WO2001025489A2

WO2001025489A2 - Diagnostic and therapeutic uses for a gene differentially expressed in prostate cancer

Info

Publication number: WO2001025489A2
Application number: PCT/US2000/027638
Authority: WO
Inventors: Pablo Garcia; Jimmy Zhang
Original assignee: Chiron Corp
Current assignee: Novartis Vaccines and Diagnostics Inc
Priority date: 1999-10-07
Filing date: 2000-10-06
Publication date: 2001-04-12
Anticipated expiration: 2002-04-07
Also published as: AU7996800A; WO2001025489A3

Abstract

The invention provides methods for detecting, diagnosing and treating cancer and hyperproliferative disease in mammalian cells, using polynucleotides that encode a secreted protein. The methods also include use of encoded polypeptides and antibodies to the polypeptides.

Description

DIAGNOSTIC AND THERAPEUTIC USES FOR A GENE DIFFERENTIALLY

EXPRESSED IN PROSTATE CANCER

TECHNICAL AREA OF THE INVENTION

This invention relates to the area of diagnosis and treatment of cancer, tumor progression, hyperproliferative cell growth, and accompanying biophysical and biological manifestations. More particularly, the invention relates to a gene which is differentially expressed in neoplastic cells.

BACKGROUND OF THE INVENTION

Genes and proteins which are differentially expressed in diseased cells are useful for diagnostic and therapeutic purposes. For example, a gene which is upreguiated in tumors or hyperproliferative cells can be used to diagnosis the onset and progression of cancer. Nucleotide sequences corresponding to genes differentially expressed in tumors or hyperproliferative cells and polypeptides encoded by these sequences can be used to develop therapeutics. Thus, there is a need in the art for genes related to human diseases.

SUMMARY OF THE INVENTION

It is an object of the invention to provide methods of diagnosing cancer, tumor progression, hyperproliferative cell growth or accompanying biological and physical manifestations. Methods of diagnosis include both nucleic acid assays and immunoassays.

One embodiment of the invention provides a diagnostic method comprising

(a) providing a polynucleotide probe that comprises a sequence capable of hybridizing to the sequence CH6001 or a complement thereof; (b) contacting a biological sample for diagnosis with the probe under hybridization conditions that permit duplex formation; and (c) determining the presence of the duplex.

Preferred embodiments utilize a polynucleotide probe comprising sequence CH6001 or a complement thereof or a polynucleotide probe comprising at least eight contiguous nucleotides of sequence CH6001 or a complement thereof. Another embodiment of the invention provides a method of diagnosing cancer, tumor progression, or hyper-proliferative cell growth by:

(a) providing a preparation comprising antibodies capable of binding to a polypeptide encoded by sequence CH6001 or a complement thereof;

(b) contacting a biological sample for diagnosis with the preparation under conditions that permit formation of an antibody-polypeptide complex; and

(c) determining the presence of said complex.

In preferred embodiments, the antibodies are monoclonal, polyclonal or single chain antibodies.

In another embodiment, the antibodies are capable of binding to a polypeptide comprising at least six contiguous amino acids of a polypeptide encoded by nucleotide sequence CH6001.

It is another object of the invention to provide diagnostic kits.

One embodiment of the invention provides a diagnostic kit comprising:

(a) a polynucleotide probe capable of hybridizing to the sequence CH6001 or a complement thereof when the sequence is present in a test biological sample;

(b) a normal biological sample;

(c) instructions for detecting differences between the levels of probe- containing duplexes in the test biological sample as compared to the normal biological sample.

In preferred embodiments, the polynucleotide probe comprises sequence CH6001 or a complement thereof or at least eight contiguous nucleotides of sequence CH6001 or a complement thereof.

Another embodiment of the invention provides a diagnostic kit comprising: (a) a preparation comprising antibodies capable of binding to a polypeptide encoded by sequence CH6001 or a complement thereof when the polypeptide is present in a test biological sample;

(b) a normal biological sample; (c) instructions for detecting differences between the levels of antibody-bound polypeptide in the test biological sample and the normal biological sample.

In preferred embodiments, the antibodies are polyclonal, monoclonal, or single chain antibodies. Another object of the invention is to provide methods of treating a mammal with cancer, tumor progression, hyperproliferative cell growth or accompanying biological and physical manifestations.

One embodiment of the invention provides a method of treating a mammal with cancer, tumor progression, hyperproliferative cell growth or accompanying biological and physical manifestations, the method comprising administering to the mammal a composition comprising a therapeutically effective amount of a polynucleotide comprising a sequence capable of hybridizing to sequence CH6001 or a complement thereof.

In a preferred embodiment, the method utilizes a polynucleotide comprising sequence CH6001 or a complement thereof or at least eight contiguous nucleotides of CH6001 or a complement thereof.

In another embodiment, the method utilizes a polynucleotide construct expressing antisense RNA of clone CH6001.

In another embodiment, the method utilizes a polynucleotide expressing a ribozyme which recognizes a clone CH6001 polynucleotide.

In another embodiment, the method utilizes a retroviral vector comprising a promoter and polynucleotides comprising sequence CH6001 or a complement thereof.

In a preferred embodiment, the retroviral vector expresses altered forms of sequence CH6001 or a complement thereof. The altered forms can be deletion mutants of the gene encoding sequence CH6001. The retroviral vectors can encode a dominant-negative mutant of a polypeptide encoded by sequence CH6001 or a complement thereof.

