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

CSTF2 for target genes of lung cancer therapy and diagnosis Download PDF

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CN102575300A
CN102575300A CN2010800477754A CN201080047775A CN102575300A CN 102575300 A CN102575300 A CN 102575300A CN 2010800477754 A CN2010800477754 A CN 2010800477754A CN 201080047775 A CN201080047775 A CN 201080047775A CN 102575300 A CN102575300 A CN 102575300A
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cstf2
gene
duplex molecule
cancer
expression
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醍醐弥太郎
角田卓也
中村佑辅
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Oncotherapy Science Inc
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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

The target gene CSTF2 of lung cancer therapy and diagnosis
Technical field
The present invention relates to bio-science field, more specifically, relate to cancer research, cancer diagnosis and field of cancer.Especially, the present invention relates to be used to detect with the method for diagnosing and be used for the experimenter's treatment with lung cancer and the method for prevention or the prognosis of assessment/mensuration.In addition, the present invention relates to screen the method for the candidate substances that is used to treat and/or prevent lung cancer.
Right of priority
The application requires the U.S. Provisional Application No.61/274 of submission on August 21st, 2009, and 800 rights and interests are through addressing its complete content income this paper.
Background technology
Primary lung cancer is the reason of cancer mortality primary in the most countries, and non-small lung cancers (NSCLC) accounts for those dead about 80% (NPL 1).The detailed molecular mechanism that lung cancer takes place is still unclear, although reported that the several genes in the lung cancer changes (NPL 2).Though surgical technic and chemotherapy have obtained progress, (NPL 1) that advanced lung cancer patient's PD is fatal often.Therefore, think that to improve patient's survival be extremely important (NPL 3) with introducing efficacious therapy more for the biology of understanding lung cancer.In recent two decades; Some cytotoxic agent newly developed such as Pa Litasai, docetaxel, gemcitabine and vinorelbine have begun for the patient with advanced NSCLC multiple treatment options to be provided; Yet; Compare with the routine treatment based on cis-platinum, the method for improving survival of patients that those schemes show is outstanding (NPL 4,5) not.
Now, the notion of specific molecular target has been applied to exploitation improvement strategy of cancer treatment, and two kinds of main ways are used for treatment: therapeutic monoclonal antibodies and small molecules agent (NPL 6).So far; II phase and the III phase of the chemotherapy of lung cancer have obtained investigation in testing to multiple targeted molecular therapy late; Tyrosine kinase inhibitor such as the ZD1939 ZD1939 and the erlotinib (erlotinib) that comprise Urogastron; The tyrosine kinase inhibitor of VEGF such as vandetanib, sorafenib, sunitinib and to monoclonal antibody such as the rhuMAb-VEGF and the Cetuximab (NPL 6-10) of Urogastron or VEGF.Yet because toxicity problem, only a limited number of patient can select these regimens, even and the treatment of using all kinds, show the better patient's of response ratio still limited (NPL 6-10).
Reference list
[non-patent literature]
[NPL?1]Ahmedin?J，Rebecca?S，Elizabeth?W，et?al.Cancer?statistics，2007.CA?Cancer?J?Clin?2007；57：43-66
[NPL?2]Sozzi?G.Molecular?biology?of?lung?cancer.Eur?J?Cancer?2001；37Suppl?7：S63-73
[NPL?3]Daigo?Y，Nakamura?Y.Gen?Thorac?Cardiovasc?Surg?2008；56：43-53
[NPL?4]Kelly?K，Crowley?J，Bunn?PA?et?al.J?Clin?Oncol?2001；19：3210-18
[NPL?5]Schiller?JH，Harrington?D，Belani?CP?et?al.N?Engl?J?Med2002；346：92-8
[NPL?6]Thatcher?Lung?Cancer?2007；57?Suppl?2：S18-23
[NPL?7]Sandler?A，Gray?R，Perry?MC，et?al.N?Engl?J?Med2006；355：2542-50
[NPL?8]Shepherd?FA，Rodrigues?Pereira?J，Ciuleanu?T，et?al.N?Engl?J?Med2005；353：123-32
[NPL?9]Thatcher?N，Chang?A，Parikh?P，et?al.Lancet?2005；366：1527-37
[NPL?10]Cesare?G，Paolo?M，Filomena?G，et?al.Oncologist2007；12：191-200
Summary of the invention
Using the systems analysis of cDNA microarray technology to thousands of expression of gene levels, is to identify relevant with the oncogenesis approach, as can to become the target molecule of the candidate that is used to develop novel treatment and diagnostic reagent a kind of effective way.In order to separate the potential molecule target that is used to diagnose and/or treat lung cancer; The inventor is previous to be used through laser capture microdissection and dissects the purified tumor cell mass by by 27; The cDNA microarray analysis that 648 kinds of genes or EST (EST) are formed the genome range gene expression profile of 101 parts of cancerous lung tissue samples (Daigo Y, Nakamura Y.Gen Thorac Cardiovasc Surg2008; 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.Hum Mol Genet 2004; 13:3029-43, Kikuchi T, Daigo Y, Ishikawa N, et al.Int J Oncol2006; 28:799-805, Taniwaki M, Daigo Y, Ishikawa N, et al.Int J Oncol2006; 29:567-75).For biology and the clinicopathlogical significance of verifying each gene product; The inventor has set up screening system (Suzuki C through the tumor tissues microarray analysis of clinical lung cancer material and the combination of RNA perturbation technique; Daigo Y; Kikuchi T, Katagiri T, Nakamura Y.Cancer Res2003; 63:7038-41, Ishikawa N, Daigo Y, Yasui W, et al.Clin Cancer Res2004; 10:8363-70, Kato T, Daigo Y, Hayama S, et al.Cancer Res2005; 65:5638-46, Furukawa C, Daigo Y, Ishikawa N, et al.Cancer Res2005; 65:7102-10, Ishikawa N, Daigo Y, Takano A, et al.Cancer Res2005; 65:9176-84, Suzuki C, Daigo Y, Ishikawa N, et al.Cancer Res2005; 65:11314-25, Ishikawa N, Daigo Y, Takano A, et al.Cancer Sci2006; 97:737-45, Takahashi K, Furukawa C, Takano A, et al.Cancer Res2006; 66:9408-19, Hayama S, Daigo Y, Kato T, et al.Cancer Res2006; 66:10339-48, Kato T, Hayama S, Yamabuki Y, et al.Clin Cancer Res2007; 13:434-42, Suzuki C, Takahashi K, Hayama S, et al.Mol Cancer Ther2007; 6:542-51, Yamabuki T, Takano A, Hayama S, et al.Cancer Res2007; 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 Res2007; 13:6624-31, Ishikawa N, Takano A, Yasui W, et al.Cancer Res2007; 67:11601-11, Mano Y, Takahashi, K, Ishikawa N, et al.Cancer Sci2007; 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, Ito T, et al.Clin Cancer Res 2009,15:256-66, Suda T, Tsunoda T, Daigo Y, Nakamura Y, 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 Cancer2008; 123:2616-25).
This systematicness strategy discloses, and cutting stimulating factor 3 ' preceding-RNA, subunit 2,64kDa (CSTF2) the frequent mistake in most of primary lung cancers expressed.CSTF2 a kind of nucleoprotein of encoding, it contains ribonucleoprotein (RNP) type RNA and combines the territory in the N petiolarea.This albumen is a member of cutting stimulating factor mixture (CSTF); Its other 2 members (Takagaki Y that in the polyadenylation of mRNA, plays a role with cutting stimulating factor; MacDonald CC, Shenk T, Manley JL.Proc.Nat.Acad.Sci 1992; 89:1403-1407).CSTF2 combines the interior element that is rich in GU (Colgan DF, the Manley JL.Genes Dev 1997 of 3 '-non-translational region of mRNA; 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-Canadillas JM, J Mol Biol 2005; 347:719-33).The amount of CSTF2 in mouse 3T6 inoblast, raise in G0 to S transition period phase (Martincic K, Campbell R, Edwalds-Gilbert G, Souan L, Lotze MT, Milcarek are C.1998; 95:11095-100).Reported that male sex-cell of mouse and rat and the strong CSTF2 in the human cancer cell express, or CSTF2 omnipresence in mouse tissue is expressed (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 S is A.2007; 104:20374-9, Huber Z, Monarez RR, Dass B, MacDonald CC.Ann N YAcad Sci.2005; 1061:163-72, Wallace AM, Denison TL, Attaya EN, MacDonald CC.Biol Reprod 2004; 70:1080-7, Dass B, Attaya EN, Michelle Wallace A, MacDonald CC.Biol Reprod 2001; 64:1722-9, Chennathukuzhi VM, Lefrancois S, Morales CR, Syed V, Hecht NB.Mol Reprod Dev 2001; 58:460-9, Dass B, McMahon KW, Jenkins NA, Gilbert DJ, Copeland NG, MacDonald CC.J Biol Chem 2001; 276:8044-50, Wallace AM, 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 Mol Biol 2009 Mar; 10:22).Although the evidence of CSTF2 in external function as CSTF member arranged, the activation of CSTF2 in the human cancer progress meaning and still do not have record as the clinical potentiality of treatment target.
As stated, the present invention relates to the effect of CSTF2 and performance in lung cancer carcinoma takes place thereof.Therefore, the present invention relates to be used to detect, diagnose, treat and/or prevent the novel composition and the method for lung cancer and the method that is used to screen useful matter.
Especially, the present invention is because of following discovery, and promptly the CSTF2 gene is crossed in cancer and expressed, but in healthy tissues not so; And the duplex molecule by particular sequence (particularly SEQ ID NO:9 and 10) formation of target CSTF2 gene can effectively suppress the cell growth of lung carcinoma cell.Particularly, the present invention provides the siRNA (siRNA) of target CSTF2 gene.These duplex molecules can utilize under separate stage, or in carrier the coding and certainly this vector expression utilize.Thereby, the carrier and the host cell that an object of the present invention is to provide this type of duplex molecule and express them.
In one aspect, the present invention provide through with duplex molecule of the present invention or vector administration in there being needed experimenter to come anticancer growth or treatment cancer, comprise the method for lung cancer.These class methods contain uses the compsn that contains one or more these duplex molecules or carrier to the experimenter.
In yet another aspect, the present invention is provided for treating the compsn of lung cancer, and it contains at least a duplex molecule of the present invention or carrier.
Also having aspect another, the present invention provides CSTF2 expression of gene level in a kind of biological sample that is derived from the experimenter through mensuration in the experimenter, to diagnose or measure the method for cancer proneness (predisposition).This experimenter of rising indication that this expression of gene level is compared with the normal control level of this gene suffers from or risky generation cancer, comprises lung cancer.In a preferred embodiment, CSTF2 expression of gene level can be through detecting the mRNA of CSTF2 gene with suitable probe or primer or measuring with anti-CSTF2 antibody test CSTF2 albumen.
In addition, the present invention relates to following discovery, promptly higher CSTF2 expression level is relevant with relatively poor survival rate.Therefore; The present invention provides a kind of and is used for patient evaluation with lung cancer or the method for measuring prognosis; This method comprises the steps: to detect CSTF2 expression of gene level, with it and predetermined confirm prognosis with reference to the expression level comparison and according to the difference between them for this patient.
Aspect another, the present invention provides a kind of screening to be used to treat and/or prevent the method for the candidate substances of cancer.This type of material can combine CSTF2 albumen, the proteic BA of reduction CSTF2, reduce the expression or the activity of the reporter gene of CSTF2 expression of gene or reduction replaced C STF2 gene.
Skilled person in the art will appreciate that one or more aspect of the present invention can meet some purpose, and one or more others can meet some other purpose.Each purpose can not be aspect it all, to be applicable to each aspect of the present invention on an equal basis.Therefore, aforementioned purpose can be considered about of the present invention any one alternative.Of the present invention these can become clearer with other purpose and characteristic after the reading following detailed is together with accompanying drawing and embodiment.Yet, be appreciated that top summary of the invention and following detailed all are preferred embodiments, but not to the restriction of the present invention or other alternative embodiment of the present invention.
The accompanying drawing summary
After the concise and to the point description of accompanying drawing and detailed description of the Invention and preferred embodiment thereof below considering, those of skill in the art expect all respects of the present invention and application easily:
[Fig. 1 ab] Fig. 1 describes the expression of CSTF2 in the lung tumor: A, the expression of CSTF2 in the 15 routine clinical lung cancer [adenocarcinoma of lung (ADC), squamous cell lung carcinoma (SCLC), and small cell lung cancer (SCC)] through sxemiquantitative RT-PCR inspection and the 15 kinds of lung cancer cell lines.Beta-actin (ACTB) expression of gene is served as the quantity contrast.B, the Western engram analysis of the proteic expression of CSTF2 in the lung cancer cell line.The quantity contrast is served as in the proteic expression of ACTB.IB, immunoblotting.
[Fig. 1 c] Fig. 1 describes the expression of CSTF2 in the lung tumor: C, through the proteic Subcellular Localization of CSTF2 of confocal microscopy inspection.
[Fig. 2 ab] Fig. 2 describes the expression of CSTF2 in the healthy tissues, and NSCLC patient's CSTF2 crosses and expresses related with poor prognosis: A, and through the expression of CSTF2 in the health adult tissue of Northern engram analysis detection.B, the expression of CSTF2 in the 5 kinds of health adult tissues detecting through the immunohistochemical staining that uses the anti-CSTF2 antibody of this family's rabbit polyclonal and the adenocarcinoma cell of lung; With haematoxylin redyeing look (x200).
[Fig. 2 cd] Fig. 2 describes the expression of CSTF2 in the healthy tissues, and NSCLC patient's CSTF2 crosses and expresses related with poor prognosis: among the C, lung ADC tissue and normal lung tissue strong, weak and do not have the CSTF2 expression typical example (initial magnification, x100).D, the Kaplan-Meier of NSCLC patient's survival analyzes (P=0.0079, sequence check).
[Fig. 3] Fig. 3 describes the inhibition to the growth of NSCLC cell to the siRNA of CSTF2: A, and response is handled (si-CSTF2-#1 or si-CSTF2-#2) to the siRNA of CSTF2 or contrasted the expression (top) of the CSTF2 of siRNA [si-enhanced green fluorescence protein (si-EGFP) or si-luciferase (si-LUC)] in the A549 that analyzes through sxemiquantitative RT-PCR and the LC319 cell.B, C is with the MTT and the CFA of the tumour cell of si-CFTF2 or contrast siRNA transfection.
[Fig. 4] Fig. 4 describes the enhancing that CSTF2 imports the mammalian cell cell growth: A, through the transient expression of CSTF2 in the COS-7 cell of Western engram analysis detection.Pair cell imports pcDNA3.1-myc-His-CSTF2 or analog carrier.B and C are through MTT and CFA assessment cell viability and colony number.Be determined in three repeating holes and carry out three times.
Detailed Description Of The Invention
Though the method for describing among any and this paper is similar with material or the method that is equal to and material can be used for implementing or test embodiment of the present invention, description preferable methods, device and material now.Yet, before describing material of the present invention and method, be appreciated that the specific size that the invention is not restricted to describe among this paper, shape, yardstick, material, method, scheme, etc., change because these can according to normal experiment and optimization.It is also understood that the term that uses during this is described is just from the purpose of describing special style or embodiment, but not intention restriction scope of the present invention, scope of the present invention only can be limited by accompanying claims.
Through addressing clearly complete income this paper of disclosure with each piece publication, patent or the patented claim mentioned in this specification sheets.Yet, nowhere may be interpreted as among this paper and admit that the present invention does not have qualification to rely on invention formerly and early than this type of openly.
If conflict is arranged,, comprise that definition is as the criterion when with this specification sheets.In addition, these materials, method and embodiment are exemplary, but not the intention restriction.
Definition
Like what use among this paper, word "/kind ", " being somebody's turn to do " and " said " mean " at least one/kind ", unless expressly stated otherwise.
Term " amino acid " refers to naturally occurring and synthetic amino acid, and with the amino acid analogue and the amino acid analog thing of naturally occurring amino acid performance identity function.Naturally occurring amino acid refers to by the genetic code amino acids coding, and in cell after translation the amino acid (for example oxyproline, Gla and O-Serine O-phosphate) through modifying.Phrase " amino acid analogue " refers to have identical Essential Chemistry structure (α carbon combines with hydrogen, carboxyl, amino and R group) with naturally occurring amino acid but has the R group of process modification or the compound (for example homoserine, nor-leucine, methionine sulfoxide, methionine(Met) methyl sulfonium) of the main chain that process is modified.Phrase " amino acid analog thing " refers to and has with general amino acid various structure but the chemical cpd of performance and the function of general amino acid similarity.
Amino acid can be censured through they known trigram symbol or one-letter symbols of being recommended by the IUPAC-IUB biochemical nomenclature commission in this article.
Like what use among this paper; Term " biological sample " refers to the subclass of complete organism or its tissue, cell or integral part (for example body fluid includes but are not limited to blood, mucus, lymph liquid, synovia, cerebrospinal fluid, saliva, amniotic fluid, Cord blood, urine, vaginal secretion and seminal fluid)." biological sample " further refers to from homogenate, lysate, extract, cell culture or the tissue culture of the subclass preparation of complete organism or its cell, tissue or integral part, or its level is divided or part.At last, " biological sample " refers to contain the substratum of cellular constituent (such as protein or polynucleotide), the nutritious soup or the gel of breeding therein such as organism.
Term " polynucleotide ", " oligonucleotide ", " nucleic acid " and " nucleic acid molecule " interchangeable in this article use refer to the polymkeric substance of nucleic acid residue, and unless expressly stated otherwise,, censure through the single-letter code that they are generally acknowledged.These terms are applicable to nucleic acid (Nucleotide) polymkeric substance that wherein one or more nucleic acid connect through ester linkage.Nucleic acid polymers can be constituted by DNA, RNA or its, and contain nucleic acid polymers that naturally occurring and non-natural exist the two.
Term " polypeptide ", " peptide " and " protein " interchangeable in this article use refer to the polymkeric substance of amino-acid residue.These terms are applicable to that wherein one or more amino-acid residues are the aminoacid polymerss through the residue of modification or the residue of non-natural existence (such as corresponding naturally occurring amino acid whose artificial chemical simulation thing), and naturally occurring aminoacid polymers.
Term " cell-proliferation activity ", " cell proliferation enhanced activity " and " active " the interchangeable in this article use of cell proliferation promotion; Refer to when polypeptide is with cells contacting or in the gene transfered cell of coded polypeptide the activity of polypeptide promotion or enhancing cell proliferation.
Represent that with term " isolating " and " purifying " of material (for example polypeptide, antibody, polynucleotide, etc.) coupling this material is substantially free of at least a material that is included in the natural origin.So; Isolating or antibody purified refers to be substantially free of cell material for example glucide, lipid or other proteinic antibody of contaminative that comes the cell or tissue of self-derived this protein (antibody) to originate, or when chemosynthesis, is substantially free of the antibody of precursor or other chemical.Term " is substantially free of cell material " and comprises polypeptide wherein and the polypeptide prepared product that is used to separate or the cellular constituent of its cell of recombinant production is separated.
So, the polypeptide that is substantially free of cell material comprises the polypeptide prepared product with the heterologous protein (being also referred to as " contaminative protein " in this article) that is less than about 30%, 20%, 10% or 5% (by dry weight basis).When polypeptide reorganization produced, in some embodiments, it also was substantially free of substratum, comprised the polypeptide prepared product with the substratum that is less than about 20%, 10% or 5% protein prepared product volume.When polypeptide produces through chemosynthesis; In some embodiments; It is substantially free of precursor or other chemical, comprises the precursor relevant with this protein synthesis with the protein prepared product volume that is less than about 30%, 20%, 10%, 5% (pressing dry weight basis) or the polypeptide prepared product of other chemical.Can be for example appearance through single band after sodium lauryl sulphate (the SDS)-polyacrylamide gel electrophoresis of protein prepared product and coomassie brilliant blue staining of gel or the like show that the specified protein prepared product contains the polypeptide of separative or purifying.In one embodiment, protein (comprising antibody of the present invention) is isolating or purifying.
