CN102575300A - CSTF2 for target genes of lung cancer therapy and diagnosis - Google Patents

Abstract

The present invention relates to the roles played by a CSTF2 gene in cancer carcinogenesis and features a method for treating or preventing cancer by administering a double-stranded molecule against the CSTF2 gene or a composition, vector or cell containing such a double stranded molecule. The present invention also features methods for diagnosing cancer or assessing/determining the prognosis of a subject with lung cancer, using the over-expressed CSTF2 gene. To that end, CSTF2 may serve as a novel prognosis biomarker for cancer, particularly lung cancer. Also, disclosed are methods of screening candidate substances for treating and preventing cancer, using as an index their effect on the expression or biological activity of CSTF2.

Description

Target gene CSTF2 for lung cancer treatment and diagnosis

Technical Field

The present invention relates to the field of bioscience, more specifically to the field of cancer research, cancer diagnosis and cancer treatment. In particular, the present invention relates to methods for detecting and diagnosing lung cancer and methods for treating and preventing or assessing/determining prognosis in a subject having lung cancer. Furthermore, the present invention relates to a method of screening a candidate substance for treating and/or preventing lung cancer.

Priority

The present application claims benefit of U.S. provisional application No.61/274,800 filed on 8/21/2009, the entire contents of which are incorporated herein by reference.

Background

Primary lung cancer is the leading cause of cancer death in most countries, and non-small lung cancer (NSCLC) accounts for approximately 80% of those deaths (NPL 1). The detailed molecular mechanism of lung cancer development remains unclear, although a number of genetic alterations in lung cancer (NPL 2) have been reported. Despite advances in surgical techniques and chemotherapy, the progression of advanced lung cancer patients is often fatal (NPL 1). Therefore, it is considered extremely important to understand the biology of lung cancer and to introduce more effective therapies to improve patient survival (NPL 3). In the last two decades, some newly developed cytotoxic agents such as paclitaxel, docetaxel, gemcitabine, and vinorelbine have begun to offer multiple treatment options for patients with advanced NSCLC, however, those regimens show insignificant survival benefit compared to cisplatin-based conventional therapies (NPL 4, 5).

The concept of specific molecular targeting has now been applied to the development of improved cancer treatment strategies, and two main approaches are used for treatment: therapeutic monoclonal antibodies and small molecule agents (NPL 6). To date, a variety of molecular targeted therapies have been investigated in phase II and phase III trials in chemotherapy of advanced lung cancer, including tyrosine kinase inhibitors of epidermal growth factor such as gefitinib and erlotinib, tyrosine kinase inhibitors of vascular endothelial growth factor such as vandetanib, sorafenib, sunitinib, and monoclonal antibodies against epidermal growth factor or vascular endothelial growth factor such as bevacizumab and cetuximab (NPL 6-10). However, due to toxicity problems, only a limited number of patients can select these treatment regimens, and even with all kinds of treatment, the proportion of patients showing a better response is still limited (NPL 6-10).

Reference meter

[ non-patent document ]

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[NPL 10]Cesare G，Paolo M，Filomena G，et al.Oncologist2007；12：191-200

Summary of The Invention

Systematic analysis of expression levels of thousands of genes using cDNA microarray technology is an effective approach to identify target molecules associated with pathways of carcinogenesis that can become candidates for the development of new therapeutic and diagnostic agents. To isolate potential molecular targets for diagnosis and/or treatment of lung Cancer, the present inventors previously analyzed genome-wide gene expression profiles of 101 lung Cancer tissue samples using tumor cell populations purified by laser microdissection with the aid of a cDNA microarray consisting of 27,648 genes or Expressed Sequence Tags (ESTs) (Daigo Y, Nakamura Y. Gen Thorac Cardiovasc Surg 2008; 56: 43-53, Kikuchi T, Daigo Y, Katagiri T, et al. oncogene 2003; 22: 2192-205, Kakiuchi S, Daigo Y, Tsunoda T, Yano S, Sone S, Nakamura Y. mol Cancer Res 2003; 1: 485-99, Kakiuchi S, Daigo Y, Ishikawa N, et al. Humlo Genet; 13: 3029-43, Kikuchi T, Ishiwa Y2006, Ishiwa J2006, Ishiwa J.Oncol J.7: Oncol J.7, Oncol J.Oncol J.7, Oncol.Oncol.J.7, Oncol.Oncol.J.O.No. No. J.Oncol.No. No. 11, Oncol.No. 9, Oncol.2006). To verify the biological and clinical pathological significance of each gene product, the inventors established a screening system by the combination of tumor tissue microarray analysis of clinical lung Cancer material with RNA interference technology (Suzuki C, Daigo Y, Kikuchi T, Katagiri T, Nakamura Y. Cancer Res 2003; 63: 7038-41, Ishikawa N, Daigo Y, Yasui W, et al. Clin Cancer Res 2004; 10: 8363-70, Kato T, Daigo Y, Hayama S, et al. Cancer Res 2005; 65: 5638-46, Furukawa C, Daigo Y, Ishikawa N, et al. Cancer Res 2005; 65: 7102-10, Dahikawa N, Isigo Y, Takano A, et al. Cancer Res 9176-84, Ishikawa C2005, Ishikawa No. 76, Ishikawa No. 65, Ishikawa No. 11314, Ishikawa No. 25, Ishiwa No. 32, takano a, et al. cancer Res 2006; 66: 9408-19, Hayama S, Daigo Y, Kato T, et al. cancer Res 2006; 66: 10339-48, Kato T, Hayama S, Yamabuki Y, et al, Clin Cancer Res 2007; 13: 434-42, Suzuki C, Takahashi K, Hayama S, et al. mol Cancer Ther 2007; 6: 542-51, Yamabuki T, Takano A, Hayama S, et al. cancer Res 2007; 67: 2517-25, Hayama S, Daigo Y, Yamabuki T, et al cancer Res 2007; 67: 4113-22, Taniwaki M, Takano A, Ishikawa N, et al, Clin Cancer Res 2007; 13: 6624-31, Ishikawa N, Takano A, Yasui W, et al. cancer Res 2007; 67: 11601-11, Mano Y, Takahashi, K, Ishikawa N, et al, cancer Sci 2007; 98: 1902-13, Kato T, Sato N, Hayama S, et al cancer Res 2007; 67: 8544-53, Kato T, Sato N, Takano A, et al.Clin Cancer Res 2008; 14: 2363-70, Dunleavy EM, Roche D, Tagami H, et al. cell 2009; 137: 485-97, Hirata D, Yamabuki T, ItoT, et al.Clin Cancer Res 2009, 15: 256-66, Suda T, Tsunoda T, Daigo Y, NakamuraY, Tahara h. cancer Sci 2007; 98: 1803-8, Mizukami Y, Kono K, Daigo Y, et al. cancer Sci 2008; 99: 1448-54, Harao M, Hirata S, Irie A, et al. int J Cancer 2008; 123: 2616-25).

This systemic strategy revealed that the cleavage stimulating factor 3' pre-RNA, subunit 2, 64kDa (CSTF2) is frequently overexpressed in most primary lung cancers. CSTF2 encodes a nucleoprotein containing a Ribonucleoprotein (RNP) -type RNA-binding domain in the N-terminal region. This protein is a member of the cleavage stimulator Complex (CSTF) and plays a role in the polyadenylation of mRNA together with the other 2 members of the cleavage stimulator (Takagaki Y, MacDonald CC, Shenk T, Manley JL.Proc.Nat.Acad.Sci 1992; 89: 1403-1407). CSTF2 binds GU-rich elements within the 3' -untranslated region of mRNA (Colgan DF, Manley JL. genes Dev 1997; 11: 2755-2766, MacDonald CC, Wilusz J, Shenk T. Mol Cell Biol 1994; 14: 6647-6654, TakagakiY, Manley JL. Mol Cell Biol 1997; 17: 3907-3914, Deka P, Rajan PK, Perez-Canadellas JM, J Mol Biol 2005; 347: 719-33). The amount of CSTF2 increased during the G0 to S phase transition in mouse 3T6 fibroblasts (Martincic K, Campbell R, Edwalds-Gilberg, Souman L, Lotze MT, Millarek C.1998; 95: 11095-100). Strong CSTF2 expression in male germ cells and human cancer cells of mice and rats, or CSTF2 ubiquitous expression in mouse tissues (Dass B, Tardif S, Park JY, Tian B, Weitlauf HM, Hess RA, Carnes K, Griswold MD, Small CL, Macdonald CC. Proc Natl Acad Sci U A.2007; 104: 20374-9, Huber Z, Monarez RR, Dass B, Macdonald CC. Ann YAcad. 2005; 1061: 163-72, Wallace AM, Denison TL, Attaya EN, Macnald CC. biol. Reprodd 2004; 70: 1080-7, Dass B, Attaya EN, Millel Chemale A, Donac CC. Bionnd J.DJ.1722, devickd W.1722, Walkund W.2001-7, Massa K J.W.W.51, Walkura J.S.W.51, Walkura.R.W.S.W.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K, dass B, Ravnik SE, Tonk V, Jenkins NA, Gilbert DJ, Copeland NG, MacDonald CC. Proc Natl Acad Sci U S A1999; 96: 6763-8, Shankarling GS, Coates PW, Dass B, Macdonald CC. BMC MolBiol 2009 Mar; 10: 22). Despite the evidence of CSTF2 function as a member of CSTF in vitro, the significance of CSTF2 activation in human cancer progression and its clinical potential as a therapeutic target has not been documented.

As mentioned above, the present invention relates to CSTF2 and its role in lung cancer carcinogenesis. Accordingly, the present invention relates to novel compositions and methods for detecting, diagnosing, treating and/or preventing lung cancer and methods for screening for useful substances.

In particular, the present invention is due to the discovery that the CSTF2 gene is overexpressed in cancer, but not in normal tissues; also, a double-stranded molecule consisting of specific sequences (particularly SEQ ID NOS: 9 and 10) targeting the CSTF2 gene can effectively inhibit cell growth of lung cancer cells. In particular, the invention provides small interfering RNA (siRNA) targeting the CSTF2 gene. These double-stranded molecules can be utilized in an isolated state or encoded in and expressed from a vector. It is therefore an object of the present invention to provide such double-stranded molecules as well as vectors and host cells expressing them.

In one aspect, the invention provides methods of inhibiting cancer cell growth or treating cancer, including lung cancer, by administering a double-stranded molecule or vector of the invention to a subject in need thereof. Such methods encompass administering to a subject a composition containing one or more of the double stranded molecules or vectors.

In another aspect, the invention provides a composition for treating lung cancer comprising at least one double-stranded molecule or vector of the invention.

In yet another aspect, the present invention provides a method of diagnosing or determining a predisposition for cancer (predisposition) in a subject by determining the expression level of the CSTF2 gene in a biological sample derived from the subject. An increase in the expression level of the gene as compared to a normal control level of the gene is indicative that the subject is suffering from or at risk of developing cancer, including lung cancer. In a preferred embodiment, the expression level of the CSTF2 gene can be determined by detecting the mRNA of the CSTF2 gene with a suitable probe or primer or the CSTF2 protein with an anti-CSTF 2 antibody.

Furthermore, the present invention relates to the finding that higher expression levels of CSTF2 correlate with poor survival. Accordingly, the present invention provides a method for assessing or determining the prognosis of a patient having lung cancer, the method comprising the steps of: the expression level of the CSTF2 gene is detected, compared to a predetermined reference expression level and a prognosis is determined for the patient based on the difference between them.

In yet another aspect, the present invention provides a method of screening a candidate substance for treating and/or preventing cancer. Such substances will bind to the CSTF2 protein, reduce the biological activity of the CSTF2 protein, reduce the expression of the CSTF2 gene or reduce the expression or activity of a reporter gene replacing the CSTF2 gene.

One skilled in the art will appreciate that one or more aspects of the present invention may serve some purposes, while one or more other aspects may serve some other purposes. Each object may not be equally applicable in all its aspects to each and every aspect of the present invention. Accordingly, the foregoing objects may be viewed as alternatives with respect to any of the present inventions. These and other objects and features of the present invention will become more apparent upon reading the following detailed description in conjunction with the accompanying drawings and embodiments. It is to be understood, however, that both the foregoing summary of the invention and the following detailed description are of preferred embodiments and are not restrictive of the invention or other alternative embodiments of the invention.

Brief Description of Drawings

Various aspects and applications of this invention will become apparent to those skilled in the art after considering the following brief description of the drawings and detailed description of the invention and the preferred embodiments thereof:

[ FIG. 1ab ] FIG. 1 depicts the expression of CSTF2 in lung tumors: a, expression of CSTF2 in 15 clinical lung cancers [ lung Adenocarcinoma (ADC), lung squamous cell carcinoma (SCLC), and small cell lung cancer (SCC) ] and 15 lung cancer cell lines examined by semi-quantitative RT-PCR. Expression of the β -Actin (ACTB) gene served as a quantitative control. B, Western blot analysis of expression of CSTF2 protein in lung cancer cell lines. Expression of ACTB protein served as a quantitative control. IB, immunoblotting.

Figure 1c figure 1 depicts the expression of CSTF2 in lung tumors: c, subcellular localization of CSTF2 protein by confocal microscopy.

[ FIG. 2ab ] FIG. 2 depicts the expression of CSTF2 in normal tissues, and the association of CSTF2 overexpression in NSCLC patients with poor prognosis: a, expression of CSTF2 in normal human tissues as detected by Northern blot analysis. B, expression of CSTF2 in 5 normal human tissues and adenocarcinoma cells of the lung detected by immunohistochemical staining using the rabbit polyclonal anti-CSTF 2 antibody; counterstaining with hematoxylin (x 200).

[ FIG. 2cd ] FIG. 2 depicts the expression of CSTF2 in normal tissues and the association of CSTF2 overexpression with poor prognosis in NSCLC patients: c, representative examples of strong, weak, and no expression of CSTF2 in pulmonary ADC tissue and normal lung tissue (initial magnification, x 100). D, Kaplan-Meier analysis of survival of NSCLC patients (P ═ 0.0079, time series assay).

Figure 3 depicts the inhibition of growth of NSCLC cells by siRNA against CSTF 2: a, expression of CSTF2 in A549 and LC319 cells analyzed by semi-quantitative RT-PCR in response to siRNA treatment against CSTF2 (si-CSTF2- #1 or si-CSTF2- #2) or control siRNAs [ si-enhanced green fluorescent protein (si-EGFP) or si-luciferase (si-LUC) ] (top). B, C, MTT and colony formation assay of tumor cells transfected with si-CFTF2 or control siRNA.

FIG. 4 depicts enhancement of cell growth by the introduction of CSTF2 into mammalian cells: transient expression of CSTF2 in COS-7 cells detected by Western blot analysis. The cells were introduced with pcDNA3.1-myc-His-CSTF2 or a mock vector. B and C, cell viability and colony number assessed by MTT and colony formation assays. The assay was performed in triplicate wells three times.

Detailed Description

Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention, the preferred methods, devices, and materials are now described. However, before the present materials and methods are described, it is to be understood that this invention is not limited to the particular sizes, shapes, dimensions, materials, methods, protocols, etc. described herein as these may vary according to routine experimentation and optimization. It is also to be understood that the terminology used in the description is for the purpose of describing the particular versions or embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

The disclosure of each publication, patent, or patent application mentioned in this specification is expressly incorporated herein in its entirety by reference. However, nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Definition of

As used herein, the words "a", "an", "the", and "the" mean "at least one", unless expressly specified otherwise.

The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to naturally occurring amino acids. Naturally occurring amino acids refer to the amino acids encoded by the genetic code, as well as amino acids that have been post-translationally modified in a cell (e.g., hydroxyproline, γ -carboxyglutamic acid, and O-phosphoserine). The phrase "amino acid analog" refers to a compound that has the same basic chemical structure as a naturally occurring amino acid (an alpha carbon bound to a hydrogen, a carboxyl group, an amino group, and an R group) but has a modified R group or a modified backbone (e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium). The phrase "amino acid mimetic" refers to a chemical compound that has a structure different from that of a general amino acid but performs a function similar to that of a general amino acid.

Amino acids may be referred to herein by their commonly known three letter symbols or one letter symbols recommended by the IUPAC-IUB Biochemical nomenclature Commission.

As used herein, the term "biological sample" refers to a subset of an intact organism or a tissue, cell, or component thereof (e.g., a bodily fluid, including but not limited to blood, mucus, lymph, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, cord blood, urine, vaginal fluid, and semen). "biological sample" further refers to a homogenate, lysate, extract, cell culture, or tissue culture, or fraction or portion thereof prepared from the whole organism or a subset of its cells, tissues, or components. Finally, "biological sample" refers to a medium containing cellular components (such as proteins or polynucleotides), such as a nutrient broth or gel in which an organism has been propagated.

The terms "polynucleotide", "oligonucleotide", "nucleic acid", and "nucleic acid molecule" are used interchangeably herein to refer to a polymer of nucleic acid residues and are referred to by their accepted single-letter codes unless otherwise specifically indicated. The terms apply to nucleic acid (nucleotide) polymers in which one or more nucleic acids are linked by ester linkages. The nucleic acid polymer may be composed of DNA, RNA, or a combination thereof, and encompasses both naturally occurring and non-naturally occurring nucleic acid polymers.

The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues are modified residues or non-naturally occurring residues, such as artificial chemical mimetics of corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers.

The terms "cell proliferation activity", "cell proliferation-enhancing activity", and "cell proliferation-promoting activity" are used interchangeably herein and refer to an activity of a polypeptide that promotes or enhances the proliferation of a cell when the polypeptide is contacted with the cell or a gene encoding the polypeptide is introduced into the cell.

The terms "isolated" and "purified" in conjunction with a substance (e.g., a polypeptide, antibody, polynucleotide, etc.) mean that the substance is substantially free of at least one substance that may be included in a natural source. As such, an isolated or purified antibody refers to an antibody that is substantially free of cellular material, such as carbohydrates, lipids, or other contaminating proteins from the cell or tissue source from which the protein (antibody) is derived, or substantially free of chemical precursors or other chemicals when chemically synthesized. The term "substantially free of cellular material" includes preparations of the polypeptide in which the polypeptide is separated from the cellular components of the cell used to isolate or recombinantly produce it.

Thus, polypeptides that are substantially free of cellular material include preparations of polypeptides having less than about 30%, 20%, 10%, or 5% (by dry weight) of heterologous protein (also referred to herein as "contaminating protein"). When the polypeptide is recombinantly produced, it is also, in some embodiments, substantially free of culture medium, including polypeptide preparations having culture medium with less than about 20%, 10%, or 5% of the volume of the protein preparation. When the polypeptide is produced by chemical synthesis, in some embodiments it is substantially free of chemical precursors or other chemicals, including polypeptide preparations having less than about 30%, 20%, 10%, 5% (by dry weight) of the volume of the protein preparation of chemical precursors or other chemicals associated with the protein synthesis. A particular protein preparation can be shown to contain isolated or purified polypeptide, for example, by the appearance of a single band following Sodium Dodecyl Sulfate (SDS) -polyacrylamide gel electrophoresis of the protein preparation, coomassie blue staining of the gel, and the like. In one embodiment, the protein (including the antibody of the invention) is isolated or purified.

An "isolated" or "purified" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. In one embodiment, the nucleic acid molecule encoding a protein of the invention is isolated or purified.

Unless otherwise defined, the term "cancer" refers to cancers that overexpress the CSTF2 gene, such as lung cancer, including Adenocarcinoma (ADC), Squamous Cell Carcinoma (SCC), Large Cell Carcinoma (LCC), and Small Cell Lung Cancer (SCLC).

CSTF2 gene or CSTF2 protein

The nucleic acid and polypeptide sequences of the human CSTF2 gene are shown in, but not limited to, SEQ ID NO: 1 and SEQ ID NO: 2. alternatively, the sequence data may be obtained via GenBank accession NM-001325.

According to one aspect of the invention, functional equivalents are considered to be "polypeptides" as defined above. As used herein, a "functional equivalent" of a protein is a polypeptide having equivalent biological activity to the protein. That is, any polypeptide that retains biological ability may be used as such functional equivalents in the present invention. For example, CSTF2 polypeptide is known to have cell proliferation enhancing activity, RNA binding activity, RNA cleavage activity, RNA polyadenylation activity, and the like. Polypeptides that retain at least one of these activities are considered functional equivalents of the CSTF2 polypeptides of the invention. Such functional equivalents include those in which one or more amino acids are substituted, deleted, added, or inserted into the naturally occurring amino acid sequence of the protein. Alternatively, a polypeptide may comprise an amino acid sequence that is at least about 80% homologous (also referred to as sequence identity), more preferably at least about 90% to 95% identical, and even more preferably 96%, 97%, 98% or 99% identical to the sequence of the corresponding protein. In other embodiments, the polypeptide may be encoded by a polynucleotide that hybridizes under stringent conditions to a naturally occurring nucleotide sequence of a gene.

The polypeptide of the present invention may vary in amino acid sequence, molecular weight, isoelectric point, presence or absence of sugar chain, or form, depending on the cell or host used to produce it, or the purification method used. In any case, it is within the scope of the present invention as long as it has a function equivalent to that of the human protein.

The phrase "stringent (hybridization) conditions" refers to conditions under which a nucleic acid molecule will hybridize to its target sequence, typically in a complex mixture of nucleic acids, but without detectable hybridization to other sequences. Stringent conditions depend on the sequence and may be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An exhaustive guide to Nucleic acid Hybridization can be found in Tijssen, Techniques in biochemistry and Molecular Biology- -Hybridization with Nucleic Probes, "Overview of principles of Hybridization and the protocol of Nucleic acid assays" (1993). Generally, stringent conditions are selected to be at a defined ionic strength and pH, versus the thermal melting point (T) of a particular sequence_m) About 5-10 deg.c lower. T is_mIs the temperature (under defined ionic strength, pH, and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (at T because the target sequence is present in excess, and therefore at _mAt equilibrium 50% of the probes are occupied). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, the positive signal is at least twice background, preferably 10 times background hybridization. Exemplary stringent hybridization conditions include the following: 50% formamide, 5 XSSC, and 1% SDS at 42 ℃ or 5 XSSC, 1% SDS at 65 ℃ with 0.2 XSSC and 0.1% SDS at 50 ℃.

In the context of the present invention, hybridization conditions for isolating DNA encoding a polypeptide functionally equivalent to the above-described human protein may be routinely selected by those skilled in the art. For example, hybridization can be performed as follows: prehybridization was performed at 68 ℃ for 30 minutes or more with a "Rapid-hyb buffer" (Amersham LIFE SCIENCE), labeled probe was added, and incubation was performed at 68 ℃ for 1 hour or more. The following washing step may be performed, for example, in low stringency conditions. An exemplary low stringency condition can include 42 ℃, 2x SSC, 0.1% SDS, preferably 50 ℃, 2x SSC, 0.1% SDS. It is often preferred to use high stringency conditions. An exemplary high stringency condition can include 3 washes in 2x SSC, 0.1% SDS for 20 minutes each at room temperature, followed by 3 washes in 1x SSC, 0.1% SDS for 20 minutes each at 37 degrees celsius, and 2 washes in 1x SSC, 0.1% SDS for 20 minutes each at 50 degrees celsius. However, several factors, such as temperature and salt concentration, can affect the stringency of hybridization, and one skilled in the art can select appropriate factors to achieve the desired stringency.

In general, it is known that modification of one or more amino acids in a protein does not affect the function of the protein. Indeed, it is known that mutated or modified proteins having an amino acid sequence modified by substitution, deletion, insertion and/or addition of one or more amino acid residues to a certain amino acid sequence retain the original biological activity (Mark et al, Proc Natl Acad Sci USA 81: 5662-6 (1984); Zollerand Smith, Nucleic Acids Res 10: 6487-500 (1982); Dalbadie-Famcland et al, Proc Natl Acad Sci USA 79: 6409-13 (1982)). Thus, those skilled in the art will recognize that individual additions, deletions, insertions, or substitutions to an amino acid sequence that alter a single amino acid or a small number of amino acids, or those modifications that are considered "conservative modifications," wherein a change in the protein results in a protein with similar function, are acceptable in the context of the present invention.

The number of amino acid mutations is not particularly limited as long as the protein activity is maintained. However, it is generally preferred to change the amino acid sequence by 5% or less. Thus, in a preferred embodiment, the number of amino acids to be mutated in such mutants is typically 30 amino acids or less, preferably 20 amino acids or less, more preferably 10 amino acids or less, more preferably 6 amino acids or less, even more preferably 3 amino acids or less.

The amino acid residue to be mutated is preferably mutated to another amino acid whose side chain properties are retained (a process called conservative amino acid substitution). Examples of amino acid side chain properties are hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), and side chains having the following common functional groups or characteristics: aliphatic side chains (G, a, V, L, I, P); side chains containing hydroxyl groups (S, T, Y); a side chain (C, M) containing a sulfur atom; side chains containing carboxylic acids and amides (D, N, E, Q); side chains containing a base (R, K, H); and aromatic-containing side chains (H, F, Y, W). Conservative substitution tables providing functionally similar amino acids are well known in the art. For example, the following 8 groups each contain amino acids that constitute conservative substitutions for one another:

1) alanine (a), glycine (G);

2) aspartic acid (D), glutamic acid (E);

3) asparagine (N), glutamine (Q);

4) arginine (R), lysine (K);

5) isoleucine (I), leucine (L), methionine (M), valine (V);

6) phenylalanine (F), tyrosine (Y), tryptophan (W);

7) serine (S), threonine (T); and

8) Cysteine (C), methionine (M) (see, e.g., Creighton, Proteins 1984).

Such conservatively modified polypeptides are included in the proteins of the invention. However, the invention is not so limited and proteins include non-conservative modifications as long as at least one biological activity of the protein is retained. In addition, modified proteins do not exclude polymorphic variants, interspecies homologs, and those encoded by alleles of such proteins.

Furthermore, the CSTF2 gene encompasses polynucleotides encoding such functional equivalents of the protein. In addition to hybridization, a polynucleotide encoding a polypeptide functionally equivalent to a protein can be isolated by using a primer synthesized based on the sequence of the above information using a gene amplification method, such as a Polymerase Chain Reaction (PCR) method. Polynucleotides and polypeptides that are functional equivalents of human genes and proteins, respectively, typically have high homology to their original nucleotide or amino acid sequences. "high homology" generally means homology of 40% or more, preferably 60% or more, more preferably 80% or more, even more preferably 90% to 95% or more, even more preferably 96%, 97%, 98%, 99% or more. The homology of a particular polynucleotide or polypeptide may follow the sequence "Wilbur and Lipman, Proc Natl Acad Sci USA 80: 726-30(1983) ".

Antibodies

As used herein, the term "antibody" is intended to include immunoglobulins and fragments thereof that can specifically react with a specified protein or peptide thereof. Antibodies may include human antibodies, primatized antibodies, chimeric antibodies, bispecific antibodies, humanized antibodies, antibodies fused to other proteins or radioactive labels, and antibody fragments. In addition, herein, antibodies are used in the broadest sense and specifically encompass intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity. "antibody" indicates all classes (e.g., IgA, IgD, IgE, IgG, and IgM).

The present invention uses antibodies against CSTF 2. These antibodies will be generated by known methods.

Exemplary techniques for generating antibodies for use in accordance with the present invention are described.

(i) Polyclonal antibodies:

polyclonal antibodies are preferably produced in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and adjuvant. It may be useful to couple the relevant antigen with a protein that is immunogenic in the species to be immunized, such as keyhole limpet, using bifunctional or derivatizing agents Hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor, and wherein a bifunctional or derivatizing agent such as maleimidobenzoic acid thiosuccinimide ester (conjugated through a cysteine residue), N-hydroxysuccinimide (conjugated through a lysine residue), glutaraldehyde, succinic anhydride, SOCl₂Or R 'N ═ C ═ NR where R and R' are different alkyl groups.