The retroviral vectors can contain polynucleotides which are at least 80% or 95% identical to sequence CH6001.

Another embodiment of the invention provides a method of treating a mammal with cancer, tumor progression, hyperproliferative cell growth or accompanying biological and physical manifestations, the method comprising administering to the mammal a composition comprising a therapeutically effective amount of polypeptides or proteins capable of binding to a polypeptide encoded by sequence CH6001 or a complement thereof.

In a preferred embodiment, the method utilizes polypeptides which are antibodies.

In another embodiment, the method utilizes polypeptides which are wild- type or mutated receptors for the protein encoded by CH6001.

In yet another embodiment, the method utilizes polypeptides which are wild-type or mutant binding partners of the protein encoded by CH6001.

The invention also provides a method of treating a mammal with cancer, tumor progression, hyperproliferative cell growth or accompanying biological and physical manifestations, the method comprising administering to the mammal a composition comprising a therapeutically effective amount of polypeptide, wherein the polypeptide is at least 75% identical to a 12 amino acid region of a polypeptide encoded by sequence CH6001 or a complement thereof.

In a preferred embodiment, this method utilizes a polypeptide which is identical to a region of a polypeptide encoded by clone CH6001.

Another embodiment of the invention provides a method of treating a mammal with cancer, tumor progression, hyperproliferative cell growth or accompanying biological and physical manifestations, the method comprising administering to the mammal a composition comprising a therapeutically effective amount of polypeptide, wherein the polypeptide comprises a mutant of a polypeptide capable of binding a polypeptide encoded by sequence CH6001 or a complement thereof.

The present invention thus provides the art with methods and compositions for diagnosing and treating cancer, tumor progression, hyperproliferative cell growth, and accompanying biophysical and biological manifestations.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 provides SDS-PAGE electrophoresis of the protein expressed by sequence CH6001.

Figure 2 provides the polynucleotide sequence of CH6001 (SEQ ID NO:l).

Figure 3 provides northern blot analysis of clone CH6001 expression in human tissues.

Figure 4 provides northern blot analysis of clone CH6001 expression in human prostate cells. Figure 5 provides the amino acid sequence of CH6001 (SEQ ID NO:2).

DETAILED DESCRIPTION OF THE INVENTION

Genes which are differentially expressed in diseased tissue are useful for diagnostic and therapeutic purposes. For example, a gene which is up-regulated in malignant or hyperproliferative cells can serve as a marker of cancer progression and be useful in determining appropriate treatment. Therefore, the discovery that the gene corresponding to sequence CH6001 is upregulated in prostate tumor cell lines is useful for diagnostic and prognostic purposes.

Modulation of misregulated genes or their protein products can be used to treat cancer, tumor progression, hyperproliferative cell growth and related physical and biological manifestations. For example, the expression of a protein encoded by genes which are upregulated in malignant or hyperproliferative cells can be decreased and/or the protein can be functionally inactivated. The polynucleotide sequence corresponding to clone CH6001 can be used to construct polynucleotide and polypeptide compositions useful for treatment, such as antisense, ribozymes, antibodies, dominant-negative mutants, and blocking peptides.

The gene corresponding to sequence CH6001 is herein identified as being up-regulated in mammalian tumor cell lines. Thus, this invention relates to methods and compositions for diagnosis and treatment of cancer.

Where percent identity is indicated, it refers to identity as calculated using the Smith-Waterman homology search algorithm as implemented in MPSRCH program (Oxford Biomolecular) using an affine gap search with the following search parameters: gap open penalty, 12; gap extension penalty, 1.

Identification and characterization of clone CH6001.

A cDNA clone comprising the sequence CH6001 was obtained by screening a cDNA library prepared from primary cultures of prostate Gleason grade 4+4 epithelial cells (Woca cells). The polypeptide expressed by clone CH6001 had an approximate molecular weight of 45 kDa (Figure 1, lane 1) and was modified by the addition of N-linked oligosaccharides in the presence of a rough endoplasmic reticulum (RER) preparation (Figure 1, lane 2).

Detailed sequence analysis of clone CH6001 (SEQ ID NO:l) indicated that it is identical to the sequence of SEQ ID NO: 12 from co-pending Patent Application Serial No. 08/988,671. There are no differences in the coding sequences or splicing patterns of the transcripts from which the clones were derived (human prostate cancer epithelial cells for clone CH6001 and human placenta for SEQ ID NO: 12 of 08/988,671, respectively). However, sequence analysis indicates the presence of multiple polyadenylation sites in the clone CH6001 gene. This difference in polyadenylation does not affect the protein coding region of the rnRNA, since the polyadenylation sites are located in the 3' direction from the open reading frame.

The amino acid sequence (SEQ ID NO:2) encoded by the clone CH6001 cDNA was disclosed in SEQ ID NO:31 of U.S. Patent Application Serial No. 09/988,671. The nucleotide sequence corresponding to the coding region of clone CH6001 was disclosed in SEQ ID NO: 12 of U.S. Patent Application Serial No. 08/988,671, which is incorporated by reference. Clones containing SEQ ID NO:12 (SEQ ID NO:l of this application) which corresponds to sequence CH6001 were deposited on December 11, 1997 with the ATCC. The deposit was designated SECP 120997. The deposit is a pool of bacterial cells (E. coli), some containing polynucleotides corresponding to SEQ ID NO:l. SEQ ID NO:l (clone CH6001) can be retrieved using an oligonucleotide probe designed from the sequence of SEQ ID NO:l using techniques familiar to the art. The polynucleotide corresponding to SEQ ID NO:l can be removed from the vector by performing a restriction digestion with EcoRI and Notl (5' site, EcoRI; 3' site, Notl). The nucleotide sequence in this deposit and the amino acid sequences they encode are controlling in the event of a discrepancy between the sequences disclosed in U.S. Patent Application Serial No. 08/988,671 and those contained in the deposit.