" isolating " or " purifying " nucleic acid molecule, for example the cDNA molecule can be substantially free of other cell material or substratum when producing through recombinant technology, or when chemosynthesis, can be substantially free of precursor or other chemical.In one embodiment, the proteic nucleic acid molecule of the present invention of encoding is isolating or purifying.
Only if definition is arranged in addition, term " cancer " referred to express the cancer of CSTF2 gene, such as lung cancer, comprised gland cancer (ADC), squamous cell carcinoma (SCC), large cell carcinoma (LCC) and small cell lung cancer (SCLC).
CSTF2 gene or CSTF2 albumen
The nucleic acid of people CSTF2 gene and peptide sequence are shown in respectively but are not limited to SEQ ID NO:1 and SEQ ID NO:2.In addition, above-mentioned sequence data also can obtain through GenBank accession number NM_001325.
According to one aspect of the present invention, think that functionally equivalent also is above-mentioned " polypeptide ".In this article, proteic " functionally equivalent " is for having the polypeptide of the BA that is equal to this albumen.Just, the polypeptide of any reservation biology ability can be used as this type of functionally equivalent among the present invention.For example, known CSTF2 polypeptide has the cell proliferation enhanced activity, RNA combines activity, RNA nicking activity, RNA polyadenylation activity or the like.Think and keep that at least a polypeptide is the functionally equivalent of CSTF2 polypeptide among the present invention in these activity.This type of functionally equivalent comprises that those are to proteicly naturally existing that aminoacid sequence substitutes, deletion, adding or insert one or more amino acid whose.Perhaps, the sequence that polypeptide can comprise with corresponding protein has at least about 80% homology (being also referred to as sequence identity), more preferably at least about 90% to 95% identity, and the further more preferably aminoacid sequence of 96%, 97%, 98% or 99% identity.In other embodiments, polypeptide can by under stringent condition with the natural polynucleotide encoding that has nucleotide sequence hybridization of gene.
Polypeptide of the present invention can aminoacid sequence, molecular weight, iso-electric point, sugar chain have or not or form aspect change, depend on the cell or the host that are used to produce it, or the purification process that uses.In any case as long as it has the function with the proteic functional equivalent of people, it just within the scope of the invention.
Phrase " strict (hybridization) condition " refers to following condition, under this condition, nucleic acid molecule can with its target sequence hybridization, usually in the nucleic acid complex mixture, but less than with the detectable hybridization of other sequence.Stringent condition depends on sequence, and can be different in different situations.Long sequence is at higher temperature specific hybrid.Detailed guidance is found in Tijssen for nucleic acid hybridization; Techniques in Biochemistry and Molecular Biology--Hybridization with Nucleic Probes, " Overview of principles of hybridization and the strategy of nucleic acid assays " (1993).Usually, stringent condition is chosen as at the ionic strength and the pH that limit, the heat fusion joint (T of bit sequencing row m) low about 5-10 ℃.T mBe following temperature (under the ionic strength, pH and the nucleic acid concentration that limit), wherein 50% with target complementary probe when the balance with target sequence hybridization (because of the excessive existence of target sequence, so at T m, 50% probe is occupied when balance).Stringent condition also can be realized through adding destabilizing agent (such as methane amide).For selectivity or specific hybrid, positive signal is the twice at least of background, 10 times of preferred background hybridization.The stringent hybridization condition of exemplary comprises as follows: 50% methane amide, 5x SSC and 1%SDS, at 42 ℃ of incubations, or 5x SSC, 1%SDS, at 65 ℃ of incubations, with 0.2x SSC and 0.1%SDS 50 ℃ of cleanings.
In background of the present invention, the hybridization conditions that is used to separate the DNA of the polypeptide that coding and above-mentioned people's albumen is equal on function can be selected by those skilled in the art are conventional.For example, can hybridize as follows: carried out 30 minutes or longer prehybridization with " Rapid-hyb buffer " (Amersham LIFE SCIENCE) at 68 degrees centigrade, add probe through mark, and at 68 degrees centigrade of incubations 1 hour or longer.Following cleaning step can for example carry out in the low strict degree condition.A kind of low strict degree condition of exemplary can comprise 42 ℃, 2x SSC, 0.1%SDS, preferred 50 ℃, 2x SSC, 0.1%SDS.The high stringent condition of usually preferred use.A kind of high stringent condition of exemplary can be included in room temperature and in 2x SSC, 0.1%SDS, clean 3 times each 20 minutes; In 1x SSC, 0.1%SDS, clean 3 times each 20 minutes then, and in 1x SSC, 0.1%SDS, cleaned 2 times each 20 minutes at 50 degrees centigrade at 37 degrees centigrade.Yet several factors such as temperature and salt concn, can influence the strict degree of hybridization, and those skilled in the art can select suitable factor to reach required strict degree.
Generally speaking, one or more amino acid whose modifications do not influence proteic function in the known protein.In fact; Known mutations or keep primary BA (Mark et al., Proc Natl Acad Sci USA 81:5662-6 (1984) through the albumen modified (it has through certain aminoacid sequence is substituted, deletes, inserts and/or adding the aminoacid sequence that one or more amino-acid residues are modified); Zoller and Smith, Nucleic Acids Res 10:6487-500 (1982); Dalbadie-McFarland et al., Proc Natl Acad Sci USA 79:6409-13 (1982)).Thereby; One skilled in the art will realize that; Aminoacid sequence is added individually, deletes, inserts or substitutes; This changes single amino acids or few amino acids, perhaps is considered to those modifications of " the conservative modification "---and the albumen that wherein proteic change generation has identity function, these all are acceptable in the background of the present invention.
As long as keep protein-active, the number of amino acid mutation does not receive special restriction.Yet, general preferred change aminoacid sequence 5% or still less.Thereby in a preferred embodiment, the amino acid number that in this type of two mutants, will suddenly change is generally 30 amino acid or still less; Preferred 20 amino acid or still less; More preferably 10 amino acid or still less, more preferably 6 amino acid or still less, even more preferably 3 amino acid or still less.
The amino-acid residue that suddenlys change preferably sports the another kind of amino acid (process that is called conservative amino acid substitutions) that the amino acid side chain characteristic keeps.The example of amino acid side chain characteristic have hydrophobic amino acid (A, I, L, M, F, P, W, Y, V), hydrophilic amino acid (R, D, N, C, E, Q, G, H, K, S, T) and side chain with following common functional group or characteristic: aliphatic lateral chain (G, A, V, L, I, P); Contain oh group side chain (S, T, Y); Contain sulphur atom side chain (C, M); Contain carboxylic acid and acid amides side chain (D, N, E, Q); Contain alkali side chain (R, K, H); With contain aromatic side chain (H, F, Y, W).Provide the amino acid whose conservative property substitution tables of functional similarity being known in the art.For example, following 8 groups constitute conservative property alternate amino acid various comprising each other:
1) L-Ala (A), glycocoll (G);
2) aspartic acid (D), L-glutamic acid (E);
3) l-asparagine (N), Stimulina (Q);
4) l-arginine (R), Methionin (K);
5) Isoleucine (I), leucine (L), methionine(Met) (M), Xie Ansuan (V);
6) phenylalanine(Phe) (F), tyrosine (Y), tryptophane (W);
7) Serine (S), Threonine (T); With
8) halfcystine (C), methionine(Met) (M) (referring to for example Creighton, Proteins 1984).
This type of conservative property modified polypeptide is included in the albumen of the present invention.Yet the present invention is not limited to this, and protein comprises that non-conservation modifies, as long as proteic at least a BA is able to keep.In addition, through the albumen of modification do not get rid of polymorphie variant, plant between homologue and those by these proteic allelotrope codings.
In addition, the CSTF2 gene is contained the polynucleotide of this type of functionally equivalent of proteins encoded.Except hybridization, can utilize gene amplification method, polymerase chain reaction (PCR) method is for example separated the polynucleotide of the polypeptide that coding and protein function be equal to through using sequence synthetic primer based on above-mentioned information.Respectively as the polynucleotide of Human genome and proteic functionally equivalent and polypeptide usually and its parent thuja acid or aminoacid sequence have high homology." high homology " is often referred to 40% or higher homology, preferred 60% or higher, and more preferably 80% or higher, even more preferably 90% to 95% or higher, even more preferably 96%, 97%, 98%, 99% or higher.The algorithm that the homology of specific polynucleotide or polypeptide can be followed in " Wilbur and Lipman, Proc Natl Acad Sci USA 80:726-30 (1983) " is confirmed.
Antibody
Like what use among this paper, term " antibody " intention comprise can with the Tegeline and the fragment thereof of specified albumen or the reaction of its peptide specific.Antibody can comprise people's antibody, long sourceization (primatized) antibody of spirit, chimeric antibody, bi-specific antibody, humanized antibody, with the antibody and the antibody fragment of other albumen or radioactively labelled substance fusion.In addition; In this article; Antibody uses with broad sense, specifically contains complete monoclonal antibody, polyclonal antibody, by multi-specificity antibody (for example bi-specific antibody) and antibody fragment that at least two kinds of complete antibodies form, needs only them and represents desired biological activity." antibody " indication all categories (for example IgA, IgD, IgE, IgG and IgM).
The present invention uses the antibody to CSTF2.These antibody can generate through currently known methods.
The exemplary technology that is used to generate the antibody that uses according to the present invention has been described.
(i) polyclonal antibody:
Polyclonal antibody is preferably through repeatedly subcutaneous (sc) or intraperitoneal (ip) injection related antigen and adjuvant produce in animal.What come in handy is, use difunctional or derivatization reagent with related antigen with in the species of treating immunity, have the coupling mutually of immunogenic albumen, wherein have for example key hole of immunogenic albumen Hemocyanin, serum albumin, bovine thyroglobulin or Trypsin inhibitor SBTI, and wherein difunctional or derivatization reagent for example maleimide phenylformic acid thiosuccimide ester (maleimidobenzoyl sulfosuccinimide ester) (through the cysteine residues coupling), N-hydroxy-succinamide (through lysine residue), LUTARALDEHYDE, succinyl oxide, SOCl 2Or R ' N=C=NR, wherein R is different alkyl with R '.
With antigen, immunogenic conjugate or verivate immune animal, mode is for example to make up the Freund's complete adjuvant of 100 or 5 μ g albumen or conjugate (being respectively applied for rabbit or mouse) and 3 times of volumes, and at this solution of multiple intradermal injections.After one month, come the animal booster immunization through subcutaneous injection at multiple spot with the peptide or the conjugate of 1/5-1/10 original vol in the Freund's complete adjuvant.After 7-14 days, animal is taken a blood sample, and measure the antibody titers of serum.The booster immunization animal reaches high platform until titre.Preferably, what be used for animal is carried out booster immunization is the conjugate of same antigen, but with different albumen couplings and/or through different cross-linked material couplings.
Conjugate also can prepare as protein fusions in reconstitution cell is cultivated.Aggregating agent prepared therefrom such as alum also is applicable to that enhancing immunity replys.
(ii) monoclonal antibody:
Monoclonal antibody obtains from the colony of the antibody of homogeneous basically, and the single antibody that promptly constitutes this colony is identical, just possibly have the sudden change of a spot of possible natural generation.So, modifier " mono-clonal " is meant such characteristic of antibody, and promptly it is not the mixture of discrete (discrete) antibody.
For example, monoclonal antibody can use hybridoma method to prepare, and this method at first is recorded in Kohler G&Milstein C.Nature.1975 Aug 7; 256 (5517): 495-7 perhaps can prepare (United States Patent(USP) No. 4,816,567) through recombinant DNA method.
In hybridoma method, the host animal that mouse or other are suitable such as hamster, produces maybe and can generation meeting specificity to combine to be used for immune proteic antibody to cause lymphocyte with this paper immunity mentioned above.Perhaps, can be at external immune lymphocyte.Use suitable fusogen such as polyoxyethylene glycol that lymphocyte and myeloma cell are merged to form hybridoma (Goding then; Monoclonal Antibodies:Principles and Practice; Pp.59-103 (Academic Press, 1986)).
Be seeded in the suitable medium hybridoma of so preparation and cultivation, said substratum preferably comprises the material of one or more parent myeloma cell who suppresses not fusion growths or survival.For example; If said parent myeloma cell lacks hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT); The substratum of so said hybridoma can comprise xanthoglobulin, methotrexate and thymus pyrimidine (HAT substratum) usually, and these materials stop the growth of HGPRT deficient cells.
Preferred myeloma cell is those efficient fusions, supports the stable high level of selected antibody produced cell to produce antibody, and responsive to certain substratum such as HAT substratum.In these; Preferred myeloma cell line is a rat bone marrow tumour system, certainly can be from cell home-delivery center of Salk institute (Salk Institute Cell Distribution Center, San Diego such as those; California USA) MOPC-21 that obtains and MPC-11 mouse tumor and can be from American type culture collection (American Type Culture Collection; Manassas, Virginia, the SP-2 or the X63-Ag8-653 that USA) obtain are cell-derived.Human myeloma and mouse-people's allos myeloma cell line is also on the books to be used to produce 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 substratum dissecting needle that hybridoma is grown therein is to the generation of antigenic monoclonal antibody.Preferably, measure the binding specificity of the monoclonal antibody that produces by hybridoma such as radioimmunoassay (RIA) or enzyme linked immunological absorption measurement method (ELISA) through immunoprecipitation or through external combination mensuration.
For example, the binding affinity of monoclonal antibody can pass through Munson PJ&Rodbard D.Anal Biochem.1980 Sep 1; 107 (1): the 30Scatchard of 220-39 analyzes and measures.
After identifying the hybridoma that produces specificity, avidity and/or active antibody with expectation; This clone can come subclone through the limiting dilution rules; And cultivate (Goding through standard method; Monoclonal Antibodies:Principles and Practice, pp.59-103 (Academic Press, 1986)).The substratum that is suitable for this purpose comprises, for example D-MEM or RPML-1640 substratum.In addition, hybridoma can come culturing in vivo as ascitic tumor in animal.
To be separated by subclone excretory monoclonal antibody and substratum, ascites or serum suitably through routine immunization sphaeroprotein purifying rules, said rules are such as for example albumin A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatography.
The DNA of coding monoclonal antibody is easy to use conventional rules, and the oligonucleotide probe of the gene through using can specificity combine to encode murine antibody heavy chain and light chain for example separates and checks order.Hybridoma is the preferred source of this type of DNA.In case separate; Just can DNA be inserted in the expression vector; Then its transfection entering itself is not produced the host cell of Tegeline in addition; In Bacillus coli cells, ape COS cell, Chinese hamster ovary (CHO) cell or myeloma cell, thereby in recombinant host cell, obtain the synthetic of monoclonal antibody.Summary paper about the DNA of recombinant expressed encoding antibody in bacterium comprises Skerra A.Curr Opin Immunol.1993 Apr; 5 (2): 256-62 and Pl ü ckthun A.Immunol Rev.1992 Dec; 130:151-88.
Immunoglobulin gene or its part were expressed, encoded to the specific antibody that another kind of generation can react with CSTF2 or the method for antibody fragment with the CSTF2 screening in bacterium expression library.For example, can use the phage expression library, the Fab fragment of The expressed, VH district and Fv district in bacterium.Referring to for example 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.Can identify the immunoglobulin fragment that can react with CSTF2 with this type of library of CSTF2 peptide screening.Perhaps, SCID-hu-mouse (can obtain from Genpharm) can be used to produce antibody or its fragment.
In another embodiment, can be from using McCafferty J, et al., Nature.1990 Dec6; 348 (6301): 552-4; Clarkson T, et al., Nature.1991 Aug 15; 352 (6336): antibody phage library separation antibody or antibody fragment that the technology of putting down in writing among the 624-8 produces; Marks JD, et al., J MoL BioL, 222:581-597 (1991) J Mol Biol.1991 Dec 5; 222 (3): 581-97 has put down in writing the use phage library respectively and has separated mouse and people's antibody.Follow-up publication has been put down in writing through chain reorganization and has been produced high-affinity (nM scope) people's antibody (Marks JD, et al., Biotechnology (N Y) .1992Jul; 10 (7): 779-83), and strategy (Waterhouse P, et al., Nucleic Acids Res.1993 May11 that the conduct of recombinating in (combinatorial infection) and the body makes up the super large phage library are infected in combination; 21 (9): 2265-6).Therefore, these technology are the technological feasible alternative methods of traditional monoclonal antibody hybridoma that are used to separate monoclonal antibody.
Also can be through heavy chain and light chain constant domain encoding sequence replacement homology mouse sequence (United States Patent(USP) No. 4,816,567 of for example choosing; Morrison SL, et al., Proc Natl Acad Sci U S is A.1984Nov; 81 (21): 6851-5), perhaps through coming modifying DNA with the whole or part encoding sequence of immunoglobulin coding sequence and NIg polypeptide is covalently bound.
Usually; With this type of NIg polypeptide replacement antibody constant domain, perhaps the variable domain with the antigen binding site of their replacement antibody comprises that with establishment an antigen binding site with certain antigen-specific has the chimeric bivalent antibody of the antigen binding site of different antigen-specifiies with another.
(iii) humanized antibody:
Be used for the humanized method of non-human antibody on the books in the art.Preferably, one or more amino-acid residues have been introduced in the humanized antibody from inhuman source.These inhuman source amino-acid residues often are called " input (import) " residue, take from certain " input " variable domain usually.Humanization can be followed Winter and co-worker's thereof method (Jones PT, et al., Nature.1986 May29-Jun 4 basically; 321 (6069): 522-5; Riechmann L, et al., Nature.1988 Mar24; 332 (6162): 323-7; Verhoeyen M, et al., Science.1988 Mar25; 239 (4847): 1534-6) implement, with the corresponding sequence of hypervariable region sequence replacement people antibody.Thereby the sub-fraction that this type of " humanization " antibody is wherein whole person's variable domain (substantially less than intact) is by the chimeric antibody of replacing from the corresponding sequence of inhuman species (United States Patent(USP) No. 4,816,567).In practice, humanized antibody is some hypervariable region residues in people's antibody normally, possibly also have some FR residues to be substituted by the residue from similar site in the rodent animal antibody and the antibody that obtains.
People's variable domain in that preparation will be used in the humanized antibody comprises light chain and heavy chain variable domain, selection be very important for reducing antigenicity.According to so-called " best-fit (best-fit) " method, screen the sequence of the variable domain of rodent animal antibody to the whole library of known person variable domain sequence.Then, acceptance and the immediate human sequence of rodent sequence are as the people's framework region (FR) that is used for 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 kind method is used the consensus sequence deutero-specific frame district from everyone antibody of light chain or the specific subgroup of heavy chain.Same framework can be used for several different humanized antibodies, and (Proc Natl Acad Sci U S is May15 A.1992 for Carter P, et al.; 89 (10): 4285-9; Presta LG, et al., J Immunol.1993 Sep 1; 151 (5): 2623-32)).
In addition, importantly, the humanization of antibody keeps antigenic high-affinity and other favourable biological characteristics.In order to realize this goal, according to a kind of preferable methods, the preparation of humanized antibody is by following process: use the three-dimensional model of parent and humanization sequence to analyze parental array and each ways makes conceptual researches humanization product.Three-dimensional Tegeline model is that those skilled in the art generally can get and are familiar with.There is the available computer program to come diagram and the possible three-dimensional conformation structure that shows selected candidate's immunoglobulin sequences.Check that these demonstrations are allowed and analyze residue possibly act in the function performance of candidate's immunoglobulin sequences, the i.e. residue of analyzing influence candidate Tegeline and its antigen bonded ability.Like this, can select the FR residue, and with the combination of acceptor and list entries, thereby realize the antibody characteristic of expectation, such as avidity to the increase of target antigen.Generally speaking, the hypervariable region residue directly and the most substantively participates in influencing the antigen combination.