Animals are immunized with antigen, immunogenic conjugate, or derivative, for example, by combining 100 or 5 μ g of protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant, and injecting the solution intradermally at multiple points. One month later, the animals were boosted by subcutaneous injection at multiple points with 1/5-1/10 of the original amount of peptide or conjugate in Freund's complete adjuvant. After 7-14 days, the animals were bled and the serum was assayed for antibody titer. Animals were boosted until titers reached a high plateau. Preferably, conjugates of the same antigen are used to boost the animal, but conjugated to a different protein and/or conjugated through a different cross-linking agent.

Conjugates can also be prepared in recombinant cell culture as protein fusions. Aggregating agents such as alum are also useful for enhancing immune responses.

(ii) Monoclonal antibodies:

monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for the possible presence of a small number of possible naturally occurring mutations. Thus, the modifier "monoclonal" refers to a feature of the antibody that is not a mixture of discrete (discrete) antibodies.

For example, monoclonal antibodies can be prepared using the hybridoma method first described in KohlerG & Milstein c.nature.1975 Aug 7; 256(5517): 495-7, or may be prepared by recombinant DNA methods (U.S. Pat. No.4,816,567).

In the hybridoma method, a mouse or other suitable host animal, such as a hamster, is immunized as described herein above to elicit the production of lymphocytes or antibodies that will specifically bind to the protein used for immunization. Alternatively, lymphocytes may be immunized in vitro. Lymphocytes are then fused with myeloma cells using a suitable fusing agent such as polyethylene glycol to form hybridoma cells (Goding, monoclonal antibodies: Principles and Practice, pp.59-103(Academic Press, 1986)).

The hybridoma cells so prepared are seeded and cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused parental myeloma cells. For example, if the parental myeloma cells lack hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium of the hybridoma will typically contain hypoxanthine, methotrexate, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.

Preferred myeloma cells are those that fuse efficiently, support stable high-level production of antibodies by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Among these, preferred myeloma Cell lines are murine myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk institute Cell Distribution Center (SalkInstitute Cell Distribution Center, San Diego, Calif. USA) and SP-2 or X63-Ag8-653 cells available from the American type culture Collection (American type culture Collection, Manassas, Virginia, USA). Human myeloma and mouse-human heteromyeloma cell lines are also described for the Production of human Monoclonal antibodies (Kozbor D, et al, J Immunol.1984 Dec; 133 (6): 3001-5; Brodeur et al, Monoclonal Antibody Production Techniques and Applications, pp.51-63(Marcel Dekker, Inc., New York, 1987)).

The medium in which the hybridoma cells are grown is analyzed for the production of monoclonal antibodies to the antigen. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay such as Radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

For example, the binding affinity of a monoclonal antibody can be determined by Munson PJ & Rodbard D.AnalBiochem.1980 Sep 1; 107(1): 220-39 by 30Scatchard analysis.

After hybridoma cells producing Antibodies with the desired specificity, affinity, and/or activity are identified, the clones may be subcloned by limiting dilution procedures and cultured by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103(Academic Press, 1986)). Suitable media for this purpose include, for example, D-MEM or RPML-1640 medium. In addition, the hybridoma cells can be cultured in vivo in animals as ascites tumors.

The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures, such as, for example, protein a-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

DNA encoding the monoclonal antibody is readily isolated and sequenced using conventional procedures, for example, by using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of murine antibodies. Hybridoma cells are a preferred source of such DNA. Once isolated, the DNA may be placed into an expression vector, which is then transfected into host cells that do not otherwise produce immunoglobulin proteins per se, such as E.coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells, to obtain synthesis of monoclonal antibodies in the recombinant host cells. A review article on recombinant expression of DNA encoding an antibody in bacteria includes Skerra a. curr Opin immunol.1993 Apr; 5(2): 256-62 and Pl ü ckthun A. Immunol Rev.1992 Dec; 130: 151-88.

Another method of generating specific antibodies or antibody fragments reactive with CSTF2 is to screen an expression library for immunoglobulin genes or portions thereof expressed in bacteria with CSTF 2. For example, phage expression libraries can be used to express the complete Fab fragments, VH regions and Fv regions in bacteria. See, e.g., Ward ES, et al, nature.1989 Oct 12; 341(6242): 544-6; huse WD, et al, science.1989 Dec 8; 246(4935): 1275-81; and McCafferty J, et al, Nature.1990Dec 6; 348(6301): 552-4. Screening of such libraries with CSTF2 peptide identified immunoglobulin fragments that were reactive with CSTF 2. Alternatively, SCID-hu-mice (available from Genpharm) can be used to produce antibodies or fragments thereof.

In yet another embodiment, the methods may be selected from the group consisting of using McCafferty J, et al, nature.1990Dec 6; 348(6301): 552-4; clarkson T, et al, nature, 1991 Aug 15; 352(6336): 624-8, or an antibody or antibody fragment; marks JD, et al, JMoL BioL, 222: 581-597(1991) J Mol biol.1991 Dec 5; 222(3): 581-97 describes the use of phage libraries to isolate mouse and human antibodies, respectively. Subsequent publications describe the generation of high affinity (nM range) human antibodies by chain shuffling (Marks JD, et al, Biotechnology (N Y) 1992 Jul; 10 (7): 779-83), as well as combinatorial infection (combinatorial infection) and in vivo recombination as strategies for constructing oversized phage libraries (Waterhouse P, et al, Nucleic Acids Res.1993 May 11; 21 (9): 2265-6). These techniques are therefore viable alternatives to traditional monoclonal antibody hybridoma techniques for isolating monoclonal antibodies.

DNA may also be modified, for example, by replacing the homologous murine sequences with human heavy and light chain constant domain coding sequences (U.S. Pat. No.4,816,567; Morrison SL, et al, Proc Natl Acad Sci U S A.1984 Nov; 81 (21): 6851-5), or by covalently linking the immunoglobulin coding sequence to all or part of the coding sequence for a non-immunoglobulin polypeptide.

Typically, antibody constant domains are replaced with such non-immunoglobulin polypeptides, or the variable domains of one antigen binding site of an antibody are replaced with them to create a chimeric diabody that includes one antigen binding site with a certain antigen specificity and another antigen binding site with a different antigen specificity.

(iii) Humanized antibody:

methods for humanizing non-human antibodies are well-known in the art. Preferably, the humanized antibody incorporates one or more amino acid residues from a non-human source. These non-human amino acid residues are often referred to as "import" residues, and are typically taken from an "import" variable domain. Humanization can be performed essentially following the method of Winter and its co-workers (Jones PT, et al, Nature.1986 May29-Jun 4; 321 (6069): 522-5; Riechmann L, et al, Nature.1988 Mar 24; 332 (6162): 323-7; Verhoeyen M, et al, science.1988 Mar 25; 239 (4847): 1534-6), replacing the corresponding sequences of a human antibody with hypervariable region sequences. Thus, such "humanized" antibodies are chimeric antibodies in which a small portion of the complete human variable domain (substitally less thanintact) is replaced with the corresponding sequence from a non-human species (U.S. Pat. No.4,816,567). In practice, humanized antibodies are typically those in which some hypervariable region residues, and possibly some FR residues, of a human antibody have been replaced by residues from analogous sites in rodent antibodies.

The choice of human variable domains, including light and heavy chain variable domains, to be used in making humanized antibodies is important for reducing antigenicity. The sequence of the variable domains of rodent antibodies is screened against the entire library of known human variable domain sequences according to the so-called "best-fit" method. Then, the human sequence closest to the rodent sequence was received as the human Framework Region (FR) for the humanized antibody (Sims MJ, et al, J Immunol.1993 Aug 15; 151 (4): 2296-308; Chothia C & Lesk AM.J Mol biol.1987 Aug 20; 196 (4): 901-17). Another approach uses specific framework regions derived from the consensus sequence of all human antibodies of a specific subset of light or heavy chains. The same framework can be used for several different humanized antibodies (Carter P, et al, Proc Natl Acad Sci U S A.1992 May 15; 89 (10): 4285-9; Presta LG, et al, J Immunol.1993 Sep 1; 151 (5): 2623-32)).

In addition, it is important that humanization of antibodies retain high affinity for the antigen and other favorable biological properties. To achieve this goal, according to a preferred method, humanized antibodies are prepared by the following process: three-dimensional models of the parental and humanized sequences were used to analyze the parental sequences and various conceptual humanized products. Three-dimensional immunoglobulin models are generally available and familiar to those skilled in the art. Computer programs are available to illustrate and display the possible three-dimensional conformational structures of selected candidate immunoglobulin sequences. Examination of these displays permits analysis of the likely role of the residues in the functional performance of the candidate immunoglobulin sequence, i.e., analysis of residues that affect the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined with the receptor and import sequences to achieve desired antibody characteristics, such as increased affinity for the target antigen. In general, hypervariable region residues are directly and most substantially involved in influencing antigen binding.

(iv) Human antibody:

as an alternative to humanization, human antibodies can be generated. For example, it is now possible to generate transgenic animals (e.g., mice) that, when immunized, are capable of producing a complete repertoire of human antibodies (repotoreires) without endogenous immunoglobulin production. For example, it has been described that homozygous deletion of the antibody heavy chain joining region (JH) gene in chimeric and germline mutant mice results in complete suppression of endogenous antibody production. Transfer of human germline immunoglobulin gene arrays into such germline mutant mice results in the production of human antibodies upon antigen challenge. See, e.g., Jakobovits a, et al, Proc Natl Acad Sci U S1993 Mar 15; 90(6): 2551-5; nature.1993 Mar 18; 362(6417): 255-8; bruggemanm, et al, Yeast Immunol.1993; 7: 33-40 parts of; and U.S. patent No.5,591,669; 5,589,369, and 5,545,807.

Alternatively, human antibodies and antibody fragments can be generated in vitro from a repertoire of immunoglobulin variable (V) domain genes (repotoreres) from an unimmunized donor using phage display technology (McCafferty J, et al, Nature.1990 Dec 6; 348 (6301): 552-4). According to this technique, antibody V domain genes are cloned in-frame (in-frame) into the major or minor coat protein genes of filamentous phage (such as M13 or fd) and displayed as functional antibody fragments on the surface of the phage particle. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selection based on the functional properties of the antibody also results in the selection of the gene encoding the antibody exhibiting those properties. Thus, the phage mimics some of the properties of the B cell. Phage display can be performed in a variety of formats, for a review of them see, e.g., Johnson KS & Chiswell dj. curr oprn Struct biol.1993; 3: 564-71. There are several sources of V gene segments available for phage display.

Clackson T, et al, Nature.1991 Aug 15; 352(6336): 624-8 a diverse panel of anti-oxazolone antibodies was isolated from a small random combinatorial library of V genes derived from the spleen of immunized mice. A complete set of V genes from non-immunized human donors can be constructed and antibodies to a diverse array of antigens (including self-antigens) can be isolated essentially following the techniques described in the following literature: marks JD, et al, Jmol biol.1991 Dec 5; 222(3): 581-97, or Griffiths AD, et al, EMBO J.1993Feb; 12(2): 725-34. See also U.S. Pat. Nos. 5,565,332 and 5,573,905.

Human antibodies can also be produced by activated B cells in vitro (see U.S. patent nos. 205,567,610 and 5,229,275). A preferred means of producing human antibodies using SCID mice is described in the co-pending application.

(v) Antibody fragment:

various techniques have been developed for the production of antibody fragments. Traditionally, these fragments have been derived by proteolytic digestion of intact antibodies (see, e.g., Morimoto K)&Inouye K.J biochem Biophys methods, 1992 Mar; 24(1-2): 107-17; brennan M, et al, science.1985jul 5; 229(4708): 81-3). However, these fragments can now also be produced directly by recombinant host cells. For example, antibody fragments can be isolated from the antibody phage libraries discussed above. Alternatively, Fab '-SH fragments can be recovered directly from E.coli and chemically coupled to form F (ab') ₂Fragments (Carter P, et al, Biotechnology (N Y) 1992 Feb; 10 (2): 163-7). According to another approach, F (ab')₂The fragments may be isolated directly from the recombinant host cell culture. Other techniques for generating antibody fragments will be apparent to the skilled practitioner. In other embodiments, the antibody of choice is a single chain Fv fragment (scFv). See WO 93/16185; U.S. Pat. Nos. 5,571,894 and 5,587,458. Antibody fragments may also be "linear antibodies", for example as described in, for example, U.S. Pat. No.5,641,870. Such linear antibody fragments may be monospecific or bispecific.

(vi) Non-antibody binding protein:

the terms "non-antibody binding protein" or "non-antibody ligand" or "antigen binding protein" are used interchangeably and refer to antibody mimetics using non-immunoglobulin scaffolds, including adnectins, avimers, single chain polypeptide binding molecules, and antibody-like binding peptide mimetics, as discussed in more detail below.

Other substances have been developed that target and bind in a similar manner to antibodies. Some of these "antibody mimetics" use a non-immunoglobulin scaffold as a surrogate protein framework for the variable region of an antibody.

For example, Ladner et al (U.S. Pat. No.5,260,203) describes single polypeptide chain binding molecules that have binding specificities similar to the light and heavy chain variable regions of antibodies, which are aggregated together but separated at the molecular level. The single chain binding molecules contain both antigen binding sites for the variable regions of the antibody heavy and light chains, which are linked by peptide linkers and fold into a structure similar to that of a two-peptide antibody. Single chain binding molecules exhibit a number of advantages over traditional antibodies, including smaller size, greater stability, and easier modification.

Ku et al (Proc Natl Acad Sci USA 92 (14): 6552-. Ku et al (1995) made a library in which two loops of cytochrome b562 were randomized and selected for binding to bovine serum albumin. Each mutant was found to selectively bind BSA in a similar manner to anti-BSA antibodies.

Lipovsek et al (U.S. Pat. Nos. 6,818,418 and 7,115,396) describe an antibody mimetic characterized by a fibronectin or fibronectin-like protein backbone and at least one variable loop. These fibronectin-based antibody mimics, called Adnectins, exhibit many of the same characteristics as natural or engineered antibodies, including high affinity and specificity for any target ligand. Any technique for developing new or improved binding proteins can be used for these antibody mimetics.

The structure of these fibronectin based antibody mimetics is similar to that of the IgG heavy chain variable region. Thus, these mimetics exhibit antigen binding characteristics similar to natural antibodies in nature and affinity. In addition, these fibronectin-based antibody mimetics also exhibit certain advantages over antibodies and antibody fragments. For example, the natural folding stability of these antibody mimetics is independent of disulfide bonds and is therefore stable under conditions that would normally destroy the antibody. Furthermore, because the structure of these fibronectin based antibody mimetics is similar to that of the IgG heavy chain, a loop randomization and shuffling process similar to that of the in vivo affinity maturation process of antibodies can be employed in vitro.

Beste et al (Proc Natl Acad Sci USA 96 (5): 1898-1903(1999)) describe an antibody mimetic based on the lipocalin skeleton (Anticalin (registered trademark)). Lipocalins comprise a β -barrel with 4 hyper-variable loops at the protein end. Beste (1999) performed random mutagenesis of the loops and selected for binding to, for example, fluorescein. Three variants exhibited specific binding to fluorescein, with one variant exhibiting similar binding to the anti-fluorescein antibody. Further analysis revealed that all randomized positions were variable, indicating that Anticalin (registered trademark) would be suitable for use as a surrogate for antibodies.

Anticalin (registered trademark) is a small single-chain peptide, usually between 160 and 180 residues, which offers several advantages over antibodies, including reduced production costs, improved storage stability and reduced immunological reactions.

Hamilton et al (U.S. Pat. No.5,770,380) describe a synthetic antibody mimetic that uses a calixarene (calixarene) rigid non-peptidic organic backbone to which multiple variable peptide loops are attached as binding sites. The peptide loops all protrude from geometrically the same side of the calixarene with respect to each other. Due to this geometric conformation, all loops are available for binding, thereby increasing the binding affinity to the ligand. However, calixarene-based antibody mimetics are not composed purely of peptides and are therefore less sensitive to protease attack than other antibody mimetics. The scaffold is also not composed purely of peptides, DNA or RNA, which means that the antibody mimetic is relatively stable in extreme environmental conditions and has a long lifetime. In addition, because the calixarene-based antibody mimetics are relatively small, their likelihood of producing an immunogenic response is reduced.

Murali et al (Cell Mol biol.49 (2): 209-216(2003)) describe a method for reducing antibodies to smaller mimetics, which are referred to as "antibody-like binding mimetics" (ABiP), and can also be used as a substitute for antibodies.

Silverman et al (Nat Biotechnol.23: 1556-. avimers were developed from the human extracellular receptor domain by in vitro exon shuffling and phage display, and are a class of binding proteins that are somewhat similar to antibodies in their affinity and specificity for various target molecules. The resulting multi-domain proteins can include multiple independent binding domains that can exhibit improved affinity (in some cases sub-nanomolar) and specificity compared to single epitope binding proteins. More details regarding the construction and methods of use of avimers are disclosed in, for example, U.S. patent application publication Nos. 20040175756, 20050048512, 20050053973, 20050089932 and 20050221384.

In addition to non-immunoglobulin frameworks, antibody properties are also mimicked in substances including, but not limited to, RNA molecules and non-natural oligomers (e.g., protease inhibitors, benzodiazepines, purine derivatives, and β -turn mimetics), all of which are suitable for use in the present invention.

(vii) Pharmaceutical formulations:

therapeutic formulations of the antibodies can be prepared for storage in lyophilized form or as an aqueous solution by mixing the antibody of the desired purity with an optional pharmaceutically acceptable carrier, excipient or stabilizer (Remington's Pharmaceutical Sciences 16th edition, Osol, a. ed. (1980)). Acceptable carriers, excipients or stabilizers at the dosages and concentrations employed The recipient is non-toxic and includes buffers such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexane diamine chloride; benzalkonium chloride, benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and/or nonionic surfactants, such as TWEEN^TM、PLURONICS^TMOr polyethylene glycol (PEG).

Lyophilized formulations suitable for subcutaneous administration are described in WO 97/04801. Such lyophilized formulations can be reconstituted with a suitable diluent to a high protein concentration and the reconstituted formulation can be administered subcutaneously to the mammal to be treated herein.

The formulations described herein may also contain more than one active agent, preferably those with complementary activities that do not adversely affect each other, as required for the particular condition being treated. For example, chemotherapeutic agents, cytokines or immunosuppressive agents may be further provided. The effective amount of such other agents will depend on the amount of antibody present in the formulation, the type of disorder or disease or treatment, and other factors discussed above. These agents are generally used at the same dosages and routes of administration as previously described, or at about 1 to 99% of the dosages used previously.

The active ingredient may also be embedded in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (e.g., hydroxymethylcellulose or gelatin-microcapsules and poly (methylmethacylate) microcapsules, respectively), in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or in macroemulsions (macroemulsions). Such techniques are disclosed, for example, in Remington's Pharmaceutical Sciences 16th edition, Osol, a.ed. (1980).

Sustained release formulations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the agent, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly (2-hydroxyethyl-methacrylate) or poly (vinyl alcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and L-glutamic acid gamma ethyl ester, non-degradable ethylene-vinyl acetate copolymers, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D- (-) -3-hydroxybutyric acid. Formulations for in vivo administration must be sterile. This can be easily achieved by filtration using sterile filtration membranes.

(x) Treatment with antibodies:

compositions comprising the present antibodies may be formulated, dosed, and administered in a form consistent with good medical practice. The antibody is preferably a human, chimeric or humanized antibody scFv, or an antibody fragment. Factors to be considered in this context include the particular lung cancer being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the condition or disease, the site to which the agent is to be delivered, the method of administration, the time course of administration and other factors known to medical practitioners. The amount of antibody administered therapeutically effective will be determined by such considerations.

As a general proposition, the parenterally administered amount of antibody should be in the range of about 0.1 to 20mg/kg of patient body weight per dose per day, with a typical starting amount of antibody ranging from about 2 to 10 mg/kg.

However, as noted above, these suggested antibody amounts are largely under therapeutic consideration. The most important factor in the selection of the appropriate dosage and time course, as mentioned above, is the result obtained.

For example, for treatment of ongoing as well as acute diseases, relatively high doses may be initially required. Depending on the disease or condition, the antibody should be administered as close as possible to the onset, diagnosis, appearance or occurrence of the disease or condition, or during the course of the recurrence of the disease or condition, in order to achieve the most effective result.

The antibody may be administered by any suitable method, including parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal administration, as well as intralesional administration if local immunosuppressive therapy is desired. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal and subcutaneous administration.

In addition, it may be suitable to administer the antibody by pulse infusion (pulse infusion), e.g., with decreasing doses of the antibody. The administration is preferably by injection, most preferably intravenous or subcutaneous injection, depending in part on whether the administration is short-term or long-term.

In addition, other agents, such as cytotoxic agents, chemotherapeutic agents, immunosuppressive agents, and/or cytokines may also be administered with the antibodies described herein. Combined administration includes simultaneous administration using different formulations or a single pharmaceutical formulation, as well as sequential administration in any order, where there is preferably a period of time in which two (or all) active agents exert their biological activities simultaneously.

In addition to administering the antibody to a patient, the present invention contemplates administering the antibody using gene therapy. The above-described method of administering a nucleic acid encoding an antibody is encompassed by the expression "administering a therapeutically effective amount of an antibody". For example, for the production of intrabodies using gene therapy, see WO96/07321, published in 1996, 3/14.

There are two main approaches to the entry of nucleic acids (optionally contained in a vector) into the cells of a patient, i.e., in vivo and ex vivo. For in vivo delivery, the nucleic acid is typically injected directly into the patient at the site where the antibody is desired. For ex vivo treatment, the patient's cells are removed, the nucleic acid is introduced into these isolated cells, and the modified cells are either administered directly to the patient, or, for example, embedded within a porous membrane and implanted into the patient (see, e.g., U.S. Pat. Nos. 4,892,538 and 5,283,187). There are a variety of techniques available for introducing nucleic acids into living cells. These techniques vary depending on whether the nucleic acid is transferred to cultured cells in vitro or to cells in vivo of the host of interest. Suitable techniques for transferring nucleic acids into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, calcium phosphate precipitation, and the like. A commonly used vector for ex vivo delivery of genes is a retrovirus.

Presently preferred in vivo nucleic acid transfer techniques include transfection with viral vectors (such as adenovirus, herpes simplex I virus or adeno-associated virus) and lipid-based systems (lipids that can be used for lipid-mediated gene transfer are e.g. DOTMA, DOPE and DC-Chol). In some instances, it is desirable to provide the nucleic acid source with an agent that targets the target cell, such as an antibody specific for a cell surface membrane protein or target cell, a ligand for a receptor on the target cell, and the like. Where liposomes are employed, proteins that bind to cell surface membrane proteins associated with endocytosis can be used to target and/or facilitate uptake, such as capsid proteins or fragments thereof that are tropic for a particular cell type, antibodies to proteins that are internalized in the circulation, and proteins that target intracellular localization and increase intracellular half-life. For example Wu et al, j.biol.chem.262: 4429-4432(1987) and Wagner et al, Proc. Natl. Acad. Sci. USA 87: 3410-3414(1990) describes receptor-mediated endocytosis. For a review of currently known gene markers and gene therapy protocols see Anderson et al, Science 256: 808-813(1992). See also WO 93/25673 and references cited therein.

In another embodiment, the invention also provides the use of an antibody to CSTF2 of the invention in the manufacture of a pharmaceutical composition for the treatment of a cancer expressing CSTF2 gene.

Alternatively, the present invention further provides the antibodies of the present invention against CSTF2 for use in the treatment of CSTF2 gene expressing cancers.

Alternatively, the present invention further provides a method or process for manufacturing a pharmaceutical composition for treating a cancer expressing CSTF2 gene, wherein the method or process comprises the step of formulating a pharmaceutically or physiologically acceptable carrier with an antibody against CSTF2 as an active ingredient.

In another embodiment, the invention also provides a method or process for manufacturing a pharmaceutical composition for treating a cancer expressing CSTF2 gene, wherein the method or process comprises the step of mixing an active component with a pharmaceutically or physiologically acceptable carrier, wherein the active component is an antibody against CSTF 2.

Double stranded molecules

As used herein, the term "isolated double-stranded molecule" refers to a nucleic acid molecule that inhibits expression of a target gene, and includes, for example, short interfering RNAs (siRNAs; e.g., double-stranded ribonucleic acids (dsRNA) or small hairpin RNAs (shRNAs)), and short interfering DNAs/RNAs (siD/R-NA; e.g., a double-stranded chimera of DNA and RNA (dsD/R-NA) or a small hairpin chimera of DNA and RNA (shD/R-NA)). Herein, "double-stranded molecule" is also referred to as "double-stranded nucleic acid", "double-stranded nucleic acid molecule", "double-stranded polynucleotide molecule", "double-stranded oligonucleotide", and "double-stranded oligonucleotide molecule".

As used herein, the term "target sequence" refers to a stretch of nucleotide sequence within the mRNA or cDNA sequence of a target gene that, if a double-stranded molecule targeted to that sequence is introduced into a cell expressing the target gene, results in the translation of the entire mRNA of the target gene being repressed. A nucleotide sequence within the mRNA or cDNA sequence of a gene can be identified as a target sequence if a double-stranded molecule comprising a sequence corresponding to the target sequence inhibits expression of the gene in a cell expressing the gene. Double-stranded polynucleotides that repress gene expression may consist of a target sequence and a 3' overhang (e.g., uu) of 2 to 5 nucleotides in length.

When the target sequence is revealed by the cDNA sequence, the sense strand sequence of the double-stranded cDNA (i.e., the sequence in which the mRNA sequence is converted into a DNA sequence) is used to define the target sequence. A double-stranded molecule comprises a sense strand (which has a sequence corresponding to a target sequence) and an antisense strand (which has a sequence complementary to the target sequence), and the antisense strand hybridizes to the sense strand at the complementary sequence to form the double-stranded molecule.

In this context, the phrase "corresponds to" means that the target sequence is converted according to the kind of nucleic acid constituting the sense strand of the double-stranded molecule. For example, when the target sequence is shown as a DNA sequence and the sense strand of a double-stranded molecule has an RNA region, the base "t" within the RNA region is replaced with the base "u". On the other hand, when the target sequence is shown as an RNA sequence and the sense strand of the double-stranded molecule has a DNA region, the base "u" in the DNA region is replaced with "t".

For example, when the target sequence is represented by the RNA sequence SEQ ID NO: 10 and the sense strand of the double-stranded molecule has a 3 ' -side half region composed of DNA, the "sequence corresponding to the target sequence" is "5'-CACUUUACUUTCTGTAACT-3'".

In addition, in the case of the antisense strand of the double-stranded molecule, the sequence complementary to the target sequence may be defined according to the kind of nucleic acid constituting the antisense strand. For example, when the target sequence is shown in the RNA sequence SEQ ID NO: 10 in which the antisense strand of the double-stranded molecule has a 5 ' -side half region composed of DNA, the "sequence complementary to the target sequence" is "3 ' -GUGAAAUGAAAGACATTGA-5 '".

On the other hand, when the double-stranded molecule consists of RNA, the double-stranded molecule has a sequence identical to SEQ ID NO: 10 is SEQ ID NO: 10, and a sequence that is identical to the RNA sequence of SEQ ID NO: 10 is the RNA sequence "3 '-GUGAAAUGAAAGACAUUGA-5'".

In addition to the sequence corresponding to the target sequence and its complement, the double-stranded molecule may have one or two 3' overhangs (e.g., uu) of 2 to 5 nucleotides in length and/or a loop sequence linking the sense and antisense strands to form a hairpin structure.

