the protein.

18. The method of claim 17, wherein the agent modulates one activity of the protein.

19. A method of identifying binding partners for a protein of claim 13, comprising:

(a) exposing said protein to a potential binding partner; and

(b) determining if the potential binding partner binds to said protein, thereby identifying binding partners for the protein.

20. A method of modulating the expression of a nucleic acid encoding a protein of claim 13, comprising: administering an effective amount of an agent which modulates the expression of a nucleic acid encoding the protein.

21. A method of modulating at least one activity of a protein of claim 13 , comprising: administering an effective amount of an agent which modulates at least one activity of the protein.

22. A non-human transgenic animal modified to contain a nucleic acid molecule of any of claims 1-4.

23. A non-human transgenic animal modified to contain a nucleic acid molecule of any of claims 1-4, wherein all or a portion of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19,

21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45 or 47 has been knocked out.

24. A method of diagnosing a disease state in a subject, comprising: determining the level of expression of a nucleic acid molecule or protein of any one of claims 1 -4 or 13.

25. The method of claim 24, wherein the disease state is a malignant neoplasm.

26. The method of claim 25, wherein the disease state is a malignant neoplasm of the cervix, endometrium, uterus, breast, colon, rectum, intestine, kidney, liver, lung, stomach, ovary, pancreas, thyroid gland, lymph node, omentum, skin, esophagus, larynx, adrenal gland, prostate, vulva, connective tissue or soft tissue.

27. A composition comprising a diluent and a polypeptide or protein selected from the group consisting of: (a) an isolated polypeptide comprising the amino acid sequence of any one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48; (b) an isolated polypeptide comprising a fragment of at least 10 contiguous amino acids of any one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48; (c) an isolated polypeptide comprising conservative amino acid substitutions of any one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48; (d) an isolated polypeptide comprising naturally occurring amino acid sequence variants of any one of SEQ LD NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48; and (e) an isolated polypeptide exhibiting at least about 60% amino acid sequence identity with any one of SEQ LD NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48.