(iv) people's antibody:
As humanized alternative, can generate people's antibody.For example, might generate such transgenic animal (for example mouse) now, they can produce the complete complete or collected works (repertoire) of people's antibody when by immunization, and do not have endogenous Tegeline to produce.For example, on the books, deletion heavy chain of antibody joining region (JH) gene that in chimeric and germ line mutation mouse, isozygotys causes endogenous antibody generation to be suppressed fully.With ethnic group is that the immunoglobulin gene array is transferred to and can be caused producing when the antigen challenge people's antibody in this type of germ line mutation mouse.For example referring to Jakobovits A, et al., Proc Natl Acad Sci U S is Mar 15 A.1993; 90 (6): 2551-5; Nature.1993 Mar 18; 362 (6417): 255-8; Br ü ggemann M, et al., Year Immunol.1993; 7:33-40; And United States Patent(USP) No. 5,591,669; 5,589,369 and 5,545,807.
Perhaps, can use display technique of bacteriophage (McCafferty J, et al., Nature.1990 Dec6; 348 (6301): 552-4) external from producing people's antibody and antibody fragment from immunoglobulin variable (V) domain gene complete or collected works (repertoire) without the donor of immunity.According to this technology, antibody V domain gene is met in the main or less important coat protein gene that frame ground (in-frame) is cloned into filobactivirus (such as M13 or fd), and on the surface of phage particle, show as the functional antibodies fragment.Because filamentous particle contains the single stranded DNA copy of phage genome, the gene of the antibody that represents those characteristics is selected so the selection of carrying out based on the functional performance of antibody also causes encoding.So, some characteristics of phage simulation B cell.Phage display can be implemented with various ways, about their summary referring to for example Johnson KS&Chiswell DJ.Curr Opin Struct Biol.1993; 3:564-71.There are several sources of V constant gene segment C to can be used for phage display.
Clackson T, et al., Nature.1991 Aug 15; 352 (6336): 624-8 has separated a series of various anti-azolactone antibody from the small-sized combinatorial library at random of spleen deutero-V gene of the immune mouse of hanging oneself.Can make up V gene complete or collected works, and can follow the technical point of putting down in writing in the following document basically and leave antibody: Marks JD, et al., J Mol Biol.1991 Dec 5 to a series of various antigens (comprising autoantigen) from not immune people's donor; 222 (3): 581-97, or Griffiths AD, et al., EMBO are J.1993Feb; 12 (2): 725-34.Also can be referring to United States Patent(USP) No. 5,565,332 and 5,573,905.
People's antibody also can produce (referring to United States Patent(USP) No. 205,567,610 and 5,229,275) by external activatory B cell.In total common pending application, put down in writing and used the SCID mouse to produce a kind of preferred means of people's antibody.
(v) antibody fragment:
Develop multiple technologies and be used to produce antibody fragment.Traditionally, these fragments be through the proteolytic digestion complete antibody deutero-(referring to for example 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).Yet these fragments also can directly be produced by recombinant host cell now.For example, can be from the antibody phage library separation antibody fragment of preceding text discussion.Perhaps, can directly reclaim Fab '-SH fragment, and chemical coupling is to form F (ab ') from intestinal bacteria 2Fragment (Carter P, et al., Biotechnology (N Y) .1992 Feb; 10 (2): 163-7).According to another kind of way, F (ab ') 2Fragment can directly be separated from the recombinant host cell culture.Other technology that is used to produce antibody fragment can be obvious to skilled practitioner.In other embodiments, the antibody of selection is strand Fv fragment (scFv).Referring to WO 93/16185; United States Patent(USP) No. 5,571,894 and 5,587,458.Antibody fragment also can be " linear antibody ", for example as for example record in the United States Patent(USP) No. 5,641,870.This type of linear antibody fragment can be monospecific or dual specific.
(vi) non-antibody is conjugated protein:
Term " non-antibody is conjugated protein " or " non-antibody part " or " antigen-binding proteins " interchangeable use; Refer to use the antibody analog of NIg skeleton; Comprise adnectins, avimers, single chain polypeptide binding molecule and antibody appearance binding peptide stand-in, discussed in detail as hereinafter.
Developed other with fixed with the similar mode target of antibody and combine the material of target thing.Some these " antibody analog " uses the alternative albumen framework of NIg skeleton as antibody variable region.
For example, Ladner et al. (United States Patent(USP) No. 5,260,203) has put down in writing the single polypeptide chain binding molecule, and it has light chain and variable region of heavy chain with antibody---they flock together but are what to separate on molecular level---similar binding specificity.The strand binding molecule contains the antigen binding site of heavy chain of antibody and variable region of light chain simultaneously, and they connect through peptide linker, and can be folded into and two similar structures of peptide antibody.The strand binding molecule is compared with traditional antibody and is showed multiple advantage, comprises that size is littler, stability is higher and is modified more easily.
(Proc Natl Acad Sci USA 92 (14): 6552-6556 (1995)) put down in writing a kind of antibody surrogate thing such as Ku based on cytochrome b5 62.Ku etc. (1995) have processed a library, and wherein two of cytochrome b5 62 rings are by randomization, and therefrom select the combination to bovine serum albumin(BSA).Find that each two mutants is with the mode selective binding BSA similar with anti-BSA antibody.
Lipovsek etc. (United States Patent(USP) No. 6,818,418 and 7,115,396) have put down in writing a kind of antibody analog, it is characterized in that having fibronectin or fibronectin appearance albumen skeleton and at least one variable loop.These antibody analogs based on fibronectin are called Adnectins, and they show characteristic many and natural or that engineered antibody is identical, comprise high-affinity and specificity to any target ligands.Anyly be used to develop protein-bonded technology new or improvement and all can be used for these antibody analogs.
These are based on the structure of the antibody analog of fibronectin and the structural similitude of IgG variable region of heavy chain.Therefore, these stand-in are illustrated in the character antigen binding characteristic similar with natural antibody with the avidity aspect.In addition, these antibody analogs based on fibronectin also show some advantage better than antibody and antibody fragment.For example, the natural folding stability of these antibody analogs does not rely on disulfide linkage, under the condition that can destroy antibody usually, is stable therefore.In addition because these are based on the structure of the antibody analog of fibronectin and the structural similitude of IgG heavy chain, so can be in external employing be similar to the body of antibody the ring randomization and the reorganization process of avidity ripening process.
(Proc Natl Acad Sci USA 96 (5): 1898-1903 (1999)) put down in writing a kind of antibody analog (Anticalin (registered trademark)) such as Beste based on the NGAL skeleton.NGAL comprises that the albumen end has the β-bucket of 4 hypermutation rings.Beste (1999) carries out random mutagenesis to ring, and selects and the combining of for example resorcinolphthalein.Three kinds of variants represent with the specificity of resorcinolphthalein and combine, and wherein a kind of variant shows and similar the combining of anti-fluorescein antibody.Further analyze and disclose, all randomization positions all are variable, show that Anticalin (registered trademark) can be suitable for the surrogate as antibody.
Anticalin (registered trademark) is small-sized strand peptide, and between 160 and 180 residues, this provides the some advantages that surpass antibody, comprises the reduction manufacturing cost usually, improves storage stability and reduces immunological response.
Hamilton etc. (United States Patent(USP) No. 5,770,380) have put down in writing a kind of synthetic antibody stand-in, and it uses the non-peptide organic backbone of calixarene (calixarene) rigidity, are attached with a plurality of variable peptide loop on the skeleton as binding site.The peptide ring is relative to each other all outstanding from geometric the same side of calixarene.Because this geometry conformation, all rings all can supply to combine, thereby the binding affinity of raising and part.Yet, compare with other antibody analog, do not constitute by peptide purely based on the antibody analog of calixarene, the susceptibility of therefore proteolytic enzyme being attacked reduces.Skeleton neither be made up of peptide, DNA or RNA purely, this means that this antibody analog is relatively stable in extreme environmental conditions, and the life-span is longer.In addition, because less relatively based on the antibody analog of calixarene, its possibility that produces immunogenic response reduces.
Murali etc. (Cell Mol Biol.49 (2): 209-216 (2003)) have put down in writing a kind of method that is used for antibody is reduced to littler simulating peptide; They are called " antibody appearance combines simulating peptide (antibody like binding peptidomemetics) " (ABiP), also can be as the surrogate of antibody.
Silverman etc. (Nat Biotechnol.23:1556-1561 (2005)) have put down in writing some fusion roteins, and they are the single chain polypeptides that comprise a plurality of territories, are called " avimers ".Receptor domain development comes avimers from the people extracellular with phage display through the reorganization of external exon, be one type they to aspect the avidity of various target molecules and the specificity with similar a bit conjugated protein of antibody.The multiple domain albumen of gained can comprise a plurality of territories that independently combine, and they can represent and the single epi-position conjugated protein affinity of comparing improvement (being inferior nmole level in some situation) and specificity.More details about avimers structure and method of use are disclosed in the for example open No.20040175756 of U.S. Patent application, 20050048512,20050053973,20050089932 and 20050221384.
Except NIg albumen framework; Analog antibody characteristic in the material that includes but not limited to RNA molecule and non-natural oligomer (for example proteinase inhibitor, BENZODIAZEPINE, purine derivative and β-corner stand-in) also, they all are applicable to the present invention.
(vii) pharmaceutical formulation:
Antibody that can be through will having expectation purity with optional pharmaceutically useful support body, vehicle or stablizer (Remington ' s Pharmaceutical Sciences 16th edition; Osol; A.Ed. (1980)) mix; With the form of the freeze-dried formulation or the aqueous solution, the treatment of preparation antibody supplies to store with preparaton.Acceptable carrier, vehicle or stablizer are nontoxic at dosage that is adopted and concentration to the recipient, comprise buffer reagent, such as phosphoric acid salt, Citrate trianion and other organic acid; Inhibitor comprises xitix and methionine(Met); Sanitas is (such as octadecyl dimethyl benzyl ammonium chloride; Chlorination hexane diamine; Benzalkonium chloride, benzethonium chloride; Phenol, butanols or phenylcarbinol; Alkyl paraben is such as methyl paraben or propyl ester; Pyrocatechol; Resorcinol; Hexalin; The 3-amylalcohol; And meta-cresol); Lower molecular weight (being less than about 10 residues) polypeptide; Protein is such as serum albumin, gelatin or Tegeline; Hydrophilic polymer is such as Vinylpyrrolidone polymer; Amino acid is such as glycocoll, Stimulina, l-asparagine, Histidine, l-arginine or Methionin; Monose, disaccharides and other glucide comprise glucose, seminose or dextrin; Sequestrant is such as EDTA; Carbohydrate is such as sucrose, N.F,USP MANNITOL, trehalose or sorbyl alcohol; The salify gegenion is such as sodium; Metal composite (for example Zn-protein complex); And/or nonionogenic tenside, such as TWEEN TM, PLURONICS TMOr polyoxyethylene glycol (PEG).
Put down in writing the freeze-dried formulation that is suitable for subcutaneous administration among the WO97/04801.But this type of freeze-dried formulation can be rebuild preparaton subcutaneous administration to increased protein concentration and reconstruction Mammals to be treated in this paper with suitable diluent.
According to the particular condition needs of need treatment, preparaton described herein also can comprise more than a kind of active substance, and preferably those have complementary activity and negative impact can not arranged mutually.For example, chemotherapeutics, cytokine or immunosuppressor can further be provided.The significant quantity of other medicament like this depends on the antibody amount that exists in the preparaton, the type of illness or disease or treatment, and the factor of other above-mentioned discussion.These medicaments usually use and aforementioned used identical dosage and route of administration, or aforementioned used dosage about 1 to 99%.
Activeconstituents for example also can be embedded in through (for example be respectively Walocel MT 20.000PV or gelatin microcapsule with gather (TEB 3K) microcapsule) in condensation technique or the micro-capsule through the interfacial polymerization preparation, in the colloid drug delivery system (for example liposome, albumin microsphere spheroid, microemulsion, nano particle and Nano capsule) or in macro emulsion (macroemulsions).This type of technology is disclosed in for example Remington ' s Pharmaceutical Sciences 16th edition, Osol, A.Ed. (1980).
Can prepare extended release preparation.The suitable example of extended release preparation comprises the solid hydrophobic property polymkeric substance semipermeability matrix that contains medicament, and this matrix is the form of moulding product, for example film or microcapsule.The example that continues release matrix comprises polyester, hydrogel (for example gather (2-hydroxyethyl-methacrylic ester) or gather (vinyl alcohol)), polylactide (USP the 3rd; 773, No. 919), L-L-glutamic acid and the multipolymer of L-glutamic acid, gamma-ethyl ester, nondegradable ethene-vinyl acetate copolymer, degradable lactic acid-ethanol copolymer such as LUPRON DEPOT (the Injectable microspheres body that constitutes by lactic acid-ethanol copolymer and TAP-144) and gather-D-(-)-3-hydroxybutyric acid.The preparaton that is used for using in the body must be aseptic.This can be easy to through using aseptic membrane filtration to realize.
(x) use Antybody therapy:
The compsn that comprises this antibody can be according to the form preparation that meets good medical practice, confirm dosage and use.This antibody is preferably the mankind, chimeric or humanized antibody scFv, or antibody fragment.The factor of in this linguistic context, considering comprise clinical setting, illness or the disease of the specific lung cancer of being treated, the specific Mammals of being treated, individual patient reason, position, application process that medicament need be delivered, use time-histories and factor that other medical practitioner knew.Effective administration of antibodies amount will be determined by these Considerations in the treatment.
As general suggestion, effective amount should arrive in the scope of 20mg/kg weight in patients every dose of about every day 0.1 on the Antybody therapy that non-digestive tract is used, and the typical initial amount ranges of antibody is about 2 to 10mg/kg.
Yet, as stated, the antibody amount of these suggestions receive to a great extent to consider on the therapeutics about.Most important factor in the selection of appropriate dose and time-histories as stated, is the result of gained.
For example, to treating ongoing and acute disease, possibly begin need higher relatively dosage.For obtaining the most effectively result, according to disease or illness, said antibody maybe be as far as possible near first million, the diagnosis of disease or illness, occur or use when taking place, or in the process of disease or illness recurrence, use.
Said antibody can be used with any suitable method, comprises in non-digestive tract, subcutaneous, intraperitoneal, the lung and intranasal administration, if also have expectation local immunity suppression therapy, in pathology, uses.The non-digestive tract infusion comprises intramuscular, intravenously, intra-arterial, intraperitoneal and subcutaneous administration.
In addition, use said antibody through pulse infusion (pulse infusion) and possibly for example, use the antibody of the dosage that successively decreases for suitable.Said administration is preferably through injection, and most preferably through intravenously or subcutaneous injection, this part depends on said that use short-term or secular.
In addition, can also use other material with antibody described herein, for example cytotoxic agent, chemotherapeutics, immunosuppressor and/or cytokine.Combined administration comprises and uses different preparatons or single medicine preparaton to use simultaneously, and the sequential application of any order, wherein preferably has such for some time, and wherein two kinds (or all) active agents are brought into play its BA simultaneously.
Except that using said antibody in the patient, the present invention has also conceived with gene therapy and has used said antibody.The above-mentioned method of using the nucleic acid of encoding antibody is covered by in " antibody of administering therapeutic significant quantity " this statement.For example, produce intracellular antibody about using gene therapy, referring to the WO96/07321 that is published on March 14th, 1996.
There are two kinds of main method to make nucleic acid (optional being included in the carrier) get into patient's cell, i.e. interior the and ex vivo (ex vivo) of body.For delivering in the body, at the position that needs antibody nucleic acid is injected directly in patient's body usually.For the ex vivo treatment, take out patient's cell, nucleic acid is imported these isolated cells; And will pass through the cell of modification or directly be applied to the patient, or for example be embedded in the porous-film and implant in patient's body (referring to for example USP the 4th, 892; No. 538 and the 5th, 283, No. 187).There are multiple technologies to can be used for nucleic acid is imported viable cell.These technology are according to being that nucleic acid is transferred to cultured cell in vitro or purpose host's cells in vivo and changes to some extent.Be suitable for comprising liposome, electroporation, microinjection, cytogamy, DEAE-Expex, the calcium phosphate precipitation method etc. used in the external technology that nucleic acid is transferred in the mammalian cell.The carrier that is usually used in ex vivo delivery gene is a retrovirus.
At present preferred nucleic acid in vivo transfer techniques comprises with virus vector (such as adenovirus, I herpes simplex virus type or adeno associated virus) and the transfection carried out based on the system of lipid (lipid that can be used for the transgenosis that lipid mediates has for example DOTMA, DOPE and DC-Chol).In some situation, hope reagent with nucleic acid source and target target cell, provide together such as the part of acceptor on the special antibody of pair cell surface membrane protein or target cell, the target cell etc.When adopting liposome; The protein bound protein of the cell surface membrane relevant with endocytosis can be used for target and/or promote to take in, for example to particular cell types have the tropism capsid protein or its fragment, in circulation by location in the proteinic antibody of internalization and the targeted cells with increase the protein of transformation period in the cell.Wu et al. for example, J.Biol.Chem.262:4429-4432 (1987) and Wagner et al. have put down in writing receptor-mediated endocytosis technology among the Proc.Natl.Acad.Sci.USA 87:3410-3414 (1990).About the summary of present known genetic marker and gene therapy scheme referring to Anderson et al., Science 256:808-813 (1992).Also can be referring to WO 93/25673 and the reference of quoting thereof.
In another embodiment, the present invention also provides the antibody that the present invention is directed to CSTF2 to be used for treating the purposes of the pharmaceutical composition of the cancer of expressing the CSTF2 gene in manufacturing.
Perhaps, the present invention further provides the antibody that the present invention is directed to CSTF2 that the cancer of confession treatment expression CSTF2 gene is used.
Perhaps; The present invention further provides method or the technology of making the pharmaceutical composition be used to treat the cancer of expressing the CSTF2 gene, and wherein this method or technology comprise pharmacy or physiology acceptable carrier with the step of preparing as the antibody to CSTF2 of active ingredient.
In another embodiment; The present invention also provides method or the technology of making the pharmaceutical composition be used to treat the cancer of expressing the CSTF2 gene; Wherein this method or technology comprise active ingredient and pharmacy or physiology acceptable carrier blended step, and wherein said active ingredient is the antibody to CSTF2.
Duplex molecule
Like what use among this paper, term " isolating duplex molecule " refers to suppress the nucleic acid molecule of expression of target gene, and comprises for example short interfering rna (siRNA; For example DsRNA (dsRNA) or bobby pin RNA (shRNA)) and the short DNA/RNA (siD/R-NA that disturbs; The bobby pin block polymer (shD/R-NA) of the double-stranded block polymer (dsD/R-NA) of DNA and RNA or DNA and RNA for example).In this article, " duplex molecule " is also referred to as " double-strandednucleic acid ", " double chain acid molecule ", " double-stranded polynucleotide ", " double-stranded polynucleotide molecule ", " double chain oligonucleotide " and " double chain oligonucleotide molecule ".
Like what use among this paper; Term " target sequence " refers to the mRNA of target gene or certain section nucleotide sequence in the cDNA sequence; If the duplex molecule of this sequence of target is imported in the cell of expressing target gene, can cause the translation of the whole mRNA of target gene to be prevented.For the mRNA of gene or certain nucleotide sequence in the cDNA sequence, express this expression of gene in the cell of this gene if comprise the duplex molecule inhibition of the sequence corresponding with target sequence, can determine that it is target sequence.The double-stranded polynucleotide that suppressor gene is expressed can be the forming of 3 ' overhang (for example uu) of 2 to 5 Nucleotide by target sequence and length.
When target sequence shows through the cDNA sequence, use the sense strand sequence (being the sequence that the mRNA sequence is transformed into dna sequence dna) of double-stranded cDNA to limit target sequence.Duplex molecule comprises sense strand (it has the sequence corresponding with target sequence) and antisense strand (it has the sequence with target complement sequence), and this antisense strand and this sense strand at the complementary sequence place hybridization to form duplex molecule.
In this article, phrase " with ... correspondence " expression changes target sequence according to the kind of the nucleic acid that constitutes the duplex molecule sense strand.For example, show with dna sequence dna and the sense strand of duplex molecule when having the RNA district that the base " t " in this RNA district is replaced with base " u " when target sequence.On the other hand, show with the RNA sequence and the sense strand of duplex molecule when having DNA district that base " u " usefulness " t " in this DNA district is replaced when target sequence.