The term "siRNA" as used herein refers to a double stranded RNA molecule that prevents translation of a target mRNA. Standard techniques for introducing siRNA into cells are used, including those in which RNA is transcribed using DNA as a template. The siRNA includes a sense nucleic acid sequence (also referred to as "sense strand"), an antisense nucleic acid sequence (also referred to as "antisense strand"), or both. The siRNA can be constructed such that a single transcript has an antisense nucleic acid sequence to which the sense nucleic acid sequence of the target gene is complementary, e.g., a hairpin structure. The siRNA may be dsRNA or shRNA.

The term "dsRNA" as used herein refers to a construct comprising two RNA molecules of mutually complementary sequences that anneal through the complementary sequences to form a double stranded RNA molecule. The nucleotide sequences of both strands may comprise not only "sense" or "antisense" RNA selected from protein coding sequences in the target gene sequence, but may also include RNA molecules having nucleotide sequences selected from non-coding regions of the target gene.

The term "shRNA" as used in the present specification means: an siRNA having a stem-loop structure comprising a first region and a second region (i.e., a sense strand and an antisense strand) that are complementary to each other. The degree and orientation of complementarity of the two regions is sufficient to allow base pairing to occur between the two regions, the first and second regions being joined by a loop region formed by the lack of base pairing between nucleotides (or nucleotide analogs) within the loop region. The loop region of the shRNA is a single-stranded region between the sense strand and the antisense strand, and may also be referred to as an "intervening single-strand"

The term "siD/R-NA" as used in this specification refers to double-stranded polynucleotide molecules comprising both RNA and DNA, including hybrids and chimeras of RNA and DNA, which prevent translation of the target mRNA. In the present specification, a hybrid means a molecule in which a polynucleotide composed of DNA and a polynucleotide composed of RNA hybridize with each other to form a double-stranded molecule; and chimeras indicate that one or both of the strands making up the double-stranded molecule may contain both RNA and DNA. Conventional techniques for introducing siD/R-NA into cells were used. The siD/R-NA includes a CSTF2 sense nucleic acid sequence (also referred to as the "sense strand"), a CSTF2 antisense nucleic acid sequence (also referred to as the "antisense strand"), or both. siD/R-NA can be constructed such that a single transcript has both a sense nucleic acid sequence and a complementary antisense nucleic acid sequence from the target gene, e.g., a hairpin. siD/R-NA can be dsD/R-NA or shD/R-NA.

The term "dsD/R-NA" as used herein refers to a construct of two molecules that comprise sequences that are complementary to each other and that have annealed together by the complementary sequences to form a double-stranded polynucleotide molecule. The nucleotide sequences of both strands may not only comprise "sense" or "antisense" polynucleotide sequences selected from the protein coding sequences of the target gene sequence, but may also comprise polynucleotides having nucleotide sequences selected from non-coding regions of the target gene. One or both of the two molecules that make up dsD/R-NA consist of both RNA and DNA (chimeric molecules), or one molecule consists of RNA and the other consists of DNA (hybrid duplex).

The term "shD/R-NA" as used herein refers to: siD/R-NA having a stem-loop structure comprising a first region and a second region complementary to each other, i.e. a sense strand and an antisense strand. The regions are sufficiently complementary and oriented to allow base pairing to occur between them, and the first and second regions are joined by a loop region formed by the lack of base pairing between nucleotides (or nucleotide analogs) within the loop region. The shD/R-NA loop region is a single-stranded region between the sense and antisense strands, and may also be referred to as an "intervening single strand".

As used herein, "isolated nucleic acid" refers to: a nucleic acid which is removed from its original environment (for example, the natural environment in the case of natural occurrence) and whose synthesis has been changed from its natural state. In the present invention, examples of the isolated nucleic acid include DNA, RNA and derivatives thereof.

The double-stranded molecule directed to the CSTF2 gene that hybridizes to the target mRNA reduces or inhibits the production of the CSTF2 protein encoded by the CSTF2 gene by binding to the normally single-stranded gene mRNA transcript, interfering with its translation, inhibiting protein expression.

Expression of the CSTF2 gene in lung cancer cell lines was inhibited by dsRNA directed against the CSTF2 gene. The present invention thus provides an isolated double stranded molecule capable of inhibiting the expression of the CSTF2 gene after introduction into a cell expressing the gene. The target sequence of the double-stranded molecule can be designed by an siRNA design algorithm, for example, as described below.

The CSTF2 target sequence includes, for example, the nucleotide sequence SEQ ID NO: 9 and 10. In other words, the present invention also provides a polypeptide whose target sequence comprises SEQ ID NO: 9 or 10 or a double-stranded molecule consisting thereof.

Specifically, the present invention provides the following double-stranded molecules [1] to [19 ]:

[1] an isolated double-stranded molecule that, when introduced into a cell, inhibits in vivo expression of the CSTF2 gene and cell proliferation, wherein said double-stranded molecule comprises a sense strand and a complementary antisense strand that hybridize to each other to form said double-stranded molecule;

[2] [1] the double-stranded molecule described in [1], wherein the double-stranded molecule acts on mRNA that hybridizes with a nucleic acid sequence selected from the group consisting of SEQ ID NO: 9 and 10 target sequence match;

[3] [1] the double-stranded molecule described in [1], wherein the sense strand comprises a nucleotide sequence corresponding to a nucleotide sequence selected from the group consisting of SEQ ID NO: 9 and 10;

[4] the double-stranded molecule of any one of [1] to [3], wherein the sense strand hybridizes to the antisense strand at the target sequence to form a double-stranded molecule having a length of less than about 100 nucleotides;

[5] the double-stranded molecule of [4], wherein the sense strand hybridizes to the antisense strand at the target sequence to form a double-stranded molecule having a length of less than about 75 nucleotides;

[6] the double-stranded molecule of [5], wherein the sense strand hybridizes to the antisense strand at the target sequence to form a double-stranded molecule having a length of less than about 50 nucleotides;

[7] the double-stranded molecule of [6], wherein the sense strand hybridizes to the antisense strand at the target sequence to form a double-stranded molecule having a length of less than about 25 nucleotides;

[8] the double-stranded molecule of [7], wherein the sense strand hybridizes to the antisense strand at the target sequence to form a double-stranded molecule having a length of about 19 to about 25 nucleotides;

[9] The double-stranded molecule of any one of [1] to [8], which consists of a single polynucleotide comprising a sense strand and an antisense strand linked together by an intervening single strand;

[10] [9] the double-stranded molecule having the general formula 5 ' - [ A ] - [ B ] - [ A ' ] -3 ', wherein [ A ] is a double-stranded molecule comprising a nucleotide sequence substantially identical to a nucleotide sequence selected from the group consisting of SEQ ID NO: 9 and 10, B is an intervening single strand consisting of 3 to 23 nucleotides, and A' is an antisense strand comprising a sequence complementary to the target sequence selected from [ A ];

[11] the double-stranded molecule according to any one of [1] to [10], which is composed of RNA;

[12] the double-stranded molecule of any one of [1] to [10], which is composed of DNA and RNA;

[13] [12] the double-stranded molecule described in [12], wherein the molecule is a hybrid of a DNA polynucleotide and an RNA polynucleotide;

[14] [13] the double-stranded molecule according to any one of [13], wherein the sense strand and the antisense strand are composed of DNA and RNA, respectively;

[15] the double-stranded molecule of [12], wherein the molecule is a chimera of DNA and RNA;

[16] the double-stranded molecule of [15], wherein the 3 ' -end of the antisense strand is flanked by RNA, or both the 5 ' -end of the sense strand and the 3 ' -end of the antisense strand are RNA;

[17] The double-stranded molecule of [16], wherein the flanking region consists of 9 to 13 nucleotides; and

[18] the double stranded molecule of any one of [1] to [17], wherein the molecule comprises one or two 3' overhangs; and

[19] the double-stranded molecule of [3], wherein the sense strand hybridizes to the antisense strand at the target sequence to form a double-stranded molecule having a length of 19 to 25 nucleotides;

the double-stranded molecules of the present invention are described in more detail below.

Methods of designing double-stranded molecules having the ability to inhibit the expression of a target gene in a cell are known (see, e.g., U.S. Pat. No.6,506,559, incorporated herein by reference in its entirety). For example, a computer program for designing siRNA can be accessed from Ambion website (Ambion. com/techlib/misc/siRNA _ finder. html on the world wide web).

The computer program can select the target nucleotide sequence of the double-stranded molecule according to the following protocol.

And (3) selecting a target:

1. the AA dinucleotide sequences were scanned downstream starting from the AUG start codon of the transcript. The occurrence of each AA and its 3' adjacent 19 nucleotides were recorded as potential siRNA targets. Tuschl et al suggest to avoid designing siRNAs for the 5 'and 3' untranslated regions (UTRs) and the region adjacent to the start codon (within 75 bases) because these regions may be richer in binding sites for regulatory proteins, while UTR binding proteins and/or translation initiation complexes may interfere with binding of siRNA endonuclease complexes.

2. Potential targets are compared to appropriate genomic databases (human, mouse, rat, etc.) and any target sequences with significant homology to other coding sequences are excluded from consideration. BLAST (Altschul SF et al, Nucleic Acids Res 1997 Sep 1, 25 (17): 3389-402) was used primarily, and can be found in NCBI servers: www.ncbi.nlm.nih.gov/BLAST/.

3. Qualified target sequences were selected for synthesis. Several target sequences are typically selected along the length of the gene to be evaluated.

Using the above protocol, the target sequence of the double stranded molecule designed for the CSTF2 gene of the present invention is SEQ ID NO: 9 and 10.

The double-stranded molecules targeted at the above target sequences were examined for their ability to suppress the growth of cells expressing the target genes, respectively. Accordingly, the present invention provides a polypeptide encoded with a sequence selected from SEQ ID NOs: 9 and 10 as a target.

Examples of double stranded molecules targeting the target sequence of the CSTF2 gene described above include isolated polynucleotides comprising a nucleic acid sequence corresponding to the target sequence and/or a sequence complementary to the target sequence. Preferred examples of polynucleotides targeting the CSTF2 gene include polynucleotides comprising a nucleotide sequence identical to SEQ ID NO: 9 or 10 and/or sequences complementary to these sequences. In one embodiment, a double-stranded molecule is comprised of two polynucleotides, one having a sequence corresponding to the target sequence, i.e., the sense strand, and the other having a sequence complementary to the target sequence, i.e., the antisense strand. The sense strand polynucleotide and the antisense strand polynucleotide hybridize to each other to form a double-stranded molecule. Examples of such double-stranded molecules include dsRNA and dsD/R-NA.

In another embodiment, a double-stranded molecule consists of one polynucleotide having both a sequence corresponding to a target sequence (i.e., the sense strand) and a sequence complementary to the target sequence (i.e., the antisense strand). Typically, the sense and antisense strands are linked by an intervening strand and hybridize to each other to form a hairpin loop structure. Examples of such double-stranded molecules include shRNA and shD/R-NA.

In other words, the double-stranded molecule of the present invention comprises a sense strand polynucleotide (which has a nucleotide sequence of a target sequence) and an antisense strand polynucleotide (which has a nucleotide sequence complementary to the target sequence), and these two polynucleotides hybridize to each other to form a double-stranded molecule. In a double-stranded molecule comprising the above polynucleotides, a portion of either or both strands of the polynucleotide may be RNA, and when the target sequence is defined by a DNA sequence, the nucleotide "t" within the target sequence and its complement is replaced with "u".

In one embodiment of the invention, such double stranded molecules of the invention comprise a stem-loop structure consisting of a sense strand and an antisense strand. The sense strand and the antisense strand may be connected by a loop. Thus, the invention also provides a double stranded molecule comprising a single polynucleotide comprising both a sense strand and an antisense strand linked by or flanked by intervening single strands.

In the present invention, the double-stranded molecule targeting the CSTF2 gene may have a sequence selected from the group consisting of SEQ ID NO: 9 and 10 as target sequences. Thus, preferred embodiments of the double-stranded molecules of the invention include those comprising a sequence identical to SEQ ID NO: 9 or 10 and polynucleotides having a sequence corresponding to SEQ id no: 9 or 10 and the complementary sequences thereof.

The double-stranded molecules of the invention may target a single CSTF2 gene sequence, or may target multiple CSTF2 gene sequences.

The double-stranded molecules of the present invention targeting the aforementioned target sequence CSTF2 gene include isolated polynucleotides comprising any nucleic acid sequence of the target sequence and/or the complement of the target sequence. Examples of polynucleotides targeted by the CSTF2 gene include those comprising the sequence SEQ ID NO: 9 or 10 and/or the complement of these nucleotides. However, the present invention is not limited to these examples, and minor modifications in the above nucleic acid sequences are acceptable as long as the modified molecules retain the ability to suppress the expression of the CSTF2 gene. Herein, the phrase "minor modification" when used in relation to a nucleic acid sequence means the substitution, deletion, addition or insertion of one, two or several nucleic acids to said sequence.

In the context of the present invention, the term "several" when used for nucleic acid substitutions, deletions, additions and/or insertions may mean 3 to 7, preferably 3 to 5, more preferably 3 to 4, even more preferably 3 nucleotide residues.

According to the invention, the double-stranded molecules of the invention can be tested for their ability using the methods used in the examples. In the examples described herein below, double-stranded molecules comprising sense strands of different portions of the mRNA of the CSTF2 gene or their complementary antisense strands were tested in vitro for their ability to reduce the production of the CSTF2 gene product in lung cancer cell lines (e.g., using a549 and SBC-5). Further, for example, the reduction of the CSTF2 gene product in cells contacted with the candidate double stranded molecule as compared to cells cultured in the absence of the candidate molecule can be determined by, for example, using the methods described in the examples: the term "semi-quantitative RT-PCR" refers to RT-PCR of primers for CSTF2 mRNA. The sequence of the CSTF2 gene product was then reduced in an in vitro cell-based assay and its inhibitory effect on cell growth was detected. The sequence that inhibits cell growth in an in vitro cell-based assay can then be tested for its corresponding ability in vivo using cancer-bearing animals (e.g., a nude mouse xenograft model) to demonstrate a reduction in production of the CSTF2 gene product and a reduction in cancer cell growth.

When the isolated polynucleotide is RNA or a derivative thereof, "t" in the nucleotide sequence should be replaced with "u". As used herein, the term "complementary" refers to Watson-Crick or Hoogsteen base pairing between nucleotide units in a polynucleotide, and the term "binding" means a physical or chemical interaction between two polynucleotides. Where the polynucleotides comprise modified nucleotides and/or non-phosphodiester linkages, the polynucleotides may likewise be combined with one another. In general, complementary polynucleotide sequences hybridize under suitable conditions to form a stable duplex containing little or no mismatches. Further, the sense strand and the antisense strand of the isolated polynucleotide of the present invention may form a double-stranded molecule or hairpin loop structure by hybridization. In a preferred embodiment, the above-described duplex contains no more than one mismatch in every 10 matches. In a particularly preferred embodiment, the strands of the duplex are fully complementary, such duplex containing no mismatches.

For CSTF2, the polynucleotide is preferably less than 1009 nucleotides in length. For example, for the gene, the polynucleotide is less than 500, 200, 100, 75, 50, or 25 nucleotides in length. The isolated polynucleotides of the present invention are useful for forming a double-stranded molecule against the CSTF2 gene, or for preparing a template DNA encoding the double-stranded molecule. When the polynucleotide is used to form a double-stranded molecule, the polynucleotide may be longer than 19 nucleotides, preferably longer than 21 nucleotides, and more preferably between about 19 and 25 nucleotides in length. Thus, the invention provides a double stranded molecule comprising a sense strand and an antisense strand, wherein the sense strand comprises a nucleotide sequence corresponding to a target sequence. In a preferred embodiment, the sense strand hybridizes to the antisense strand at the target sequence to form a double-stranded molecule 19 to 25 nucleotide pairs in length.

When introduced into a cell, the double-stranded molecule serves as a guide for the recognition of homologous sequences in the mRNA for the RNA-induced silencing complex (RISC). The recognized target RNA is cleaved and degraded by Dicer nuclease activity, whereby the double-stranded molecule ultimately reduces or inhibits production (expression) of the polypeptide encoded by the RNA. Thus, the double-stranded molecules of the present invention may be defined by their ability to produce single strands that specifically hybridize under stringent conditions to the mRNA of the CSTF2 gene. The portion of the mRNA that hybridizes to a single strand generated from a double-stranded molecule is referred to herein as a "target sequence" or "target nucleic acid" or "target nucleotide". In the present invention, the nucleotide sequence of the "target sequence" can be shown not only using the RNA sequence of mRNA but also using the DNA sequence of cDNA synthesized from mRNA.

The double-stranded molecules described herein may comprise one or more modified nucleotides and/or non-phosphodiester linkages. Chemical modifications well known in the art can increase the stability, availability and/or cellular uptake of the double-stranded molecule. It will be apparent to the skilled person that other types of chemical modification of the molecules of the invention may be introduced (WO 03/070744; WO 2005/045037). In one embodiment, modifications may be used to provide improved resistance to degradation or improved intake. Examples of such modifications include, but are not limited to, phosphorothioate linkages, 2 '-O-methyl ribonucleotides (particularly on the sense strand of a double-stranded molecule), 2' -deoxy-fluoro ribonucleotides, 2 '-deoxyribonucleotides, "universal base" nucleotides, 5' -C-methyl nucleotides, and incorporation of inverted deoxyabasic residues (US 20060122137).

In another embodiment, modifications may be used to enhance the stability of the double-stranded molecule or to increase targeting efficiency. Such modifications include, but are not limited to, chemical cross-linking between the two complementary strands of a double-stranded molecule, chemical modifications of the 3 ' or 5 ' end of one strand of a double-stranded molecule, sugar modifications, nucleobase modifications and/or backbone modifications, 2-fluoro modified ribonucleotides and 2 ' -deoxyribonucleotides (WO 2004/029212). In another embodiment, modifications can be utilized to increase or decrease the affinity for complementary nucleotides in the target mRNA and/or in the complementary double stranded molecular chains (WO 2005/044976). For example, an unmodified pyrimidine nucleotide may be replaced with a 2-thio, 5-alkynyl (5-alkinyl), 5-methyl or 5-propynyl (5-propylnyl) pyrimidine. In addition, unmodified purines may be substituted with 7-deaza (7-deaza), 7-alkyl or 7-alkenyl purines. In another embodiment, when the double-stranded molecule is a double-stranded molecule having a 3 'overhang, the overhanging nucleotide of the 3' -terminal nucleotide can be replaced by a deoxyribonucleotide (Elbashir SM et al, Genes Dev 2001 Jan 15, 15 (2): 188-200). For further details, publications such as US20060234970 may be utilized. The present invention is not limited to these examples, and any known chemical modification may be applied to the double-stranded molecule of the present invention as long as the resulting molecule retains the ability to inhibit expression of a target gene.

Furthermore, the double-stranded molecules of the invention may comprise both DNA and RNA, for example dsD/R-NA or shD/R-NA. Specifically, hybrid polynucleotides formed from a DNA strand and an RNA strand or DNA-RNA chimeric polynucleotides exhibit improved stability. The stability of the double-stranded molecule can be enhanced by forming a mixture of DNA and RNA, i.e., a hybrid double-stranded molecule consisting of a DNA strand (polynucleotide) and an RNA strand (polynucleotide), or a chimeric double-stranded molecule comprising both DNA and RNA on either single strand (polynucleotide) or both single strands (polynucleotides), and the like.

The hybrid of a DNA strand and an RNA strand may have a structure in which the sense strand is DNA and the antisense strand is RNA, or vice versa, as long as it is capable of inhibiting the expression of a target gene after introduction into a cell expressing the gene. Preferably, the sense strand polynucleotide is DNA and the antisense strand polynucleotide is RNA. Also, the chimeric double-stranded molecule may have a structure in which both the sense strand and the antisense strand are composed of DNA and RNA, or either of the sense strand or the antisense strand is composed of DNA and RNA, as long as the double-stranded molecule has an activity of inhibiting the expression of a target gene when introduced into a cell expressing the gene. In order to improve the stability of the double-stranded molecule, it is preferable that the molecule contains as much DNA as possible; in order to induce the expression inhibition of the target gene, it is required that the molecule is RNA within a certain range to sufficiently induce the expression inhibition.

As a preferred example of a chimeric double-stranded molecule, the region of the upstream portion of the double-stranded molecule (i.e., the region flanking the target sequence or its complement located in the sense strand or antisense strand) is RNA. Preferably, the upstream part region represents the 5 '-side (5' -end) of the sense strand and the 3 '-side (3' -end) of the antisense strand. Alternatively, the region flanking the 5 'end of the sense strand or the 3' end of the antisense strand is referred to as the upstream part region. That is, in preferred embodiments, the region flanking the 3 ' end of the antisense strand consists of RNA, or the region flanking the 5 ' end of the sense strand and the region flanking the 3 ' end of the antisense strand consist of RNA. For example, the chimeric or hybrid double-stranded molecules of the invention comprise the following combinations.

Sense strand:

5’-[---DNA---]-3’

3’-(RNA)-[DNA]-5’

: the antisense strand of the nucleic acid sequence is,

sense strand:

5’-(RNA)-[DNA]-3’

3’-(RNA)-[DNA]-5’

: an antisense strand, and

sense strand:

5’-(RNA)-[DNA]-3’

3’-(---RNA---)-5’

: the antisense strand.

The upstream part region is preferably a domain consisting of 9 to 13 nucleotides, counted from the end of the target sequence or its complement within the sense strand or antisense strand of the double-stranded molecule. Further, preferred examples of such chimeric double-stranded molecules include examples of: the chain length is 19-21 nucleotides, wherein at least the upstream half of the polynucleotide (5 'side region for sense strand and 3' side region for antisense strand) is RNA and the other half is DNA. In such chimeric double-stranded molecules, the effect of suppressing the expression of a target gene is much stronger than when the antisense strand is RNA as a whole (US 20050004064).

In the present invention, the double-stranded molecules may form hairpin structures, such as short hairpin RNAs (shRNAs) and short hairpins consisting of DNA and RNA (shD/R-NA). shRNA or shD/R-NA is an RNA sequence or a mixed sequence of RNA and DNA that forms a tight hairpin turn that can be used to silence gene expression through RNA interference. The shRNA or shD/R-NA comprises a sense target sequence and an antisense target sequence on a single strand, wherein the sequences are separated by a loop sequence. Typically, the hairpin structure is cleaved by cellular machinery into either dsRNA or dsD/R-NA, which is further combined with RISC or dsD/R-NA. This complex binds to and cleaves mRNA that matches the target sequence of the dsRNA or dsD/R-NA.

In order to form the hairpin loop structure, a loop sequence composed of an arbitrary nucleotide sequence may be provided between the sense sequence and the antisense sequence. Thus, the present invention also provides double stranded molecules having the general formula 5 ' - [ A ] - [ B ] - [ A ' ] -3 '. Wherein [ A ] is a sense strand comprising a sequence corresponding to a target sequence; [B] is an intervening single chain; [ A' ] is an antisense strand comprising a sequence complementary to a target sequence. The target sequence may be selected, for example, from SEQ id nos: 9 and 10.

The present invention is not limited to these examples, and the target sequence in [ a ] may be a sequence modified on the basis of these examples, provided that the double-stranded molecule retains the ability to inhibit the expression of CSTF2 gene as a target. The region [ A ] and [ A' ] hybridize to form a loop composed of the region [ B ]. The intervening single-stranded portion [ B ], i.e., the loop sequence, may preferably be 3 to 23 nucleotides in length. For example, the loop sequence may be selected from the group consisting of http:// www.ambion.com/techlib/tb/tb _506. html. Furthermore, loop sequences consisting of 23 nucleotides also provide active siRNAs (Jacqe JM et al, Nature 2002 Jul25, 418 (6896): 435-8, Epub 2002 Jun 26):

CCC, CCACC or CCACACC: jacqe JM et al, Nature 2002 Jul 25, 418 (6896): 435-8, Epub 2002 Jun 26;

UUCG: lee NS et al, Nat Biotechnol 2002 May, 20 (5): 500-5; fruscoconi Pet al, Proc Natl Acad Sci USA 2003 Feb 18, 100 (4): 1639-44, Epub 2003 Feb 10; and

UUCAAGAGA：Dykxhoorn DM et al.，Nat Rev Mol Cell Biol 2003 Jun，4(6)：457-67。

examples of preferred double-stranded molecules having hairpin loop structures of the invention are shown below. In the following structures, the loop sequence may be selected from the group consisting of AUG, CCC, UUCG, CCACC, CTCGAG, AAGCUU, CCACACC, and UUCAAGAGA; however, the present invention is not limited thereto:

GGCUUUAGUCCCGGGCAGA- [ B ] -UCUGCCCGGGACUAAAGCC (for the target sequence SEQ ID NO: 9);

CACUUUACUUUCUGUAACU- [ B ] -AGUUACAGAAAGUAAAGUG (for the target sequence SEQ ID NO: 10);

in addition, in order to enhance the inhibitory activity of the double-stranded molecule, several nucleotides may be added to the 3 'end of the sense strand and/or the antisense strand of the target sequence as 3' overhangs. The number of nucleotides added is at least 2, usually 2 to 10, preferably 2 to 5. The added nucleotides form a single strand at the 3' end of the antisense strand of the double-stranded molecule. The nucleotides used for the 3' overhangs are preferably, but not limited to, "u" or "t". When the double-stranded molecule has a hairpin loop structure, a 3 'overhang is added to the 3' end of the antisense strand.

The method for preparing the double-stranded molecule is not particularly limited, but a chemical synthesis method well known in the art is preferably used. According to the chemical synthesis method, sense and antisense single-stranded polynucleotides are synthesized separately and then annealed together by an appropriate method to obtain a double-stranded molecule. Specific examples of annealing include those wherein the synthesized single stranded polynucleotides are mixed in a molar ratio of preferably at least about 3: 7, more preferably about 4: 6, and most preferably substantially equimolar amounts (i.e., a molar ratio of about 5: 5). The mixture is then heated to a temperature at which the double stranded molecules dissociate and then gradually cooled. The annealed double-stranded polynucleotide can be purified by a conventional method known in the art. Examples of purification methods include: a method using agarose gel electrophoresis, or a method in which remaining single-stranded polynucleotides are optionally removed (e.g., degraded using an appropriate enzyme).

The regulatory sequences flanking the CSTF2 sequence may be the same or different, so that their expression can be regulated independently, or in a temporal or spatial manner. The double-stranded molecule can be transcribed intracellularly by cloning the CSTF2 gene template into a vector, such as a vector containing an RNA polymerase III transcription unit from small nuclear RNA (snRNA) U6 or the human H1RNA promoter.

Alternatively, the double-stranded molecule may be transcribed intracellularly by cloning its coding sequence into a vector containing regulatory sequences adjacent to the coding region that direct expression of the double-stranded molecule in an appropriate cell, such as the rnalyiii transcription unit from small nuclear RNA (snRNA) U6 or the human H1RNA promoter. The regulatory sequences flanking the coding sequence of the double-stranded molecule may be identical or different, such that their expression can be regulated independently, or in a temporal or spatial manner. Details of the vector capable of producing a double-stranded molecule will be described below.

Vector encoding the double-stranded molecule of the invention:

the invention also includes vectors encoding one or more of the double stranded molecules described herein, and cells comprising the vectors.

Specifically, the present invention provides the following vectors [1] to [10 ].

[1] A vector encoding a double-stranded molecule that, when introduced into a cell, inhibits in vivo expression of the CSTF2 gene and proliferation of the cell, wherein said double-stranded molecule comprises a sense strand and an antisense strand complementary thereto, which hybridize to each other to form said double-stranded molecule.