Analysis of the protein sequence encoded by clone CH6001 reveals that it contains two stretches of hydrophobic amino acids (amino acids 26-52 and 315-348) that could function as signal peptides and/or transmembrane domains. In addition, the sequence indicates that this protein contains two potential N-glycosylation sites (NXS/T) at amino acids 212 and 347. These observations are consistent with the results of Figure 1 which indicate CH6001 encodes a secreted and/or membrane protein. These results are also consistent with the previous identification of SEQ ID NO: 12 from U.S. Patent Application Serial No. 08/988,671 as a polynucleotide encoding a secreted polypeptide.

CH6001 rnRNA is preferentially expressed at high levels in human cancers of multiple origins. The mRNA is expressed in a restricted, tissue-specific pattern in normal human tissues (Figure 3, lanes 8-15). No expression was detected in peripheral blood leukocytes (lane 8), colon (lane 9), small intestine (lane 10), thymus (lane 14) and spleen (lane 15). Modest expression was observed in normal ovaries (lane 11) and prostate (lane 13). Significant expression was observed in testis (lane 12). In contrast, significant expression of the RNA was observed in cell lines derived from several types of cancer (Figure 3, lanes 1-7). Expression was observed in melanoma cells (lane 1), lung carcinoma cells (lane 2), colorectal adenocarcinoma cells (lane 3), lymphoblastic leukemia cells (lane 4), myelogenous leukemia cells (lane 5), HeLa cells (lane 6), and promyelocytic leukemia cells (lane 7).

CH6001 mRNA was also expressed in several human cell lines and primary cultures from both normal prostate and prostate cancer cells (Figure 4). Normal (FFpz, lane 1) and Gleason grade 3+3 (FFca, lane 2) primary culture cells showed moderate levels of expression of CH6001, in accordance with the observation in normal prostate tissues (Figure 3). In contrast, HPV immortalized prostate cells HPV-7 (lane 3) and HPV- 10 (lane 4) as well as the Gleason grade 5+4 primary culture cells (lane 5) showed high levels of expression. Thus, clone CH6001 mRNA levels correspond with the transformed phenotype. Therefore, polynucleotides and polypeptides derived from the sequence of clone CH6001 can be used in the diagnosis and treatment of cancer, proliferative disorders and related conditions.

Diagnostic and prognostic use of clone CH6001.

Subgenomic polynucleotides corresponding to clone CH6001 and complements thereof can be used as a marker to diagnose and determine the prognosis of cancer, tumor progression, hyperproliferative cell growth or accompanying biological and physical manifestations. For example, the clone CH6001 polynucleotides and encoded polypeptides can be utilized to determine the occurrence of melanomas, leukemias and prostatic disorders such as benign prostate hyperplasia (BPH) or localized prostate cancer.

To determine the occurrence of cancer, tumor progression, hyperproliferative growth, and/or accompanying biological or physical manifestations, the levels of clone CH6001 polynucleotides or polypeptides in a sample are compared to the levels in a normal control sample. The normal control can include a pool of cells from a particular organ or tissue or tissues and/or cells from throughout the body. Immunoassays or nucleic acid assays described below can be used for such measurements.

Any observed difference between the sample and normal control can indicate the occurrence of disease or disorder. Typically, if the levels of clone CH6001 polynucleotides and the encoded polypeptides are higher than those found in the normal control, the results indicate the occurrence of cancer, tumor progression, hyperproliferative growth, and/or accompanying biological or physical manifestations. In addition, polynucleotides corresponding to clone CH6001 and antibodies which bind polypeptides encoded by clone CH6001 can be used to diagnose the severity as well as the occurrence of cancer, tumor progression, hyperproliferative growth, and/or accompanying biological or physical manifestations, including leukemias and prostate disorders. For example, the greater the difference observed in the sample versus the normal control of clone CH6001 polynucleotides or encoded polypeptides, the greater the severity of the disorder.