For example, when target sequence shows with RNA sequence SEQ ID NO:10 and the sense strand of duplex molecule when having the 3 ' side that is made up of DNA and partly distinguishing, " sequence corresponding with target sequence " is " 5 '-CACUUUACUUTCTGTAACT-3 ' ".
Also have,, can limit according to the kind of the nucleic acid that constitutes antisense strand with the sequence of target complement sequence for the antisense strand of duplex molecule.For example, when target sequence is presented among the RNA sequence SEQ ID NO:10, and the antisense strand of duplex molecule is when having the 5 ' side that is made up of DNA and partly distinguishing, and " with the sequence of target complement sequence " is " 3 '-GUGAAAUGAAAGACATTGA-5 ' ".
On the other hand; When duplex molecule is made up of RNA; With the corresponding sequence of target sequence shown in the SEQ ID NO:10 is the RNA sequence of SEQ ID NO:10, and is RNA sequence " 3 '-GUGAAAUGAAAGACAUUGA-5 ' " with the corresponding complementary sequence of target sequence shown in the SEQ ID NO:10.
Except the sequence and complementary sequence thereof corresponding with target sequence, duplex molecule can have the ring sequence of the 3 ' overhang that one or two length is 2 to 5 Nucleotide (for example uu) and/or connection sense strand and antisense strand to form hairpin structure.
" siRNA " refers to stop the double stranded rna molecule of said target mrna translation to the term that uses among this paper.Use is the standard technique of siRNA transfered cell, comprises wherein being those of template transcribe rna with DNA.Said siRNA includes phosphorothioate odn sequence (also using " sense strand " to refer to), anti sense nucleotide sequence (also using " antisense strand " to refer to) or both.Said siRNA can so make up make that single transcript has a target gene phosphorothioate odn sequence and its complementary anti sense nucleotide sequence arranged, for example, hairpin structure.Said siRNA can be dsRNA or shRNA.
The term that uses among this paper " dsRNA " refers to comprise the construct of two RNA molecules of mutual complementary sequence, said two RNA molecules through said complementary sequence annealing to form double stranded rna molecule.The nucleotide sequence of said two chains can not only comprise " justice is arranged " or " antisense " RNA that is selected from protein coding sequence in the target-gene sequence, also can comprise the RNA molecule with the nucleotide sequence that is selected from said target gene non-coding region.
The term " shRNA " that uses in this manual is meant: have the siRNA of stem-ring structure, it comprises first district complimentary to one another and second district (being sense strand and antisense strand).The complementary degree in two districts and direction are enough to make between two districts base pairing take place, and said first district and second district link together through the ring district, and said ring forms because lack base pairing between the Nucleotide (or nucleotide analog) in the ring district.The ring district of shRNA is the strand district between sense strand and antisense strand, may also be referred to as " interleaving strand (intervening single-strand) "
The term " siD/R-NA " that uses in this manual is meant and comprises the two double-stranded polynucleotide molecule of RNA and DNA, comprises heterozygote and the mosaic of RNA and DNA, the translation of its prevention said target mrna.In this manual, heterozygote is represented such molecule, wherein by DNA polynucleotide of forming and the polynucleotide of forming by RNA mutually mutual cross form duplex molecule; And or two of representing to form in the chain of said duplex molecule of mosaic can be contained RNA and DNA simultaneously.Use is with the routine techniques of siD/R-NA transfered cell.Said siD/R-NA comprises that CSTF2 has phosphorothioate odn sequence (also using " sense strand " to refer to), CSTF2 anti sense nucleotide sequence (also using " antisense strand " to refer to) or both.SiD/R-NA can make up like this, and single transcript is had simultaneously has phosphorothioate odn sequence and complementary anti sense nucleotide sequence, for example a hair clip from target gene.SiD/R-NA can be dsD/R-NA or shD/R-NA.
The term " dsD/R-NA " that uses in this article is meant the construct of such two molecules, and said two molecules comprise sequence complimentary to one another and by the double-stranded polynucleotide molecule of said complementary sequence annealing formation together.Article two, the nucleotide sequence of chain can not only comprise " justice is arranged " or " antisense " polynucleotide sequence of the albumen coded sequence that is selected from target-gene sequence, can also comprise the polynucleotide with the nucleotide sequence that is selected from the target gene non-coding region.The two forms (mosaic molecule) by RNA and DNA to form in two molecules of dsD/R-NA one or two, and perhaps molecule is made up of RNA and another forms (heterozygosis two strands) by DNA.
The term " shD/R-NA " that uses in this article is meant: have the siD/R-NA of stem-ring structure, it comprises first district complimentary to one another and second district, i.e. sense strand and antisense strand.The complementary degree in said district and direction are enough to make base pairing take place between them, and first district is connected through the ring district with second district, and said ring is because the shortage base pairing forms between the Nucleotide (or nucleotide analog) in the ring district.The ring district of shD/R-NA is the strand district between sense strand and antisense strand, may also be referred to as " interleaving strand ".
" the isolating nucleic acid " that uses in this specification sheets is meant: from original environment when Lock-in (for example physical environment), be removed, compare the nucleic acid of the change that synthetic property has taken place with its state of nature.In the present invention, the example of isolating nucleic acid comprises DNA, RNA and their verivate.
With said target mrna hybridization to the duplex molecule of CSTF2 gene through with normal circumstances under for the gene mRNA transcript of strand combines, disturb its translation, the expression of arrestin matter reduces or suppresses the proteic generation by the CSTF2 of CSTF2 genes encoding.
The CSTF2 expression of gene can be suppressed by the dsRNA to the CSTF2 gene in the lung cancer cell line.Therefore the invention provides the separation duplex molecule that can after importing the cell of expressing the CSTF2 gene, suppress this genetic expression.The target sequence of said duplex molecule can design through the siRNA algorithm for design, and is for example following.
The CSTF2 target sequence comprises for example nucleotide sequence SEQ ID NO:9 and 10.In other words, the present invention also provides its target sequence to comprise SEQ ID NO:9 or 10 or by its duplex molecule of forming.
Particularly, the invention provides following duplex molecule [1] to [19]:
[1] a kind of isolating duplex molecule, it suppresses the expression in vivo and the cell proliferation of CSTF2 gene after being imported into cell, and wherein said duplex molecule comprises sense strand and complementary antisense strand with it, and the two is hybridized each other and forms said duplex molecule;
[2] duplex molecule described in [1], wherein said duplex molecule are to the mRNA effect, and said mRNA matees with the target sequence that is selected from SEQ ID NO:9 and 10;
[3] duplex molecule described in [1], wherein said sense strand contains the sequence corresponding to the target sequence that is selected from SEQ ID NO:9 and 10;
[4] [1] to [3] duplex molecule described in arbitrary, wherein hybridization is to form duplex molecule at the target sequence place for sense strand and antisense strand, and this duplex molecule has the length that is less than about 100 Nucleotide;
[5] duplex molecule described in [4], wherein hybridization is to form duplex molecule at the target sequence place for sense strand and antisense strand, and this duplex molecule has the length that is less than about 75 Nucleotide;
[6] duplex molecule described in [5], wherein hybridization is to form duplex molecule at the target sequence place for sense strand and antisense strand, and this duplex molecule has the length that is less than about 50 Nucleotide;
[7] duplex molecule described in [6], wherein hybridization is to form duplex molecule at the target sequence place for sense strand and antisense strand, and this duplex molecule has the length that is less than about 25 Nucleotide;
[8] duplex molecule described in [7], wherein hybridization is to form duplex molecule at the target sequence place for sense strand and antisense strand, and this duplex molecule has about 19 length to about 25 Nucleotide;
[9] [1] to [8] duplex molecule described in arbitrary, it is made up of single polynucleotide, and said polynucleotide comprise through interleaving sense strand and the antisense strand that strand links together;
[10] duplex molecule described in [9]; It has general formula 5 '-[A]-[B]-[A ']-3 '; Wherein, [A] for comprising the sense strand of the sequence corresponding with the target sequence that is selected from SEQ ID NO:9 and 10, and [B] for to interleave strand by what 3～23 Nucleotide constituted, [A '] for comprising and the antisense strand of the sequence of target complement sequence of [A] middle selection;
[11] [1] to [10] duplex molecule described in arbitrary, it is made up of RNA;
[12] [1] to [10] duplex molecule described in arbitrary, it is made up of DNA and RNA;
[13] duplex molecule described in [12], wherein said molecule are the heterozygotes of DNA polynucleotide and RNA polynucleotide;
[14] duplex molecule described in [13], wherein said sense strand and antisense strand are made up of DNA and RNA respectively;
[15] duplex molecule described in [12], wherein said molecule are the mosaic of DNA and RNA;
[16] duplex molecule described in [15], wherein the zone of antisense strand 3 ' the distolateral wing is RNA, perhaps the zone of the zone of sense strand 5 ' the distolateral wing and antisense strand 3 ' the distolateral wing is RNA;
[17] duplex molecule described in [16], wherein said flank region is made up of 9 to 13 Nucleotide; With
[18] [1] to [17] duplex molecule described in arbitrary, wherein said molecule comprises one or two 3 ' overhang; With
[19] duplex molecule described in [3], wherein hybridization is to form duplex molecule at the target sequence place for sense strand and antisense strand, and this duplex molecule has the length of 19 to 25 Nucleotide;
Duplex molecule of the present invention will more be described in detail below.
It is known (for example see, United States Patent(USP) No. 6,506,559, this paper incorporates its full content into through carrying stating) that design has the method that suppresses the duplex molecule of expression of target gene ability in the cell.For example, can (ambion.com/techlib/misc/siRNA_finder.html on the World Wide Web) visit the computer program that is used to design siRNA from the Ambion website.
This computer program can be selected the target nucleotide sequences of duplex molecule according to following rules.
The selection of target spot:
1. the AUG initiator codon from transcript begins to scan downstream search AA dinucleotide sequence.Write down 19 adjacent Nucleotide of appearance and the 3 ' side thereof of each AA as potential siRNA target spot.Zone (within 75 bases) the design siRNA to 5 ' and 3 ' non-translational region (UTR) and contiguous initiator codon is avoided in suggestions such as Tuschl; Regulate the combination of proteins site because these zones possibly more are rich in, but and UTR is conjugated protein and/or the combination of translation initiation complex interfere siRNA endonuclease enzyme complex.
2. potential target spot and suitable genome database (people, mouse, rat etc.) are compared, the target sequence that any and other encoding sequences is had remarkable homology is got rid of outside considering.(Altschul SF etc., Nucleic Acids Res 1997 Sep 1,25 (17): 3389-402), it is found in NCBI server: www.ncbi.nlm.nih.gov/BLAST/ to main use BLAST.
3. select qualified target sequence to be used to synthesize.Usually select several target sequences along the length of gene to be assessed.
Use above-mentioned rules, the target sequence that is directed against the duplex molecule of CSTF2 gene among design the present invention is SEQ ID NO:9 and 10.
Respectively to above-mentioned target sequence being its ability of preventing expression target gene cell to be grown of duplex molecule inspection of target.Therefore, to provide with the sequence that is selected from SEQ ID NO:9 and 10 be the duplex molecule of target in the present invention.
The example of the duplex molecule of target CSTF2 gene mentioned above target sequence comprise contain the nucleotide sequence corresponding with target sequence and/or with the isolating polynucleotide of the sequence of target complement sequence.The preference of the polynucleotide of target CSTF2 gene comprise contain the sequence corresponding with SEQ ID NO:9 or 10 and/or with the polynucleotide of these sequence complementary sequences.In one embodiment, duplex molecule is made up of two polynucleotide, and polynucleotide have the sequence corresponding with target sequence, i.e. sense strand, and another polynucleotide have the sequence with target complement sequence, i.e. antisense strand.Sense strand polynucleotide and antisense strand polynucleotide are hybridized to form duplex molecule each other.The example of this type of duplex molecule comprises dsRNA and dsD/R-NA.
In another embodiment, duplex molecule is made up of polynucleotide, its have the sequence corresponding (being sense strand) with target sequence and with the sequence (being antisense strand) of target complement sequence the two.Usually, sense strand is connected through interleaving chain with antisense strand, and hybridizes each other to form hairpin ring structure.The example of this type of duplex molecule comprises shRNA and shD/R-NA.
In other words, duplex molecule of the present invention comprises sense strand polynucleotide (it has the nucleotide sequence of target sequence) and antisense strand polynucleotide (it has the nucleotide sequence with target complement sequence), and these two kinds of polynucleotide are hybridized to form duplex molecule each other.In comprising the duplex molecule of above-mentioned polynucleotide, the part of the polynucleotide of arbitrary chain or two chains can be RNA, and when target sequence limits with dna sequence dna, Nucleotide " t " usefulness " u " replacement in target sequence and the complementary sequence thereof.
In one embodiment of the invention, this type of duplex molecule of the present invention comprises stem-ring structure, and said stem-ring structure is made up of sense strand and antisense strand.Sense strand can be connected through ring with antisense strand.Thereby the present invention also provides the duplex molecule that comprises single polynucleotide, these polynucleotide contain sense strand and antisense strand the two, this sense strand and antisense strand through interleave strand and be connected or flank for interleaving strand.
In the present invention, the duplex molecule of target CSTF2 gene can have be selected from SEQ ID NO:9 and 10 sequence as target sequence.Thereby the preference of duplex molecule of the present invention is included in the polynucleotide of the sequence corresponding with SEQ ID NO:9 or 10 and complementary sequence place thereof hybridizing each other and has and SEQ ID NO:9 or the 10 corresponding sequences and the polynucleotide of complementary sequence thereof.
Duplex molecule of the present invention can the single CSTF2 gene order of target, or can a plurality of CSTF2 gene orders of target.
The separation polynucleotide that comprise any nucleotide sequence of the complementary sequence that contains target sequence and/or target sequence with the above-mentioned target sequence CSTF2 gene duplex molecule of the present invention that is target.With the CSTF2 gene is that the polynucleotide example of target comprises that those contain sequence SEQ ID NO:9 or 10 and/or the polynucleotide of the complementary sequence of these Nucleotide.Yet the present invention is not limited in these examples, and less important modification is an acceptable in the above-mentioned nucleotide sequence, as long as the said ability of preventing CSTF2 genetic expression that keeps through decorating molecule.Among this paper, phrase " less important modification " is represented one, two or several nucleic acid are replaced, lack, add or inserted to said sequence when relevant with nucleotide sequence when being used for.
In background of the present invention, term " several " can mean 3-7 when being used for nucleic acid replacement, disappearance, adding and/or inserting, and preferred 3-5, more preferably 3-4, further is more preferably 3 nucleotide residues.
According to the present invention, duplex molecule of the present invention can use the method for using among the embodiment to detect its ability.Among the following in this article embodiment, it (for example uses A549 and SBC-5) and reduces the ability that the CSTF2 gene product produces in lung cancer cell line in the duplex molecule test of external sense strand or its complementary antisense strand to the mRNA different piece that comprises the CSTF2 gene.Further; For example, than there not being cultured cells under the candidate molecules situation, with cell that candidate's duplex molecule contacts in the CSTF2 gene product minimizing can by; For example, use at embodiment: the RT-PCR that is directed against the primer of CSTF2mRNA in " sxemiquantitative RT-PCR " item detects.Then, in the external sequence that reduces the CSTF2 gene product in based on the analysis of cell, can detect its restraining effect to the cell growth.Can use cancer stricken animal (for example nude mouse heteroplastic transplantation model) to detect its intravital respective capabilities to cytostatic sequence in external analysis then, reduce with generation reduction and the growth of cancer cells that confirms the CSTF2 gene product based on cell.
When said separation polynucleotide were the RNA or derivatives thereof, " t " in the nucleotide sequence should replace with " u ".The term that uses among this paper " complementation " refers to Watson-Crick between the nucleotide units or Hoogsteen base pairing in the polynucleotide, and term " combination " means interaction two physics between the polynucleotide or chemistry.When Nucleotide and/or the non-phosphodiester that comprises modification when said polynucleotide connected, these polynucleotide can also likewise mutually combine.Generally speaking, the complementary polynucleotide sequence is hybridized the stable duplex that comprises seldom or do not have mispairing with formation under conditions suitable.Further, the sense strand and the antisense strand that separate polynucleotide described in the present invention can pass through hybridization formation duplex molecule or hairpin ring structure.In preferred embodiments, above-mentioned duplex comprises in per 10 couplings and is no more than a mispairing.In particularly preferred embodiments, the chain of duplex is complementary fully, and such duplex does not comprise mispairing.
As far as CSTF2, said polynucleotide length preferably is less than 1009 Nucleotide.For example, to described gene, polynucleotide length is less than 500,200,100,75,50 or 25 Nucleotide.Separate polynucleotide described in the present invention to the duplex molecule of formation, or the template DNA of the said duplex molecule of preparation coding is useful to the CSTF2 gene.When said polynucleotide were used to form duplex molecule, said polynucleotide can be longer than 19 Nucleotide, preferably were longer than 21 Nucleotide, and were more preferably length between about 19 and 25 Nucleotide.Thereby, the invention provides the duplex molecule that comprises sense strand and antisense strand, wherein sense strand comprises and the target sequence corresponding nucleotide sequences.In preferred embodiments, hybridization is the duplex molecule of 19 to 25 nucleotide pairs to form length at the target sequence place for sense strand and antisense strand.
In the time of in the duplex molecule transfered cell, duplex molecule is the guide that the reticent mixture of RNA inductive (RISC) serves as homologous sequence among the identification mRNA.The target RNA that is identified receives the cutting and the degraded of Dicer nuclease, and final reduction of said thus duplex molecule or inhibition are by the generation (expression) of this RNA encoded polypeptides.Therefore, duplex molecule of the present invention can limit through its ability that is created under the stringent condition with the mRNA specific hybrid strand of CSTF2 gene.The part of hybridizing with the strand that generates from duplex molecule among the mRNA in this article, is called " target sequence " or " target nucleic acid " or " target nucleotide ".In the present invention, the nucleotide sequence of " target sequence " not only can use the RNA sequence of mRNA to show, but also can use the dna sequence dna from mRNA synthetic cDNA to show.
Duplex molecule described in the present invention can comprise one or more modified nucleotides and/or the connection of non-phosphodiester.Chemically modified well-known in the art can increase stability, operability and/or the cell of said duplex molecule and take in.Those skilled in the art understand the chemically modified (WO03/070744 of other type that can introduce molecule of the present invention; WO2005/045037).In one embodiment, can use modification with the anti-degradation property that improvement is provided or the absorption of improvement.The example of above-mentioned modification includes but are not limited to, the mixing of thiophosphatephosphorothioate connection, 2 '-O-methyl ribonucleotides (particularly on the sense strand of duplex molecule), 2 '-deoxidation-fluoro ribonucleotide, 2 '-deoxyribonucleotide, " universal base " Nucleotide, 5 '-C-methyl nucleotide and reversing deoxidation dealkalize base residue (US20060122137).
In another embodiment, can use and modify the stability that strengthens duplex molecule or increase target-seeking efficient.Such modification includes but not limited to ribonucleotide and 2 '-deoxyribonucleotide (WO2004/029212) that 3 ' or 5 ' terminal chemically modified of chemically crosslinked, a chain of duplex molecule between two complementary strands of duplex molecule, sugar-modified, nuclear base modification and/or backbone modification, 2-fluoro are modified.In another embodiment, can utilize to modify to increase or to reduce and be directed against in the said target mrna and/or the avidity (WO2005/044976) of complementary nucleotide in the complementary duplex molecule chain.For example, the pyrimidine nucleotide of unmodified can use 2-sulphur, 5-alkynyl (5-alkynyl), 5-methyl or 5-proyl (5-propynyl) pyrimidine to substitute.In addition, the purine of unmodified can use 7-denitrogenation (7-deza), 7-alkyl or 7-thiazolinyl purine to replace.In another embodiment; When duplex molecule is when having the duplex molecule of 3 ' overhang; Can replace to deoxyribonucleotide (Elbashir SM etc., Genes Dev 2001 Jan 15,15 (2): 188-200) to the outstanding Nucleotide of 3 '-terminal nucleotide.About further details, can utilize open source literature such as US20060234970.The invention is not restricted to these instances, can any known chemical be modified and be applied to duplex molecule of the present invention, as long as the gained molecule keeps the ability that suppresses expression of target gene.