[2] [1] the vector encoding a double-stranded molecule which acts on mRNA which hybridizes with a nucleic acid sequence selected from the group consisting of SEQ ID NO: 9 and 10 target sequence match;

[3] The vector of [1], wherein the sense strand comprises a nucleotide sequence corresponding to a nucleotide sequence selected from the group consisting of SEQ ID NO: 9 and 10;

[4] the vector of any one of [1] to [3], which encodes a double-stranded molecule, wherein a sense strand of the double-stranded molecule hybridizes to an antisense strand at a target sequence to form a double-stranded molecule of less than about 100 nucleotide pairs in length;

[5] the vector of [4], which encodes a double-stranded molecule, wherein the sense strand of the double-stranded molecule hybridizes to the antisense strand at the target sequence to form a double-stranded molecule of less than about 75 nucleotide pairs in length;

[6] the vector of [5], which encodes a double-stranded molecule, wherein the sense strand of the double-stranded molecule hybridizes to the antisense strand at the target sequence to form a double-stranded molecule of less than about 50 nucleotide pairs in length;

[7] the vector of [6], which encodes a double-stranded molecule, wherein the sense strand of the double-stranded molecule hybridizes to the antisense strand at the target sequence to form a double-stranded molecule of less than about 25 nucleotide pairs in length;

[8] the vector of [7], which encodes a double-stranded molecule, wherein the sense strand and the antisense strand of the double-stranded molecule hybridize at the target sequence to form a double-stranded molecule having a length of about 19 to about 25 nucleotide pairs;

[9] the vector of any one of [1] to [8], wherein the double-stranded molecule consists of a single polynucleotide comprising a sense strand and an antisense strand linked together by an intervening single strand;

[10] The vector of [9] encoding a double-stranded molecule having the general formula 5 ' - [ a ] - [ B ] - [ a ' ] -3 ', wherein [ a ] is a double-stranded molecule comprising a nucleotide sequence substantially identical to a sequence selected from the group consisting of SEQ ID NOs: 9 and 10, [ B ] is an intervening single strand consisting of 3 to 23 nucleotides, [ a' ] is an antisense strand comprising a sequence complementary to the target sequence selected in [ a ];

the vector of the invention preferably encodes a double-stranded molecule of the invention in an expressible form. In the present specification, the term "in an expressible form" means that the vector, when introduced into a cell, will express the molecule. In a preferred embodiment, the vector comprises regulatory elements necessary for the expression of the double-stranded molecule. Thus, in one embodiment, the expression vector encodes and is suitable for expressing the nucleic acid sequence of the present invention. Such vectors of the invention may be used to produce the double-stranded molecules of the invention, and may also be used directly as active ingredients in cancer therapy.

The vector of the present invention can be produced by the following method: for example, sequences encoding the sense and antisense strands of a double-stranded molecule directed against the CSTF2 gene are cloned into an expression vector, and regulatory sequences are operably linked to the sequences encoding the strands to allow expression of both strands (by transcription of the DNA molecule) (LeeNS et al, Nat Biotechnol 2002 May, 20 (5): 500-5). For example, an RNA molecule that is antisense to an mRNA is transcribed from a first promoter (e.g., a promoter sequence flanking the 3 'end of the cloned DNA) and an RNA molecule that is sense-stranded with respect to the mRNA is transcribed from a second promoter (e.g., a promoter sequence flanking the 5' end of the cloned DNA). The sense and antisense strands hybridize in vivo to produce a double-stranded molecular construct for silencing the gene. Alternatively, two vector constructs encoding the sense and antisense strands of a double-stranded molecule, respectively, are used to express the sense and antisense strands, respectively, followed by formation of a double-stranded molecule construct. Furthermore, the cloned sequence may encode a construct with secondary structure (e.g., a hairpin), i.e., a single transcript of the vector contains both the sense and complementary antisense sequences of the target gene.

The vector of the invention may also be configured so that it effects stable insertion into the genome of the target Cell (see Thomas KR & Capecchi MR, Cell 1987, 51: 503-12 for a description of homologous recombination cassette vectors). Reference may be made to, for example: wolff et al, Science 1990, 247: 1465-8; U.S. Pat. Nos. 5,580,859, 5,589,466, 5,804,566, 5,739,118, 5,736,524, 5,679,647 and WO 98/04720. Examples of DNA-based delivery techniques include: "naked DNA", assisted (bupivicaine, polymer, peptide-mediated) delivery, cationic lipid complex, and particle-mediated delivery ("gene gun"), or pressure-mediated delivery (see, e.g., U.S. patent No. 5,922,687).

The vector of the present invention includes, for example, a viral vector or a bacterial vector. Examples of expression vectors include attenuated viral hosts such as vaccinia or fowlpox (see U.S. Pat. No. 4,722,848). This strategy involves, for example, the use of vaccinia virus as a vector to express nucleotide sequences encoding double-stranded molecules. The recombinant vaccinia virus, when introduced into cells expressing the target gene, expresses the molecule and thereby inhibits proliferation of the cell. Other examples of vectors that can be used include bacillus calmette-guerin (BCG). BCG vectors are described in Stover et al, Nature 1991, 351: 456 to 60, respectively. A wide variety of other vectors can be used for therapeutic administration and production of the duplex molecule, examples include adenoviral and adeno-associated viral vectors, retroviral vectors, Salmonella typhi (Salmonella typhi) vectors, detoxified anthrax toxin vectors, and the like. See, e.g., Shata et al, Mol Med Today 2000, 6: 66-71; shedlock et al, J Leukoc Biol 2000, 68: 793-806; and Hipp et al, In Vivo 2000, 14: 571-85.

Methods of using the double-stranded molecules of the invention to inhibit or reduce cancer cell growth or to treat cancer:

the present invention provides methods of inhibiting the growth of cancer cells, such as lung cancer cells, by inhibiting the expression of CSTF2 to induce dysfunction of the CSTF2 gene. CSTF2 gene expression can be inhibited by any of the inventive double stranded molecules described above that specifically target the CSTF2 gene or the inventive vector that can express at least one of the double stranded molecules.

The ability of the double-stranded molecules and vectors of the invention described above to inhibit the growth of cancerous cells suggests that they may be used in methods of treating cancer. Accordingly, the present invention provides a method for treating a patient suffering from lung cancer without adverse effects by administering a double-stranded molecule against CSTF2 gene or a vector expressing the molecule, because the gene is hardly detected in normal organs.

Specifically, the present invention provides the following processes [1] to [36 ]:

[1] a method of treating or preventing cancer in a subject comprising administering to the subject a pharmaceutically effective amount of a double-stranded molecule directed against the CSTF2 gene or a vector encoding the double-stranded molecule, wherein the double-stranded molecule, when introduced into a cell, inhibits expression of the CSTF2 gene;

[2] [1] wherein the double-stranded molecule acts on mRNA that hybridizes to a nucleic acid sequence selected from the group consisting of SEQ ID NO: 9 and 10 target sequence match;

[3] the method of [1], wherein the sense strand comprises a nucleotide sequence identical to a nucleotide sequence selected from SEQ ID NO: 9 and 10;

[4] the method of any one of [1] to [3], wherein a plurality of double-stranded molecules are administered;

[5] the method of any of [1] to [4], wherein the sense strand and the antisense strand of the double-stranded molecule hybridize at the target sequence to form a double-stranded molecule that is less than about 100 nucleotide pairs in length;

[6] the method of [5], wherein the sense strand and the antisense strand of the double-stranded molecule hybridize at the target sequence to form a double-stranded molecule that is less than about 75 nucleotide pairs in length;

[7] the method of [6], wherein the sense strand and the antisense strand of the double-stranded molecule hybridize at the target sequence to form a double-stranded molecule that is less than about 50 nucleotide pairs in length;

[8] the method of [7], wherein the sense strand and the antisense strand of the double-stranded molecule hybridize at the target sequence to form a double-stranded molecule that is less than about 25 nucleotide pairs in length;

[9] the method of [9], wherein the sense strand and the antisense strand of the double-stranded molecule hybridize at the target sequence to form a double-stranded molecule that is about 19 to about 25 nucleotide pairs in length;

[10] The method of any one of [1] to [9], wherein the double-stranded molecule is composed of a single polynucleotide comprising a sense strand and an antisense strand linked together by an intervening single strand;

[11] [10] wherein the double stranded molecule has the general formula 5 ' - [ A ] - [ B ] - [ A ' ] -3 ', wherein [ A ] is a double stranded molecule comprising a double stranded sequence substantially identical to a sequence selected from the group consisting of SEQ ID NOs: 9 and 10, B is an intervening single strand consisting of 3 to 23 nucleotides, and A' is an antisense strand comprising a sequence complementary to the target sequence selected from [ A ];

[12] the method of any one of [1] to [11], wherein the double-stranded molecule is RNA;

[13] the method of any one of [1] to [11], wherein the double-stranded molecule comprises DNA and RNA;

[14] the method of [13], wherein the double-stranded molecule is a hybrid of a DNA polynucleotide and an RNA polynucleotide;

[15] the method of [14], wherein the sense and antisense strand polynucleotides are comprised of DNA and RNA, respectively;

[16] the method of [13], wherein the double-stranded molecule is a chimera of DNA and RNA;

[17] the method of [16], wherein the region flanking the 3 ' end of the antisense strand consists of RNA, or the region flanking the 5 ' end of the sense strand and the region flanking the 3 ' end of the antisense strand consist of RNA;

[18] The method of [17], wherein the flanking region consists of 9 to 13 nucleotides;

[19] the method of any one of [1] to [18], wherein the double-stranded molecule comprises one or more 3' overhangs;

[20] the method of any one of [1] to [19], wherein the double-stranded molecule is contained in a composition comprising a transfection-enhancing agent and a pharmaceutically acceptable carrier in addition to the molecule.

[21] The method of any one of [1] to [20], wherein the double-stranded molecule is encoded by a vector;

[22] [21] wherein the double-stranded molecule encoded by the vector acts on mRNA that hybridizes to a nucleic acid sequence selected from the group consisting of SEQ ID NO: 9 and 10 target sequence match;

[23] the method of [22], wherein the sense strand of the double-stranded molecule encoded by the vector comprises a nucleotide sequence identical to a sequence selected from the group consisting of SEQ ID NOs: 9 and 10;

[24] the method of [23], wherein a plurality of double-stranded molecules are administered;

[25] the method of any one of [21] to [24], wherein the sense strand of the double-stranded molecule encoded by the vector is less than about 100 nucleotide pairs in length;

[26] the method of [25], wherein the sense strand and the antisense strand of the double-stranded molecule hybridize at the target sequence to form a double-stranded molecule that is less than about 75 nucleotide pairs in length;

[27] The method of [26], wherein the sense strand and the antisense strand of the double-stranded molecule hybridize at the target sequence to form a double-stranded molecule that is less than about 50 nucleotide pairs in length;

[28] the method of [27], wherein the sense strand and the antisense strand of the double-stranded molecule hybridize at the target sequence to form a double-stranded molecule that is less than about 25 nucleotide pairs in length;

[29] the method of [28], wherein the sense strand and the antisense strand of the double-stranded molecule hybridize at the target sequence to form a double-stranded molecule that is between about 19 and about 25 nucleotide pairs in length;

[30] the method of any one of [21] to [29], wherein the double-stranded molecule encoded by the vector is composed of a single polynucleotide comprising a sense strand and an antisense strand linked together by an intervening single strand;

[31] [30] wherein said double stranded molecule encoded by said vector has the general formula 5 ' - [ A ] - [ B ] - [ A ' ] -3 ', wherein [ A ] is a double stranded molecule comprising a double stranded sequence substantially identical to a sequence selected from the group consisting of SEQ ID NOs: 9 and 10, [ B ] is an intervening single strand consisting of 3 to 23 nucleotides, [ a' ] is an antisense strand comprising a sequence complementary to the target sequence selected in [ a ];

[34] The method of any one of [21] to [31], wherein the double-stranded molecule encoded by the vector is contained in a composition comprising a transfection-enhancing agent and a pharmaceutically acceptable carrier in addition to the molecule;

[35] the method of any one of [1] to [34], wherein the cancer is lung cancer; and

[36] [35] the method, wherein the lung cancer is adenocarcinoma, squamous cell carcinoma, large cell carcinoma or small cell lung cancer.

The process of the present invention will be described in more detail below.

Growth of cells expressing the CSTF2 gene can be inhibited by contacting the cells with a double-stranded molecule directed against the CSTF2 gene, a vector expressing the molecule, or a composition comprising the same. The cells may be further contacted with a transfection agent. Suitable transfection agents are known in the art. The phrase "inhibiting cell growth" means that the cell proliferates at a lower rate or has reduced viability compared to a cell not exposed to the molecule. Cell growth can be determined by techniques known in the art, for example using an MTT cell proliferation assay.

Any kind of cell can be repressed according to the present method, so long as the cell expresses or overexpresses the target gene of the double-stranded molecule of the present invention. Exemplary cells include lung cancer cells, such as NSCLC and SCLC.

Thus, for patients suffering from or at risk of developing diseases associated with CSTF2, such as cancer, a treatment may be performed by administering at least one inventive double-stranded molecule, at least one vector expressing at least one such molecule, or at least one composition comprising at least one such molecule. For example, a lung cancer patient may be treated according to the methods of the present invention. The type of cancer can be identified by standard methods based on the particular type of tumor diagnosed. Lung cancer can be diagnosed, for example, by carcinoembryonic antigen (CEA), CYFRA, pro-GRP, and the like as lung cancer markers, or by chest X-ray and/or sputum cytology. More preferably, the patient treated by the method of the invention is selected by: expression of the CSTF2 gene is detected in a biopsy specimen obtained from the patient by methods known in the art, such as RT-PCR or immunoassay. Preferably, prior to treatment of the present invention, the overexpression of the CSTF2 gene is confirmed by methods known in the art, for example, immunohistochemical analysis or RT-PCR, on said biopsy specimen from the subject.

According to the methods of the invention, when multiple such double-stranded molecules (or vectors expressing the same molecule or compositions containing the same molecule) are administered in order to inhibit cell growth and thereby treat cancer, each such molecule may have a different structure, but act on mrnas that match the same target sequence. Alternatively, multiple double stranded molecules can act on mrnas that match different target sequences of the same gene, or on mrnas that match different target sequences of different genes. For example, the method may use double stranded molecules directed against different target sequences of the CSTF2 gene. Alternatively, for example, the present methods may utilize double stranded molecules directed against one, two or more target sequences of the CSTF2 gene and other genes.

To inhibit cell growth, the double-stranded molecules of the invention can be introduced directly into the cell in a form that permits binding of the molecule to the corresponding mRNA transcript. Alternatively, as described above, DNA encoding the double-stranded molecule can be introduced into cells as a vector. For introducing the double-stranded molecule and the vector into cells, transfection enhancers such as FuGENE (Roche diagnostics), Lipofectamine 2000(Invitrogen), Oligofectamine (Invitrogen) and nucleofector (Wako pure chemical) can be used.

A treatment is considered "effective" when it results in a clinical benefit, such as a decrease in CSTF2 gene expression, a decrease in the size, prevalence (prevalence), or metastatic potential of the cancer in the subject. When applied prophylactically to a treatment, "effective" means that it delays or prevents the formation of cancer, or prevents or alleviates the clinical symptoms of cancer. Effectiveness is determined in conjunction with any known diagnostic or therapeutic method for a particular tumor type.

To the extent that the methods and compositions of the present invention are used in the context of "prevention" and "prophylaxis," such terms are used interchangeably herein to refer to any activity that reduces the mortality or morbidity burden resulting from a disease. Prevention and prophylaxis can occur at "primary, secondary, and tertiary prevention levels". Primary prevention and prophylaxis avoids the occurrence of disease, while secondary and tertiary prevention and prophylaxis levels encompass activities aimed at preventing and preventing the progression of disease and the appearance of symptoms, as well as reducing the negative effects of established disease by restoring function and alleviating disease-related complications. Alternatively, prevention and prophylaxis may include a wide range of prophylactic therapies aimed at lessening the severity of a particular condition (e.g., reducing the proliferation and metastasis of tumors, etc.).

Treating and/or preventing cancer and/or preventing postoperative recurrence thereof includes any of the steps such as surgically removing cancer cells, inhibiting cancerous cell growth, tumor regression or regression, inducing cancer regression and suppressing carcinogenesis, tumor regression, and reducing or inhibiting metastasis. Effective treatment and/or prevention of cancer can reduce mortality and improve prognosis in individuals with cancer, reduce the levels of tumor markers in their blood, and reduce detectable symptoms associated with cancer. For example, a reduction or amelioration of symptoms constitutes an effective treatment and/or prevention, including a 10%, 20%, 30% or more reduction, or a stable condition.

It is understood that the double stranded molecules of the present invention degrade sub-stoichiometric CSTF2 mRNA. Without wishing to be bound by any theory, it is believed that the double stranded molecules of the invention cause degradation of the target mRNA in a catalytic manner. Thus, there are far fewer double stranded molecules that need to be delivered to or near the site of cancer to effect a therapeutic effect as compared to conventional cancer therapies.

One skilled in the art can readily determine the effective amount of the double-stranded molecule of the invention, taking into account the weight, age, sex, type of disease, symptoms and other conditions of the subject, the route of administration, and whether it is administered locally or systemically. Generally, an effective amount of a double-stranded molecule of the invention is at an intercellular concentration of about 1 nanomolar (nM) to about 100nM, preferably about 2nM to about 50nM, and more preferably about 2.5nM to about 10nM at or near the cancer site. It is contemplated that greater or lesser amounts of double stranded molecules may be used. The exact dosage required in a particular situation can be readily and routinely determined by one of ordinary skill in the art.

The methods of the invention are useful for inhibiting the growth or metastasis of cancers that express the CSTF2 gene, such as lung cancer, including NSCLC and SCLC. Specifically, the polypeptide comprising the target sequence SEQ ID NO: the 9 or 10 double-stranded molecule is particularly preferred for the treatment of lung cancer.

For the treatment of cancer, the double-stranded molecules of the invention may also be administered to a subject in combination with an agent different from the double-stranded molecule. Alternatively, the double-stranded molecules of the invention may also be administered to a subject in combination with other therapeutic methods intended for the treatment of cancer. For example, the double-stranded molecules of the invention can be administered in combination with current therapeutic methods for treating cancer or preventing cancer metastasis (e.g., radiation therapy, surgery, and treatment with chemotherapeutic agents such as cisplatin, carboplatin, cyclophosphamide, 5-fluorouracil, doxorubicin, daunorubicin (daunorubicin), or tamoxifen (tamoxifen)).

In the methods of the invention, the double-stranded molecule may be administered to the subject as a naked double-stranded molecule, in combination with a delivery substance, or as a recombinant plasmid or viral vector expressing the double-stranded molecule.

Suitable delivery materials for administration in combination with a duplex molecule of the invention include Mirus TransitTKO lipophilic materials, Lipofectin, Lipofectamine, Cellffectin, or polycations (e.g.polylysine), or liposomes. One preferred delivery material is a liposome.

Liposomes can help deliver the duplex to a specific tissue, such as lung tumor tissue, and can also increase the blood half-life of the duplex. Liposomes suitable for use in the present invention are formed from conventional vesicle-forming lipids (vesicular-forming lipids), which typically include neutral or negatively charged phospholipids, as well as sterols, such as cholesterol. Consideration of several factors may generally provide guidance for the selection of lipids, such as the desired liposome size and half-life of the liposomes in the bloodstream. Various methods for preparing liposomes are known, for example, Szoka et al, Ann Rev Biophys Bioeng1980, 9: 467; U.S. Pat. nos. 4,235,871; nos. 4,501,728; nos. 4,837,028; and No. 5,019,369; the entire contents of the above documents are incorporated herein by reference.

Preferably, the liposome encapsulating the double-stranded molecule of the invention comprises a ligand molecule capable of delivering the liposome to the cancer site. Preferred ligands are ligands that bind to receptors commonly found in tumor or vascular endothelial cells, such as monoclonal antibodies that bind to tumor antigens or endothelial cell surface antigens.

Particularly preferably, the liposomes encapsulating the double-stranded molecules of the invention are modified so as not to be cleared by mononuclear macrophages and the reticuloendothelial system, for example, due to the surface-bound opsonization inhibition moieties (opsonization inhibition moieties) of their structure. In one embodiment, the liposomes of the invention may comprise both an opsonization-inhibiting moiety and a ligand.

The opsonization-inhibiting moiety used to prepare the liposomes of the invention is typically a large hydrophilic polymer associated with the liposome membrane. As used in this specification, for example, an opsonization-inhibiting moiety is said to "bind" to a liposomal membrane when the opsonization-inhibiting moiety chemically or physically bridges the liposomal membrane, for example by insertion of a lipid-soluble anchor (anchor) into the membrane itself, or by direct binding to a reactive group of the membrane lipid. These opsonization-inhibiting hydrophilic polymers form a protective skin that significantly reduces liposome uptake by the macrophage-monocyte system ("MMS") and reticuloendothelial system ("RES"); this is described, for example, in U.S. patent No. 4,920,016, the entire disclosure of which is incorporated herein by reference. Thus, liposomes modified with opsonization-inhibiting moieties can be retained in the blood circulation for significantly longer periods of time than unmodified liposomes. For the above reasons, such liposomes are sometimes also referred to as "stealth" (stealth) liposomes.

Stealth liposomes are known to accumulate in tissues supplied by means of a porous or "leaky" microvasculature. Therefore, in target tissues characterized by such defects in the microvasculature, such as solid tumors, these liposomes accumulate with high efficiency. See Gabizon et al, Proc Natl Acad Sci USA 1988, 18: 6949-53. Furthermore, the reduction in RES uptake prevents significant accumulation of stealth liposomes in the liver and spleen, thereby reducing the toxicity of stealth liposomes. Thus, liposomes of the invention modified with opsonization-inhibiting moieties are capable of delivering the double-stranded molecules of the invention to tumor cells.

Is suitable for conditioning modified liposomeThe inhibiting moiety is preferably a water soluble polymer having a molecular weight of from about 500 to about 40,000 daltons, more preferably from about 2,000 to about 20,000 daltons. Such polymers include polyethylene glycol (PEG) or polypropylene glycol (PPG) derivatives; for example, methoxy PEG or PPG, and PEG or PPG stearate; synthetic polymers such as polyacrylamide or poly-N-vinylpyrrolidone; linear, branched or dendritic polyamidoamines (polyaminoamides); polyacrylic acid; polyalcohols (polyalchohols), such as polyvinyl alcohol and polyxylitol, to which carboxyl or amino groups are chemically bound, and gangliosides, such as ganglioside GM₁. Interpolymers of PEG, methoxy PEG, or methoxy PPG or derivatives thereof are also suitable. In addition, the opsonization-inhibiting polymer can be a block copolymer of PEG and any of a polyamino acid, a polysaccharide, a polyamidoamine, a polyethyleneamine, or a polynucleotide. The opsonization-inhibiting polymer can also be a natural polysaccharide containing amino acids or carboxylic acids, such as galacturonic acid, glucuronic acid, mannuronic acid, hyaluronic acid, pectic acid, neuraminic acid, alginic acid, carrageenan; aminated polysaccharides or oligosaccharides (linear or branched); or a carboxylated polysaccharide or oligosaccharide, for example, a carboxylated polysaccharide or oligosaccharide having a carboxyl group bonded thereto by reaction with a carbonic acid derivative.

Preferably, the opsonization-inhibiting moiety is PEG, PPG, or a derivative thereof. Liposomes modified with PEG or PEG derivatives are sometimes referred to as "pegylated liposomes".

The opsonization-inhibiting moiety can be bound to the liposome membrane by any of a number of well-known techniques. For example, the N-hydroxysuccinimide ester of PEG can be conjugated to a phosphatidylethanolamine lipid-soluble anchor (lipid-soluble anchor) and then conjugated to a membrane. Similarly, dextran polymers can be derivatized with stearamide lipid soluble anchors by reductive amination using Na (CN) BH₃And mixed solvents, such as a 30: 12 ratio mixture of tetrahydrofuran and water at 60 deg.C.

Vectors expressing the double-stranded molecules of the invention are discussed above. Such a vector expressing at least one double-stranded molecule of the invention may also be administered directly or in combination with suitable delivery agents including Mirus Transit LT1 lipophilic agents, Lipofectin, Lipofectamine, cellfectin, polycations (e.g. polylysine) or liposomes. Methods for delivering a recombinant viral vector expressing a double-stranded molecule of the invention to a cancer region of a patient are within the skill of the art.

The double-stranded molecules of the invention can be administered to a subject by any means suitable for delivering the double-stranded molecule to a cancer site. For example, the double-stranded molecule can be administered by gene gun, electroporation, or other suitable parenteral or enteral routes of administration.

Suitable enteral routes of administration include oral, rectal, or intranasal delivery.

Suitable routes of parenteral administration include intravascular administration (e.g., intravenous bolus, intravenous infusion, intra-arterial bolus, intra-arterial infusion, and catheter instillation to the vascular network), peri-and intra-tissue injection (e.g., peri-and intra-tumoral injection), subcutaneous injection or deposition, including subcutaneous infusion (e.g., using an osmotic pressure pump), direct application to the site of cancer or an area near it, e.g., via a catheter or other placement device (e.g., a suppository or implant comprising a porous, non-porous, or gelatinous material), and inhalation. Preferably, the double-stranded molecule or vector is administered to or near the cancer site by injection or infusion.

The double-stranded molecules of the invention may be administered in a single dose or in divided doses. When the administration of the double-stranded molecule of the invention is by infusion, the infusion may be a single continuous dose, or may be administered by multiple infusions. It is preferred to inject the agent directly into the tissue at or near the site of cancer. It is particularly preferred to inject the agent multiple times into the tissue at or near the site of the cancer.

One skilled in the art can readily determine an appropriate dosage regimen for administering a double-stranded molecule of the invention to a given subject. For example, the double-stranded molecule can be administered to the subject at one time, e.g., in a single injection or deposition, at or near the site of the cancer. Alternatively, the double stranded molecule may be administered to the subject once or twice daily for a period of about 3 to about 28 days, more preferably about 7 to about 10 days. In a preferred dosage regimen, the double stranded molecule can be injected once a day at or near the cancer site over a 7 day period. When the dosage regimen comprises multiple administrations, it is understood that the effective amount of the double stranded molecule administered to the subject may comprise the total amount of the double stranded molecule administered throughout the dosage regimen.

In the present invention, a cancer overexpressing CSTF2 may be treated with at least one active ingredient selected from the group consisting of:

(a) the double-stranded molecules of the present invention,

(b) DNA encoding them, and

(c) vectors encoding them.

Such cancers include, but are not limited to, lung cancer. Thus, prior to administration of the double-stranded molecule of the present invention as an active ingredient, it is preferably confirmed whether the expression level of CSTF2 in the cancer cell or tissue to be treated is increased as compared to normal cells of the same organ. Thus, in one embodiment, the present invention provides a method of treating a cancer that (over) expresses CSTF2, which method may comprise the steps of:

i) Detecting the expression level of CSTF2 in cancer cells or tissues obtained from a subject having a cancer to be treated;

ii) comparing said expression level of CSTF2 to a normal control; and are

iii) administering to a subject having a cancer that overexpresses CSTF2 as compared to a normal control at least one member selected from the group consisting of:

(a) the double-stranded molecules of the present invention,

(b) DNA encoding them, and

(c) vectors encoding them.

Alternatively, the present invention also provides a pharmaceutical composition comprising at least one ingredient selected from the group consisting of:

(a) the double-stranded molecules of the present invention,

(b) DNA encoding them, and

(c) vectors encoding them.

In other words, the invention further provides a method for identifying a subject to be treated with:

(a) the double-stranded molecules of the present invention,

(b) DNA encoding them, or

(c) The vectors encoding them are those which, when used,

the method may comprise the step of determining the expression level of CSTF2 in a cancer cell or tissue derived from a subject, wherein an increase in said level compared to the genonormal control is indicative of the subject having a cancer treatable by:

(a) the double-stranded molecules of the present invention,

(b) DNA encoding them, or

(c) Vectors encoding them.

The method of treating cancer according to the present invention will be described in more detail below.

The subject to be treated with the present method is preferably a mammal. Exemplary mammals include, but are not limited to, for example, humans, non-human primates, mice, rats, dogs, cats, horses, and cows.