Nucleic acid assays utilize subgenomic polynucleotides capable of hybridizing under stringent conditions to clone CH6001 polynucleotides or complements thereof. Polynucleotide probes comprising at least 10 contiguous nucleotides selected from the nucleotide sequence of CH6001 are labeled, for example, with a radioactive, fluorescent, biotinylated, or chemiluminescent label, and detected by well known methods appropriate for the particular label selected. Subgenomic polynucleotides are preferably intron-free. Polynucleotides corresponding to clone CH6001 can be introduced into vectors and propagated in suitable hosts. Plasmids can be introduced into host cells using techniques available in the art. These techniques include, but are not limited to, electroporation and calcium phosphate-mediated transfection. They can be isolated and purified from DNA vectors by standard techniques, such as restriction enzyme digestion and gel electrophoresis or chromatography. The polynucleotides can also be produced using the polymerase chain reaction according to techniques well known in the art. The subgenomic polynucleotides can be used to compare related genes in normal control tissue and suspected diseased tissue by any means known in the art. For example, the CH6001 gene from a suspected diseased tissue can be sequenced and compared with the CH6001 gene sequence in the normal tissue. The polynucleotide- related genes, or portions thereof, in the two tissues are amplified, for example using nucleotide primers based on the nucleotide sequence of CH6001, using the polymerase chain reaction. The amplified genes or portions of genes are hybridized to nucleotide probes selected from the same nucleotide sequence and sequenced. A difference in the nucleotide sequence of the polynucleotide-related gene in the tissue suspected of being diseased compared with the normal nucleotide sequence suggests a role of the polynucleotide-encoded proteins in the disease, and provides a lead for preparing a therapeutic agent. The nucleotide probes or nucleotides incorporated during sequencing are labeled by a variety of methods, such as radiolabeling, biotinylation, or labeling with fluorescent or chemiluminescent tags, and detected by standard methods known in the art. Alternatively, CH6001 mRNA levels in normal and suspected diseased tissues are compared. PolyA⁺ RNA is isolated from the two tissues as is known in the art. For example, one of skill in the art can readily determine differences in the size or amount of polynucleotide-related mRNA transcripts between the two tissues by Northern blot analysis, primer extension, SI nuclease protection, reverse transcription- polymerase chain reaction (RT-PCR), or in situ hybridization using polynucleotide probes corresponding to clone CH6001 or a complement thereof. Increased or decreased expression of a polynucleotide-related mRNA in a tissue sample suspected of being diseased, compared with the expression of the same polynucleotide-related mRNA in a normal tissue, suggests that the expressed protein has a role in the disease, and also provides a lead for preparing a therapeutic agent.

Clone CH6001 gene expression can also be examined using polynucleotide arrays. Polynucleotide arrays provide a high throughput technique that can assay a large number of polynucleotide sequences in a sample. This technology can be used as a diagnostic and as a tool to test for differential expression to determine function of an encoded protein.

To create arrays, polynucleotide probes are spotted onto a substrate in a two-dimensional matrix or array. Samples of polynucleotides can be labeled and then hybridized to the probes. Double stranded polynucleotides, comprising the labeled sample polynucleotides bound to probe polynucleotides, can be detected once the unbound portion of the sample is washed away. Techniques for constructing arrays and methods of using these arrays are described in EP No. 0 799 897; PCT No. WO 97/29212; PCT No. WO 97/27317; EP No. 0 785 280; PCT No. WO 97/02357; U.S. Pat. No. 5,593,839; U.S. Pat. No. 5,578,832; EP No. 0 728 520; U.S. Pat. No. 5,599,695; EP No. 0 721 016; U.S. Pat. No. 5,556,752; PCT No. WO 95/22058; and U.S. Pat. No. 5,631,734.

Antibodies which bind polypeptides encoded by clone CH6001 and complements thereof can be used in diagnosing and determining the prognosis of cancer, tumor progression, hyperproliferative cell growth or accompanying biological and physical manifestations. These antibodies may be monoclonal, polyclonal or single chain antibodies and are produced by methods well known in the art. Antibodies which bind the polypeptide encoded by clone CH6001 are described in U.S. Patent Application Serial No. 08/988,671.

Any method known in the art can be used to compare clone CH6001 encoded proteins from normal control samples and suspected diseased samples. The size of the proteins in the two tissues can be compared, for example, using antibodies against polypeptides encoded by clone CH6001 to detect CH6001 polypeptides by western blot. Alterations in the size of the CH6001 protein in a tissue suspected of being diseased compared with the level in a normal control sample indicate the protein is abnormal, possibly due to truncation, deletion or altered post-translational modification. Size alterations are indicative that CH6001 has a role in the disease and provides a lead for preparing a therapeutic agent. Other changes, such as protein expression levels and subcellular localization can also be detected immunologically, for example by using antibodies directed against polypeptides encoded by clone CH6001 for western blot or immuno fluorescence. A higher or lower level of CH6001 encoded protein in a tissue suspected of being diseased, or in conditioned media of cells derived from said tissue, compared with the level in a normal control sample is indicative that CH6001 has a role in the disease and provides another lead for preparing a therapeutic agent. Similarly, changes in subcellular localization of CH6001 protein also indicates CH6001 has a role in the disease. Reagents specific for CH6001 polynucleotides and polypeptides, such as antibodies and nucleotide probes, can be supplied in a kit for detecting the presence of an expression product in a biological sample. The kit can also contain buffers or labeling components, detection reagents, instructions for using reagents to detect and quantify expression products in biological samples and control normal biological samples. Normal biological samples may be in any form suitable for the particular method of detection utilized by the kit. For example, normal biological samples can be polynucleotides, polypeptides, cellular extracts or tissue sections.

Identification of Binding Partners and Peptide Agonists and Antagonists Polypeptides encoded by clone CH6001 can be used to screen peptide libraries to identify binding partners from among the encoded polypeptides. Candidate binding partners for the clone CH6001 protein include cell surface receptors and proteins which bind the CH6001 protein to form multimeric protein complexes. Such binding partners can be useful in treating cancer, hyperproliferation and related conditions. For example, peptides and antibodies capable of binding to a CH6001 binding partner or receptor can block CH6001 binding to said partner or receptor and thereby inhibit the biological activity of CH6001. Polypeptide binding partners can be identified by any method available in the art such as expression cloning using a labeled CH6001 polypeptide probe or yeast two-hybrid screening using a fusion protein comprising a CH6001 polypeptide as bait. Yeast two-hybrid screens, also called interaction trap assays, are described, for example, in Gyuris et al., Cell 74:791-803 (1993). Binding partners can also be identified by low stringency immunoprecipitation using an antibody directed against a CH6001 polypeptide followed by sequencing of coprecipitating polypeptides. Peptide agonists and antagonists are screened using any available method, such as signal transduction, antibody binding, receptor binding, mitogenic assays, chemotaxis assays, etc. A library of peptides may be synthesized following the methods disclosed in U.S. Pat. No. 5,010,175 and in PCT WO 91/1723. The assay conditions ideally should resemble the conditions under which the native activity is exhibited in vivo, that is, under physiologic pH, temperature, and ionic strength. Suitable agonists or antagonists will exhibit strong inhibition or enhancement of the native activity at concentrations that do not cause toxic side effects in the subject.