In addition, duplex molecule of the present invention can comprise DNA and RNA the two, for example dsD/R-NA or shD/R-NA.Particularly, heterozygosis polynucleotide or the DNA-RNA mosaic polynucleotide by DNA chain and RNA chain formation show the stability of raising.Can form the mixing of DNA and RNA; Heterozygous duplex molecule of promptly forming by DNA chain (polynucleotide) and RNA chain (polynucleotide) or the mosaic type duplex molecule that on arbitrary strand (polynucleotide) or two strands (polynucleotide), comprises DNA and RNA simultaneously; Like that, strengthen the stability of said duplex molecule.
The heterozygote of DNA chain and RNA chain can have such structure, and wherein sense strand is DNA and antisense strand is RNA, and is perhaps opposite, as long as it can suppress this genetic expression after in importing the cell of expressing target gene.Preferably, the sense strand polynucleotide are DNA and the antisense strand polynucleotide are RNA.Equally; The mosaic type duplex molecule can have such structure; Wherein sense strand and antisense strand are formed by DNA and RNA; Perhaps arbitrary in sense strand or the antisense strand is made up of DNA and RNA, and when needing only in importing the cell of expressing target gene, said duplex molecule has the activity that suppresses this genetic expression and gets final product.In order to improve the stability of duplex molecule, preferably comprise DNA as much as possible in the molecule; And in order to induce target gene expression to suppress, requiring molecule is RNA within the specific limits, with abduction delivering inhibition fully.
As a preferred embodiment of mosaic type duplex molecule, the upstream portion of duplex molecule zone (promptly being positioned at the zone of the flank of sense strand or antisense intrachain target sequence or its complementary sequence) is RNA.Preferably, said upstream portion subregion is represented the 5 ' side (5 ' end) of sense strand and the 3 ' side (3 ' end) of antisense strand.The zone that perhaps, will be positioned at sense strand 5 ' terminal flank or antisense strand 3 ' terminal flank is called the upstream portion zone.In other words, in preferred embodiments, the zone of antisense strand 3 ' terminal flank is made up of RNA, and perhaps the zone of the zone of sense strand 5 ' terminal flank and antisense strand 3 ' terminal flank is formed by RNA.For example, mosaic type of the present invention or heterozygous duplex molecule comprise following combination.
Sense strand:
5’-[---DNA---]-3’
3’-(RNA)-[DNA]-5’
: antisense strand,
Sense strand:
5’-(RNA)-[DNA]-3’
3’-(RNA)-[DNA]-5’
: antisense strand and
Sense strand:
5’-(RNA)-[DNA]-3’
3’-(---RNA---)-5’
: antisense strand.
The territory that the upstream portion subregion preferably is made up of 9-13 Nucleotide is counted from the sense strand of duplex molecule or the end of antisense intrachain target sequence or its complementary sequence.In addition; The preferred embodiment of this mosaic type duplex molecule comprises such instance: chain length is a 19-21 Nucleotide; Wherein half of the upper reaches at least of polynucleotide district (for sense strand be 5 ' regions and be 3 ' regions for antisense strand) be RNA, and second half is DNA.In such mosaic type duplex molecule, when the effect of inhibition expression of target gene is RNA than antisense strand is whole much better than (US20050004064).
In the present invention, duplex molecule can form hairpin structure, for example short hairpin RNA (shRNA) and the bob folder (shD/R-NA) be made up of DNA and RNA.ShRNA or shD/R-NA are the mixed sequences of RNA sequence or RNA and DNA, and it forms hair clip turning closely, can be used for disturbing silencer to express through RNA.ShRNA or shD/R-NA include adopted target sequence and antisense target sequence on single chain, wherein said each sequence is encircled sequence and separated.Usually, hairpin structure is cut into dsRNA or dsD/R-NA by cell mechanism, and said dsRNA or dsD/R-NA further combine with RISC.The mRNA of the target sequence coupling of this mixture combination and cutting and said dsRNA or dsD/R-NA.
In order to form hairpin ring structure, can the ring sequence that setting is made up of any nucleotide sequence between adopted sequence and the antisense sequences arranged.Therefore, the present invention also provides and has general formula 5 '-duplex molecule of [A]-[B]-[A ']-3 '.In the formula, [A] is sense strand, comprises the sequence corresponding with target sequence; [B] is for interleaving strand; [A '] is antisense strand, comprises the sequence with target complement sequence.Target sequence for example can be selected from for example SEQ ID NO:9 and 10.
The invention is not restricted to these instances, keep to suppress under the prerequisite as the ability of the CSTF2 expression of gene of target at duplex molecule, the target sequence in [A] can be on the basis of these instances, to modify and the sequence that obtains.Zone [A] and [A '] hybridization and form the ring that constitutes by zone [B].Interleave strand part [B], promptly encircle sequence, length preferably can be 3～23 Nucleotide.For example, the ring sequence can be selected from the group of being made up of following sequence (http://www.ambion.com/techlib/tb/tb_506.html).In addition, the ring sequence of being made up of 23 Nucleotide also provides active siRNA (Jacque JM et al., Nature 2002 Jul25,418 (6896): 435-8, Epub 2002 Jun 26):
CCC, CCACC or CCACACC:Jacque 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; Fruscoloni P et al., Proc Natl Acad Sci USA 2003 Feb 18,100 (4): 1639-44, Epub 2003 Feb10; With
UUCAAGAGA：Dykxhoorn?DM?et?al.，Nat?Rev?Mol?Cell?Biol?2003?Jun，4(6)：457-67。
The duplex molecule instance that preferably has hairpin ring structure of the present invention is as follows.In the structure below, the ring sequence can be selected from the group of being made up of AUG, CCC, UUCG, CCACC, CTCGAG, AAGCUU, CCACACC and UUCAAGAGA; But the invention is not restricted to this:
GGCUUUAGUCCCGGGCAGA-[B]-UCUGCCCGGGACUAAAGCC (to target sequence SEQ ID NO:9);
CACUUUACUUUCUGUAACU-[B]-AGUUACAGAAAGUAAAGUG (to target sequence SEQ ID NO:10);
In addition, active for the inhibition that strengthens duplex molecule, can add several Nucleotide to the sense strand of target sequence and/or 3 ' end of antisense strand, as 3 ' overhang.The number of the Nucleotide that adds is at least 2, is generally 2-10, is preferably 2-5.The Nucleotide that adds is at 3 ' the terminal strand that forms of the antisense strand of duplex molecule.The Nucleotide that is used for 3 ' overhang is preferred but be not limited to " u " or " t ".When duplex molecule has hairpin ring structure, 3 ' overhang is added into 3 ' end of antisense strand.
Preparing method for duplex molecule does not have particular restriction, but preferably adopts chemical synthesis process well-known in the art.According to chemical synthesis process, synthesizing respectively has justice and antisense strand polynucleotide, adopts appropriate means that they are annealed to together then, obtains duplex molecule.The annealed object lesson comprises wherein synthetic strand polynucleotide with preferably at least about 3: 7, more preferably from about 4: 6, the mixed in molar ratio of equimolar amount (promptly about 5: 5 mol ratio) basically most preferably.Then, mixture heating up to the dissociated temperature of duplex molecule, is cooled off gradually again.Can adopt domestic method well-known in the art to come purifying through the double-stranded polynucleotide of annealed.The example of purification process comprises: utilize the method for agarose gel electrophoresis, perhaps wherein randomly remove the method for remaining strand polynucleotide (for example utilizing suitable enzyme to degrade).
The regulating and controlling sequence of the said CSTF2 of being positioned at sequence flank can be identical or different, so that their expression can perhaps be regulated and control with timeliness or spatiality mode independently.Said duplex molecule can be through being cloned into the CSTF2 gene template in carrier (for example contain from the RNA polymerase III transcriptional units of small nuclear rna (snRNA) U6 or the carrier of human H1RNA promotor) in born of the same parents, to transcribe.
Perhaps, duplex molecule can be transcribed in born of the same parents through its encoding sequence being cloned in the carrier that contains adjacent with the coding region adjusting sequence that instructs duplex molecule in suitable cell, to express (for example from small nuclear rna (snRNA) U6 RNApoly III transcriptional units or people H1RNA promotor).The adjusting sequence of the encoding sequence flank of duplex molecule can be identical or different, makes their expression independently to regulate and control, and perhaps regulates and control with time or space mode.The details of the carrier that can generate duplex molecule can be described below.
The carrier of code book invention duplex molecule:
The present invention also comprises the carrier of one or more duplex molecules as herein described of encoding and the cell that comprises this carrier.
Particularly, the invention provides following carrier [1] to [10].
[1] a kind of carrier, it is encoded after being imported into cell, suppresses the expression in vivo and the duplex molecule of cell proliferation of CSTF2 gene, and wherein said duplex molecule comprises sense strand and complementary antisense strand with it, and the two hybridizes the said duplex molecule of formation each other.
[2] carrier described in [1], its duplex molecule of encoding, said duplex molecule be to the mRNA effect, said mRNA and the target sequence coupling that is selected from SEQ ID NO:9 and 10;
[3] carrier described in [1], wherein sense strand comprises the sequence corresponding to the target sequence that is selected from SEQ ID NO:9 and 10;
[4] [1] to [3] carrier described in arbitrary, its duplex molecule of encoding, wherein the sense strand of duplex molecule and antisense strand at the target sequence place hybridization to form the duplex molecule of length less than about 100 nucleotide pairs;
[5] carrier described in [4], its duplex molecule of encoding, wherein the sense strand of duplex molecule and antisense strand at the target sequence place hybridization to form the duplex molecule of length less than about 75 nucleotide pairs;
[6] carrier described in [5], its duplex molecule of encoding, wherein the sense strand of duplex molecule and antisense strand at the target sequence place hybridization to form the duplex molecule of length less than about 50 nucleotide pairs;
[7] carrier described in [6], its duplex molecule of encoding, wherein the sense strand of duplex molecule and antisense strand at the target sequence place hybridization to form the duplex molecule of length less than about 25 nucleotide pairs;
[8] carrier described in [7], its duplex molecule of encoding, wherein hybridization is about 19 duplex molecules to about 25 nucleotide pairs to form length at the target sequence place for the sense strand of duplex molecule and antisense strand;
[9] [1] to [8] carrier described in arbitrary, wherein said duplex molecule is made up of single polynucleotide, and said polynucleotide comprise through interleaving sense strand and the antisense strand that strand links together;
[10] carrier described in [9]; Its coding have general formula 5 '-[A]-[B]-[A ']-3 ' duplex molecule; Wherein, [A] for comprising the sense strand of the sequence corresponding with the target sequence that is selected from SEQ ID NO:9 and 10, and [B] for to interleave strand by what 3 to 23 Nucleotide constituted, [A '] for comprising and the antisense strand of the sequence of target complement sequence of [A] middle selection;
Carrier optimized encoding of the present invention is in the duplex molecule of the present invention of effable form.In this manual, term " is in effable form ", when being meant that this carrier is in being imported into cell, will express this molecule.In preferred embodiments, carrier comprises duplex molecule and expresses necessary regulatory element.Thereby, in one embodiment, expression vector codes nucleotide sequence of the present invention and the said nucleotide sequence of suitable expression.This carrier of the present invention can be used to produce duplex molecule of the present invention, also can directly be used as the activeconstituents of cancer therapy.
Carrier of the present invention can generate through following method: for example; Coding is gone in the expression vector to the sense strand of the duplex molecule of CSTF2 gene and the sequence clone of antisense strand; Be connected in the sequence of the said chain of coding with making the regulating and controlling sequence operability; With expression (through transcribing of dna molecular) (Lee NS et al., Nat Biotechnol 2002 May, 20 (5): 500-5) that allow two chains.For example; Transcribe by first promotor (for example being positioned at the promoter sequence of 3 ' terminal flank of cloned DNA) with the RNA molecule of mRNA antisense, mRNA is transcribed by second promotor (for example being positioned at the promoter sequence of 5 ' terminal flank of cloned DNA) for sense strand RNA molecule.Sense strand and antisense strand are hybridized in vivo, produce the duplex molecule construct that is used for reticent this gene.Perhaps, use encode the respectively sense strand of duplex molecule and two vector construction bodies of antisense strand to come to express respectively sense strand and antisense strand, form the duplex molecule construct subsequently.And clone's sequence codified has the construct of secondary structure (for example hair clip), that is, the single transcript of carrier comprise simultaneously the adopted sequence of having of target gene and complementary antisense sequences the two.
Also can dispose carrier of the present invention makes and it is implemented in stable insertion in the genome of target cell (about the explanation of homologous recombination box carrier, referring to Thomas KR&Capecchi MR, Cell 1987,51:503-12).Can reference example like Wolff etc., Science 1990,247:1465-8; USP the 5th, 580, No. 859, the 5th, 589, No. 466, the 5th, 804, No. 566, the 5th, 739, No. 118, the 5th, 736, No. 524, the 5th, 679, No. 647 and WO 98/04720.Example based on the conveying technology of DNA comprises: " naked DNA ", auxiliary (bupivacaine (bupivicaine), polymkeric substance, peptide-mediated type) are carried, cation lipid complex body and particle mediation type is delivered (" particle gun ") or pressure-mediated type is carried (for example with reference to USP the 5th; 922, No. 687).
Carrier of the present invention comprises, for example, and virus vector or bacteria carrier.The example of expression vector comprises attenuated virus hosts (with reference to USP the 4th, 722, No. 848) such as cowpox or chicken pox.This strategy relate to for example use vaccinia virus as carrier express the coding duplex molecule nucleotide sequence.Recombined vaccinia virus is expressed this molecule and is suppressed the propagation of cell thus when being imported into the cell of expressing target gene.Other example of spendable carrier comprises BCG-CWS (BCG).The BCG carrier is at Stover etc., and Nature 1991, and is on the books among the 351:456-60.Other diversified carrier can be used for the therapeutic administration and the production of duplex molecule, and example comprises that adenovirus carrier and gland are with the anthrax toxin carrier of companion's virus vector, retroviral vector, salmonella typhi (Salmonella typhi) carrier, detoxification etc.Reference example such as Shata etc., Mol Med Today 2000,6:66-71; Shedlock etc., J Leukoc Biol 2000,68:793-806; And Hipp etc., In Vivo 2000,14:571-85.
Use duplex molecule of the present invention to suppress or reduction growth of cancer cells or treatment method for cancer:
The invention provides anticancer growth, the method for lung carcinoma cell growth for example, said method is induced the dysfunction of CSTF2 gene through the expression that suppresses CSTF2.CSTF2 genetic expression can suppress through the carrier of the present invention that the duplex molecule of the present invention of any selectively targeted CSTF2 gene mentioned above maybe can be expressed at least a said duplex molecule.
The invention described above duplex molecule and carrier suppress the ability of the cell growth of cancerous cells and point out it to can be used for treating method for cancer.Therefore, invention provides through using to treat to the duplex molecule of CSTF2 gene or the carrier of expressing said molecule and suffers from the method that patients with lung cancer does not have undesirable action, because almost detect less than this gene in the normal organ.
Particularly, the present invention provides the method for following [1] to [36]:
[1] a kind of in the experimenter treatment or the method for preventing cancer; Comprise the experimenter is used the duplex molecule that is directed against the CSTF2 gene of pharmacy effective dose or the carrier of this duplex molecule of encoding, suppress the CSTF2 expression of gene when wherein this duplex molecule is in transfered cell;
[2] method of [1], wherein said duplex molecule are to the mRNA effect, and said mRNA matees with the target sequence that is selected from SEQ ID NO:9 and 10;
[3] method of [1], wherein sense strand comprises the sequence corresponding with the target sequence that is selected from SEQ ID NO:9 and 10;
[4] [1] to [3] arbitrary method is wherein used multiple duplex molecule;
[5] [1] to [4] arbitrary method, hybridization is to form duplex molecule at the target sequence place for the sense strand of wherein said duplex molecule and antisense strand, and this duplex molecule length is less than about 100 nucleotide pairs;
[6] method of [5], hybridization is to form duplex molecule at the target sequence place for the sense strand of wherein said duplex molecule and antisense strand, and this duplex molecule length is less than about 75 nucleotide pairs;
[7] method of [6], hybridization is to form duplex molecule at the target sequence place for the sense strand of wherein said duplex molecule and antisense strand, and this duplex molecule length is less than about 50 nucleotide pairs;
[8] method of [7], hybridization is to form duplex molecule at the target sequence place for the sense strand of wherein said duplex molecule and antisense strand, and this duplex molecule length is less than about 25 nucleotide pairs;
[9] method of [9], the sense strand of wherein said duplex molecule and antisense strand in target sequence place hybridization forming duplex molecule, this duplex molecule length be about 19 to about 25 nucleotide pairs;
[10] [1] to [9] arbitrary method, wherein said duplex molecule is made up of single polynucleotide, and said polynucleotide comprise through interleaving sense strand and the antisense strand that strand links together;
[11] method of [10]; Wherein said duplex molecule have general formula 5 '-[A]-[B]-[A ']-3 '; Wherein, [A] for comprise be selected from SEQ ID NO:9 and 10 in the sense strand of the corresponding sequence of target sequence, [B] for to interleave strand by what 3～23 Nucleotide constituted, [A '] is for comprising and the antisense strand of the sequence of target complement sequence of [A] middle selection;
[12] [1] to [11] arbitrary method, wherein said duplex molecule is RNA;
[13] [1] to [11] arbitrary method, wherein said duplex molecule comprises DNA and RNA;
[14] method of [13], wherein said duplex molecule are the heterozygotes of DNA polynucleotide and RNA polynucleotide;
[15] method of [14], wherein said have justice and antisense strand polynucleotide to be made up of DNA and RNA respectively;
[16] method of [13], wherein said duplex molecule are the mosaics of DNA and RNA;
[17] method of [16], wherein the zone of antisense strand 3 ' the distolateral wing is made up of RNA, and perhaps the zone of the zone of sense strand 5 ' the distolateral wing and antisense strand 3 ' the distolateral wing constitutes by RNA;
[18] method of [17], wherein said flank region is made up of 9 to 13 Nucleotide;
[19] [1] to [18] arbitrary method, wherein said duplex molecule comprises one or more 3 ' overhangs;
[20] [1] to [19] arbitrary method, wherein said duplex molecule is contained in the compsn, and said compsn also comprises transfection toughener and pharmaceutically acceptable carrier except that said molecule.
[21] [1] to [20] arbitrary method, wherein said duplex molecule is by vector encoded;
[22] method of [21], wherein by the said duplex molecule of said vector encoded to the mRNA effect, said mRNA and the target sequence coupling that is selected from SEQ ID NO:9 and 10;
[23] method of [22], wherein the sense strand by the said duplex molecule of said vector encoded comprises the sequence corresponding with the target sequence that is selected from SEQ ID NO:9 and 10;
[24] method of [23] is wherein used multiple duplex molecule;
[25] [21] to [24] arbitrary method, wherein by the sense strand length of the said duplex molecule of said vector encoded less than about 100 nucleotide pairs;
[26] method of [25], hybridization is to form duplex molecule at the target sequence place for the sense strand of wherein said duplex molecule and antisense strand, and this duplex molecule length is less than about 75 nucleotide pairs;
[27] method of [26], hybridization is to form duplex molecule at the target sequence place for the sense strand of wherein said duplex molecule and antisense strand, and this duplex molecule length is less than about 50 nucleotide pairs;
[28] method of [27], hybridization is to form duplex molecule at the target sequence place for the sense strand of wherein said duplex molecule and antisense strand, and this duplex molecule length is less than about 25 nucleotide pairs;
[29] forming duplex molecule, this duplex molecule length is between about 19 and about 25 nucleotide pairs in target sequence place hybridization for the method for [28], the sense strand of wherein said duplex molecule and antisense strand;
[30] [21] to [29] arbitrary method, wherein the said duplex molecule by said vector encoded is made up of single polynucleotide, and said polynucleotide comprise through interleaving sense strand and the antisense strand that strand links together;
[31] method of [30]; Wherein by the said duplex molecule of said vector encoded have general formula 5 '-[A]-[B]-[A ']-3 '; Wherein, [A] for comprising the sense strand of the sequence corresponding with the target sequence that is selected from SEQ ID NO:9 and 10, and [B] for to interleave strand by what 3 to 23 Nucleotide constituted, [A '] for comprising and the antisense strand of the sequence of target complement sequence of [A] middle selection;
[34] [21] to [31] arbitrary method, wherein the said duplex molecule by said vector encoded is contained in the compsn, and said compsn also comprises transfection toughener and pharmaceutically acceptable carrier except that said molecule;
[35] [1] to [34] arbitrary method, wherein said cancer is a lung cancer; With
[36] method of [35], wherein said lung cancer are gland cancer, squamous cell carcinoma, large cell carcinoma or small cell lung cancer.