According to the present invention, the expression level of CSTF2 in cancer cells or tissues obtained from a subject is determined. The expression level can be determined at the level of the transcription (nucleic acid) product using methods known in the art. For example, the mRNA of CSTF2 can be quantified using probes by hybridization methods (e.g., Northern hybridization). The detection may be performed on a chip or array. For detecting the expression level of CSTF2, the use of an array is preferred. The sequence information of CSTF2 can be used by one skilled in the art to prepare the above probes. For example, the cDNA of CSTF2 can be used as a probe. If desired, the probe may be labeled with a suitable label such as a dye, a fluorescent substance and an isotope, and the expression level of the gene may be detected as the intensity of the label to which hybridization has occurred.

Further, the transcript of CSTF2 (e.g., SEQ ID NO: 1) can be quantified by amplification-based detection methods (e.g., RT-PCR) using primers. Such primers can be prepared based on available sequence information of the gene.

Specifically, the probes or primers used in the present method hybridize to the mRNA of CSTF2 under stringent, medium or low stringency conditions. As used herein, the phrase "stringent (hybridization) conditions" refers to conditions under which a probe or primer will hybridize to its target sequence, but not to other sequences. Stringent conditions are sequence dependent and will be different in different circumstances. Specific hybridization of longer sequences is observed at higher temperatures than shorter sequences. Generally, the temperature of stringent conditions is selected to be about 5 ℃ lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of probes complementary to their target sequence hybridize to the target sequence at equilibrium. Since the target sequence is generally present in excess, at Tm, 50% of the probes are occupied at equilibrium. Typically, stringent conditions will be such that: wherein the salt concentration is less than about 1.0M sodium ion, typically about 0.01-1.0M sodium ion (or other salt), pH7.0-8.3, and the temperature is at least about 30 ℃ for shorter probes or primers (e.g., 10-50 nucleotides) and at least about 60 ℃ for longer probes or primers. Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide.

Or,the translation products can be detected for the diagnosis of the present invention. For example, the amount of CSTF2 protein (SEQ ID NO: 2) can be determined. Methods for determining the amount of a protein as a translation product include immunoassays that use an antibody that specifically recognizes the protein. The antibody may be monoclonal or polyclonal. Furthermore, any fragment or modification of an antibody (e.g., chimeric antibody, scFv, Fab, F (ab')₂Fv, etc.) can be used for detection, so long as the fragment or modified antibody retains binding ability to the CSTF2 protein. Methods for preparing these kinds of antibodies for detecting proteins are well known in the art, and any method may be used in the present invention to prepare these antibodies and their equivalents.

As another method for detecting the expression level of CSTF2 gene based on the translation product of CSTF2, the intensity of staining can be measured by immunohistochemical analysis using an antibody against CSTF2 protein. That is, in this measurement, strong staining indicates an increase in the presence/level of the protein and, at the same time, a high expression level of the CSTF2 gene.

For the expression level of a target gene (e.g., CSTF2 gene) in a cancer cell, the level may be determined to be increased if the level is increased, for example, by 10%, 25% or 50%, or by more than 1.1-fold, more than 1.5-fold, more than 2.0-fold, more than 5.0-fold, more than 10.0-fold, or more, as compared to a control level of the target gene (e.g., a level in a normal cell).

The control level can be determined concurrently with the cancer cells using samples previously collected and stored from subjects whose disease state (cancerous or non-cancerous) is known. In addition, normal cells obtained from a noncancerous region of an organ having the cancer to be treated can be used as a normal control. Alternatively, the control level may be determined by statistical methods based on results obtained by analyzing previously determined expression levels of the CSTF2 gene from samples from subjects with known disease states. Further, the control level may be an expression pattern database from previously tested cells. Also, according to one aspect of the invention, the expression level of the CSTF2 gene in the biological sample may be compared to a plurality of control levels determined from a plurality of reference samples. Preferably a control level determined from a reference sample from a tissue type similar to that of the subject-derived biological sample is used. Also, preferably, standard values for the expression levels of the CSTF2 gene in populations with known disease states are used. Standard values can be obtained by any method known in the art. For example, a range of the mean ± 2s.d. or the mean ± 3s.d. may be used as the standard value.

In the context of the present invention, a control level determined from a biological sample known to be non-cancerous is referred to as a "normal control level". On the other hand, if the control level is determined from a cancerous biological sample, it is referred to as a "cancerous control level".

If the expression level of the CSTF2 gene is increased compared to a normal control level or is similar/equivalent to a cancerous control level, the subject may be diagnosed as having the cancer to be treated.

Compositions comprising the double-stranded molecules of the invention:

in addition to the above, the present invention also provides pharmaceutical compositions comprising at least one double-stranded molecule of the invention or a vector encoding such a molecule. Specifically, the present invention provides the following compositions [1] to [34 ]:

[1] a composition for inhibiting the growth of cancer cells and/or treating or preventing cancer, wherein said composition comprises at least one isolated double-stranded molecule directed to the CSTF2 gene or a vector encoding the double-stranded molecule, wherein the double-stranded molecule, when introduced into a cell, inhibits expression of CSTF2 and cell proliferation, wherein the double-stranded molecule comprises a sense strand and an antisense strand complementary thereto, which hybridize to each other to form the double-stranded molecule, said composition further comprising a pharmaceutically acceptable carrier;

[2] [1] wherein the double-stranded molecule acts on mRNA that hybridizes to a nucleic acid sequence selected from the group consisting of SEQ ID NO: 9 and 10 target sequence match;

[3] the composition of [1], wherein the double stranded molecule, wherein the sense strand comprises a double-stranded sequence identical to a sequence selected from SEQ id nos: 9 and 10.

[4] The composition of [1], wherein the composition comprises a plurality of the double-stranded molecules;

[5] the composition of any of [1] to [3], wherein the sense strand and the antisense strand of the double-stranded molecule hybridize at the target sequence to form a double-stranded molecule that is less than about 100 nucleotide pairs in length;

[6] the composition of [5], wherein the sense strand and the antisense strand of the double-stranded molecule hybridize at the target sequence to form a double-stranded molecule that is less than about 75 nucleotide pairs in length;

[7] the composition of [6], wherein the sense strand and the antisense strand of the double-stranded molecule hybridize at the target sequence to form a double-stranded molecule that is less than about 50 nucleotide pairs in length;

[8] the composition of [7], wherein the sense strand and the antisense strand of the double-stranded molecule hybridize at the target sequence to form a double-stranded molecule that is less than about 25 nucleotide pairs in length;

[9] the composition of [8], wherein the sense strand and the antisense strand of the double-stranded molecule hybridize at the target sequence to form a double-stranded molecule that is about 19 to about 25 nucleotide pairs in length;

[10] The composition of any of [1] to [9], wherein the double-stranded molecule consists of a single polynucleotide comprising a sense strand and an antisense strand linked together by an intervening single strand;

[11] [10] wherein the double stranded molecule has the general formula 5 ' - [ A ] - [ B ] - [ A ' ] -3 ', wherein [ A ] is a double stranded molecule comprising a double stranded sequence substantially identical to a sequence selected from the group consisting of SEQ ID NOs: 9 and 10, B is an intervening single strand consisting of 3 to 23 nucleotides, and A' is an antisense strand comprising a sequence complementary to the target sequence selected from [ A ];

[12] the composition of any one of [1] to [11], wherein the double-stranded molecule is RNA;

[13] the composition of any one of [1] to [11], wherein the double-stranded molecule is DNA and/or RNA;

[14] the composition of [13], wherein the double-stranded molecule is a hybrid of a DNA polynucleotide and an RNA polynucleotide;

[15] [14] the composition, wherein the sense strand polynucleotide and the antisense strand polynucleotide consist of DNA and RNA, respectively;

[16] the composition of [13], wherein the double-stranded molecule is a chimera of DNA and RNA;

[17] the composition of [16], wherein the region flanking the 3 ' end of the antisense strand consists of RNA, or the region flanking the 5 ' end of the sense strand and the region flanking the 3 ' end of the antisense strand consist of RNA;

[18] The composition of [17], wherein the flanking region consists of 9 to 13 nucleotides;

[19] the composition of any of [1] to [18], wherein the double-stranded molecule comprises one or two 3' overhangs;

[20] the composition of any one of [1] to [19], wherein the composition further comprises a transfection-enhancing agent.

[21] The composition of any of [1] to [20], wherein the double-stranded molecule is encoded by a vector and contained in the composition;

[22] [21] wherein the double-stranded molecule encoded by the vector acts on mRNA that hybridizes with a nucleic acid sequence selected from the group consisting of SEQ ID NO: 9 and 10 target sequence match;

[23] [21] wherein the sense strand of the double stranded molecule encoded by the vector comprises a nucleotide sequence that is identical to a sequence selected from the group consisting of SEQ ID NO: 9 and 10;

[24] the composition of any of [21] to [23], wherein a plurality of the double-stranded molecules are administered;

[25] the composition of any of [21] to [24], wherein the sense strand and the antisense strand of the double-stranded molecule hybridize at the target sequence to form a double-stranded molecule that is less than about 100 nucleotide pairs in length;

[26] the composition of [25], wherein the sense strand and the antisense strand of the double-stranded molecule hybridize at the target sequence to form a double-stranded molecule that is less than about 75 nucleotide pairs in length;

[27] The composition of [26], wherein the sense strand and the antisense strand of the double-stranded molecule hybridize at the target sequence to form a double-stranded molecule that is less than about 50 nucleotide pairs in length;

[28] the composition of [27], wherein the sense strand and the antisense strand of the double-stranded molecule hybridize at the target sequence to form a double-stranded molecule that is less than about 25 nucleotide pairs in length;

[29] the composition of [28], wherein the sense strand and the antisense strand of the double-stranded molecule hybridize at the target sequence to form a double-stranded molecule that is about 19 to about 25 nucleotide pairs in length;

[30] the composition of any one of [21] to [29], wherein the double-stranded molecule encoded by the vector consists of a single polynucleotide comprising both a sense strand and an antisense strand linked together by an intervening single strand;

[31] [30] wherein the double stranded molecule has the general formula 5 ' - [ A ] - [ B ] - [ A ' ] -3 ', wherein [ A ] is a double stranded molecule comprising a double stranded sequence substantially identical to a sequence selected from the group consisting of SEQ ID NOs: 9 and 10, B is an intervening single strand consisting of 3 to 23 nucleotides, and A' is an antisense strand comprising a sequence complementary to the target sequence selected from [ A ];

[32] The composition of any one of [21] to [31], wherein the composition further comprises a transfection-enhancing agent;

[33] the composition of any one of [1] to [32], wherein the cancer is lung cancer. And

[34] [33] the composition of, wherein the lung cancer is adenocarcinoma, squamous cell carcinoma, large cell carcinoma or small cell lung cancer.

Suitable compositions of the present invention are described in more detail below.

The double-stranded molecules of the invention are preferably formulated into pharmaceutical compositions prior to administration to a subject according to techniques well known in the art. The pharmaceutical compositions of the present invention are characterized as being at least sterile and pyrogen free. As used herein, "pharmaceutical formulation" includes formulations suitable for use in human and veterinary medicine. Methods for preparing the Pharmaceutical compositions of the present invention are within the ordinary skill in the art, and are described, for example, in Remington's Pharmaceutical Science, 17th ed., Mack Publishing Company, Easton, Pa. (1985), the entire disclosure of which is incorporated herein by reference.

The pharmaceutical formulations of the invention comprise at least one double-stranded molecule of the invention or a carrier encoding them (for example, from 0.1% to 90% by weight), or a physiologically acceptable salt of said molecule, in admixture with a physiologically acceptable carrier medium. The physiologically acceptable carrier medium is preferably water, buffered water, physiological saline, 0.4% saline, 0.3% glycine, hyaluronic acid and the like.

According to the invention, the composition may comprise a plurality of double-stranded molecules, each of which may be directed against a different target sequence of the same gene or a different target sequence of a different gene. For example, the composition may comprise a double-stranded molecule directed against a CSTF2 gene target sequence. Alternatively, for example, the composition may comprise a double stranded molecule directed against one, two or more target sequences of the CSTF2 gene and other genes.

Furthermore, the present compositions may comprise a vector encoding 1 or more double stranded molecules. For example, the vector may encode 1, 2 or several instant double-stranded molecules. Alternatively, the present compositions may comprise a plurality of vectors, each encoding a different double-stranded molecule.

Furthermore, the instant double-stranded molecules may be included in the present compositions as liposomes. Details of liposomes can be found in the section "method for inhibiting or reducing cancer cell growth or treating cancer using the double-stranded molecules of the invention".

The composition of the invention may be a pharmaceutical composition. The pharmaceutical compositions of the present invention may also contain conventional pharmaceutical excipients and/or additives. Suitable pharmaceutical excipients include stabilizers, antioxidants, osmotic pressure regulators, buffers, and pH regulators. Suitable additives include: a physiologically biocompatible buffer (e.g., trometamol hydrochloride), supplemental chelator (e.g., DTPA or DTPA-bisamide, etc.), or a calcium chelator complex (e.g., calcium DTPA, CaNaDTPA-bisamide), or, optionally, supplemental calcium or sodium salt (e.g., calcium chloride, calcium ascorbate, calcium gluconate, or calcium lactate). The pharmaceutical compositions of the present invention may be packaged for use as a liquid or may be lyophilized.

For solid compositions, conventional non-toxic solid carriers may be used; for example, pharmaceutical grades of mannitol, lactic acid, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.

For example, any of the carriers and excipients listed above, as well as 10-95%, preferably 25-75% of one or more double stranded molecules of the invention may be included in a solid pharmaceutical composition for oral administration. Pharmaceutical compositions for aerosol (inhalation) administration may comprise 0.01-20 wt.%, preferably 1-10 wt.%, of one or more of the double-stranded molecules of the invention coated in liposomes as described above, and a propellant. Carriers such as lecithin for intranasal delivery and the like may also be included as desired.

In addition to the above, other pharmaceutically active ingredients may be included in the present compositions, as long as they do not inhibit the in vivo function of the present double-stranded molecule. For example, the above composition may contain a chemotherapeutic agent conventionally used for cancer treatment.

In another embodiment, the invention also provides the use of a double stranded nucleic acid molecule of the invention in the preparation of a pharmaceutical composition for the treatment of lung cancer characterized by expression of CSTF 2. For example, the present invention relates to the use of the following double stranded nucleic acid molecules for the preparation of a pharmaceutical composition for the treatment of lung cancer expressing CSTF 2: the molecule inhibits expression of the CSTF2 gene in a cell, and the molecule comprises a sense strand and an antisense strand complementary thereto, which hybridize to each other to form the double-stranded nucleic acid molecule and which is expressed as a nucleotide sequence selected from the group consisting of SEQ id nos: 9 and 10 are targeted.

Alternatively, the present invention further provides the double stranded nucleic acid molecule of the invention for use in the treatment of lung cancer expressing the CSTF2 gene.

In addition, the present invention provides a method or process for the manufacture of a pharmaceutical composition for the treatment of lung cancer characterized in part by the expression of CSTF 2. The method or process comprises the step of formulating a pharmaceutically or physiologically acceptable carrier with, as an active ingredient, a double-stranded nucleic acid molecule that inhibits expression of CSTF2 in a cell and that comprises a sense strand and an antisense strand complementary thereto, which hybridize to each other to form the double-stranded nucleic acid molecule and which is expressed as a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 9 and 10 are targeted.

In another embodiment, the present invention also provides a method or process for the manufacture of a pharmaceutical composition for the treatment of lung cancer characterized by expression of CSTF2, wherein said method or process comprises the step of admixing an active ingredient with a pharmaceutically or physiologically acceptable carrier, wherein said active ingredient is a double stranded nucleic acid molecule that inhibits expression of CSTF2 in a cell overexpressing the CSTF2 gene, the molecule comprising a sense strand and an antisense strand complementary thereto, which hybridize to each other to form a double stranded nucleic acid molecule and are separated from each other by a linker sequence selected from the group consisting of SEQ ID NOs: 9 and 10 are targeted.

Method for detecting or diagnosing lung cancer

Expression of the CSTF2 gene was found to be specifically elevated in lung cancer cells (FIGS. 1A-1C). Thus, the genes identified herein and their transcription and translation products can be used as markers for lung cancer and lung cancer can be diagnosed by measuring the expression of the CSTF2 gene in a cell sample. In particular, the present invention provides methods for diagnosing lung cancer by determining the expression level of CSTF2 in a subject. Lung cancers that can be diagnosed by the present methods include NSCLC and SCLC. Further, NSCLC, including lung Adenocarcinoma (ADC), lung Squamous Cell Carcinoma (SCC), and Large Cell Carcinoma (LCC), can also be diagnosed or detected by the present invention.

According to the present invention, intermediate results for examining the condition of a subject may be provided. The intermediate results may be combined with other information to assist a physician, nurse or other practitioner in diagnosing that the patient is suffering from the disease. That is, the present invention provides the diagnostic marker CSTF2 for the examination of cancer. Alternatively, the present invention provides a method for detecting or identifying cancer cells in a subject-derived lung tissue sample, said method comprising the step of determining the expression level of CSTF2 gene in a subject-derived biological sample, wherein an increase in said expression level compared to a normal control level of said gene is indicative for or suspected of the presence of cancer cells in the tissue. Such results may be combined with additional information to aid a doctor, nurse, or other healthcare practitioner in diagnosing that a subject has a disease. In other words, the present invention can provide useful information to a physician to diagnose a subject with a disease. For example, in accordance with the present invention, when there is a question regarding the presence of cancer cells in tissue obtained from a subject, clinical decisions can be made by considering the expression level of the CSTF2 gene, plus other different aspects of the disease, including histopathology, levels of known tumor markers in the blood, and the clinical course of the subject, among others. For example, some well-known diagnostic lung tumor markers in blood are IAP, ACT, BFP, CA19-9, CA50, CA72-4, CA130, CEA, KMO-1, NSE, SCC, SP1, Span-1, TPA, CSLEX, SLX, STN, and CYFRA. That is, in this embodiment of the invention, the results of the gene expression analysis serve as intermediate results for further diagnosing the disease state in the subject.

Specifically, the present invention provides the following processes [1] to [11 ]:

[1] a method of diagnosing lung cancer or a predisposition for developing lung cancer in a subject, comprising the steps of:

(a) detecting the expression level of the CSTF2 gene in a biological sample derived from the subject;

(b) correlating the detected increase in expression level compared to a normal control level for the gene with the presence of disease in the subject;

[2] [1] the method, wherein the expression level is at least 10% higher than a normal control level;

[3] the method of [1] or [2], wherein the expression level is detected by a method selected from the group consisting of:

(a) detecting mRNA encoded by the CSTF2 gene;

(b) detecting the protein encoded by the CSTF2 gene;

(c) detecting the biological activity of the protein encoded by the CSTF2 gene;

[4] the method of [3], wherein the expression level is determined by detecting hybridization of a probe to mRNA encoded by CSTF2 gene;

[5] the method of [3], wherein the expression level is determined by detecting binding of an antibody against the protein encoded by CSTF2 gene and the protein encoded by CSTF2 gene;

[6] the method of any one of [1] to [5], wherein the biological sample comprises a biopsy sample, sputum or blood.

[7] The method of any one of [1] to [6], wherein the patient-derived biological sample comprises epithelial cells.

[8] The method of any one of [1] to [7], wherein the patient-derived biological sample comprises cancerous cells.

[9] The method of [8], wherein the subject-derived biological sample comprises cancerous epithelial cells.

[10] The method of any one of [1] to [9], wherein the subject-derived biological sample comprises lung tissue or lung cells.

[11] The method of any one of [1] to [10], wherein the lung cancer is NSCLC or SCLC.

Methods for diagnosing lung cancer are described in more detail below.

The subject diagnosed by the present method is preferably a mammal. Examples of mammals include, but are not limited to, for example, humans, non-human primates, mice, rats, dogs, cats, horses, and cows.

To carry out the diagnosis, a biological sample is preferably taken from the subject to be diagnosed. Any biological material can be used as the biological sample for the assay as long as it includes the target transcription or translation product of the CSTF2 gene. Such biological samples include, but are not limited to, body tissues and fluids, such as blood, sputum, pleural effusion and urine, that are intended to be diagnosed or suspected of having cancer. The biological sample preferably contains a population of cells that includes epithelial cells, more preferably cancerous epithelial cells or epithelial cells derived from tissue suspected to be cancerous. Further, if necessary, the cells can be purified from the obtained body tissues and body fluids and used as a biological sample. In a preferred embodiment, the biological sample contains lung tissue or lung cells. Such biological samples may be obtained by collecting lung tissue or lung cells from a region of the subject's lung suspected of being cancerous, e.g., by biopsy.

According to the invention, the expression level of the CSTF2 gene in the patient-derived biological sample is determined. Expression levels can be determined at the level of the transcript (mRNA) using methods well known in the art. For example, mRNA of the CSTF2 gene can be quantified by hybridization methods (e.g., Northern hybridization) using probes. The detection may be performed on a chip or array. For detecting the expression levels of multiple genes (e.g., multiple cancer specific genes), including the CSTF2 gene, it is preferred to use an array. The sequence information of the CSTF2 gene (SEQ ID NO: 1; GenBank accession No.: NM-001325) can be used by those skilled in the art to prepare the above-described probes. For example, a cDNA of CSTF2 gene can be used as a probe. If desired, the probe may be labelled with a suitable label, for example a dye, fluorescence or an isotope, and the level of expression of the gene may be detected as the intensity of the label at which hybridisation occurs.

Further, the transcript of the CSTF2 gene can be quantified by amplification-based detection techniques (e.g., RT-PCR) using primers. The above primers can also be prepared based on known sequence information of the gene. For example, the primers or probes used in the examples (SEQ ID NOS: 3, 4, 7 and 8) can be used for detection by RT-PCR or Northern blotting, but the present invention is not limited thereto.

Specifically, the probes or primers used in the present method hybridize to the mRNA of the CSTF2 gene under stringent conditions, medium stringent conditions, and low stringent conditions. As defined above, the phrase "stringent (hybridization) conditions" refers to conditions under which a probe or primer will hybridize to its target sequence, but not to other sequences. Stringent conditions are sequence dependent and will vary under different circumstances. Specific hybridization of longer sequences occurs at higher temperatures than shorter sequences. Generally, the temperature of stringent conditions is selected to be about 5 ℃ below the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium. Since the target sequence is generally present in excess, at Tm, 50% of the probes are occupied at equilibrium. Typically, stringent conditions are such that: wherein the salt concentration is less than about 1.0M sodium ion, typically about 0.01-1.0M sodium ion (or other salt), pH7.0-8.3, and the temperature is at least about 30 ℃ for shorter probes or primers (e.g., 10-50 nucleotides) and at least about 60 ℃ for longer probes or primers. Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.

Alternatively, the diagnosis of the present invention can be carried out by detecting the translation product. For example, the amount of CSTF2 protein can be determined. Methods of determining the amount of a protein as a translation product include immunoassays, and such methods use antibodies that specifically recognize the protein. The antibody may be monoclonal or polyclonal. Moreover, any fragment or modification of the antibody (e.g., chimeric antibody, scFv, Fab, F (ab') 2, Fv, etc.) can be used for detection, so long as the fragment retains the binding ability to the CSTF2 protein. Methods for preparing these types of antibodies for detecting proteins are well known in the art, and any method can be used to prepare these antibodies and their equivalents in the present invention.

As another method for detecting the gene thereof based on the translation product of CSTF2, the intensity of staining thereof can be observed by immunohistochemical analysis using an antibody against CSTF2 protein. That is, the observation of strong staining indicates an increase in the presence of the protein and at the same time a high expression level of CSTF 2.

In addition, in addition to the expression level of the CSTF2 gene, the expression level of other cancer-related genes, such as genes known to be differentially expressed in lung cancer, can also be determined to improve the accuracy of the diagnosis.

For a cancer marker gene, including the CSTF2 gene, its expression level in a biological sample is considered to be increased if it is increased, for example, by 10%, 25%, or 50% compared to the control level of the corresponding cancer marker gene, or by more than 1.1-fold, more than 1.5-fold, more than 2.0-fold, more than 5.0-fold, more than 10-fold, or more.

The control level can be determined simultaneously with the testing of the biological sample, using samples previously collected and stored from subjects whose disease state (cancerous or non-cancerous) is known. Alternatively, the control level may be determined by statistical methods based on results obtained by analyzing previously determined expression levels of the CSTF2 gene from samples from subjects with known disease states. Further, the control level may be an expression pattern database from previously tested cells. Also, according to one aspect of the invention, the expression level of the CSTF2 gene in the biological sample may be compared to a plurality of control levels determined from a plurality of reference samples. Preferably a control level determined from a reference sample from a tissue type similar to the tissue type of the sample derived from the patient is used. Also, preferably, standard values for the expression levels of the CSTF2 gene in populations with known disease states are used. Standard values can be obtained by any method known in the art. For example, a range of the mean +/-2S.D. or the mean +/-3S.D. can be used as standard values.

In the context of the present invention, a control level determined from a biological sample known to be non-cancerous is referred to as a "normal control level". On the other hand, if the control level is determined from a cancerous biological sample, it is referred to as a "cancerous control level".

When the expression level of the CSTF2 gene is increased compared to the normal expression level or is similar to a cancerous control level, the subject may be diagnosed as suffering from or at risk of developing cancer. Further, when comparing the expression levels of a plurality of cancer-associated genes, the similarity in gene expression pattern between the sample and the cancerous reference indicates that the subject is suffering from or at risk of developing cancer.

The difference between the expression level of the test biological sample and the control level can be normalized to the expression level of a control nucleic acid (e.g., housekeeping gene) whose expression level is known not to change with the cancerous or non-cancerous state of the cell. Exemplary control genes include, but are not limited to, β -actin, glyceraldehyde-3-phosphate dehydrogenase, and ribosomal protein P1.

Alternatively, the present invention provides the use of an agent for the preparation of a diagnostic agent for the diagnosis of cancer. In some embodiments, the agent may be selected from the group consisting of:

(a) reagents for detecting mRNA of CSTF2 gene;

(b) Reagents for detecting CSTF2 protein; and

(c) reagents for detecting the biological activity of CSTF2 protein.

In particular, such reagents are oligonucleotides that hybridize to CSTF2 polynucleotides or antibodies that bind to CSTF2 polypeptides.

In the present invention, it is disclosed that CSTF2 is not only a useful diagnostic marker, but also a suitable target for cancer therapy. Thus, cancer therapy targeting CSTF2 can be achieved by the present invention. In the present invention, cancer therapy targeting CSTF2 refers to suppression or inhibition of CSTF2 activity and/or expression in cancer cells. Any anti-CSTF 2 agent can be used for cancer therapy targeting CSTF 2. In the present invention, the anti-CSTF 2 agent includes the following as active ingredients:

(a) the double-stranded molecules of the present invention,

(b) DNA encoding it, and

(c) a vector encoding the same.

Thus, in a preferred embodiment, the present invention provides the following process: (i) diagnosing whether a subject has a cancer to be treated with an anti-CSTF 2 agent, and/or (ii) selecting a subject for cancer treatment targeting CSTF2, the method comprising the steps of:

(a) determining the expression level of the CSTF2 gene in cancer cells or tissues obtained from a subject suspected of having a cancer to be treated;

(b) Comparing the expression level of the CSTF2 gene with a normal control level;

(c) diagnosing the subject as having a cancer to be treated if the expression level of CSTF2 is increased as compared to a normal control level; and are

(d) Selecting the subject for cancer treatment if the subject is diagnosed in step (c) as having cancer to be treated.

Alternatively, such methods comprise the steps of:

(a) determining the expression level of the CSTF2 gene in cancer cells or tissues obtained from a subject suspected of having a cancer to be treated;

(b) comparing the expression level of the CSTF2 gene to a cancerous control level;

(c) diagnosing the subject as having the cancer to be treated if the expression level of the CSTF2 gene is similar or equivalent to a cancerous control level; and are

(d) Selecting the subject for cancer treatment if the subject is diagnosed in step (c) as having cancer to be treated.