The end results of such screening and experimentation will be at least one novel CH6001 protein binding partner, such as a receptor and at least one peptide agonist or antagonist of the novel binding partner. Such agonists and antagonists can be used to modulate, enhance, or inhibit CH6001 protein function in cells.

Therapeutic use of clone CH6001.

Pharmaceutical compositions can comprise polypeptides, antibodies or polynucleotides. Therapeutics, whether polynucleotide or polypeptide or small molecule, can be tested, for example, in the mouse tumor assay described in Pei et al., Mol. Endo. ii:433-441 (1997). Other models for testing polynucleotides, polypeptides, antibodies, or small molecules useful for treatment include animal models and cell lines disclosed in Bosland, Encyclopedia of Cancer, Volume II, pages 1283 to 1296 (1997), Academic Press. Other useful cell lines are described in Brothman, Encyclopedia of Cancer, Volume II, pages 1303 to 1313 (1997), Academic Press.

The term "therapeutically effective amount" as used herein refers to an amount of a therapeutic agent to treat, ameliorate, or prevent a specific disease or condition, or to exhibit a detectable therapeutic or preventative effect. The effect can be detected by, for example, chemical markers or antigen levels. The effects also include reduction in physical symptoms. The effective amount for a given situation can be determined by routine experimentation and is within the judgment of the clinician. The precise effective amount will vary depending on factors including, but not limited to, the subject's size and health, the nature and extent of the condition, and the therapeutics selected for administration. For purposes of the present invention, an effective dose will be from about 0.01 mg/ kg to 50 mg/kg or 0.05 mg/kg to about 10 mg kg of the polynucleotide, polypeptide or antibody compositions in the individual to which it is administered. A pharmaceutical composition can also contain a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, such as antibodies, polypeptides, polynucleotides and other therapeutic agents. Suitable carriers and pharmaceutically acceptable salts are well known to those of ordinary skill in the art. A thorough discussion of pharmaceutically acceptable excipients is available in Remington 's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991).

Once formulated, the polynucleotide and polypeptide compositions of the invention can be (1) administered directly to the subject; (2) delivered ex vivo, to cells derived from the subject; or (3) delivered in vitro for expression of recombinant proteins. Direct delivery of the compositions will generally be accomplished by injection, either subcutaneously, intraperitoneally, intravenously or intramuscularly, or delivered to the interstitial space of a tissue. The compositions can also be administered into a tumor or lesion. Other modes of administration include oral and pulmonary administration, suppositories, and transdermal applications, needles, and gene guns or hyposprays. Dosage treatment may be a single dose schedule or a multiple dose schedule.

Methods for the ex vivo delivery and reimplantation of transformed cells into a subject are known in the art and described in e.g., International Publication No. WO 93/14778. Generally, delivery of nucleic acids for both ex vivo and in vitro applications can be accomplished by, for example, dextran-mediated transfection, calcium phosphate precipitation transfection, viral infection, polybrene mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei, all well known in the art. Neoplasias that are treated with pharmaceutical compositions include, but are not limited to, prostate cancers, cervical cancers, melanomas, colorectal adenocarcinomas, Wilms' tumor, retinoblastoma, sarcomas, myosarcomas, lung carcinomas, leukemias, such as chronic myelogenous leukemia, promyelocytic leukemia, monocytic leukemia, and myeloid leukemia, and lymphomas, such as histiocytic lymphoma. Proliferative disorders that are treated with the therapeutic composition include disorders such as anhydric hereditary ectodermal dysplasia, congenital alveolar dysplasia, epithelial dysplasia of the cervix, fibrous dysplasia of bone, and mammary dysplasia. Hyperplasias, for example, endometrial, adrenal, breast, prostate, or thyroid hyperplasias or pseudoepitheliomatous hyperplasia of the skin, are treated with antisense therapeutic compositions. Even in disorders in which mutations in the corresponding gene are not implicated, downregulation or inhibition of gene expression can have therapeutic application. For example, decreasing gene expression can help to suppress tumors in which enhanced expression of the gene is implicated.

Examples of polynucleotide therapeutic agents include ribozymes, antisense RNA, and mammalian expression vectors.

Trans-cleaving catalytic RNAs (ribozymes) are RNA molecules possessing endoribonuclease activity. Ribozymes are engineered to cleave any RNA species site-specifically in the background of cellular RNA. The cleavage event renders the mRNA unstable and prevents protein expression. Ribozyme design and therapeutic uses are disclosed in Usman et al., Current Opin. Struct. Biol. 5:527-533 (1996), which is incorporated by reference.