The inventive method is more detailed the description below.
Can be through with cell and the growth that contacts the cell that suppresses to express the CSTF2 gene to the duplex molecule of CSTF2 gene, the compsn of expressing the carrier of this molecule or comprising same molecule.Can further said cell be contacted with transfection agents.Suitable transfection agents is known in this area.The said cell of phrase " cell growth inhibiting " expression than the cell that is not exposed to said molecule with lower speed propagation or have the viability of reduction.The cell growth can be passed through technical measurement known in the art, for example uses the MTT cell proliferating determining.
The growth of the cell of any kind of all can be prevented according to present method, needs only said cell expressing or crosses the target gene of expressing duplex molecule according to the invention.The cell that can be used as example comprises lung carcinoma cell, such as NSCLC and SCLC.
Therefore; For just suffering from or risky generation and CSTF2 diseases associated; For example the patient of cancer can treat through at least a carrier of using at least a duplex molecule of the present invention, at least a said molecule of expression or at least a compsn that comprises at least a said molecule.For example, patients with lung cancer can be treated according to the method for the invention.Cancer types can be through identifying according to the standard method of the particular type tumour of being diagnosed.Lung cancer can be passed through, and for example, CEACAMS (CEA), CYFRA, pro-GRP or the like be as the lung cancer sign, or diagnoses through chest x-ray and/or sputum cytology.More preferably, the patient with the method for the invention treatment selects with such method: through methods known in the art, for example RT-PCR or immunoassay detect the CSTF2 expression of gene in the biopsy specimen that obtains from the patient.Preferably, before treatment of the present invention, for from experimenter's said biopsy specimen through method known in the art, for example, immunohistochemical analysis or RT-PCR confirm that crossing of CSTF2 gene express.
According to the method for the invention; For cell growth inhibiting is also treated cancer by this; When using multiple said duplex molecule (or express the carrier of same molecule or contain the compsn of same molecule), each said molecule can have different structure, but acts on the mRNA with identical target sequence coupling.Perhaps, multiple duplex molecule can act on the mRNA that matees with the different target sequences of homologous genes, or acts on the mRNA that matees with heterogeneic different target sequences.For example, said method can be used the duplex molecule to the different target sequences of CSTF2 gene.Perhaps, for example, the duplex molecule of present method a kind of, two kinds or more kinds of target sequences to CSTF2 gene and other gene capable of using.
Be cell growth inhibiting, duplex molecule of the present invention can be directly can realize this molecule and corresponding mRNA transcript bonded form transfered cell.In addition, as stated, the DNA of coding duplex molecule can be used as the carrier transfered cell.For with duplex molecule and carrier transfered cell; Can use the transfection toughener, for example FuGENE (Roche diagnostics), Lipofectamine 2000 (Invitrogen), Oligofectamine (Invitrogen) and Nucleofector (Wako pure Chemical).
When treatment causes clinical benefit, during such as the reduction of size, morbidity (prevalence) or the metastatic potential of cancer among the minimizing of CSTF2 genetic expression, the experimenter, can think that this treatment is " effectively ".When prophylactically being suitable for treatment, " effectively " is meant its delay or prevents that cancer from forming, perhaps prevent or alleviate the clinical symptom of cancer.Validity combines any known diagnosis or the treat-ment of specific tumors type to confirm.
With regard to the linguistic context that method and composition of the present invention is used for " prevention " and " strick precaution ", the interchangeable in this article use of this type of term refers to reduce any activity of the mortality ratio that is derived from disease or sickness rate burden.Prevention and strick precaution can betide " one-level, secondary and tertiary prevention level ".Primary prevention with take precautions against the generation avoid disease, and secondary and tertiary prevention and strick precaution level contain appearance and the activity that reduces the negative impact of the disease of having set up through the restore funcitons and the related complication that palliates a disease that is intended to prevent and takes precautions against progress and the symptom of disease.Perhaps, prevention and take precautions against can comprise the preventative widely therapy of the seriousness that is intended to alleviate particular condition (for example reducing propagation and the transfer of tumour etc.).
Treat and/or prevent cancer and/or prevent its recurrence after operation to comprise any following step, such as the exenterate cancer cells, suppress cancerous cells growth, tumour decline or disappear, induce cancer to go down and prevent cancer generation, tumor regression, and reduce or suppress and shift.Effectively treating and/or preventing of cancer can reduce trouble cancer individual death rate and improve its prognosis, reduces the level of tumor markers in its blood, and alleviates the detected symptom that it follows cancer.For example, the alleviating or improve to constitute of symptom effectively treats and/or prevents, and comprises 10%, 20%, 30% or more the reduction more, or stable disease.
Should be understood that the said duplex molecule of the present invention substoichiometric CSTF2mRNA that degrades.Though be reluctant to arrest in any theory, think that said duplex molecule of the present invention causes the degraded of said target mrna with catalytic way.Therefore, compare with the cancer therapy of routine, the treatment effect need be transported to the position of cancer or near the duplex molecule it is wanted much less in order to implement.
Those skilled in the art are in the body weight of having considered the experimenter, age, sex, disease type, symptom and other condition, route of administration and be on the basis of factors such as topical application or systemic administration, can easily confirm the significant quantity of duplex molecule of the present invention.Generally speaking, the significant quantity of duplex molecule of the present invention be cancer location or near it intercellular concentration be about 1 nmole (nM) to about 100nM, preferably approximately 2nM is to about 50nM, more preferably approximately 2.5nM arrives about 10nM.Consideration can be used the duplex molecule of more or less amount.Persons skilled in the art can reach easily confirms required definite dosage under the particular case routinely.
Method of the present invention can be used for suppressing to express the cancer of CSTF2 gene, and for example lung cancer comprises NSCLC and SCLC, growth or transfer.Particularly, the duplex molecule that contains target sequence SEQ ID NO:9 or 10 especially preferably is used for treating lung cancer.
In order to treat cancer, can also duplex molecule of the present invention be made up with the medicament that is different from said duplex molecule and be applied to the experimenter.Perhaps, duplex molecule of the present invention can also make up with other treat-ment that is intended to be used for cancer therapy and be applied to the experimenter.For example; Duplex molecule according to the invention can with (for example be used to treat treat-ment that cancer or preventing cancer shift now; Radiotherapy; Surgical operation, and use chemotherapeutics, like the treatment of cis-platinum, carboplatin, endoxan, 5 FU 5 fluorouracil, Zorubicin, daunorubicin (daunorubicin) or tamoxifen (tamoxifen) etc.) combined administration.
In the method for the invention, the form that duplex molecule can naked duplex molecule, with deliver the combined form of material, perhaps with the administered of the recombinant plasmid of expressing duplex molecule or virus vector in the experimenter.
Be used for comprising Mirus Transit TKO lipophilic substance, Lipofectin, Lipofectamine, Cellfectin or polycation (for example polylysine) or liposome with the suitable delivery material of duplex molecule combined administration of the present invention.A kind of preferred delivery material is a liposome.
Liposome can help duplex molecule is delivered in specific tissue such as the lung tumor tissue, can also increase the transformation period in the blood of duplex molecule.The liposome that is fit to use in the present invention is that the vesica formation property lipid (vesile-forming lipids) by routine forms, and vesica formation property lipid generally includes neutrality or electronegative phosphatide, and sterol, such as SUV.Consideration to some factors can provide guidance for the selection of lipid usually, like the liposome size of expectation and the transformation period of the liposome in blood flow etc.It is known that the multiple method for preparing liposome is arranged, Szoka etc. for example, Ann Rev Biophys Bioeng1980,9:467; USP the 4th, 235, No. 871; The 4th, 501, No. 728; The 4th, 837, No. 028; With the 5th, 019, No. 369; The full content of above-mentioned document quoted incorporate this specification sheets into.
Preferably, the liposome that encapsulates duplex molecule of the present invention comprises the ligand molecular that can liposome be delivered to cancer location.Preferred ligands be with tumour or vascular endothelial cell in the part of common receptors bind, for example with tumour antigen or surface endothelial cell antigens bonded monoclonal antibody.
Particularly preferably; The liposome that encapsulates duplex molecule of the present invention has passed through to be modified in order to avoid removed by monokaryon scavenger cell and reticuloendothelial system; For example, because the surface bonding of its structure has opsonization to suppress part (opsonization inhibition moities).In one embodiment, liposome of the present invention can comprise simultaneously that opsonization suppresses part and part.
The opsonization that is used to prepare liposome of the present invention suppress part normally with the large-scale hydrophilic polymer of liposome membrane bonded.As it is employed in this manual; For example; When opsonization suppresses partly chemically or physically to overlap on liposome membrane; For example insert film itself, perhaps when directly combining, claim that then opsonization suppresses partly " to combine " with liposome membrane with the reactive group of membrane lipid through fat-soluble anchor (anchor).These opsonization inhibition hydrophilic polymers form protectiveness top layers, and this top layer is reduced huge biting-monocyte system (" MMS ") and reticuloendothelial system (" RES significantly ") to the absorption of liposome; On the books to this in No. the 4th, 920,016, USP for example, whole disclosures of the latter are quoted and are incorporated this specification sheets into.Therefore, compare with the liposome of unmodified, it is significantly longer to be suppressed the time that the liposome of part modified can be retained in the blood circulation by opsonization.From above reason, such liposome also is called as " stealth " (stealth) liposome sometimes.
Known hidden liposome is accumulated in the tissue that relies on porousness or the supply of " seepage property " capillary blood vessel system.Therefore, damaged with such capillary blood vessel system be the target tissue of characteristic, for example in the solid tumor, these liposomes can be accumulated expeditiously.Referring to Gabizon etc., Proc Natl Acad Sci USA 1988,18:6949-53.In addition, the minimizing of the absorption of RES stops hidden liposome significantly accumulating in liver and spleen, thereby reduces the toxicity of hidden liposome.Therefore, the liposome of the present invention that suppresses partly to modify with opsonization can be delivered to tumour cell with duplex molecule of the present invention.
The opsonization that is applicable to modified liposome suppresses part and is preferably about 500～about 40,000 dalton of molecular weight, 2,000～about 20,000 daltonian water-soluble polymerss more preferably from about.Comprise polyoxyethylene glycol (PEG) or W 166 (PPG) verivate in such polymkeric substance; For example, methoxyl group PEG or PPG and PEG or PPG stearate; Synthetic polymer such as SEPIGEL 305 or poly N-vinyl pyrrolidone; Straight chain shape, the dendritic or dendritic polyamide amine (polyamidoamine) of branch; ROHM; Polyalcohols (polyalchohols) has carboxyl or amino Z 150PH and polyxylose alcohol such as Chemical bond, and Sphingolipids,sialo, such as Sphingolipids,sialo GM 1The interpolymer of PEG, methoxyl group PEG or methoxyl group PPG or derivatives thereof also is fit to.In addition, the polymkeric substance that suppresses opsonization can be PEG and polyamino acid, polysaccharide, daiamid, gather any segmented copolymer in ethyleneamines or the polynucleotide.The polymkeric substance that suppresses opsonization also can be the natural polysaccharide that contains amino acid or carboxylic acid, for example galacturonic acid, glucuronic acid, mannuronic acid, mucinase, pectic acid, neuraminic acid, Lalgine, carrageenin; Amination polyose or oligosaccharides (the straight chain shape perhaps divides dendritic); Perhaps carboxylated polysaccharide or oligosaccharides for example, have generated carboxylated polyose of carboxyl bonded or oligosaccharides through the derivatives reaction with carbonic acid.
Preferably, opsonization inhibition part is PEG, PPG or derivatives thereof.Liposome with PEG or PEG derivative modified is sometimes referred to as " PEGization liposome ".
Opsonization suppresses part can be through any being attached on the liposome membrane in many known technologies.For example, the N-hydroxy-succinamide ester of PEG can combine with the fat-soluble anchor of phosphatidylethanolamine (lipid-soluble anchor), and then is attached on the film.Similarly, can pass through reductive amination, with the dextran polymer derivatize, use Na (CN) BH in the said reductive amination with the fat-soluble anchor of stearylamide 3And mixed solvent, like 30: 12 mixed things of 60 ℃ THFs and water.
Preceding text have been discussed the carrier of expressing duplex molecule of the present invention.The carrier of so at least a duplex molecule of the present invention of expression also can directly be used or use with suitable delivery combinations of substances, and said suitable delivery reagent comprises Mirus Transit LT1 lipophilic substance, Lipofectin, Lipofectamine, cellfectin, polycation (for example polylysine) or liposome.The method of cancerous area that the recombinant viral vector of expressing duplex molecule of the present invention is transported to the patient is in the technical scope in present technique field.
Duplex molecule of the present invention can be used to the experimenter through any means that are suitable for duplex molecule is delivered to cancer location.For example, duplex molecule can be used through route of administration in particle gun, electroporation or other suitable non-digestive tract or the intestines.
Route of administration comprises oral cavity, rectum or intranasal delivery in the suitable intestines.
Suitable non-digestive tract route of administration comprises in the blood vessel and using (for example intravenous push, intravenous infusion, intra-arterial are injected, endoarterial infusion and instil to the conduit of blood vessel network); Inject (for example injecting around the tumour and in the tumour) around organizing with in organizing; Subcutaneous injection or deposition; Comprise h inf (for example utilize soak into press pump), be applied directly to cancer location or near the zone it, for example (for example by conduit or other apparatus for placing; The suppository or the implant that comprise porousness, imporosity or gelatin-like material), and suck.Preferably through injection or infusion with duplex molecule or vector administration near cancer location or its.
Duplex molecule of the present invention can or divide a plurality of dosage to use with single dose.When duplex molecule of the present invention use to the infusion mode time, infusion can be single lasting dosage, perhaps uses through infusion repeatedly.Preferably medicament is injected directly in cancer location or near the tissue it.Particularly preferably be the medicament multiple injection in cancer location or near the tissue it.
Those skilled in the art can easily confirm to be used for given experimenter is used the appropriate dose scheme of duplex molecule of the present invention.For example, duplex molecule can be disposable employed be given the experimenter, for example with single injection or sedimentary administered near cancer location or its.Perhaps, duplex molecule can about 3～about 28 days, more preferably from about 7～about 10 days during in once a day or secondary use to the experimenter.In the preferred dosage scheme, duplex molecule once-a-day is expelled in can be during 7 days near cancer location or its.When dosage comprises when repeatedly using, it should be understood that the significant quantity of the duplex molecule of using to the experimenter, can be included in the total amount of this duplex molecule of using in the whole dosage.
In the present invention, can treat the cancer of expressing CSTF2 with at least a activeconstituents that is selected from down group:
(a) duplex molecule of the present invention,
(b) encode they DNA and
(c) vectors encoding them.
Said cancer includes but not limited to lung cancer.Thereby, use duplex molecule of the present invention as activeconstituents before, whether the expression level of CSTF2 is compared with the normal cell of homolog and is raise in cancer cells that preferred affirmation will be treated or the tissue.So, in one embodiment, the invention provides the method for cancer that CSTF2 is expressed in a kind of treatment (mistake), this method can comprise the following steps:
I) detect the expression level of CSTF2 in cancer cells that the experimenter with the cancer that will treat obtains or tissue;
Ii) said CSTF2 expression level and normal control are compared; And
Iii) use at least a composition that is selected from down group to having the experimenter who compared the cancer of expressing CSTF2 with normal control:
(a) duplex molecule of the present invention,
(b) encode they DNA and
(c) vectors encoding them.
Perhaps, the present invention also provides a kind of pharmaceutical composition, and it comprises at least a composition that is selected from down group, is used to be applied to the experimenter with cancer of expressing CSTF2:
(a) duplex molecule of the present invention,
(b) encode they DNA and
(c) vectors encoding them.
In other words, the present invention further provides a kind of method that is used to identify the experimenter that will use following each item treatment:
(a) duplex molecule of the present invention,
(b) encode their DNA, or
(c) vectors encoding them,
This method can comprise the step of measuring the expression level of CSTF2 in the cancer cells be derived from the experimenter or the tissue, and this experimenter of rising indication that wherein said level is compared with this gene normal control has the cancer of available following each item treatment:
(a) duplex molecule of the present invention,
(b) encode their DNA, or
(c) vectors encoding them.
Can describe the present invention in further detail below and treat method for cancer.
To be preferably Mammals with the experimenter of present method treatment.The Mammals of exemplary includes but are not limited to the for example mankind, non-human primates, mouse, rat, dog, cat, horse and ox.
According to the present invention, be determined at the expression level of CSTF2 in cancer cells that the experimenter obtains or tissue.Use means known in the art, can transcribe definite expression level on (nucleic acid) product level.For example, can pass through the mRNA that hybridizing method (for example, Northern hybridization) uses probe quantitative CSTF2.Said detection can be implemented on chip or array.As far as detecting the expression level of CSTF2, preferably use array.The sequence information of those skilled in the art CSTF2 capable of using prepares above-mentioned probe.For example, the cDNA of CSTF2 can be used as probe.Like needs, said probe can come mark with suitable affinity tag, for example dyestuff, fluorescent substance and isotropic substance, and said expression of gene level can be used as the intensity detection of the affinity tag that hybridization has taken place.
Further, can be through (for example, RT-PCR) using the transcription product (for example SEQ ID NO:1) of the quantitative CSTF2 of primer based on the detection method of amplification.But above-mentioned primer can be based on the calling sequence information preparation of said gene.
Particularly, used probe or the primer of present method hybridized with the mRNA of CSTF2 under stringent condition, medium stringent condition or low stringency condition.As used herein, phrase " strict (hybridization) condition " is meant such condition, and under this condition, probe or primer will be hybridized with its target sequence, but not with other sequence hybridization.Stringent condition is sequence-dependent, and can be different under different environment.The specific hybridization of longer sequence is compared under comparatively high temps with shorter sequence and is observed.Usually, the temperature of the stringent condition ionic strength of selecting the bit sequencing to be listed in qualification is hanged down about 5 ℃ with the heat fusion joint under the pH (Tm).Tm has temperature 50% and probe its target complement sequence and target sequence hybridization under (under the ionic strength, pH and the nucleic acid concentration that limit) equilibrium state.Because the general excessive existence of target sequence, therefore at Tm, 50% probe is occupied during balance.Typically; Stringent condition can be such: wherein salt concn is less than about 1.0M sodium ion; Typically about 0.01-1.0M sodium ion (or other salt); PH7.0-8.3, temperature is about at least 30 ℃ for short probe or primer (for example 10-50 Nucleotide), is about at least 60 ℃ for long probe or primer.Stringent condition also can be through adding destabilizing agent, and for example methane amide is realized.
Perhaps, can detect translation product to carry out diagnosis of the present invention.For example, can confirm the amount of CSTF2 albumen (SEQ ID NO:2).Mensuration comprises immunoassay as the method for the protein content of translation product, and it uses the said proteic antibody of specific recognition.Antibody can be mono-clonal or polyclonal.And, any fragment of antibody or modification (for example chimeric antibody, scFv, Fab, F (ab ') 2, Fv etc.) all can be used for detecting, as long as this fragment or keep the proteic binding ability of CSTF2 through modified antibodies.The method that is used to detect proteic antibody for preparing these kinds is well-known in the art, and can use any these antibody of method preparation and their Equivalent in the present invention.