Method for assessing cancer prognosis

The present invention relates in part to the novel finding that CSTF2 expression is significantly associated with poor prognosis in patients. Accordingly, the present invention provides a method for determining or assessing the prognosis of a patient suffering from cancer, particularly lung cancer, by detecting the expression level of CSTF2 in a biological sample from the patient; comparing the measured expression level to a control level; and determining an elevated expression level compared to the control level as an indication of a poor prognosis (poor survival). In particular, the present invention provides a method of assessing or determining the prognosis of a subject having lung cancer, the method comprising the steps of:

(a) Detecting the expression level of the CSTF2 gene in a biological sample derived from the subject;

(b) comparing the detected expression level to a control level; and are

(c) Determining a prognosis for the subject based on the comparison of (b).

Herein, the term "prognosis" refers to the possible outcome as well as the prospect of recovery from the disease as indicated by the nature and symptoms of the case with respect to the disease. Accordingly, adverse, negative, poor prognosis is defined as lower post-treatment survival duration and survival rate. Conversely, a positive, favorable, or good prognosis is defined as an increase in survival or survival after treatment.

The term "assessing prognosis" refers to the ability to predict, or correlate certain measures or measures with the future outcome of a patient's cancer (e.g., malignancy, likelihood of curing the cancer, survival rate, etc.). For example, determining the expression level of the CSTF2 gene over time makes it possible to predict patient outcome (e.g., increase or decrease in malignancy, increase or decrease in cancer grade, likelihood of curing cancer, survival rate, etc.).

In the context of the present invention, the phrase "assessing (or determining) prognosis" is intended to cover the prediction and likelihood analysis of cancer, progression, in particular cancer recurrence, metastatic spread and disease recurrence. The methods of assessing prognosis herein are intended for clinical use in making decisions regarding treatment modalities, including therapeutic intervention, diagnostic criteria such as staging of disease, and disease monitoring and surveillance for metastasis and recurrence of neoplastic disease.

The patient-derived biological sample used in the present method may be any sample derived from the subject to be evaluated, as long as the CSTF2 gene can be detected in the sample. The biological sample is preferably a lung cell (a cell obtained from the lung). Further, the biological sample may include a bodily fluid such as sputum, blood, serum, or plasma. In addition, the sample may be cells purified from a tissue. Biological samples can be obtained from a patient at various points in time, including before, during, and/or after treatment. For example, lung cancer cells obtained from a subject to be evaluated are preferred biological samples.

According to the present invention, it was shown that the higher the expression level of the CSTF2 gene in the patient-derived biological sample, the worse the prognosis of disease remission, recovery and/or survival after treatment and the higher the likelihood of adverse clinical consequences. Thus, according to the present methods, the "control level" used as a comparison may be, for example, the expression level of any CSTF2 gene in an individual or a population of individuals that shows a good or positive prognosis for cancer after treatment, referred to herein as the "good prognosis control level". In addition, the "control level" can be, for example, the expression level of any CSTF2 gene in an individual or a population of individuals who exhibit a poor or negative prognosis for cancer after treatment, referred to herein as a "poor prognosis control level". The "control level" is a single expression pattern derived from a single reference population, or a plurality of expression patterns. Thus, the control level may be determined based on the expression level of the CSTF2 gene prior to any kind of treatment in a cancer patient or population of cancer patients whose disease state (good or poor prognosis) is known. In the context of the present invention, the cancer is lung cancer. It is preferable to use a standard value for the expression level of the CSTF2 gene in a patient group whose disease state is known. The standard value can be obtained by any method known in the art. For example, a range of +/-2 standard deviations from the mean or +/-3 standard deviations from the mean may be used as standard values.

The control level can be determined simultaneously with the biological sample being tested by using samples previously collected and stored from patients (control or control group) whose disease state (good or poor prognosis) is known prior to receiving any kind of treatment.

Alternatively, the control level may be determined by statistical methods based on the expression level of the CSTF2 gene by analyzing samples previously collected or stored from a control group. Further, the control level may be a database of expression patterns from previously tested cells.

Additionally, according to one aspect of the invention, the expression level of the CSTF2 gene in the biological sample can be compared to a plurality of control levels, which are determined from a plurality of reference samples. Preferably, a control level determined from a reference sample obtained from a tissue type similar to the patient-derived biological sample is used.

According to the present invention, the similarity of the expression level of the CSTF2 gene to a good prognosis control level indicates a more desirable prognosis for the patient, while an increase in the expression level relative to a good prognosis control level indicates a less desirable, less desirable prognosis for post-treatment remission, recovery, survival and/or clinical outcome. On the other hand, a decrease in the expression level of CSTF2 as compared to a poor prognosis control level indicates a more desirable prognosis for the patient, while an expression level similar to the poor prognosis control level indicates a less desirable, less desirable prognosis for the remission, recovery, survival and/or clinical outcome after treatment. For example, lung cancer cells obtained from a subject who shows a better or poorer prognosis of cancer after treatment are preferred biological samples at a better or worse prognosis control level, respectively.

The expression level of CSTF2 gene in a biological sample is considered altered when the expression level varies more than 1.0, 1.5, 2.0, 5.0, 10.0 or more fold relative to the control level.

The difference in expression levels between the biological sample being tested and the control level can be normalized relative to a control, such as a housekeeping gene. For example, polynucleotides whose expression levels are known to be invariant in cancerous and non-cancerous cells, including those encoding β -actin, glyceraldehyde-3-phosphate dehydrogenase, and ribosomal protein P1, can be used to normalize CSTF2 gene expression levels.

Expression levels can be determined by detecting gene transcripts in patient-derived biological samples using techniques well known in the art. The gene transcripts detected by the present method include both transcription and translation products, such as mRNA and protein.

For example, the transcription product of the CSTF2 gene can be detected by hybridization, such as Northern blot hybridization analysis, using a CSTF2 gene probe directed against the gene transcript. The detection may be performed on a chip or array. For detecting the expression level of the CSTF2 gene, an array is preferably used. As another example, amplification-based detection methods, such as reverse transcription-based polymerase chain reaction using primers specific for the CSTF2 gene, can be used for detection (see examples). CSTF2 gene specific probes or primers can be designed and prepared using conventional techniques with reference to the full sequence of the CSTF2 gene (SEQ ID NO: 1). For example, the primers (SEQ ID NOS: 3 and 4) used in the examples can be used for detection by RT-PCR, but the present invention is not limited thereto.

Specifically, the probes or primers used in the present method hybridize to the mRNA of CSTF2 under stringent, medium or low stringency conditions.

Alternatively, the evaluation of the present invention may be carried out by detecting the translation product. For example, the amount of CSTF2 protein can be determined. The method of determining the amount of protein as a translation product includes an immunoassay method using an antibody specifically recognizing CSTF2 protein. The antibody may be monoclonal or polyclonal. Further, fragments or modifications of any of the antibodies (e.g., chimeric antibodies, scFv, Fab, F (ab') 2, Fv, etc.) can be used for detection, so long as the fragment retains the binding ability to the CSTF2 protein. Methods for making such antibodies for detecting proteins are well known in the art, and any method can be used in the present invention to make such antibodies and equivalents thereof.

As another method for detecting the expression level of the CSTF2 gene based on the translation product of the gene, the intensity of staining thereof can be observed by immunohistochemical analysis using an antibody against the CSTF2 protein. That is, strong staining was observed indicating an increase in the amount of CSTF2 present and, at the same time, a high expression level of the CSTF2 gene.

Further, the CSTF2 protein is known to have cell proliferation activity. Therefore, the expression level of the CSTF2 gene can be determined using the above cell proliferation activity as an index. For example, cell proliferation activity of a biological sample can be determined by preparing and culturing cells expressing CSTF2 in the presence of the biological sample and then measuring the proliferation rate or measuring the cell cycle or colony forming ability.

In addition, in addition to the expression level of the CSTF2 gene, the expression level of other lung cancer-related genes, such as genes known to be differentially expressed in lung cancer, can also be determined to improve the accuracy of the assessment. Examples of such other lung cell-associated genes include those described in WO2004/031413 and WO 2005/090603. The contents of which are incorporated herein by reference.

Alternatively, according to the present invention, intermediate results for assessing the prognosis of a subject may be provided, in addition to other test results. The intermediate results may assist a physician, nurse or other practitioner in evaluating, determining or estimating the prognosis of the subject. Other information that may be considered in connection with the intermediate results obtained by the present invention include the clinical symptoms and physical condition of the subject.

In other words, the expression level of the CSTF2 gene is a useful prognostic marker for assessing, predicting or determining the prognosis of a subject suffering from lung cancer (e.g., NSCLC). Accordingly, the present invention also provides a method for detecting a prognostic marker for assessing, predicting or determining the prognosis of a subject suffering from lung cancer (including NSCLC), comprising the steps of:

a) detecting or determining the expression level of CSTF2 gene in a biological sample derived from the subject, and

b) correlating the expression level detected or determined in step a) with the prognosis of the subject.

In particular, according to the present invention, an elevated expression level compared to a control level indicates a likelihood or suspicion of a poor prognosis (poor survival).

The patient whose prognosis of cancer is to be assessed according to the method is preferably a mammal, including humans, non-human primates, mice, rats, dogs, cats, horses and cattle.

Alternatively, the invention provides the use of an agent for the preparation of a reagent for the assessment of the prognosis of cancer. In some embodiments, the agent is selected from the group consisting of:

(a) reagents for detecting mRNA of CSTF2 gene;

(b) reagents for detection of CSTF 2; and

(c) reagents for detecting the biological activity of CSTF2 protein.

In particular, such reagents are oligonucleotides that hybridize to CSTF2 polynucleotides or antibodies that bind to CSTF2 polypeptides.

Kit for diagnosing cancer or assessing cancer prognosis:

the present invention provides kits for diagnosing cancer or assessing the prognosis of cancer. Alternatively, the invention also provides kits for determining a subject suffering from a cancer treatable with a double-stranded molecule of the invention or a vector encoding it, which may also be used to assess and/or monitor the effect of a cancer treatment. In a preferred embodiment, the cancer is lung cancer. In particular, the kit comprises at least one substance or reagent for detecting the expression of the CSTF2 gene in a biological sample derived from a patient, which substance may be selected from the group consisting of:

(a) a substance or reagent for detecting mRNA of CSTF2 gene;

(b) a substance or reagent that detects protein of CSTF 2;

(c) substances or reagents for detecting the biological activity of the protein of CSTF 2.

Suitable materials or reagents for detecting the mRNA of the CSTF2 gene include nucleic acids that specifically bind to or identify the CSTF2mRNA, such as oligonucleotides having a sequence complementary to a portion of the CSTF2 mRNA. Examples of such oligonucleotides are primers and probes specific for CSTF2 mRNA. Such oligonucleotides can be prepared based on methods well known in the art. If necessary, a substance or reagent for detecting CSTF2mRNA may be immobilized on a solid substrate. In addition, more than one substance or reagent for detecting CSTF2mRNA may be included in the kit.

The probe or primer may be of a particular size. The size is selected from the group consisting of: at least 10 nucleotides, at least 12 nucleotides, at least 15 nucleotides, at least 20 nucleotides, at least 25 nucleotides, at least 30 nucleotides, and probes and primers can range in size from 5-10 nucleotides, 10-15 nucleotides, 15-20 nucleotides, 20-25 nucleotides, and 25-30 nucleotides.

In another aspect, the substance or reagent suitable for detection of CSTF2 protein comprises an antibody directed against CSTF2 protein. The antibody may be monoclonal or polyclonal. Further, fragments or modifications of any of the antibodies (e.g., chimeric antibodies, scFv, Fab, F (ab')₂Fv, etc.) can be used as the substance or reagentAs long as the fragment retains the binding ability to the CSTF2 protein. Methods for making such antibodies for detecting proteins are well known in the art, and any method may be used in the present invention to make such antibodies and equivalents thereof. Further, the antibody may be labeled with a signal-generating molecule by direct linkage or indirect labeling techniques. Labels and labeled antibodies and methods of detecting binding of an antibody to its target are well known in the art, and any label and method can be used in the present invention. In addition, more than one substance or reagent for detecting CSTF2 protein may be included in the kit.

Further, the biological activity can be determined, for example, by measuring cell proliferation activity in the biological sample due to expression of CSTF2 protein. For example, the cell proliferation activity of a biological sample derived from a patient is determined by culturing the cells in the presence of the biological sample and then detecting the rate of proliferation, or measuring the cell cycle or colony forming ability. If desired, the substance or reagent for detecting CSTF2mRNA can be immobilized on a solid substrate. In addition, more than one substance for detecting the biological activity of the CSTF2 protein may be included in the kit.

The kit may comprise more than one of the foregoing substances or reagents. Further, the kit may include a solid substrate and a substance for binding a probe against CSTF2 gene or an antibody against CSTF2 protein, a medium and a container for culturing cells, positive and negative control substances or reagents, and a second antibody for detecting the antibody against CSTF2 protein. For example, a tissue sample obtained from a patient with a good or poor prognosis may be used as a useful control substance or reagent. The kits of the invention may further comprise other commercially and user desirable materials, including buffers, diluents, filters, needles, syringes, and packing slips with instructions for use (e.g., written, magnetic tape, CD-ROM, etc.). These substances or reagents and the like are contained in a container with a label. Suitable containers include bottles, vials (vitamins), and test tubes. The container may be made from a variety of materials, such as glass or plastic.

As an embodiment of the present invention, when the substance or reagent is a probe for CSTF2mRNA, the substance or reagent may be immobilized on a solid substrate such as a porous strip to form at least one detection site. The measurement or detection region of the porous strip may comprise a plurality of sites, each comprising a nucleic acid (probe). The test strip may also include sites for negative and/or positive controls. Alternatively, the control site may be located on a different strip than the test strip. Optionally, different detection sites may comprise different amounts of immobilized nucleic acid, i.e., a greater amount at a first detection site and a lesser amount at subsequent sites. After addition of the test sample, the number of sites displaying a detectable signal provides a quantitative indication of the amount of CSTF2mRNA present in the sample. The detection sites may be configured to have any suitably detectable shape, typically a stripe or dot across the width of the test strip.

The kit of the present invention may further comprise a positive control or CSTF2 standard sample. The positive control samples of the present invention can be prepared by collecting CSTF2 positive blood samples and subsequently determining their CSTF2 levels. Alternatively, purified CSTF2 protein or polynucleotide may be added to serum that does not contain CSTF2 to form the positive sample or CSTF2 standard.

Screening of anti-Lung cancer substance

In the context of the present invention, the substance to be identified by the present screening method may be any substance or a composition comprising several substances. Furthermore, the test substance exposed to the cells or proteins according to the screening method of the present invention may be a single substance or a combination of substances. When a combination of substances is used in the process, the substances may be contacted sequentially or simultaneously.

Any test substance, for example, cell extracts, cell culture supernatants, fermentation microbial products, marine organism extracts, plant extracts, purified or crude proteins, peptides, non-peptide substances, synthetic micromolecular substances (including nucleic acid constructs such as antisense RNA, siRNA, ribozymes, aptamers, and the like), and natural substances, can be used in the screening method of the present invention. The test substances of the present invention can also be obtained using any of a number of combinatorial library methods well known in the art, including (1) biological libraries, (2) spatially addressable parallel solid phase or liquid phase libraries, (3) synthetic library methods requiring deconvolution, (4) one-bead one-substance library methods, and (5) synthetic library methods using affinity chromatography selection. The biological library approach using affinity chromatography selection is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of substances (Lam (1997) Anticancer drug Des.12: 145-67). Examples of methods for synthesizing molecular libraries can be found in the prior art (DeWitt et al, Proc Natl Acad Sci USA 1993, 90: 6909-13; Erb et al, Proc Natl Acad Sci USA 1994, 91: 11422-6; Zuckermann et al, J Med Chem 37: 2678-85, 1994; Choet al, Sci 1993, 261: 1303-5; Carell et al, Angew Chem Int Ed Engl 1994, 33: 2059; Carell et al, Angew Chem Int Ed Engl 1994, 33: 2061; Gallop Chem et al 1994, JMed Chem 1994, 37: 1233-51). The substance libraries may be provided in solution (see Houghten, Bio/Techniques 1992, 13: 412-21) or on beads (Lam, Nature 1991, 354: 82-4), on chips (Fodor, Nature 1993, 364: 555-6), on bacteria (U.S. Pat. No. 5,223,409), on spores (U.S. Pat. No. 5,571,698; 5,403,484 and 5,223,409), on plasmids (Cull et al, ProcNatl Acad Sci USA 1992, 89: 1865-9) or on phages (Scott and Smith, Science 249, 1990: 386-90; Devlin, Science1990, 404: 6; Cwirla et al, Proc Natl Acad Sci USA 1990, 87: 6378-82; Felici, J Mol Biol 1991, 222: 301-10; U.S. patent application 2002103360).

Substances in which a part of the structure of a substance screened by any of the screening methods of the present invention is converted by addition, deletion, and/or substitution are included in the substances identified by the screening methods of the present invention.

Further, when the test substance to be screened is a protein, in order to obtain a DNA encoding the protein, the entire amino acid sequence of the protein may be determined to presume the nucleic acid sequence encoding the protein, or a partial amino acid sequence of the resulting protein may be analyzed, an oligo DNA prepared based on the sequence as a probe, and a cDNA library screened with the probe to obtain a DNA encoding the protein. The obtained DNA was confirmed to be useful for the preparation of a candidate test substance for the treatment or prevention of cancer.

The test sample useful for the screening described herein may also be an antibody that specifically binds to CSTF2 protein or a partial peptide thereof lacking the in vivo biological activity of the original protein.

Although the construction of test agent libraries is well known in the art, further guidance in identifying test substances and constructing libraries of such test substances for use in the present screening methods is provided below.

In the present invention, it is disclosed that repressing the expression level and/or biological activity of CSTF2 results in the repression of cancer cell growth. Thus, when an agent represses the expression and/or activity of CSTF2, the repression is indicative of a potential therapeutic effect in the subject. In the present invention, a potential therapeutic effect means having a reasonably expected clinical benefit. In the present invention, such clinical benefits include:

(a) The reduction in the expression of the CSTF2 gene,

(b) a reduction in the size, prevalence, or metastatic potential of the cancer in the subject,

(c) preventing the occurrence of cancer, or

(d) Preventing or alleviating the clinical symptoms of cancer.

(i) Molecular modeling:

knowledge of the molecular structure of the substance with the property of interest and/or the molecular structure of CSTF2 facilitates the construction of test agent libraries. One of the methods of prescreening test agents for suitability for further evaluation is computer modeling of the interaction of the test agent with its target.

Computer modeling techniques provide the possibility of visualizing the three-dimensional atomic structure of a selected molecule and the rational design of new substances that will interact with the molecule. Three-dimensional construction typically relies on data derived from x-ray crystal analysis or NMR imaging of selected molecules. Molecular dynamics require force field data. Computer graphics systems offer the possibility of predicting how new substances will attach to target molecules, and of manipulating the structure of substances and target molecules experimentally to perfect binding specificity. To predict what a molecule-substance interaction is when a small change in one or both of the molecule and substance occurs, molecular mechanics software and computationally intensive computers are required, which are typically coupled with a user-friendly, menu-driven interface between the molecular design program and the user.

One example of a molecular modeling system generally described above includes CHARMm and QUANTA programs, Polygen Corporation, Waltham, Mass. CHARMm performs energy minimization and molecular dynamics functions. QUANTA performs construction, graphical modeling and molecular structural analysis. Interactive construction, modification, visualization and analysis of molecular interactions can be performed using QUANTA.

There are several documents reviewing computer modeling of drugs interacting with specific proteins, such as rotivine et al acta Pharmaceutica Fennica 1988, 97: 159 to 66; ripka, NewScientist 1988, 54-8; McKinlay & Rossmann, Annu Rev Pharmacol Toxicol 1989, 29: 111-22; perry & Davies, Prog Clin Biol Res 1989, 291: 189-93; lewis & Dean, Proc R Soc Lond 1989, 236: 125-40, 141-62; and Askew et al, JAm Chem Soc 1989, 111: 1082-90.

Other computer programs that can screen and graphically describe chemicals are available from companies such as BioDesign, Inc., of Mississauga, Ontario, Canada, Pasadena, Calif., Allelix, Inc., Cambridge, Hypercube, Inc. of Ontario. See, e.g., DesJarlais et al, J Med Chem1988, 31: 722-9; meng et al, J Computer Chem 1992, 13: 505 to 24; meng et al, Proteins 1993, 17: 266 to 78; shoichet et al, Science 1993, 259: 1445-50.

Once a putative inhibitor is identified, any number of variants can be constructed based on the chemical structure of the identified putative inhibitor using combinatorial chemistry techniques, as described below. The resulting library of putative inhibitors, or "test substances," can be screened using the methods of the invention to identify test agents for treating or preventing lung cancer.

(ii) And (3) combinatorial chemical synthesis:

combinatorial libraries of test substances can be prepared as part of rational drug design programs that involve knowledge about the core structures present in known inhibitors. This strategy allows the library to be kept on a reasonable scale, facilitating high throughput screening. Alternatively, a simple, particularly short, polymeric molecular library can be constructed by simply synthesizing all permutations of the molecular families that make up the library. An example of the latter method is a peptide library consisting of all 6 amino acids in length. This peptide library contained all 6 amino acid sequence permutations. This type of library is called a linear combinatorial chemical library.

The preparation of combinatorial chemical libraries is well known to those skilled in the art and can be generated by chemical or biosynthetic means. Combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Pat. No. 5,010,175; Furka, Int J Pept Prot Res 1991, 37: 487-93; Houghten et al, Nature1991, 354: 84-6). Other chemistries for generating chemical diversity libraries may also be used. These include, but are not limited to, peptides (e.g., PCT publication WO 91/19735), encoded peptides (e.g., WO93/20242), random biological oligomers (e.g., WO 92/00091), benzodiazepines (e.g., U.S. Pat. No. 5,288,514), divermers such as hydantoins, benzodiazepines and dipeptides (DeWittet al, Proc Natl Acad Sci USA 1993, 90: 6909-13), polyalkyleneols (Villogous) polypeptides (Hagihara et al, J Amper Chem Soc 1992, 114: 6568), non-peptide mimetics having a glucose backbone (scaffold) (Hirschmann, J Amper Chem Soc 1992, 114: 9217-8), mimetic organic syntheses of small compound libraries (analogous organic synthesis of small compound libraries) (Chem., J. Ampter Chem. C1992., 261: 1303-D, 1303. and oligophosphoids) (peptide phosphonate, 1994: 2000, oligophosphoid et al, peptide phosphonate, peptide synthesis, peptide, JOrg Chem 1994, 59: 658) nucleic acid libraries (see augmentations of Ausubel, Current Protocols in molecular biology 1995; sambrook et al, Molecular Cloning: a Laboratory Manual, 1989, Cold Spring Harbor Laboratory, New York, USA), peptide nucleic acid libraries (see, e.g., U.S. patent 5,539,083), antibody libraries (see, e.g., Vaughan et al, Nature Biotechnology 1996, 14 (3): 309-14 and PCT/US96/10287), carbohydrate libraries (see e.g. Liang et al, Science1996, 274: 1520-22; us patent 5,593,853), and libraries of small organic molecules (see, e.g., benzodiazepines, Gordon em. curr Opin biotechnol.1995 Dec 1; 6(6): 624-31; isoprenoids (isoprenoids), U.S. patent 5,569,588; thiazolidinones (thiazolididinones) and thiamazanones (methiazanone), U.S. patent No. 5,549,974; pyrrolidines (pyrrolidines), U.S. Pat. nos. 5,525,735 and 5,519,134; morpholino compounds, U.S. patent No. 5,506,337; benzodiazepine, 5,288,514; etc.).

Devices for preparing combinatorial libraries are commercially available (see, e.g., 357MPS, 390MPS, Advanced Chem Tech, Louisville KY, Symphony, Rainin, Woburn, MA, 433AApplied Biosystems, Foster City, CA, 9050 Plus, Millipore, Bedford, MA). In addition, a variety of combinatorial libraries are commercially available per se (see, e.g., ComGenex, Princeton, n.j., Tripos, inc., st.louis, MO, 3D Pharmaceuticals, Exton, PA, Martek Biosciences, Columbia, MD, etc.).

(iii) Other candidates

Another approach is to use recombinant bacteriophages to generate libraries. Very large libraries (e.g., 106-. Still another means mainly uses a chemical Method, and examples thereof include the Geysen Method (Geysen et al, Molecular Immunology 1986, 23: 709-15; Geysen et al, J Immunology Method 1987, 102: 259-74) and the Method of Fodor et al (Science 1991, 251: 767-73). Furka et al (14th International conformation of biochemistry 1988, Volume #5, Abstract FR: 013; Furka, Int J Peptide Protein Res1991, 37: 487-93), Houghten (U.S. Pat. No. 4,631,211) and Rutter et al (U.S. Pat. No. 5,010,175) describe methods of generating Peptide mixtures that can be tested as agonists or antagonists.

Aptamers are macromolecules composed of nucleic acids that can bind tightly to specific molecular targets. Tuerk and Gold (science.249: 505- & 510(1990)) disclose the SELEX (systematic evolution of ligands by exponential enrichment) method for selecting aptamers. In the SELEX method, a large library of nucleic acid molecules (e.g., 10)¹⁵Species different molecules) can be used for screening.

Screening for CSTF 2-binding substance

In the present invention, over-expression of the CSTF2 gene was detected in lung cancer, but not in normal organs (fig. 1 and 2). In addition, suppression of CSTF2 gene expression by siRNA directed against the CSTF2 gene induced suppression of cancer cell growth (fig. 3). These results indicate that the CSTF2 gene plays a crucial role in cancer cells. Accordingly, the present invention provides a method for screening for substances binding to CSTF2 polypeptide using the CSTF2 gene, protein encoded by the gene. Due to the expression of the CSTF2 gene in lung cancer, substances bound to the CSTF2 polypeptide are expected to suppress the proliferation of lung cancer cells and thus are useful for treating or preventing lung cancer. Accordingly, the present invention also provides a method of screening a candidate substance for suppressing proliferation of lung cancer cells using the CSTF2 polypeptide, and a method of screening a candidate substance for treating or preventing lung cancer. Specifically, in one embodiment of the method of screening for a candidate substance for treating or preventing cancer or inhibiting the growth of cancer cells, the method comprises the steps of:

(a) Contacting a test substance with a CSTF2 polypeptide or fragment thereof;

(b) detecting the binding activity between the polypeptide or fragment thereof and the test substance; and

(c) selecting a test substance that binds to the polypeptide or a fragment thereof as a candidate substance for treating or preventing cancer.

In another embodiment, the present invention also provides a method of using a CSTF2 polypeptide or fragment thereof for screening candidate substances for treating or preventing cancer or inhibiting the growth of cancer cells, comprising the steps of:

(a) contacting a test agent with a CSTF2 polypeptide or functional fragment thereof;

(b) detecting the binding activity between the polypeptide or fragment thereof and the test agent in step (a); and are

(c) Correlating the binding activity of (b) with the therapeutic effect of the test substance.

Alternatively, the potential therapeutic effect of a test substance or compound on the treatment or prevention of cancer may also be assessed or estimated in accordance with the present invention. In some embodiments, the present invention provides methods for assessing or evaluating the therapeutic effect of a test substance on treating or preventing cancer or inhibiting cancer associated with CSTF2 overexpression, the method comprising the steps of:

(a) contacting a test agent with a polypeptide encoded by a CSTF2 polynucleotide;

(b) Detecting the binding activity between the polypeptide and the test substance; and are

(c) Correlating the potential therapeutic effect with a test substance, wherein the substance exhibits the potential therapeutic effect when bound to the polypeptide.