The CH6001 polynucleotide sequence provides adequate sequence for constructing an effective ribozyme. A target cleavage site is selected in the target sequence, and a ribozyme is constructed based on the 5' and 3' nucleotide sequences that flank the cleavage site. Retroviral vectors are engineered to express monomeric and multimeric hammerhead ribozymes targeting the mRNA of clone CH6001 coding sequence. These monomeric and multimeric ribozymes are tested in vitro for an ability to cleave the CH6001 mRNA. A cell line is stably transduced with the retroviral vectors expressing the ribozymes, and the transduction is confirmed by Northern blot analysis and reverse-transcription polymerase chain reaction (RT-PCR). The cells are screened for inactivation of the target mRNA by such indicators as reduction of expression of disease markers or reduction of the gene product of the target mRNA.

Antisense nucleic acids are designed to specifically bind to RNA, resulting in the formation of RNA-DNA or RNA-RNA hybrids, with an arrest of DNA replication, reverse transcription or messenger RNA translation. Antisense polynucleotides based on a selected sequence can interfere with expression of the corresponding gene. Antisense polynucleotides are typically generated within the cell by expression from antisense constructs that contain the antisense strand as the transcribed strand. Antisense polynucleotides will bind and/or interfere with the translation of the corresponding mRNA. The expression products of control cells and cells treated with the antisense construct are compared to detect the protein product of the gene corresponding to the polynucleotide. The protein is isolated and identified using routine biochemical methods.

Antisense therapy for a variety of cancers is in clinical phase and has been discussed extensively in the literature. Given the extensive background literature and clinical experience in antisense therapy, one skilled in the art can use antisense CH6001 polynucleotides as therapeutics. The dosage and means of administration are determined based on the specific qualities of the composition, the patient, the progression of the disease and other relevant factors. Preferably, the therapeutic antisense composition contains an expression construct comprising a promoter upstream of a polynucleotide segment of at least 12, 22, 25, 30, or 35 contiguous nucleotides of clone CH6001 in the antisense orientation. Therapeutic antisense agents may be administered locally or systemically by a variety of methods known in the art. Examples cited include those mentioned above and receptor-mediated targeted delivery. Therapeutic compositions containing antisense CH6001 polynucleotides are administered in a range of about 100 ng to about 200 mg of polynucleotides for local administration in a gene therapy protocol, as discussed later. In all cases, routine experimentation in clinical trials will determine specific ranges for optimal therapeutic effects. Dominant negative mutations are readily generated for proteins whose biological activity requires binding to another protein. A mutant polypeptide will interact with wild-type proteins and form a non-functional multimer. A mutation is, for example, in a substrate-binding domain, a catalytic domain, or a cellular localization domain. Dominant negative mutants may contain point mutations or deletions. Preferably, the mutant polypeptide will be overproduced. General strategies are available for making dominant negative mutants. See Herskowitz, Nature 329:219-222 (1987). Since clone CH6001 expresses a secreted protein, potential dominant negatives include mutants which lack secretory signals, mutants exhibiting reduced or enhanced binding to a receptor or binding partner, and mutants containing deletion of domains required for biological function.

Polypeptide compositions can also include antibodies and peptides. The effective dosages for therapeutic compositions containing protein, polypeptide or antibody are in the range of about 5 μg to about 50 μg/kg of patient body weight, about 50 μg to about 5 mg/kg, about 100 μg to about 500 μg/kg of patient body weight, and about 200 to about 250 μg/kg.

Antibodies may be polyclonal, monoclonal or single-chain antibodies prepared by methods known in the field. Methods of generating the antibodies are described in U.S. Patent Application Serial No. 08/988,671. Antibodies specific to polypeptides encoded by clone CH6001 bind the protein and inhibit the protein from functioning in the cell. For example, the antibodies can prevent polypeptides encoded by clone CH6001 from binding protein partners or receptors. Such antibodies can also induce conformation changes in the polypeptides they bind. The invention also pertains to antibodies directed against protein partners and receptors for clone CH6001 -encoded polypeptides. Such antibodies can disrupt protei protein interactions required for cellular function of clone CH6001. They can also inhibit downstream signaling by the receptor protein. Techniques for purifying antibodies are available in the art. For example, the antibodies can be passed over a column to which CH6001 polypeptide is bound. Bound antibodies are then eluted, for example, with a buffer having a high salt concentration. Therapeutic compositions and methods comprising peptide agonists and antagonists are also included in the invention. The peptides can effect the function of polypeptides encoded by clone CH6001 or their binding partners and receptors. For example, the peptides may block protei protein interactions or cause conformational changes which diminish or enhance clone CH6001's normal functional activity. The peptides may also alter clone CH6001's activity or specificity in a therapeutically useful manner.

The therapeutic polynucleotides and polypeptides of the present invention may be utilized in gene delivery vehicles. The gene delivery vehicle may be of viral or non-viral origin (see generally, Jolly, Cancer Gene Therapy 7:51-64 (1994); Kimura, Human Gene Therapy 5:845-852 (1994); Connelly, Human Gene Therapy 7:185-193 (1995); and Kaplitt, Nature Genetics 5:148-153 (1994)). Gene therapy vehicles for delivery of constructs including a coding sequence of clone CH6001 can be administered either locally or systemically. These constructs can utilize viral or non- viral vector approaches. Expression of such coding sequences can be induced using endogenous mammalian or heterologous promoters. Expression of the coding sequence can be either constitutive or regulated.