As the method for another kind based on the translation product detection CSTF2 gene expression dose of CSTF2, the proteic antibody of CSTF2 that is directed against capable of using is measured painted intensity through immunohistochemical analysis.Anticipate promptly, in this measured, strong dyeing showed that said proteinic existence/level increases, and shows the high expression level of CSTF2 gene simultaneously.
For target gene (for example CSTF2 gene) for the expression level in the cancer cells; If this level has for example increased by 10%, 25% or 50% than the control level (the for example level in normal cell) of target gene; Or be increased to and surpass 1.1 times, surpass 1.5 times, surpass 2.0 times, surpass 5.0 times, surpass 10.0 times or more words, can confirm that this level increases.
Control level can be confirmed with cancer cells simultaneously, uses the sample of before having collected and having preserved from the known experimenter of morbid state (carcinous or non-carcinous).In addition, have the normal cell that the non-carcinous district of the organ of the cancer that will treat obtains certainly and can be used as normal control.Perhaps, control level can be by statistical method, based on confirming through analyzing the result that the previous CSTF2 gene expression dose of measuring from the known experimenter's of morbid state sample obtains.Further, control level can be the expression pattern database of the cell crossed from first Pretesting.And, according to an aspect of the present invention, can CSTF2 expression of gene level in the biological sample and a plurality of control level of confirming from a plurality of reference samples be compared.Preferably use the control level of confirming from from the reference sample of the types of organization similar with the biological sample types of organization that is derived from the experimenter.And, preferably, use the standard value of CSTF2 gene expression dose in the colony with known morbid state.Standard value can obtain through any method known in the art.For example, the scope of MV ± 2S.D. or MV ± 3S.D. can be used as standard value.
In background of the present invention, the control level of confirming from known non-carcinous biological sample is called " normal control level ".On the other hand, if control level confirms that from carcinous biological sample then it is called " carcinous control level ".
If CSTF2 expression of gene level has improved or similar with carcinous control level/has been equal to than the normal control level, then diagnosable experimenter has the cancer that will treat.
The compsn that comprises duplex molecule of the present invention:
Except above-mentioned points, the present invention also provides the pharmaceutical composition of the carrier that comprises at least a duplex molecule of the present invention or this molecule of encoding.Particularly, the present invention provides the compsn of following [1] to [34]:
[1] a kind of compsn that is used for anticancer growth and/or treatment or preventing cancer; Wherein said compsn comprises at least a isolating to the duplex molecule of CSTF2 gene or the carrier of this duplex molecule of encoding; Suppress expression and the cell proliferation of CSTF2 when wherein this duplex molecule is in transfered cell; Wherein this duplex molecule comprise sense strand and with its complementary antisense strand, the two phase mutual cross is to form duplex molecule, said compsn also comprises the pharmaceutically acceptable body that supports;
[2] compsn in [1], wherein said duplex molecule acts on mRNA, said mRNA and the target sequence coupling that is selected from SEQ ID NO:9 and 10;
[3] compsn of [1], wherein said duplex molecule, wherein sense strand comprises and the corresponding sequence of target sequence that is selected from SEQ ID NO:9 and 10.
[4] compsn of [1], wherein said compsn comprise multiple said duplex molecule;
[5] [1] to [3] arbitrary compsn, hybridization is to form duplex molecule at the target sequence place for the sense strand of wherein said duplex molecule and antisense strand, and this duplex molecule length is less than about 100 nucleotide pairs;
[6] compsn of [5], hybridization is to form duplex molecule at the target sequence place for the sense strand of wherein said duplex molecule and antisense strand, and this duplex molecule length is less than about 75 nucleotide pairs;
[7] compsn of [6], hybridization is to form duplex molecule at the target sequence place for the sense strand of wherein said duplex molecule and antisense strand, and this duplex molecule length is less than about 50 nucleotide pairs;
[8] compsn of [7], hybridization is to form duplex molecule at the target sequence place for the sense strand of wherein said duplex molecule and antisense strand, and this duplex molecule length is less than about 25 nucleotide pairs;
[9] hybridization is to form duplex molecule at the target sequence place for the compsn of [8], the sense strand of wherein said duplex molecule and antisense strand, and this duplex molecule length is about 19 and arrives about 25 nucleotide pairs;
[10] [1] to [9] arbitrary compsn, wherein said duplex molecule is made up of single polynucleotide, and said polynucleotide comprise through interleaving sense strand and the antisense strand that strand links together;
[11] compsn of [10]; Wherein said duplex molecule have general formula 5 '-[A]-[B]-[A ']-3 '; Wherein, [A] for comprising the sense strand sequence of the sequence corresponding with the target sequence that is selected from SEQ ID NO:9 and 10, and [B] for to interleave strand by what 3～23 Nucleotide constituted, [A '] for comprising and the antisense strand of the sequence of target complement sequence of [A] middle selection;
[12] [1] to [11] arbitrary compsn, wherein said duplex molecule is RNA;
[13] [1] to [11] arbitrary compsn, wherein said duplex molecule is DNA and/or RNA;
[14] compsn of [13], wherein said duplex molecule are the heterozygotes of DNA polynucleotide and RNA polynucleotide;
[15] compsn of [14], wherein said sense strand polynucleotide and antisense strand polynucleotide are made up of DNA and RNA respectively;
[16] compsn of [13], wherein said duplex molecule are the mosaics of DNA and RNA;
[17] compsn of [16], wherein the zone of antisense strand 3 ' the distolateral wing is made up of RNA, and perhaps the zone of the zone of sense strand 5 ' the distolateral wing and antisense strand 3 ' the distolateral wing is formed by RNA;
[18] compsn of [17], wherein said flank region is made up of 9 to 13 Nucleotide;
[19] [1] to [18] arbitrary compsn, wherein said duplex molecule comprises one or two 3 ' overhang;
[20] [1] to [19] arbitrary compsn, wherein said compsn further comprises the transfection toughener.
[21] [1] to [20] arbitrary compsn, wherein said duplex molecule is by vector encoded and be contained in the compsn;
[22] compsn in [21], wherein the said duplex molecule by said vector encoded acts on mRNA, said mRNA and the target sequence coupling that is selected from SEQ ID NO:9 and 10;
[23] compsn of [21], wherein the sense strand by the said duplex molecule of said vector encoded comprises and the corresponding sequence of target sequence that is selected from SEQ ID NO:9 and 10;
[24] [21] to [23] arbitrary compsn is wherein used multiple said duplex molecule;
[25] [21] to [24] arbitrary compsn, hybridization is to form duplex molecule at the target sequence place for the sense strand of wherein said duplex molecule and antisense strand, and this duplex molecule length is less than about 100 nucleotide pairs;
[26] compsn of [25], hybridization is to form duplex molecule at the target sequence place for the sense strand of wherein said duplex molecule and antisense strand, and this duplex molecule length is less than about 75 nucleotide pairs;
[27] compsn of [26], hybridization is to form duplex molecule at the target sequence place for the sense strand of wherein said duplex molecule and antisense strand, and this duplex molecule length is less than about 50 nucleotide pairs;
[28] compsn of [27], hybridization is to form duplex molecule at the target sequence place for the sense strand of wherein said duplex molecule and antisense strand, and this duplex molecule length is less than about 25 nucleotide pairs;
[29] compsn of [28], the sense strand of wherein said duplex molecule and antisense strand in target sequence place hybridization forming duplex molecule, this duplex molecule length be about 19 to about 25 nucleotide pairs;
[30] [21] to [29] arbitrary compsn, wherein the said duplex molecule by said vector encoded is made up of single polynucleotide, said polynucleotide comprise through interleave sense strand that strand links together and antisense strand the two;
[31] compsn of [30]; Wherein said duplex molecule have general formula 5 '-[A]-[B]-[A ']-3 '; Wherein, [A] for comprising the sense strand of the sequence corresponding with the target sequence that is selected from SEQ ID NO:9 and 10, and [B] for to interleave strand by what 3～23 Nucleotide constituted, [A '] for comprising and the antisense strand of the sequence of target complement sequence of [A] middle selection;
[32] [21] to [31] arbitrary compsn, wherein said compsn further comprises the transfection toughener;
[33] [1] to [32] arbitrary compsn, wherein said cancer is a lung cancer.With
[34] compsn of [33], wherein said lung cancer are gland cancer, squamous cell carcinoma, large cell carcinoma or small cell lung cancer.
The suitable compsn of the present invention will be described below more with detailing.
Said duplex molecule of the present invention preferably was formulated as pharmaceutical composition according to technology well-known in the art before being applied to the experimenter.Pharmaceutical composition of the present invention is characterized as to be aseptic at least and not to contain pyrogen." pharmaceutical formulation " used herein comprises the preparaton that is applicable to that the mankind and animal doctor use.The method for preparing pharmaceutical composition of the present invention belongs to this area general technology, for example, is described in Remington ' s Pharmaceutical Science; 17th ed., Mack Publishing Company, Easton; Pa. (1985), its whole disclosures are contained among this paper by reference.
Pharmaceutical formulation of the present invention comprises at least a duplex molecule of the present invention or vectors encoding them (for example, being 0.1% to 90% by weight), or acceptable salt on the physiology of said molecule, mixes with physiologically acceptable mounting medium.Acceptable carrier medium preferably water, buffered water, saline water, 0.4% salt solution, 0.3% glycocoll, mucinase and analogue on the physiology.
According to the present invention, said compsn can comprise multiple duplex molecule, and wherein each can be to the different target sequences or the heterogeneic different target sequence of homologous genes.For example, said compsn can comprise the duplex molecule to CSTF2 gene target sequence.Perhaps, for example, said compsn can comprise the duplex molecule to a kind of, two kinds or more kinds of target sequences of CSTF2 gene and other gene.
And this compsn can comprise the carrier of one or more duplex molecules of encoding.For example, can encode a kind, 2 kinds or several these duplex molecules of said carrier.Perhaps, this compsn can comprise variety carrier, and the different duplex molecule of each vector encoded.
And the form that this duplex molecule can be used as liposome is included in this compsn.The detailed content of liposome can be with reference to " using duplex molecule of the present invention to suppress or reduction growth of cancer cells or treatment method for cancer " item.
Compsn of the present invention can be a pharmaceutical composition.Can also comprise traditional pharmaceutical excipient and/or additive in the pharmaceutical composition of the present invention.Suitable pharmaceutical excipient comprises stabilization agent, inhibitor, soaks into and press regulator, buffer reagent and pH regulator agent.Suitable additive comprises: the buffer reagent of physiology biocompatibility (for example tromethane hydrochloride), add sequestrant (for example DTPA or DTPA-bisamide etc.); Or calcium sequestrant mixture (for example, calcium DTPA, CaNaDTPA-bisamide); Perhaps; Randomly, add calcium or sodium salt (for example calcium chloride, ascobic acid calcium, calcium gluconate or lactic acid ca).Pharmaceutical composition of the present invention can be packed so that use as liquid, perhaps also in addition lyophilize.
For solids compsn, can use conventional nontoxic solid-state carrier; For example, the N.F,USP MANNITOL of pharmaceutical grade, lactic acid, starch, Magnesium Stearate, soluble saccharin, talcum, Mierocrystalline cellulose, glucose, sucrose, magnesiumcarbonate etc.
For example, be used for Orally administered solid composite medicament and can comprise above-mentioned any carrier and the vehicle of enumerating, and 10-95%, one or more duplex molecules of the present invention of preferred 25-75%.What be used for that pharmaceutical composition that aerosol (suction) uses can comprise 0.01-20 weight %, preferred 1-10 weight % encapsulates one or more duplex molecules of the present invention in above-mentioned liposome and propelling agent.Can also comprise carrier as required, for example be used for the Yelkin TTS of delivery in the nose etc.
Except that above-mentioned, can also comprise other pharmacy activity component in this compsn, as long as they do not suppress the interior function of body of this duplex molecule.For example, can comprise the chemotherapeutics that routine is used for cancer therapy in the above-mentioned compsn.
In another embodiment, also to provide double chain acid molecule of the present invention to be used for treating to express CSTF2 in preparation be the purposes of the lung cancer drugs compsn of characteristic in the present invention.For example; The present invention relates to following double chain acid molecule is used for treating the lung cancer drugs compsn of expressing CSTF2 in preparation purposes: this molecule suppresses the CSTF2 expression of gene in cell; And this molecule comprises sense strand and complementary antisense strand with it; The two is hybridized each other and forms this double chain acid molecule, and this molecule is a target with the sequence that is selected from SEQ ID NO:9 and 10.
Perhaps, the present invention further is provided at treatment and expresses the double chain acid molecule of the present invention that uses in the lung cancer of CSTF2 gene.
In addition, the present invention also provides and produces that to be used to treat part be the lung cancer drugs method for compositions or the technology of characteristic to express CSTF2.This method or technology comprise pharmacy or physiology acceptable carrier with the step as the following double chain acid molecule preparationization of activeconstituents; Wherein said double chain acid molecule suppresses the expression of CSTF2 in the cell; And this molecule comprises sense strand and complementary antisense strand with it; The two is hybridized each other and forms this double chain acid molecule, and this molecule is a target with the sequence that is selected from SEQ ID NO:9 and 10.
In another embodiment; It is the lung cancer drugs method for compositions or the technology of characteristic that the present invention also provides production to be used to treat to express CSTF2; Wherein said method or technology comprise activeconstituents and acceptable carrier blended step pharmaceutically or on the physiology; Wherein said activeconstituents is such double chain acid molecule, and it suppresses the expression of CSTF2 in crossing the cell express the CSTF2 gene, this molecule comprise sense strand with its complementary antisense strand; The two phase mutual cross to be forming double chain acid molecule, and is target with the sequence that is selected from SEQ ID NO:9 and 10.
The method of detection or diagnosing
The specific raising in lung carcinoma cell of discovery CSTF2 expression of gene (Figure 1A-1C).Therefore, the gene that this paper identifies and transcribe and can be used as the lung cancer sign with translation product and be used for diagnosis, and through measuring the diagnosable lung cancer of CSTF2 expression of gene in the cell sample.Particularly, the invention provides through confirming the method for CSTF2 expression level diagnosing among the experimenter.The lung cancer of available present method diagnosis comprises NSCLC and SCLC.Further, NSCLC comprises adenocarcinoma of lung (ADC), squamous cell lung carcinoma (SCC) and large cell carcinoma (LCC), also can or detect through the present invention's diagnosis.
According to the present invention, can be provided for checking the intermediate result of experimenter's situation.Said intermediate result can with out of Memory combine with assist a physician, nurse or other practitioner's diagnosing patients suffer from said disease.In other words, the present invention is provided for checking the diagnosis marker CSTF2 of cancer.Perhaps; The invention provides a kind of method that is used for detecting or identifying the lung tissue sample cancer cells that is derived from the experimenter; Said method comprises the step of measuring CSTF2 gene expression dose in the biological sample that is derived from the experimenter, exists or suspect in the rising indication tissue that wherein said expression level is compared with the normal control level of said gene to have cancer cells.This type of result can help doctor, nurse or other health care practitioner with other information combination and diagnose the experimenter to suffer from disease.In other words, the present invention can provide useful information to diagnose the experimenter to suffer from disease to the doctor.For example; According to the present invention; When about cancer cells in the tissue that the experimenter obtains have query the time, through considering CSTF2 expression of gene level, add other different aspects of disease; Comprise the level of the known cancer mark in histopathology, the blood and experimenter's CC etc., can make clinical decision.For example, the diagnostic lung tumor mark in some known blood is IAP, ACT, BFP, CA19-9, CA50, CA72-4, CA130, CEA, KMO-1, NSE, SCC, SP1, Span-1, TPA, CSLEX, SLX, STN and CYFRA.In other words, in this specific embodiments of the present invention, the result of gene expression analysis is used for further diagnosing experimenter's morbid state as intermediate result.
Particularly, the invention provides the method for following [1] to [11]:
[1] a kind of in the experimenter method of diagnosing or lung cancer occurence tendency property, said method comprises the steps:
(a) detection resources CSTF2 expression of gene level in experimenter's biological sample;
(b) raising of detected expression level than the normal control level of this gene is associated with the existence of disease among this experimenter;
[2] method of [1], wherein said expression level is than normal control level height at least 10%;
[3] method of [1] or [2], wherein said expression level detects through the method that is selected from down group:
(a) detection is by the mRNA of CSTF2 genes encoding;
(b) detection is by the protein of CSTF2 genes encoding;
(c) detection is by the proteinic BA of CSTF2 genes encoding;
[4] method of [3], wherein said expression level be through detection probes with confirm by the hybridization of the mRNA of CSTF2 genes encoding;
[5] method of [3], wherein said expression level be through detect to by the proteinic antibody of CSTF2 genes encoding with confirm by the combination of proteins of CSTF2 genes encoding;
[6] [1] to [5] arbitrary method, wherein said biological sample comprises biopsy samples, phlegm or blood.
[7] [1] to [6] arbitrary method, the wherein said patient's of being derived from biological sample comprises epithelial cell.
[8] [1] to [7] arbitrary method, the wherein said patient's of being derived from biological sample comprises cancerous cells.
[9] method of [8], the wherein said experimenter's of being derived from biological sample comprises carcinous epithelial cell.
[10] [1] to [9] arbitrary method, the wherein said experimenter's of being derived from biological sample comprises lung tissue or pneumonocyte.
[11] [1] to [10] arbitrary method, wherein said lung cancer is NSCLC or SCLC.
The method of diagnosing is more detailed the narration below.
Experimenter with present method diagnosis is preferably Mammals.Mammiferous example includes but are not limited to, for example, and the mankind, non-human primates, mouse, rat, dog, cat, horse and ox.
For implementing diagnosis, preferably gather biological sample from the experimenter that will diagnose.Any biologic material all can be used as biological sample and is used for measuring, as long as it comprises the CSTF2 genetic transcription or the translation product of target.Said biological sample include but not limited to, and wants to diagnose or suspect bodily tissue and the body fluid of suffering from cancer, for example blood, phlegm, hydrothorax and urine.Biological sample preferably contains such cell colony, and this colony comprises epithelial cell, more preferably carcinous epithelial cell or be derived from the epithelial cell of suspecting for carcinous tissue.Further, if necessary, can be from the bodily tissue of gained and body fluid the said cell of purifying, and it used be biological sample.In a preferred embodiment, biological sample contains lung tissue or pneumonocyte.This type of biological sample can be through suspecting that in experimenter's lung lung tissue is collected in carcinous zone or pneumonocyte obtains, for example through biopsy.
According to the present invention, be determined at CSTF2 expression of gene level in the said patient's of being derived from the biological sample.Expression level can be confirmed in transcription product (mRNA) level, use method well known in the art.For example, the mRNA of CSTF2 gene can pass through hybridizing method (for example, Northern hybridization) use probe quantitative.Can on chip or array, implement said detection.To detecting a plurality of genes (for example, multiple cancer specific gene), comprise the CSTF2 gene, expression level, preferably use array.Those skilled in the art's CSTF2 gene capable of using (SEQ ID NO:1; The GenBank accession number: sequence information NM_001325) prepares above-mentioned probe.For example, the cDNA of CSTF2 gene can be used as probe.Like needs, said probe can be used for example dyestuff, fluorescence or coordination mark usually of suitable affinity tag, and the intensity that said expression of gene level can be used as the affinity tag that hybridization takes place detects.
Further, CSTF2 gene transcription product can (for example, RT-PCR) use primer quantitative through the detection technique based on amplification.Above-mentioned primer also can be based on said gene known sequences information preparation.For example, the primer or the probe (SEQ ID NO:3,4,7 and 8) that are used for embodiment can be used for the detection through RT-PCR or Northern trace, but the present invention is not limited to this.