In the present invention, the therapeutic effect may be correlated with the binding activity of the CSTF2 polypeptide or functional fragment thereof. For example, when a test substance binds to CSTF2 polypeptide or a functional fragment thereof, the test substance can be identified or selected as a candidate substance having a therapeutic effect. Alternatively, when a test agent does not bind to CSTF2 polypeptide or functional fragment thereof, the test agent or compound may be identified as an agent with no significant therapeutic effect.

The process of the present invention will be described in more detail below.

The CSTF2 polypeptide to be used for screening may be a recombinant polypeptide or a protein derived from nature or a partial peptide thereof. The polypeptide contacted with the test substance may be, for example, a purified polypeptide, a soluble protein, a form bound to a carrier, or a fusion protein fused to another polypeptide.

As a method for screening proteins using the CSTF2 polypeptide, for example, proteins binding to the CSTF2 polypeptide, various methods known to those skilled in the art can be used. Such screening can be carried out, for example, by immunoprecipitation methods, specifically, as described below. The gene encoding the CSTF2 polypeptide is expressed in a host (e.g., animal) cell or the like by inserting the gene into a foreign gene expression vector such as pSV2neo, pcDNAI, pcdna3.1, pCAGGS, and pCD 8.

The promoter used for this expression may be any commonly used promoter, including, for example, SV40 early promoter (Rigby in Williamson (ed.), Genetic Engineering, vol.3.academic Press, London, 83-141(1982)), EF-alpha promoter (Kim et al, Gene 91: 217-23(1990)), CAG promoter (Niwa et al, Gene 108: 193(1991)), RSV LTR promoter (Cullen, Methods in Enzymology 152: 684-704(1987)), SR alpha promoter (Takebe et al, Mol Cell Biol 8: 466(1988)), CMV immediate early promoter (Seed and Cell Biol 42: 3365-9(1987)), and SV40 promoter (liver Cell J94, Mol et al, Mol late adenovirus J946, Mol Cell J94: 1989, and Mol late adenovirus (1989: 1987)), and SV 385 (1989, Mol et al J94: 1989, Mol et al J1989, and Mol adenovirus 01: 1987), HSV TK promoter and the like.

The introduction into a host Cell to express a foreign gene can be carried out according to any method, for example, electroporation method ((Chu et al, Nucleic Acids Res 15: 1311-26(1987)), calcium phosphate method (Chenand Okayama, Mol Cell Biol 7: 2745-52(1987)), DEAE dextran method (Lopata et al, Nucleic Acids Res 12: 5707-17 (1984); Sussman and Milman, Mol Cell Biol 4: 1641-3(1984)), Lipofectin method (Derija B, Cell 76: 1025-37 (1994); Lamb et al, Nature Genetics 5: 22-30 (1993): Rabidran et al, Science 259: 230-4(1993)), and the like.

For the polypeptide encoded by the CSTF2 gene, a recognition site (epitope) of a monoclonal antibody whose specificity is known can be introduced into the N-or C-terminus of the polypeptide, thereby expressing the polypeptide as a fusion protein comprising the epitope. A commercially available epitope-antibody system can be used (Experimental Medicine 13: 85-90 (1995)). Vectors capable of expressing fusion proteins with, for example, β -galactosidase, maltose binding protein, glutathione S-transferase, and Green Fluorescent Protein (GFP) using their multiple cloning sites are commercially available. In addition, fusion proteins prepared by introducing small epitopes consisting of only a few to twelve (a dozen) amino acids so that the fusion does not alter the properties of the CSTF2 polypeptide have also been reported. Epitopes such as polyhistidine (His-tag), influenza lectin HA, human c-myc, FLAG, vesicular stomatitis virus glycoprotein (VSV-GP), T7 gene 10 protein (T7-tag), human herpes simplex virus glycoprotein (HSV-tag), E-tag (epitope on monoclonal phage), and monoclonal antibodies recognizing them can be used as epitope-antibody systems for screening proteins binding to CSTF2 polypeptide (Experimental medicine 13: 85-90(1995))

In immunoprecipitation, these antibodies are added to cell lysates prepared with a suitable detergent to form immune complexes. The immunocomplex consists of a CSTF2 polypeptide, a polypeptide comprising the ability to bind to the polypeptide, and an antibody. In addition to using antibodies directed against the above epitopes, immunoprecipitation can also be performed using antibodies directed against the CSTF2 polypeptide, and such antibodies can be prepared as described above. The immune complex can be precipitated, for example, by protein a sepharose or protein Gsepharose when the antibody is a mouse IgG antibody. If the polypeptide encoded by the CSTF2 gene is prepared as a fusion protein with epitopes (e.g., GST), an immune complex can be formed in the same manner as that using an antibody against the CSTF2 polypeptide using a substance specifically binding to these epitopes, e.g., glutathione-sepharose 4B.

Immunoprecipitation can be performed following or according to, for example, methods in the literature (Harlow and Lane, Antibodies, 511-52, Cold Spring Harbor Laboratory publications, New York (1988)).

Immunoprecipitated proteins are commonly analyzed using SDS-PAGE, and using a gel of appropriate concentration, the molecular weight of the bound protein can be used to analyze the protein. Since proteins bound to CSTF2 polypeptide are difficult to detect by common staining methods such as coomassie blue staining or silver staining, the detection sensitivity of proteins can be improved by the following methods: in the presence of a radioisotope ³⁵S-methionine or³⁵Culturing the cells in a culture medium of S-cysteine, labeling the protein in the cells, and detecting the protein. When the molecular weight of the protein is known, the target protein can be purified directly from SDS-polyacrylamide gel and its sequence determined.

As a method for screening a protein binding to the CSTF2 polypeptide, for example, West-Western blot analysis (Skolnik et al, Cell 65: 83-90(1991)) can be used. Specifically, the protein binding to the CSTF2 polypeptide can be obtained by preparing a cDNA library from cultured cells (e.g., LC176, LC319, A549, NCI-H23, NCI-H226, NCI-H522, PC3, PC9, PC14, SK-LU-1, EBC-1, RERF-LC-AI, SK-MES-1, SW900 and 157SW 3) expected to express the protein binding to the CSTF2 polypeptide using a phage vector (e.g., ZAP), expressing the protein on LB agarose, immobilizing the expressed protein on a filter, reacting the purified and labeled CSTF2 polypeptide with the above filter, and detecting plaques expressing the protein binding to the CSTF2 polypeptide according to the label. The polypeptides of the present invention may be labeled using a binding between biotin and avidin, or using an antibody that specifically binds to CSTF2 polypeptide or a peptide or polypeptide fused to CSTF2 polypeptide (e.g., GST). Methods utilizing radioactive isotopes, fluorescence, or the like may also be used.

Alternatively, in another embodiment of the screening method of the present invention, a Two-Hybrid System ("MATCHMAKER Two-Hybrid System", "MammalianMCHMAKER Two-Hybrid Assay Kit", "MATCHMAKER one-Hybrid System" (Clontech), "Hybrid ZAP Two-Hybrid Vector System" (Stratagene), references "Dalton and Treisman, Cell 68: 597-612 (1992)", "Fields and Sterngland z, Trends Genet 10: 286-92 (1994)") using cells may be used.

In a two-hybrid system, a polypeptide of the invention is fused to an SRF-binding region or GAL 4-binding region and expressed in yeast cells. A cDNA library is prepared from cells expected to express a protein that binds to a polypeptide of the invention, and when expressed, is fused to the VP16 or GAL4 transcriptional activation region. Then, a cDNA library is introduced into the above-mentioned yeast cells, and cDNA derived from the library is isolated from the detected positive clones (when a protein capable of binding to the polypeptide of the present invention is expressed in the yeast cells, the binding of both activates the reporter gene, making the positive clones detectable). The protein encoded by the above-isolated cDNA can be prepared by introducing the cDNA into E.coli and expressing the protein. As the reporter gene, for example, Ade2 gene, lacZ gene, CAT gene, luciferase gene, and the like can be used in addition to the HIS3 gene.

Affinity chromatography may also be used to screen for substances that bind to the polypeptide encoded by the CSTF2 gene. For example, the polypeptide of the present invention may be immobilized on a support of an affinity column, and a test substance containing a protein capable of binding to the polypeptide of the present invention may be applied to the column. The test substance herein may be, for example, a cell extract, a cell lysate, etc. After loading with the test substance, the column is washed, whereby a substance that binds to the polypeptide of the present invention can be prepared. When the test substance is a protein, the amino acid sequence of the resulting protein is analyzed, an oligo DNA is synthesized based on the sequence, and a cDNA library is screened using the oligo DNA as a probe, thereby obtaining a DNA encoding the protein.

A biosensor using the surface plasmon resonance phenomenon may be used as a device for detecting or quantifying a binding substance in the present invention. When such a biosensor is used, the interaction between the polypeptide of the present invention and the test substance can be observed in real time in the form of a surface plasmon resonance signal using only a minute amount of a polypeptide (e.g., BIAcore, Pharmacia) which is not labeled. Thus, using a biosensor, such as BIAcore, one can assess binding between a polypeptide of the invention and a test substance.

Methods for screening molecules for binding when immobilized CSTF2 polypeptide is exposed to synthetic chemicals or natural substance libraries or random phage peptide display libraries, and methods using combinatorial chemistry based high throughput screening methods (Wright et al, Science 273: 458-64 (1996); Verdine, Nature 384: 11-13 (1996); Hogan, Nature 384: 17-9(1996)) to isolate not only proteins that bind to CSTF2 protein, but also substances that bind to CSTF2 protein, including agonists and antagonists, are well known to those skilled in the art.

In addition to the CSTF2 polypeptide, fragments of the polypeptide may also be used in the screening of the present invention, as long as they retain at least one biological activity of the naturally occurring CSTF2 polypeptide.

The polypeptide or fragment thereof may be further linked to other substances, so long as the polypeptide and fragment retain at least one of its biological activities. Useful substances include: peptides, lipids, sugars and sugar chains, acetyl groups, natural and synthetic polymers, and the like. These types of modifications can be made to confer additional functionality or to stabilize the polypeptide and fragments.

The polypeptides or fragments used in the methods of the invention may be obtained as naturally occurring proteins from nature by conventional purification methods, or by chemical synthesis based on the selected amino acid sequence. For example, conventional peptide synthesis methods that can be used for the synthesis include:

1)Peptide Synthesis，Interscience，New York，1966；

2)The Proteins，Vol.2，Academic Press，New York，1976；

3) Peptide Synthesis (japanese), Maruzen co., 1975;

4) basics and experience of Peptide Synthesis (japanese), Maruzen co., 1985;

5) development of Pharmaceuticals (volume II) (Japanese), Vol.14(peptide synthesis), Hirokawa, 1991;

6) WO 99/67288; and

7)Barany G.&Merrifield R.B.，Peptides Vol.2，“Solid Phase Peptide Synthesis”，Academic Press，New York，1980，100-118。

alternatively, the protein may be obtained by any known genetic engineering method for producing a polypeptide (e.g., Morrison J., J Bacteriology 1977, 132: 349-51; Clark-Curtiss & Curtiss, Methods in Enzymology (eds. Wu et al.)1983, 101: 347-62). For example, a suitable vector comprising a polynucleotide encoding a protein of interest in an expressible form (e.g., downstream of a regulatory sequence comprising a promoter) is first prepared, then transformed into a suitable host cell, and the host cell is then cultured to produce the protein. More specifically, the gene encoding the CSTF2 polypeptide is expressed in a host (e.g., animal) cell or the like by inserting the gene into a vector for expressing a foreign gene such as pSV2neo, pcDNAI, pcdna3.1, pCAGGS or pCD 8. Promoters may be used for expression. Any commonly used promoter may be used, including for example the SV40 early promoter (Rigby in Williamson (eds.), genetic engineering, vol.3.academic Press, London, 1982, 83-141), EF- α promoter (Kimet et al, Gene 1990, 91: 217-23), CAG promoter (Niwa et al, Gene 1991, 108: 193), RSV LTR promoter (Cullen, Methods in Enzymology 1987, 152: 684-704), SR α promoter (Takebe et al, Mol Cell Biol 1988, 8: 684-9), immediate early promoter (Seed et al, Proc Natl Acad Sci 1987, 84: 3365-9), SV40 late promoter (Ghesen et al, J Mol Appl applied Gene 1982, 1: 385-94), adenovirus promoter (CMV et al USA 1987, HSV 1989), HSV Cell et al 946, HSV Cell et al 466, HSV II. The vector may be introduced into the host Cell to express the CSTF2 gene according to any method, for example, electroporation (Chu et al, Nucleic Acids Res 1987, 15: 1311-26), calcium phosphate (Chen et al, Mol Cell Biol 1987, 7: 2745-52), DEAE dextran (Lopata et al, Nucleic Acids Res 1984, 12: 5707-17; Sussman et al, Mol Cell Biol 1985, 4: 1641-3), lipofection (Derijard B, Cell 1994, 7: 1025-37; Lamb et al, Nature Genetics 1993, 5: 22-30; Rabndran et al, Science 1993, 259: 230-4), and the like.

The CSTF2 protein can also be produced in vitro using an in vitro translation system.

The CSTF2 polypeptide to be contacted with the test substance may be, for example, a purified polypeptide, a soluble protein or a fusion protein fused to other polypeptides.

In the present invention, it was revealed that suppression of CSTF2 gene expression decreased cell growth. Thus, by screening candidate substances that bind to CSTF2 polypeptide, candidate substances useful for treating or preventing cancer can be identified. These candidate substances or agents can be evaluated for the potential to treat or prevent cancer by secondary and/or further screening to identify therapeutic substances for cancer.

Screening of substances inhibiting the biological Activity of CSTF2

The present invention provides methods of screening for substances that can suppress the proliferation of cancer cells, and methods of screening for substances useful for treating or preventing cancer, including lung cancer. Accordingly, the present invention provides a method for screening a substance for treating or preventing cancer or inhibiting the growth of cancer cells using a polypeptide encoded by CSTF2 gene, comprising the steps of:

(a) contacting a test substance with a CSTF2 polypeptide;

(b) detecting the biological activity of the polypeptide of step (a); and are

(c) Selecting a test agent that suppresses the biological activity of the CSTF2 polypeptide compared to the biological activity of the polypeptide in the absence of the test agent.

In another embodiment, the present invention also provides a method for screening a substance for treating or preventing cancer or for inhibiting the growth of cancer cells using a polypeptide encoded by CSTF2 gene, comprising the steps of:

(a) contacting a test substance with a CSTF2 polypeptide; and are

(b) Detecting the biological activity of the polypeptide of step (a); and are

(c) Correlating the biological activity of (b) with the therapeutic effect of the test substance.

Alternatively, in some embodiments, the present invention provides a method for assessing or evaluating the therapeutic effect of a test substance for treating or preventing cancer or inhibiting cancer associated with CSTF2 overexpression, the method comprising the steps of:

(a) contacting a test agent with a polypeptide encoded by a polynucleotide of the CSTF2 gene;

(b) detecting the biological activity of the polypeptide of step (a); and are

(c) Correlating the potential therapeutic effect with the test agent, wherein the agent exhibits the potential therapeutic effect when the biological activity of the polypeptide encoded by the polynucleotide of the CSTF2 gene is suppressed as compared to the biological activity of the polypeptide detected in the absence of the test agent.

In the present invention, the therapeutic effect may be correlated with the biological activity of the CSTF2 polypeptide. For example, a test substance may be identified or selected as a candidate substance having a therapeutic effect when the test substance inhibits or inhibits the biological activity of the CSTF2 polypeptide as compared to the level detected in the absence of the test substance. Alternatively, a test agent or compound may be identified as one that does not suppress or inhibit the biological activity of the CSTF2 polypeptide when compared to the level detected in the absence of the test agent, the test agent or compound may be identified as one that has no significant therapeutic effect.

The process of the present invention will be described in more detail below.

Any CSTF2 polypeptides may be used for screening as long as they contain the biological activity of the CSTF2 protein. Such biological activities include cell proliferation activity, RNA binding activity, mRNA cleavage activity, and mRNA polyadenylation activity of the CSTF2 protein. For example, the CSTF2 protein may be used, as may a polypeptide functionally equivalent to the CSTF2 protein. The polypeptides may be expressed endogenously or exogenously by the cell.

The substances isolated by this screen are candidates for antagonists to the polypeptide encoded by the CSTF2 gene. The term "antagonist" refers to a molecule that inhibits the function of a polypeptide by binding to the polypeptide. The term also refers to molecules that can reduce or inhibit the expression of the gene encoding CSTF 2. Moreover, the substances isolated by this screen are candidates for substances that inhibit the in vivo interaction of CSTF2 polypeptide with molecules including DNA, RNA, and proteins.

When the biological activity to be detected in the present method is cell proliferation, the detection can be carried out, for example, by the following method: preparing cells expressing CSTF2 polypeptide, culturing the cells in the presence of a test substance, determining the rate of cell proliferation, measuring the cell cycle, etc., and by measuring the viable cells or colony forming ability, as shown, for example, in fig. 3 or 4. Selecting a substance that reduces the proliferation rate of cells expressing CSTF2 as a candidate substance for the treatment or prevention of cancer, including lung cancer.

In the present invention, it was revealed that suppression of CSTF2 gene expression decreased cell growth. Thus, by screening candidate substances that reduce the biological activity of the CSTF2 polypeptide, candidate substances useful for treating or preventing cancer can be identified. The potential of these candidate substances to treat or prevent cancer can be assessed by secondary and/or further screening to identify therapeutic substances for cancer.

More specifically, the method comprises the steps of:

(a) contacting a test agent with a cell that overexpresses the CSTF2 gene;

(b) measuring cell proliferation activity; and are

(c) Selecting a test agent that reduces cell proliferative activity compared to cell proliferative activity in the absence of the test agent.

In a preferred embodiment, the method of the present invention may further comprise the steps of:

(d) test substances were selected that had little or no effect on cells expressing CSTF 2.

"suppression of biological activity" is defined herein as a preferred at least 10% suppression, more preferably at least 25%, 50% or 75% suppression, most preferably at least 90% suppression of the biological activity of CSTF2, as compared to the absence of the agent.

In a preferred embodiment, control cells that do not express CSTF2 polypeptide are used. Thus, the present invention also provides a method of screening a candidate substance for inhibiting cell growth or a candidate substance for treating or preventing a CSTF 2-related disease using a CSTF2 polypeptide or a fragment thereof, comprising the steps of:

(a) Culturing cells expressing CSTF2 polypeptide or functional fragment thereof and control cells not expressing CSTF2 polypeptide or functional fragment thereof in the presence of a test substance;

(b) detecting the biological activity of the cells expressing the protein and the control cells; and are

(c) Selecting a test agent that inhibits a biological activity in cells expressing the protein as compared to proliferation detected in control cells and in the absence of the test agent.

In some embodiments, RNA binding activity, mRNA cleavage activity, or mRNA polyadenylation activity can be used as the biological activity of the CSTF2 polypeptide to be detected in the screening methods of the invention. Methods for detecting these activities are well known in the art. When any of these activities are detected in the screen, it may be preferred to use a polypeptide comprising the RNA recognition motif of the CSTF2 polypeptide as a functional equivalent of the CSTF2 polypeptide. For example, a polypeptide having the amino acid sequence of SEQ ID NO: 2 is the RNA recognition motif of the CSTF2 polypeptide consisting of SEQ ID NO: 2, amino acids 17 to 90.

Screening for Agents that alter expression of CSTF2

The present invention provides a method for screening a substance inhibiting the expression of CSTF2 gene. Substances that inhibit the expression of the CSTF2 gene are expected to suppress the proliferation of lung cancer cells and are therefore useful for treating or preventing lung cancer. Therefore, the present invention also provides a method for screening a candidate substance for suppressing proliferation of lung cancer cells, and a method for screening a candidate substance for treating or preventing lung cancer. In the context of the present invention, the above screening may comprise, for example, the following steps:

(a) Contacting a test substance with a cell expressing a CSTF2 gene;

(b) detecting the expression level of the CSTF2 gene; and are

(c) Selecting a test agent that reduces the expression level of the CSTF2 gene as compared to the expression level detected in the absence of the test agent.

In another embodiment, the present invention also provides a method of screening for a candidate substance that inhibits the proliferation of cancer cells and a method of screening for a candidate substance for treating or preventing a disease associated with CSTF 2.

In the context of the present invention, such screening may comprise, for example, the following steps:

(a) contacting a test agent with a cell expressing a CSTF2 gene;

(b) detecting the expression level of the CSTF2 gene; and are

(c) Correlating the expression level of (b) with the therapeutic effect of the test substance.

Alternatively, in some embodiments, the present invention also provides a method for evaluating or evaluating the therapeutic effect of a test substance for treating or preventing cancer or inhibiting cancer associated with CSTF2 overexpression, the method comprising the steps of:

(a) contacting a test agent with cells expressing CSTF 2;

(b) a potential therapeutic effect is associated with the test substance, wherein the potential therapeutic effect is indicated when the test substance reduces the expression level of CSTF2 as compared to the control.

In the present invention, the therapeutic effect can be correlated with the expression level of CSTF2 gene. For example, a test substance may be identified or selected as a candidate substance having a therapeutic effect when the test substance reduces the expression level of CSTF2 gene as compared to the level detected in the absence of the test substance. Alternatively, a test substance may be identified as one that has no significant therapeutic effect when it does not decrease the expression level of CSTF2 gene as compared to the level detected in the absence of the test substance.

The process of the present invention will be described in more detail below.

Cells expressing the CSTF2 gene include, for example, cell lines established from lung cancer; such cells can be used in the above-described screening of the present invention (e.g., A427, A549, LC319, PC14, PC3, PC9, NCI-H1373, NCI-H1781, NCI-H358, NCI-H226, NCI-H520, NCI-H1703, NCI-H2170, EBC-1, RERF-LC-AI, LX1, DMS114, DMS273, SBC-3, SBC-5, NCI-H196, NCI-H446, SK-MES-1, LU 61). Expression levels can be estimated by methods well known to those skilled in the art, such as RT-PCR, Northern blot analysis, Western blot analysis, immunostaining, and flow cytometry analysis. As defined herein, a "reduced expression level" is preferably a level that reduces the expression level of at least CSTF2 gene by at least 10%, more preferably by at least 25%, 50% or 75%, most preferably by 95% as compared to the expression level in the absence of the agent. The substance herein includes a chemical substance, a double-stranded nucleotide, and the like. The preparation of double-stranded nucleotides is described above. In the screening method, a substance that decreases the expression level of CSTF2 gene may be selected as a candidate substance for treating or preventing lung cancer.

In the present invention, it was revealed that suppression of CSTF2 gene expression decreased cell growth. Therefore, by screening for substances that decrease the expression level of the CSTF2 gene, candidate substances useful for treating or preventing cancer can be identified. These candidate substances can be used to evaluate the potential for treating or preventing cancer by secondary and/or further screening to identify therapeutic substances for cancer.

Alternatively, the screening method of the present invention may comprise the steps of:

(a) contacting a test substance with a cell into which a vector comprising a transcription regulatory region of CSTF2 gene and a reporter gene expressed under the control of the transcription regulatory region is introduced;

(b) measuring the expression level or activity of a reporter gene; and are

(c) Selecting a test agent that reduces the level of expression or activity of the reporter gene.

In another embodiment, the present invention also provides a method of screening a candidate substance for inhibiting the proliferation of cancer cells and a method of screening a candidate substance for treating or preventing a disease associated with CSTF 2.

In accordance with another aspect, the present invention provides a method comprising the steps of:

(a) contacting a test substance with a cell into which a vector comprising a transcription regulatory region of CSTF2 gene and a reporter gene expressed under the control of the transcription regulatory region is introduced;

(b) Detecting the expression or activity of the reporter gene; and are

(c) Correlating the expression level of (b) with the therapeutic effect of the test agent.

Alternatively, in some embodiments, the present invention also provides a method for evaluating or evaluating the therapeutic effect of a test substance for treating or preventing cancer or inhibiting cancer associated with CSTF2 overexpression, the method comprising the steps of:

a) contacting a test substance with a cell having introduced therein a vector comprising a transcription regulatory region of CSTF2 gene and a reporter gene expressed under the control of the transcription regulatory region

b) Measuring the expression or activity of the reporter gene; and are

c) Correlating the potential therapeutic effect with a test agent, wherein the potential therapeutic effect is exhibited when the test agent decreases expression or activity of the reporter gene.

In the present invention, the therapeutic effect may be correlated with the expression level or activity of the reporter gene. For example, a test agent can be identified or selected as a candidate agent for therapeutic effect when the agent reduces the expression level or activity of the reporter gene as compared to the level detected in the absence of the test agent. Alternatively, a test agent can be identified as one that has no significant therapeutic effect when the agent does not decrease the expression level or activity of the reporter gene as compared to the level detected in the absence of the test agent.

Suitable reporter genes and host cells are well known in the art. For example, the reporter genes are luciferase, Green Fluorescent Protein (GFP), shiitake coral (Discosoma sp.) red fluorescent protein (DsRed), Chloramphenicol Acetyltransferase (CAT), Laz and β -Glucuronidase (GUS), and the host cells are COS7, HEK293, HeLa, etc. The reporter construct required for the screen can be prepared by linking the reporter gene sequence to the transcriptional regulatory region of the CSTF2 gene. The transcriptional regulatory region of CSTF2 described herein is a region from the start codon up to at least 500bp upstream, preferably 1,000bp, more preferably 5000 or 10,000bp upstream. Nucleotide fragments containing the transcriptional regulatory region can be isolated from genomic libraries or can be amplified by PCR. The reporter constructs required for such screening can be prepared by linking a reporter gene sequence to a transcriptional regulatory region of any of these genes. Methods for identifying transcriptional regulatory regions, and assay protocols therefor, are well known (molecular cloning, third edition, Chapter 17, 2001, Cold Springs Harbor Laboratory Press).

Host cells are infected with a vector containing the reporter construct and the expression or activity of the reporter gene is detected by methods well known in the art (e.g., using a luminometer, absorption spectrometer, flow cytometer, etc.). As defined herein, "reduced expression or activity" is that the expression or activity of the reporter gene is preferably reduced by at least 10%, more preferably by 25%, 50% or 75%, most preferably by at least 95% as compared to in the absence of the agent.

In the present invention, it was revealed that suppression of CSTF2 gene expression decreased cell growth. Thus, by screening candidate substances that decrease reporter gene expression or activity, candidate substances that are potentially useful for treating or preventing cancer can be identified. These candidate substances can be used to evaluate the potential for treating or preventing cancer by secondary and/or further screening to identify therapeutic substances for cancer.

Aspects of the invention are described in the following examples, which are not intended to limit the scope of the invention described in the claims.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Suitable methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention.

The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

Examples

Materials and methods

Cell lines and tissue samples.

The 15 human lung cancer cell lines used in this study included 5 adenocarcinomas (NCI-H1781, NCI-H1373, LC319, A549, and PC-14), 5 squamous cell carcinomas (SK-MES-1, NCI-H520, NCI-H1703, NCI-H2170, and LU61), 1 large cell carcinoma (LX1), and 4 small cell lung carcinomas (SBC-3, SBC-5, DMS114, and DMS 273). All cells were cultured as monolayers in appropriate medium supplemented with 10% FCS and maintained at 37 ℃ in humidified air containing 5% CO 2. Human Small Airway Epithelial Cells (SAEC), used as normal controls, were cultured in optimized medium (cambrex bioscience, Inc). Primary NSCLC tissue samples and their corresponding normal tissue adjacent to the resection margin were obtained from patients not treated with anti-cancer prior to tumor resection, as early as informed (Kikuchi T, Daigo Y, Katagiri T, et al oncogene 2003; 22: 2192-205, Taniwakim, Daigo Y, Ishikawa N, et al. int J Oncol 2006; 29: 567-75, Kato T, Daigo Y, Hayama S, et al. cancer Res 2005; 65: 5638-46). All tumors were staged based on the pathological tumor-lymph node-metastasis classification of the International Union of cancer (Table 1; Sobin L, Wittekind CH. TNMclaisiocation of malignant tumors.6th ed. New York: Wiley-Liss; 2002). Formalin-fixed primary lung tumor and adjacent normal lung tissue samples for immunostaining on tissue microarrays were obtained from 327 patients (196 adenocarcinomas, 98 squamous cell carcinomas, 23 large cell carcinomas, and 10 adenosquamous carcinomas; 99 female and 228 male patients; median age 64.7 years, range 29-85 years) who underwent surgery in the saitmama cancer center. These patients who received their primary cancer resection did not receive any pre-operative treatment, and only those with positive lymph node metastasis received platinum-based adjuvant chemotherapy after their surgery. The study and the use of all the clinical materials were approved by various scientific ethics committees.