Any gene delivery method known in the art can be utilized. For example, the present invention can employ recombinant retroviruses which are constructed to carry or express a selected nucleic acid molecule of interest. The present invention also employs alphavirus-based vectors and parvovirus, that can function as gene delivery vehicles. Other gene delivery vehicles and methods may be employed, including polycationic condensed DNA linked or unlinked to killed adenovirus alone, for example Curiel, Hum. Gene Ther. 3:147-154 (1992); ligand linked DNA, for example see Wu, J. Biol. Chem. 264:16985-16987 (1989); eukaryotic cell delivery vehicles cells, for example see U.S. Patent Application Serial No. 08/240,030, filed

May 9, 1994, and U.S. Patent Application Serial No. 08/404,796; deposition of photopolymerized hydrogel materials; hand-held gene transfer particle gun, as described in U.S. Patent No. 5,149,655; ionizing radiation as described in U.S. Patent No. 5,206,152 and in WO 92/11033; nucleic charge neutralization or fusion with cell membranes. Additional approaches are described in Philip, Mol. Cell Biol. 74:2411-

2418 (1994), and in Woffendin, Proc. Natl. Acad. Sci. 97:1581-1585 (1994).

Non-viral delivery methods include, but are not limited to, mechanical delivery systems such as the approach described in Woffendin et al., Proc. Natl. Acad. Sci. USA 91(24):\ 1581-11585 (1994) and naked DNA protocols. Exemplary naked DNA introduction methods are described in WO 90/11092 and U.S. Patent No. 5,580,859.

Packaging cell lines suitable for use with the above-described retroviral vector constructs may be readily prepared (see PCT publications WO 95/30763 and WO 92/05266), and used to create producer cell lines (also termed vector cell lines) for the production of recombinant vector particles.

EXAMPLES

The following examples provide data and experimental procedures.

However, the invention is not limited to the examples. The invention is defined in the specification as a whole, which includes the claims.

EXAMPLE 1 IDENTIFICATION AND CHARACTERIZATION OF CLONE CH6001

A cDNA clone comprising the sequence CH6001 (SEQ ID NO:l) was obtained by screening a cDNA library from primary cultures of prostate Gleason grade 4+4 epithelial cells (Woca cells). The protein encoded by cDNA clone CH6001 was expressed in vitro in the absence (Figure 1, lane 1) or presence (lane 2) of a Rough Endoplasmic Reticulum (RER) preparation. The expressed proteins were resolved by SDS-PAGE gel electrophoresis. A primary translation product of about 43-45 kDa was observed (lane 1). In the presence of RER, this protein was modified by the addition of N-linked oligosaccharides which resulted in an increased molecular weight (lane 2).

EXAMPLE 2 SEQUENCE ANALYSIS OF CLONE CH6001

Detailed sequence analysis of clone CH6001 indicated that it is identical to SEQ ID NO: 12 of U.S. Patent Application Serial No. 08/988,671. The clone comprising SEQ ID NO: 12 of '671 was derived from human placenta. The polynucleotide sequence of clone CH6001 is shown in Figure 2. There were no differences in the coding regions of the two transcripts from which the clones were derived. In contrast, clone CH6001 lacks approximately 240 bp at the 3' end of the mRNA with respect to SEQ ID NO: 12. Sequence analysis indicates that this difference is due to the use of multiple polyadenylation sites in the gene encoding this protein. This difference in polyadenylation does not affect the protein coding region of the mRNA, since the polyadenylation sites are located in the 3' direction from the open reading frame. Analysis of the protein sequence encoded by clone CH6001 reveals that it does not contain a signal peptide sequence cleavable by signal peptidase. However, it contains two stretches of hydrophobic amino acids (amino acids 26-52 and 315-348) that could function as signal peptides and/or transmembrane domains. In addition, the sequence indicates this protein contains two potential N-glycosylation sites (NXS/T) at amino acids 212 and 347. These observations are consistent with the results of Figure 1 which indicate CH6001 encodes a secreted and/or membrane protein.

EXAMPLE 3 NORTHERN BLOT ANALYSIS OF CLONE CH6001 EXPRESSION IN NORMAL AND CANCER HUMAN TISSUES

Northern blot analysis from multiple tissues indicates that clone CH6001 mRNA is preferentially expressed at high levels in human cancers of multiple origins. The mRNA is expressed in a restricted, tissue-specific pattern in normal human tissues (Figure 3, lanes 8-15). No expression was detected in peripheral blood leukocytes (lane 8), colon (lane 9), small intestine, (lane 10), thymus (lane 14) and spleen (lane 15). Modest expression was observed in normal ovaries (lane 11) and prostate (lane 13). Significant expression was observed in testis (lane 12). In contrast, significant expression of the mRNA was observed in cell lines derived from several types of cancer (Figure 3, lanes 1-7). Expression was observed in melanoma cells (lane 1), lung carcinoma cells (lane 2), colorectal adenocarcinoma cells (lane 3), lymphoblastic leukemia cells (lane 4), myelogenous leukemia cells (lane 5), HeLa cells (lane 6), and promyelocytic leukemia cells (lane 7).

EXAMPLE 4

NORTHERN BLOT ANALYSIS OF CLONE CH6001 EXPRESSION IN HUMAN PROSTATE CELLS

To examine clone CH6001 mRNA expression in the development of prostate cancer, northern blot analysis was performed on several human cell lines and primary cultures from both normal prostate and prostate cancer cells (Figure 4). Normal (FFpz, lane 1) and Gleason grade 3+3 (FFca, lane 2) primary culture cells showed moderate levels of expression of CH6001, in accordance with the observation in normal prostate tissues (Figure 3). In contrast, HPV immortalized prostate cells HPN-7 (lane 3) and HPN-10 (lane 4) as well as the Gleason grade 5+4 primary culture cells (lane 5) showed high levels of expression.

Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such specific embodiments and equivalents are intended to be encompassed by the following claims. All patents, published application, and publications cited herein are incorporated by reference as if set forth fully herein.

Claims

CLAIMSWe claim:

1. A method of diagnosing cancer, tumor progression, hyperproliferative cell growth or accompanying biological and physical manifestations comprising:

(a) providing a polynucleotide probe that comprises a sequence capable of hybridizing to the sequence CH6001 or a complement thereof;

(b) contacting a biological sample for diagnosis with the probe under hybridization conditions that permit duplex formation; and

(c) determining the presence of the duplex.

2. The method of claim 1, wherein the polynucleotide probe comprises sequence CH6001 or the complement thereof.

3. The method of claim 1, wherein the polynucleotide probe comprises at least eight contiguous nucleotides of sequence CH6001 or a complement thereof.

4. A method of diagnosing cancer, tumor progression, or hyperproliferative cell growth comprising:

(b) contacting a biological sample for diagnosis with the preparation under binding conditions that permit formation of an antibody-polypeptide complex; and

(c) determining the presence of said complex.

5. The method of claim 4, wherein the antibodies are selected from the group consisting of monoclonal antibodies, polyclonal antibodies and single chain antibodies.

6. The method of claim 4, wherein the antibodies are capable of binding to a polypeptide comprising at least six contiguous amino acids of a polypeptide encoded by nucleotide sequence CH6001.

7. A diagnostic kit comprising:

(a) a polynucleotide probe that comprises a sequence capable of hybridizing to the sequence CH6001 or a complement thereof when the sequence is present in a test biological sample;

(b) instructions for detecting differences between the levels of probe- containing duplexes in the test biological sample as compared to a normal biological sample.

8. The kit of claim 7, wherein the polynucleotide probe comprises sequence CH6001 or a complement thereof.

9. The kit of claim 7, wherein the polynucleotide probe comprises at least eight contiguous nucleotides of sequence CH6001 or a complement thereof.

10. A diagnostic kit comprising:

(a) a preparation comprising antibodies capable of binding to a polypeptide encoded by sequence CH6001 or a complement thereof when the polypeptide is present in a test biological sample

(b) instructions for detecting differences between the levels of antibody-bound polypeptide in the test biological sample and a normal biological sample.

11. The kit of claim 10, wherein the antibodies are selected from the group consisting of monoclonal antibodies, polyclonal antibodies and single chain antibodies.

12. A method of treating a mammal with cancer, tumor progression, hyperproliferative cell growth or accompanying biological and physical manifestations, the method comprising administering to the mammal a composition comprising a therapeutically effective amount of a polynucleotide comprising a sequence capable of hybridizing to sequence CH6001 or a complement thereof.

13. The method of claim 12, wherein the polynucleotide comprises sequence CH6001 or a complement thereof.

14. The method of claim 12, wherein the polynucleotide comprises at least eight contiguous nucleotides of CH6001 or a complement thereof.

15. The method of claim 12, wherein the polynucleotide is selected from the group consisting of an antisense construct and a ribozyme construct.

16. The method of claim 12, wherein the polynucleotide is a retroviral vector comprising a promoter and polynucleotides comprising sequence CH6001 or a complement thereof.

17. The method of claim 12, wherein the polynucleotide is an altered form of sequence CH6001 or a complement thereof.

18. The method of claim 17, wherein the polynucleotide contains a deletion of a region of sequence CH6001 or a complement thereof.

19. The method of claim 17, wherein the polynucleotide encodes a dominant-negative mutant of a polypeptide encoded by sequence CH6001 or a complement thereof.

20. The method of claim 17, wherein the polynucleotide is at least 80% identical to sequence CH6001.

21. The method of claim 17, wherein the polynucleotide is at least 95% identical to sequence CH6001.

22. A method of treating a mammal with cancer, tumor progression, hyperproliferative cell growth or accompanying biological and physical manifestations, the method comprising administering to the mammal a composition comprising a therapeutically effective amount of a protein capable of binding to a polypeptide encoded by sequence CH6001 or a complement thereof.

23. The method of claim 22, wherein the protein is an antibody.

24. The method of claim 22, wherein the protein is a wild-type or mutated receptor for the protein encoded by CH6001 or a complement thereof.

25. The method of claim 22, wherein the protein is a wild-type or mutant binding partner of the protein encoded by CH6001 or a complement thereof.

26. A method of treating a mammal with cancer, tumor progression, hyperproliferative cell growth or accompanying biological and physical manifestations, the method comprising administering to the mammal a composition comprising a therapeutically effective amount of protein, wherein the protein is at least 75% identical to a 12 amino acid region of a polypeptide encoded by sequence CH6001 or a complement thereof.

27. The method of claim 26, wherein the protein is identical to a region of a polypeptide encoded by clone CH6001.

28. A method of treating a mammal with cancer, tumor progression, hyperproliferative cell growth or accompanying biological and physical manifestations, the method comprising administering to the mammal a composition comprising a therapeutically effective amount of protein, wherein the protein is a mutant of a polypeptide capable of binding a polypeptide encoded by sequence CH6001 or a complement thereof.