[ Table 1]

Association between CSTF2 positivity in NSCLC tissue and patient characteristics (n ═ 327)

ADC, adenocarcinoma

non-ADC, squamous cell carcinoma, enlarged cell carcinoma and adenosquamous cell carcinoma

^*P < 0.05 (Fisher's true test)

Semi-quantitative reverse transcription-PCR.

Aliquots of a total of 3. mu.g of mRNA from each sample were reverse transcribed into single stranded cDNA using random primers (Roche Diagnostics) and Superscript II (Invitrogen). Semi-quantitative reverse transcription-PCR (RT-PCR) experiments were performed with synthetic primers specific for human CSTF2 (gene accession No. NM — 001325) or β -Actin (ACTB) specific primers used as internal controls for each of the following groups: CSTF2, 5'-GTCATGCAGGGAACAGGAAT-3' (SEQ ID NO: 3) and 5'-TGAGTCATTCAAGGGTTAGGATG-3' (SEQ ID NO: 4); ACTB, 5 'GAGGTGATAGCATTGCTTTCG-containing 3' (SEQ ID NO: 5) and 5'-CAAGTCAGTGTACAGGTAAGC-3' (SEQ ID NO: 6). The number of cycles of the PCR reaction was optimized to ensure that the product intensity was within the linear phase of amplification.

Northern blot analysis.

Human multi-tissue blots covering 23 tissues (BD Bioscience) were hybridized with the 32P-labeled 521-bp PCR product of CSTF2 (which was prepared as a probe using primers 5'-CGAGGCTTGTTAGGAGATGC-3' (SEQ ID NO: 7) and 5'-CCCCCATGTTAAGGACTG-3' (SEQ ID NO: 8)). Prehybridization, hybridization, and washing were performed following the manufacturer's recommendations. The blot was autoradiographed at-80 ℃ for 14 days with an enhanced screen.

Western blotting.

Lysing the tumor cells in a lysis buffer; 50mmol/L Tris-HCl (pH 8.0), 150mmol/L NaCl, 0.5% NP40, 0.5% sodium deoxycholate, and protease inhibitor cocktail group III (Calbiochem). The protein content of each lysate was determined by the Bio-Rad protein assay kit, with bovine serum albumin as standard. 10. mu.g of each lysate was resolved on 7.5% to 12% denaturing polyacrylamide gel (with 3% polyacrylamide stacking gel) and electrophoretically transferred to nitrocellulose membrane (GE Healthcare Biosciences). After blocking with 5% nonfat milk powder in TBST (Tris buffered saline with Tween 20), the membranes were incubated with rabbit polyclonal antibodies for 1 hour at room temperature. Commercial rabbit polyclonal anti-CSTF 2 antibody was purchased from ATLAS and was probed to be specific for human CSTF2 by Western blot analysis using lysates of lung cancer cell lines. Immunoreactive proteins were incubated with horseradish peroxidase-conjugated secondary antibodies (GE Healthcare Bio-sciences) for 1 hour at room temperature. After washing with TBST, the reaction was developed using an enhanced chemiluminescence kit (GEHealthcare Bio-sciences).

Immunofluorescence analysis.

The cultured cells were washed twice with PBS (-), fixed in 4% formaldehyde solution at 4 ℃ for 60 minutes, and rendered permeable by treatment with PBS (-) containing 0.1% Triton X-100 for 5 minutes. Cells were covered with caslock (zymed) for 10 min to block non-specific binding, followed by a primary antibody reaction. The cells were then incubated with either an antibody against human CSTF2 or a c-myc tagged protein.

Immunohistochemistry and tissue microarrays.

To investigate the clinicopathological significance of CSTF2 protein in formalin-fixed and paraffin block-embedded clinical lung cancer samples, sections were stained using Envision + kit/horseradish peroxidase (DakoCytomation) in the following manner. For antigen retrieval, slides were soaked in target retrieval solution pH 9(DakoCytomation) and boiled in an autoclave at 108 ℃ for 15 minutes. After blocking endogenous peroxidase and protein, rabbit polyclonal anti-human CSTF2 antibody (0.06 microgram/ml; ATLAS) was added to each slide and the sections were incubated with horseradish peroxidase-labeled anti-rabbit IgG [ Histofine Simple Stain MAX PO (G), Nichirei ] as a secondary antibody. Substrate-chromogen was added and the specimen was counterstained with hematoxylin.

327 parts of formalin-fixed primary NSCLC were used to construct tumor tissue microarrays; these primary NSCLCs were obtained by a single institution (see above) following the same protocol for tissue collection, fixation, and preservation after resection (Chin S F, Daigo Y, Huang HE, et al. Mol Pathol 2003; 56: 275-9, Callagy G, Cattaneo E, Daigo Y, et al. Diagn. Mol Pathol 2003; 12: 27-34, CallagyG, Pharoah P, Chin SF, et al. J Pathol 2005; 205: 388-96). The tissue regions sampled were selected based on visual alignment with corresponding H & E stained sections on the slides, taking into account the histological heterogeneity of individual tumors. 3, 4, or 5 tissue cores (diameter, 0.6 mm; depth, 3-4mm) from one donor tumor mass were placed into a recipient paraffin mass using a tissue microarray instrument (Beecher Instruments). 1 core of normal tissue was punched out of each case and 5 micron sections of the resulting microarray blocks were used for immunohistochemical analysis. CSTF2 positivity was semi-quantitatively assessed by 3 independent investigators without prior knowledge of clinical pathology data. Since the staining intensity within the core of each tumor tissue is mostly uniform, the staining intensity of CSTF2 was semi-quantitatively evaluated using the following criteria: strong positive (score 2+), dark brown staining in > 50% of tumor cells, making the nucleus and cytoplasm completely obscured; weakly positive (1+), any lower degree of brown staining is detectable in the tumor cell nucleus and cytoplasm; none (score 0), no detectable staining in tumor cells. Cases were accepted as strongly positive only if the investigator defined them as strongly positive independently.

And (5) carrying out statistical analysis.

Statistical analysis was performed using the StatView statistical program (SaS). Fisher confirmatory test was used to assess the association of strong CSTF2 immunoreactivity with clinical pathological variables such as age, gender, pathological tumor-lymph node-metastasis stage, and histological type. Data tumor-specific survival curves were calculated from the day of surgery to NSCLC-associated death or to the last follow-up observation. Calculating a Kaplan-Meier curve for each relevant variable and for CSTF2 expression; differences in survival time between patient subgroups were analyzed using a time series test. Univariate and multivariate analyses were performed using Cox proportional hazards regression models to determine the association between clinical pathology variables and cancer-related deaths. First, the association between death and possible prognostic factors including age, gender, histology, pT classification, and pN classification was analyzed, taking into account one factor at a time. Second, multivariate analysis was applied in a reverse (stepwise) procedure, which always forced strong CSTF2 expression into the model along with any variables that met the entry level P < 0.05. As the model continues to add factors, the independent factors do not exceed the exit level P < 0.05.

An RNA interference assay.

To evaluate the biological function of CSTF2 in lung and esophageal cancer cells, small interfering rna (sirna) duplexes (SIGMA) against the target gene were used. The target sequences of the synthetic oligonucleotides for RNA interference are as follows: si-CSTF2- #1, 5'-GGCUUUAGUCCCGGGCAGA-3' (SEQ ID NO: 9); si-CSTF2- #2, 5'-CACUUUACUUUCUGUAACU-3' (SEQ ID NO: 10), control 1: (EGFP, enhanced green fluorescent protein [ GFP ] gene, a mutant of Veronica aequorea (Aequoricegoria) GFP), 5'-GAAGCAGCACGACUUCUUC-3' (SEQ ID NO: 11); control 2(LUC, luciferase gene from firefly (Photinus pyralis)), 5'-CGUACGCGGAAUACUUCGA-3' (SEQ ID NO: 12). The lung cancer cell lines a549 and LC319 were plated onto 10-cm disks (8.0 x105 per disk) and transfected with each siRNA oligonucleotide (100nmol/L) using 30 microliters of Lipofectamine2000(Invitrogen) according to the manufacturer's instructions. After 7 days of incubation, the cells were stained by Giemsa solution to assess colony formation and cell viability was assessed by 3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide (MTT) assay.

Results

Expression of CSTF2 in lung cancer and normal tissues.

To identify novel target molecules for the development of therapeutics and/or biomarkers for lung Cancer, first, genome-wide gene expression profiling was performed on 101 lung cancers using a cDNA microarray consisting of 27,648 genes or ESTs (Kikuchi T, Daigo Y, Katagiri T, et al. oncogene 2003; 22: 2192. sup. 205, Kakiuchi S, Daigo Y, Tsunoda T, Yano S, Sone S, Nakamura Y. mol Cancer Res 2003; 1: 485-99, Kakiuchi S, Daigo Y, Ishikawa N, et al. HumMol Genet 2004; 13: 3029-43, Kikuchi. sup. T, Daigo Y, Ishikawa N, Ishikawa J Oncol 2006; 28: 799, Taniwaki M, Daiko Y, Ishiwa J Oncol J51. sup. 7. sup. 5675). The CSTF2 transcript was identified to be overexpressed (3-fold or higher) in most of the lung cancer samples examined, whereas CSTF2 was rarely expressed in all 29 normal tissues (except testis). As a result, CSTF2 was considered a better candidate gene for a new molecular target. It was confirmed by semi-quantitative RT-PCR experiments that CSTF2 was overexpressed in the 12/15 lung cancer tissues examined and in the 15/15 lung cancer cell lines (fig. 1A). In addition, expression of CSTF2 protein was confirmed in 9 lung cancer cell lines by Western blot analysis using anti-CSTF 2 antibody (fig. 1B). To determine the subcellular localization of endogenous CSTF2 in lung cancer cells, immunofluorescence analysis was performed using anti-CSTF 2 antibody, which found its staining in the nucleus of SBC5 cells (fig. 1C).

Northern blot analysis using CSTF2cDNA as probe identified a 2.6-kb transcript only in testis in 16 normal human tissues examined (FIG. 2A). In addition, CSTF2 protein expression was examined in 5 normal tissues (heart, lung, liver, kidney, and testis) as well as lung cancer using anti-CSTF 2 antibody. Positive staining with CSTF2 was observed in the nuclei of testicular cells, but not in other normal tissues (fig. 2B).

CSTF2 overexpression is associated with a poor prognosis in NSCLC patients.

To validate the biological and clinical pathological significance of CSTF2 in the development of lung cancer, immunohistochemical staining was performed on a tissue microarray containing primary NSCLC tissue from 327 patients undergoing curative surgical resection. Positive staining of CSTF2 was observed for the anti-CSTF 2 polyclonal antibody in the nucleus of lung cancer cells, but negative in any of its stromal cells adjacent to normal lung cells or surrounding tumor cells. CSTF2 expression levels on the tissue array were classified ranging from none (score 0) to weak/strong positives (score 1+ to 2+) (fig. 2C). In 327 NSCLC, CSTF2 stained strongly in 77 (24%; score 2+), weakly in 165 (50%; score 1+), and not in 85 (26%: score 0; details are shown in Table 1). Next, the association of CSTF2 expression levels (strong positive versus weak positive/none) with various clinical pathological variables was examined, and strong CSTF2 expression was found to be associated with poor prognosis in NSCLC patients after primary tumor resection (P ═ 0.0079, time series test; fig. 2D), but not with any other clinical pathological variables. In addition, univariate analysis was examined to assess patient prognosis and the association between several clinical pathology factors (including age (═ 65 vs. < 65 years), gender (male vs. female), histology (non-adenocarcinoma vs. adenocarcinoma), smoking history (smoker vs. non-smoker), pT stage (tumor size; T2-T3 vs. T1), pN stage (lymph node metastasis; N1-N2 vs. N0), and CSTF2 expression (score 2+ vs. 0, 1 +). All these parameters, except the history of smoking, were significantly correlated with poor prognosis (table 2). Multivariate analysis using the Cox proportional hazards model indicated that pT stage, pN stage, age, and strong CSTF2 positivity were independent prognostic factors for NSCLC (table 2).

[ Table 2]

Cox's proportional risk model analysis of prognostic factors in NSCLC patients

ADC, adenocarcinoma

non-ADC, squamous cell carcinoma, enlarged cell carcinoma and adenosquamous cell carcinoma

Inhibition of lung cancer cell growth by siRNA against CSTF 2.

To assess whether the up-regulation of CSTF2 plays a role in the growth or survival of lung cancer cells, synthetic oligonucleotides directed to sirnas of CSTF2 (si-CSTF2- #1 and si-CSTF2- #2) along with control sirnas (si-LUC and si-EGFP) were transfected into a549 and LC319 cells endogenously overexpressing CSTF 2. The mRNA levels of CSTF2 in cells transfected with si-CSTF2- #1 and si-CSTF2- #2 were significantly reduced compared to cells transfected with either control siRNA (FIG. 3A). Cell viability and colony numbers investigated by MTT assay and colony formation assay were significantly reduced in cells transfected with si-CSTF2- #1 and si-CSTF2- #2 (fig. 3B and 3C).

Activation of mammalian cell proliferation by CSTF 2.

To examine the potential role of CSTF2 in tumorigenesis, a plasmid designed to express CSTF2 (pcDNA3.1/myc-His-CSTF2) was constructed and transfected into COS-7 cells. Exogenous CSTF2 expression was confirmed by Western blot analysis (fig. 4A). MTT and colony formation assays were performed and it was found that the growth of COS-7 cells transfected with CSTF2 was significantly enhanced compared to COS-7 cells transfected with the mock vector (FIGS. 4B and 4C).

Discussion of the related Art

In order to develop a molecular targeted anticancer drug with high specificity to malignant cells and lowest risk of adverse reactions, a powerful target screening system was established to identify proteins specifically activated in lung cancer cells and their interacting proteins. First, 101 lung cancer samples were analyzed for genome-wide expression profiling by a genome-wide cDNA microarray system containing 27,648 genes coupled with laser microdissection. After confirming that such genes are expressed in normal organs with little or no expression by cDNA microarray analysis and multi-tissue Northern blot analysis, hundreds of clinical samples are analyzed on tissue microarrays for protein expression of candidate targets, followed by investigation of loss-of-function phenotypes using RNA interference systems and further determination of the biological function of the protein. Through these analyses, candidate genes for the development of novel diagnostic biomarkers, therapeutic drugs, and/or immunotherapy that are upregulated in Cancer cells but are not expressed in normal organs other than testis, placenta, and/or fetal tissues were identified (Daigo Y, Nakamura Y. Gen Thorac cardiovascular Surg 2008; 56: 43-53, Kikuchi T, Daigo Y, Katagiri T, et al Oncogene 2003; 22: 2192. sup. 205, Kakiuchi S, Daigo Y, Tsunoda T, Yano S, Sone S, Nakamura Y. Mol Cancer Res 2003; 1: 485-99, Kauchi S, DaigoY 2006, Ishikawa N, Ishiwa. M. Mol Genet 2004; 13: 3029-43, Kikuchi T, Ishiwa Y, Ishiwa J797, Ishiwa. sup. Oncogene 2006; Ishiwa J797: Ishikawa J567, suzuki C, Daigo Y, Kikuchi T, Katagiri T, Nakamura Y. cancer Res 2003; 63: 7038-41, Ishikawa N, Daigo Y, Yasui W, et al, Clin Cancer Res 2004; 10: 8363-70, Kato T, Daigo Y, Hayama S, et al. cancer Res 2005; 65: 5638-46, Furukawa C, Daigo Y, Ishikawa N, et al. cancer Res 2005; 65: 7102-10, Ishikawa N, Daigo Y, Takano A, et al, cancer Res 2005; 65: 9176-84, Suzuki C, Daigo Y, Ishikawa N, et al cancer Res 2005; 65: 11314-25, Ishikawa N, Daigo Y, Takano A, et al cancer Sci 2006; 97: 737-45, Takahashi K, Furukawa C, Takano A, et al. cancer Res 2006; 66: 9408-19, Hayama S, Daigo Y, Kato T, et al. cancer Res 2006; 66: 10339-48, Kato T, Hayama S, Yamabuki Y, et al, Clin Cancer Res 2007; 13: 434-42, Suzuki C, Takahashi K, Hayama S, et al. mol Cancer Ther 2007; 6: 542-51, Yamabuki T, Takano A, Hayama S, et al. cancer Res 2007; 67: 2517-25, Hayama S, Daigo Y, Yamabuki T, et al cancer Res 2007; 67: 4113-22, Taniwaki M, Takano A, Ishikawa N, et al, Cancer. Clin Cancer Res 2007; 13: 6624-31, Ishikawa N, Takano A, Yasui W, et al. cancer Res 2007; 67: 11601-11, Mano Y, Takahashi, K, Ishikawa N, et al, cancer Sci 2007; 98: 1902-13, Kato T, Sato N, Hayama S, et al cancer Res 2007; 67: 8544-53, Kato T, Sato N, Takano A, et al.Clin Cancer Res 2008; 14: 2363-70, Dunleavy EM, Roche D, Tagami H, et al. cell 2009; 137: 485-97, Hirata D, Yamabuki T, ItoT, et al.Clin Cancer Res 2009, 15: 256-66, Suda T, Tsunoda T, Daigo Y, NakamuraY, Tahara h. cancer Sci 2007; 98: 1803-8, Mizukami Y, Kono K, Daigo Y, et al. cancer Sci 2008; 99: 1448-54).

As described above, CSTF2 (which encodes a member of the cleavage stimulating factor) is overexpressed at a higher frequency in lung cancer and is likely to play an important role in the growth of lung cancer. Knockdown of CSTF2 expression by siRNA suppressed the growth of lung cancer cells. Furthermore, clinical pathology evidence obtained by our tissue microarray experiments showed that NSCLC patients with strong positive expression of CSTF2 had shorter cancer-specific survival than NSCLC patients with weak positive/negative expression of CSTF 2. The results obtained by in vitro and in vivo assays strongly suggest that CSTF2 is likely to be an important growth factor and is associated with a more malignant phenotype of lung cancer cells.

In conclusion, the CSTF2 gene may play an important role in the growth/progression of lung cancer. For lung cancer patients who may have a poor prognosis, overexpression of CSTF2 in the resection specimen may be a useful indicator of adjuvant therapy.

Industrial applicability

Gene expression analysis of cancer using genome-wide cDNA microarrays as described herein identifies specific genes as targets for cancer prevention and treatment. The present invention provides molecular diagnostic markers for the identification and detection of cancer, particularly lung cancer, based on the expression of the differentially expressed gene, CSTF 2.

The data provided herein increase the overall understanding of cancer, facilitate the development of novel diagnostic strategies, and provide clues for identifying molecular targets for therapeutic and prophylactic agents. Such information helps to understand tumorigenesis more deeply and provides instructions for developing new strategies for diagnosis, treatment, and ultimately prevention of cancer.

As demonstrated herein, cell growth is suppressed by a double-stranded molecule that specifically targets the CSTF2 gene. Therefore, these novel double-stranded molecules are useful as anticancer drugs.

The expression of the CSTF2 gene is significantly elevated in cancer, particularly lung cancer, compared to normal organs. Thus, the gene can be conveniently used as a diagnostic marker for cancer, particularly lung cancer, and the protein encoded thereby can be used in diagnostic assays for cancer. In addition, it was found that the prognosis of cancer patients with higher expression levels of CSTF2 gene tended to be worse. Thus, the CSTF2 gene can be used to assess the prognosis of cancer patients.

In addition, the present invention provides novel therapeutic approaches for the treatment of cancer, including lung cancer. The CSTF2 gene is a useful target for the development of anticancer drugs.

All patents, patent applications, and publications cited herein are incorporated by reference in their entirety.

Additionally, while the invention has been described in detail and with reference to specific embodiments thereof, it will be understood that the foregoing description is illustrative and explanatory in nature and is intended to illustrate the invention and preferred embodiments thereof. Those skilled in the art will readily recognize from routine experimentation that various changes and modifications can be made therein without departing from the spirit and scope of the invention. As such, it is intended that the invention not be limited by the above description, but be defined by the appended claims and their equivalents.

Claims (30)

1. A method for diagnosing cancer or a predisposition for developing cancer in a subject, wherein the method comprises the steps of:

(a) determining the expression level of the CSTF2 gene in the subject-derived biological sample by any one method selected from the group consisting of:

(i) the mRNA of the CSTF2 gene was detected,

(ii) detecting the protein encoded by the CSTF2 gene, and

(iii) detecting the biological activity of the protein encoded by the CSTF2 gene; and are

(b) Correlating the increase in expression level determined in step (a) compared to a normal control level of the CSTF2 gene with the presence of cancer in the subject.

2. The method of claim 1, wherein the expression level determined in step (a) is at least 10% greater than the normal control level.

3. The method of claim 1, wherein the subject-derived biological sample comprises a biopsy sample, sputum, blood, pleural effusion, or urine.

4. A method for assessing or determining the prognosis of a subject with cancer, wherein the method comprises the steps of:

(a) detecting the expression level of the CSTF2 gene in a biological sample derived from the subject;

(b) comparing the detected expression level to a control level; and are

(c) Determining a prognosis for the subject based on the comparison of (b).

5. The method of claim 4, wherein the control level is a good prognosis control level and an increase in the expression level compared to the control level is indicative of a poor prognosis.

6. The method of claim 5, wherein the increase is at least 10% greater than the control level.

7. The method of claim 4, wherein the expression level is determined by any one method selected from the group consisting of:

(a) detecting mRNA of CSTF2 gene;

(b) detecting the protein encoded by the CSTF2 gene; and

(c) the biological activity of the protein encoded by the CSTF2 gene was examined.

8. The method of claim 4, wherein the subject-derived biological sample comprises a biopsy sample, sputum or blood, pleural effusion or urine.

9. A kit for diagnosing cancer or assessing or determining prognosis in a subject with cancer comprising an agent selected from the group consisting of:

(a) reagents for detecting mRNA of CSTF2 gene;

(b) reagents for detecting the protein encoded by the CSTF2 gene; and

(c) reagents for detecting the biological activity of the protein encoded by the CSTF2 gene.

10. The kit of claim 9, wherein the reagent comprises a probe or primer directed to a gene transcript of the CSTF2 gene or an antibody directed to a translation product of the CSTF2 gene.

11. An isolated double stranded molecule that inhibits expression of the CSTF2 gene in vivo and cell proliferation when introduced into a cell, wherein the molecule comprises a sense strand and its complementary antisense strand, wherein the strands hybridize to each other to form the double stranded molecule.

12. The double-stranded molecule of claim 11, wherein the sense strand comprises a nucleotide sequence identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 9 and 10.

13. The double-stranded molecule of claim 11 or 12, wherein the sense strand hybridizes to the antisense strand at the target sequence to form the double-stranded molecule, the double-stranded molecule being 19-25 base pairs in length.

14. The double stranded molecule of any one of claims 11 to 13 consisting of a single polynucleotide comprising both the sense and antisense strands connected by an intervening single strand.

15. The double stranded molecule of claim 14 having the general formula 5 ' - [ a ] - [ B ] - [ a ' ] -3 ', wherein [ a ] is a sense strand comprising a nucleotide sequence that is identical to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 9 and 10, [ B ] is an intervening single strand consisting of 3 to 23 nucleotides, and [ a' ] is an antisense strand comprising the complement of the target sequence selected in [ a ].

16. A vector encoding the double stranded molecule of any one of claims 11 to 15.

17. A method of treating or preventing cancer in a subject, wherein the method comprises administering to the subject a pharmaceutically effective amount of a double-stranded molecule directed against the CSTF2 gene or a vector encoding the double-stranded molecule, wherein the double-stranded molecule, when introduced into a cell expressing the CSTF2 gene, inhibits the expression of the CSTF2 gene.

18. The method of claim 17, wherein the double stranded molecule is a double stranded molecule of any one of claims 11 to 15.

19. The method of claim 17, wherein the vector is the vector of claim 16.

20. A composition for treating a cancer expressing CSTF2 gene, wherein the composition comprises

At least one isolated double-stranded molecule directed against the CSTF2 gene or a vector encoding the double-stranded molecule, wherein the double-stranded molecule inhibits the expression of the CSTF2 gene when introduced into a cell expressing the CSTF2 gene; and

a pharmaceutically acceptable carrier.

21. The composition of claim 20, wherein the double-stranded molecule is a double-stranded molecule of any one of claims 11 to 15.

22. The composition of claim 20, wherein the carrier is the carrier of claim 16.

23. A method of screening a candidate substance for treating or preventing cancer or inhibiting the growth of cancer cells, wherein the method comprises the steps of:

(a) Contacting a test agent with a CSTF2 polypeptide or fragment thereof;

(b) detecting the binding activity between the polypeptide or fragment and the test agent; and are

(c) Selecting a test substance that binds to the polypeptide or fragment as a candidate substance for treating or preventing cancer.

24. A method of screening a candidate substance for treating or preventing cancer or inhibiting the growth of cancer cells, wherein the method comprises the steps of:

(a) contacting a test agent with a CSTF2 polypeptide or fragment thereof;

(b) detecting the biological activity of the polypeptide or fragment;

(c) comparing the biological activity of the polypeptide or fragment to the biological activity detected in the absence of the test substance; and are

(d) Selecting a test substance that suppresses the biological activity of the polypeptide as a candidate substance for treating or preventing cancer.

25. The method of claim 24, wherein the biological activity is cell proliferation activity, RNA binding activity, mRNA cleavage activity, or mRNA polyadenylation activity.

26. A method of screening a candidate substance for treating or preventing cancer or inhibiting the growth of cancer cells, wherein the method comprises the steps of:

(a) contacting a test agent with a cell expressing a CSTF2 gene; and are

(b) Selecting a test agent that reduces the expression level of the CSTF2 gene compared to the expression level detected in the absence of the test agent.

27. A method of screening a candidate substance for treating or preventing cancer or inhibiting the growth of cancer cells, wherein the method comprises the steps of:

(a) contacting the test substance with a cell having a vector introduced therein, the vector comprising a transcription regulatory region of CSTF2 gene and a reporter gene expressed under the control of the transcription regulatory region;

(b) measuring the expression or activity of the reporter gene; and are

(c) Selecting a test agent that reduces the level of expression or activity of the reporter gene as compared to the level of expression or activity detected in the absence of the test agent.

28. A method for treating or preventing cancer in a subject comprising administering to the subject an anti-CSTF 2 antibody or immunologically active fragment thereof.

29. The method of any one of claims 1 to 8, 17 to 19 and 23 to 28, the kit of claim 9 or 10, or the composition of any one of claims 20 to 22, wherein the cancer is lung cancer.

30. A vector comprising any one of a polynucleotide combination comprising a sense strand nucleic acid and an antisense strand nucleic acid, wherein the sense strand nucleic acid comprises a nucleotide sequence identical to SEQ ID NO: 9 or 10 and the antisense strand nucleic acid consists of a sequence complementary to the sense strand, wherein transcripts of the sense strand and the antisense strand hybridize to each other to form a double-stranded molecule, and wherein the vector, when introduced into a cell expressing a CSTF2 gene, inhibits cell proliferation.

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