Particularly, used probe or the primer of present method hybridized with the mRNA of CSTF2 gene under stringent condition, medium stringent condition and low stringency condition.Like preceding text definition, phrase " strict (hybridization) condition " is meant such condition, and under this condition, probe or primer will be hybridized with its target sequence, but not with other sequence hybridization.Stringent condition is sequence-dependent, can be different under different environment.The specific hybridization of longer sequence is compared with shorter sequence under comparatively high temps and is taken place.Usually, the temperature of stringent condition is chosen as low about 5 ℃ of ionic strength and the heat fusion joint under the pH (Tm) that the bit sequencing is listed in qualification.Tm has temperature 50% and probe target complement sequence and target sequence hybridization under (under the ionic strength, pH and the nucleic acid concentration that limit) equilibrium state.Because the general excessive existence of target sequence, therefore under Tm, 50% probe is occupied during balance.Typically; Stringent condition is such: wherein salt concn is less than about 1.0M sodium ion; Typically about 0.01-1.0M sodium ion (or other salt); PH7.0-8.3, temperature is about at least 30 ℃ for short probe or primer (for example 10-50 Nucleotide), being used for long probe or primer is about at least 60 ℃.Stringent condition also can through add destabilizing agent for example methane amide realize.
Perhaps, diagnosis of the present invention can be carried out through detecting translation product.For example, can confirm the proteic amount of CSTF2.Mensuration comprises immunoassay as the method for the protein content of translation product, and these class methods are used the said proteic antibody of specific recognition.Antibody can be mono-clonal or polyclonal.And, any fragment of antibody or modification (for example chimeric antibody, scFv, Fab, F (ab ') 2, Fv etc.) all can be used for detecting, as long as this fragment keeps the proteic binding ability of CSTF2.The method that is used to detect proteic antibody for preparing these types is well-known in the art, and can use any these antibody of method preparation and their Equivalent in the present invention.
As the method for another kind based on its gene of translation product detection of CSTF2, the proteic antibody of CSTF2 that is directed against capable of using is observed its painted intensity through immunohistochemical analysis.That is, observe strong dyeing and show that said proteinic existence increases, and show the high expression level of CSTF2 simultaneously.
In addition, except that CSTF2 expression of gene level, also can confirm other cancer related gene, the expression of gene level of for example known variant expression in lung cancer is to improve the accuracy of said diagnosis.
For the cancer marker gene that comprises the CSTF2 gene, if its control level than corresponding cancer marker gene has increased for example 10%, 25% or 50% words; Or be increased to above 1.1 times; Surpass 1.5 times, surpass 2.0 times, above 5.0 times; Surpass 10 times or more, can think that then its expression level in biological sample increases.
Control level can be confirmed with the test organisms sample simultaneously, uses the sample of before having collected and having preserved from the known experimenter of morbid state (carcinous or non-carcinous).Perhaps, control level can be by statistical method, according to confirming through analyzing the result that the previous CSTF2 gene expression dose of measuring from the known experimenter's of morbid state sample obtains.Further, control level can be the expression pattern DB of the cell crossed from first Pretesting.And, according to an aspect of the present invention, can CSTF2 expression of gene level in the biological sample and a plurality of control level of confirming from a plurality of reference samples be compared.Preferably use the control level of confirming from from the reference sample of the types of organization similar with the sample tissue type that is derived from the patient.And, preferably, use the standard value of CSTF2 gene expression dose in the colony with known morbid state.Standard value can obtain through any method known in the art.For example, MV+/-2S.D. or MV+/-scope of 3S.D. can be used as standard value.
In background of the present invention, the control level of confirming from known non-carcinous biological sample is called " normal control level ".On the other hand, if control level is definite from carcinous biological sample, then be called " carcinous control level ".
Compare that the normal expression level increases or similar with carcinous control level when CSTF2 expression of gene level, then the experimenter is diagnosable for just suffering from or risky generation cancer.Further, when the expression level of more multiple cancer related gene, the similarity of gene expression pattern shows that the experimenter is just suffering from or risky generation cancer between sample and the carcinous reference.
The in addition stdn of the expression level of test organisms sample and the difference between control level, the expression level of the contrast nucleic acid (for example house-keeping gene) that can known relatively expression level can not change along with the cancer or the non-cancer state of cell.The example crt gene include but not limited to, beta-actin, glyceraldehyde-3-phosphate dehydrogenase and ribosomal protein P1.
Perhaps, the present invention provides reagent preparation to be used for the purposes of the diagnostic reagent of diagnosing cancer.In some embodiments, this reagent can be selected from down group:
(a) be used to detect the reagent of the mRNA of CSTF2 gene;
(b) be used to detect the proteic reagent of CSTF2; With
(c) be used to detect the reagent of the proteic BA of CSTF2.
Particularly, this type of reagent be with the oligonucleotide of CSTF2 multi-nucleotide hybrid or with CSTF2 polypeptide bonded antibody.
In the present invention, disclosed CSTF2 and be not only useful diagnosis marker, but also be the suitable target thing of cancer therapy.Therefore, the cancer therapy of target CSTF2 can realize through the present invention.In the present invention, the cancer therapy of target CSTF2 refer to prevent or anticancer in CSTF2 active and/or express.Any anti-CSTF2 agent all can be used for the cancer therapy of target CSTF2.In the present invention, anti-CSTF2 agent comprises that following substances is as active ingredient:
(a) duplex molecule of the present invention,
(b) encode it DNA and
(c) encode its carrier.
Thereby in a preferred embodiment, the present invention provides following method: (i) whether the diagnosis experimenter has the cancer that will use anti-CSTF2 agent treatment, and/or (ii) is the cancer therapy selection experimenter of target CSTF2, and this method comprises the steps:
(a) measure CSTF2 expression of gene level in the cancer cells of suspecting experimenter's acquisition or tissue with the cancer that will treat;
(b) CSTF2 expression of gene level and normal control level are compared;
(c), this experimenter is diagnosed as has the cancer that to treat if the expression level of CSTF2 is compared rising with the normal control level; And
(d) if in step (c), this experimenter is diagnosed as and has the cancer that to treat, select this experimenter to carry out cancer therapy.
Perhaps, these class methods comprise the steps:
(a) measure CSTF2 expression of gene level in the cancer cells of suspecting experimenter's acquisition or tissue with the cancer that will treat;
(b) CSTF2 expression of gene level and carcinous control level are compared;
(c), this experimenter is diagnosed as has the cancer that to treat if CSTF2 expression of gene level is similar with carcinous control level or be equal to; And
(d) if in step (c), this experimenter is diagnosed as and has the cancer that to treat, select this experimenter to carry out cancer therapy.
Estimate the method for cancer prognosis
The present invention partly relates to following newly discovered, and promptly CSTF2 expresses and patient's poor prognosis significant correlation.Therefore, the present invention provides definite or estimates cancer, the especially method of patients with lung cancer prognosis suffered from, and said method detects the CSTF2 expression level in patient's biological sample; Expression level that records and control level are compared; And definite expression level of comparing rising with control level is the indication of poor prognosis (bad survival).Particularly, the present invention provides a kind of method of estimating or confirming the experimenter's with lung cancer prognosis, and said method comprises the steps:
(a) detection resources CSTF2 expression of gene level in experimenter's biological sample;
(b) detected expression level and control level are compared; And
(c) based on the prognosis of relatively confirming this experimenter of (b).
At this, term " prognosis " and refer to by the character of case and symptom show about the possible outcome of said disease and the prospect of from disease, recovering.Correspondingly, disadvantageous, negative, bad prognosis is defined as after the lower treatment between survival time and survival rate.On the contrary, positive, favourable or good prognosis is defined as between the survival time of treatment back or survival rate improves.
Term " evaluation of prognosis " refers to predict, the result in future of advance notice and patient's cancer (for example, pernicious, cure the possibility, survival rate of cancer etc.), or with the ability that future, the result interrelated of certain detection or measurement and patient's cancer.For example, confirm the CSTF2 gene through the time expression level make prediction patient result (for example, virulent increases or reduces, and the possibility, survival rate of cancer etc. are cured in the increase of cancer grade or minimizing) become possibility.
In background of the present invention, phrase " is estimated (or confirming) prognosis " and is intended to contain cancer, progress, particularly cancer return, shifts the prediction and the probability analysis of spreading with palindromia.The method of the evaluation of prognosis here is intended to be used for clinical in to making decision about the treatment pattern, said treatment pattern comprises that treatment gets involved, Case definition for example disease by stages, and to the transfer of tumor disease and the surveillance of disease and the monitoring of recurrence.
The biological sample that is derived from the patient that present method is used can be any sample that is derived from the experimenter that will estimate, as long as the CSTF2 gene can detect in sample.The preferred pneumonocyte of said biological sample (from the cell of lung acquisition).Further, said biological sample can comprise body fluid for example phlegm, blood, serum or blood plasma.In addition, said sample can be from organizing the cell of purifying.Biological sample can obtain from the patient at different time points, comprise treatment before, in the treatment and/or the treatment after.The lung carcinoma cell that the experimenter that for example, will assess certainly obtains is preferred biological sample.
According to the present invention, it is high more to be presented in the biological sample that is derived from the patient CSTF2 expression of gene level, the treatment back state of an illness relax, recover and/or the prognosis of survival just more for bad, and the possibility of bad clinical consequences is just high more.Therefore; According to present method, can be with " control level " of making comparisons, for example; After treatment, show the individuality of good or positive cancer prognosis or any CSTF2 expression of gene level in the individual colony that forms, be referred to as " good prognosis control level " at this.In addition, said " control level " can be, and for example, after treatment, shows the individuality of bad or passive cancer prognosis or any CSTF2 expression of gene level in the individual colony that forms, and is referred to as " poor prognosis control level " at this.Said " control level " is the single expression pattern that is derived from from single reference group, or multiple expression pattern.Therefore, said control level can be based in its morbid state (good or poor prognosis) known cancer patient or cancer patients's the colony, and the CSTF2 expression of gene level before carrying out any kind of treatment is confirmed.In background of the present invention, cancer is a lung cancer.The preferred standard value of using the CSTF2 expression of gene level in the known patient's set of morbid state.Said standard value can obtain through any methods known in the art.For example, the scope of MV+/-2 times standard deviation or MV+/-3 times standard deviation can be used as standard value.
Said control level can confirm with the biological sample of being tested simultaneously, and this realizes through using before the sample of before the treatment of accepting any kind of, gathering and storing from patient's (contrast or control group) of known its morbid state (good or poor prognosis).
Perhaps, said control level can be through statistical method based on confirming through analyzing previous CSTF2 gene expression dose from the sample that control group is collected or stored.Further, said control level can be the DB from the expression pattern of the cell of first Pretesting.
In addition, according to an aspect of the present invention, can CSTF2 expression of gene level in the biological sample and multiple control level be compared, said control level is confirmed with reference to sample from multiple.Preferred use from the control level of confirming with reference to sample of the similar types of organization of the biological sample that is derived from patient gained.
According to the present invention; The similarity of CSTF2 expression of gene level and good prognosis control level shows the comparatively ideal prognosis of said patient, and relatively the increase of good prognosis control level expression level shows the prognosis of state of an illness mitigation after more unfavorable, the worse treatment, recovery, survival and/or clinical consequences.On the other hand; Show the comparatively ideal prognosis of patient than the minimizing of poor prognosis control level CSTF2 expression level, and the expression level similar with the poor prognosis control level shows the prognosis of state of an illness mitigation after more comparatively ideal, the not bad treatment, recovery, survival and/or clinical consequences.For example, leisure treatment back shows that the lung carcinoma cell that the experimenter of better or relatively poor cancer prognosis obtains is respectively the preferred biological sample of better or poor prognosis control level.
When relative comparison horizontal expression level change to surpass 1.0,1.5,2.0,5.0,10.0 or more times the time, the CSTF2 gene expression dose in the biological sample can be thought to have changed.
The difference of expression level can be with respect to contrast between the biological sample that tries and control level, housekeeping gene for example, stdn in addition.For example, known its expression level is constant polynucleotide in carcinous and non-cancerous cells, comprise the gene of those codings beta-actin, glyceraldehyde-3-phosphate dehydrogenase and ribosomal protein P1, can be used for stdn CSTF2 gene expression dose.
Expression level can be confirmed through utilizing technology well-known in the art in being derived from patient's biological sample, to detect the genetic transcription thing.The said genetic transcription thing that detects through present method had both comprised that transcription product also comprised translation product, for example mRNA and protein.
For example, CSTF2 gene transcription product can be hybridized through using to the CSTF2 gene probe of genetic transcription thing, and for example the Northern blot hybridization is analyzed to detect.Said detection can be carried out on chip or array.To detecting CSTF2 expression of gene level, preferably use array.As another example, based on the detection method of amplification, the primer that for example uses the CSTF2 gene specific can be used for detecting (referring to embodiment) based on the polymerase chain reaction of rt.CSTF2 gene-specific probe or primer can use complete sequence (SEQ ID NO:1) design and the preparation of routine techniques with reference to the CSTF2 gene.For example, the primer (SEQ ID NO:3 and 4) that uses in an embodiment can be used for detecting through RT-PCR, but this aspect is not limited in this.
Particularly, used probe or the primer of present method hybridized with the mRNA of CSTF2 under stringent condition, medium stringent condition or low stringency condition.
Perhaps, evaluation of the present invention also can be carried out through detecting translation product.For example, can confirm the proteic amount of CSTF2.Confirm to comprise the method for immunity that uses the proteic antibody of specific recognition CSTF2 as the method for the proteinic amount of translation product.Said antibody can be monoclonal or polyclonal.Further, the fragment of any said antibody or modification (for example, chimeric antibody, scFv, Fab, F (ab ') 2, Fv or the like) all can be used for detecting, as long as said fragment keeps and the proteic binding ability of CSTF2.Prepare this type of method that is used to detect proteic antibody and be well known in the art, can use any method to prepare such antibody and Equivalent thereof among the present invention.
Detect the method for this expression of gene level as another kind based on CSTF2 gene translation product, can observe its painted intensity to the proteinic antibody of CSTF2 through the immunohistochemical analysis utilization.That is, observe the amount increase that strong dyeing shows CSTF2, and show the high expression level of CSTF2 gene simultaneously.
Further, known CSTF2 albumen has cell-proliferation activity.Therefore, CSTF2 expression of gene level can use above-mentioned cell-proliferation activity to confirm as index.For example, the cell of preparation and culture expression CSTF2 in the presence of biological sample forms ability through detecting rate of propagation or mensuration cell cycle or colony subsequently, can confirm the cell-proliferation activity of said biological sample.
In addition, except that CSTF2 expression of gene level, also can confirm other lung cancer related gene, the gene of for example known variant expression in lung cancer, expression level, to improve the accuracy of said evaluation.Above-mentioned other the instance of pneumonocyte genes involved comprises the gene that those are described in WO2004/031413 and WO2005/090603.Their content is included in this paper with way of reference.
Perhaps, according to the present invention, except that other test result, also can be provided for estimating the intermediate result of experimenter's prognosis.Said intermediate result can assist a physician, nurse or other practitioner estimate, confirm or estimate experimenter's prognosis.The clinical symptom and the physical appearance that can comprise the experimenter with the out of Memory that gained intermediate result of the present invention is taken into consideration.
In other words, CSTF2 expression of gene level is used to the useful prognostic marker of experimenter's evaluation, prediction or the definite prognosis of suffering from lung cancer (for example NSCLC).Therefore, the present invention also provides a kind of prognostic marker that is used to detect, and is the method for experimenter's evaluation, prediction or the definite prognosis of suffering from lung cancer (comprising NSCLC), and it comprises the steps:
A) detect or measure CSTF2 expression of gene level in the biological sample that is derived from the experimenter, and
B) expression level that detects in the step a) or be measured to and this experimenter's prognosis are associated.
Especially, according to the present invention, compare the possibility or the suspection of the expression level indication poor prognosis (relatively poor survival) of rising with control level.
To be preferably Mammals according to the patient that present method is estimated cancer prognosis, comprise people, non-human primates, mouse, rat, dog, cat, Ma Heniu.
Perhaps, the present invention provides reagent preparation to be used to estimate the purposes of the reagent of cancer prognosis.In some embodiments, this reagent is selected from down group:
(a) be used to detect the reagent of the mRNA of CSTF2 gene;
(b) be used to detect the reagent of CSTF2; With
(c) be used to detect the reagent of the proteic BA of CSTF2.
Particularly, this type of reagent be with the oligonucleotide of CSTF2 multi-nucleotide hybrid or with CSTF2 polypeptide bonded antibody.
The test kit of diagnosing cancer or evaluation cancer prognosis:
The invention provides the test kit of diagnosing cancer or evaluation cancer prognosis.Perhaps, the present invention also is provided for confirming to suffer from available duplex molecule of the present invention or the experimenter's of the cancer that its carrier of encoding is treated test kit, and it also can be used for assessing and/or the effect of monitoring cancer therapy.In a preferred embodiment, said cancer is a lung cancer.Particularly, said test kit comprises at least a material or the reagent that in being derived from patient's biological sample, detects CSTF2 genetic expression, and said material can be selected from down group:
(a) material or the reagent of the mRNA of detection CSTF2 gene;
(b) proteinic material or the reagent of detection CSTF2;
(c) material or the reagent of the proteinic BA of detection CSTF2.
The material or the reagent that are suitable for detecting the mRNA of CSTF2 gene comprise that specificity combines or identify the nucleic acid of CSTF2mRNA, such as the oligonucleotide that has with a part of complementary sequence of CSTF2mRNA.It is specific primer and probe that the example of this class oligonucleotide has couple CSTF2mRNA.Can be based on this class oligonucleotide of method preparation well-known in the art.If desired, can be with the material that is used to detect CSTF2mRNA or immobilization of reagentsization at solid substrate.In addition, in said test kit, can comprise material or reagent more than a kind of CSTF2mRNA of detection.
Probe or primer can be specific sizes.This size is selected from down group: at least 10 Nucleotide, at least 12 Nucleotide, at least 15 Nucleotide, at least 20 Nucleotide, at least 25 Nucleotide, at least 30 Nucleotide, and also the magnitude range of probe and primer can be 5-10 Nucleotide, a 10-15 Nucleotide, a 15-20 Nucleotide, a 20-25 Nucleotide and 25-30 Nucleotide.
On the other hand, being suitable for detecting proteinic material of CSTF2 or reagent comprises to the proteinic antibody of CSTF2.Said antibody can be monoclonal or polyclonal.Further, the fragment of any said antibody or modification (for example, chimeric antibody, scFv, Fab, F (ab ') 2, Fv or the like) all can be used as said material or reagent, as long as said fragment keeps and the proteic binding ability of CSTF2.For the method that detects this antibody-like of protein Preparation is well known in the art, and can use any method to prepare above-mentioned antibody and Equivalent thereof in the present invention.Further, the molecule of said antibody available energy generation signal carries out mark through direct connection or indirect labelling technology.The bonded method of affinity tag and traget antibody and detection antibody and its target is well known in the art, and the present invention can use any affinity tag and method.In addition, in said test kit, can comprise more than a kind of proteinic material of detection CSTF2 or reagent.
Further, said BA can pass through, and for example, measures the cell-proliferation activity that causes owing to the CSTF2 protein expression in the said biological sample and confirms.For example, the said cell of cultivation in the presence of the biological sample that is derived from the patient, the speed of breeding through detection then, or measure cell cycle or colony formation ability, can confirm the cell-proliferation activity of said biological sample.Like needs, can be with the material that is used to detect CSTF2mRNA or immobilization of reagentsization at solid substrate.In addition, in said test kit, can comprise material more than the proteinic BA of a kind of CSTF2 of detection.
Said test kit can comprise more than a kind of aforesaid material or reagent.Further; Said test kit can comprise solid substrate and be used to combine to the probe of CSTF2 gene or to the material of the proteinic antibody of CSTF2; The substratum and the container that are used for culturing cell; The positive and negative control material or reagent, and be used to detect SA to the proteinic antibody of CSTF2.For example, the tissue sample that obtains from the patient with good prognosis or poor prognosis can be used as useful contrast material or reagent.Test kit of the present invention can further comprise other material from commercial and user perspective expectation, comprises damping fluid, diluent, filter, syringe needle, syringe and has the unit packing list (for example, written, tape, CD-ROM or the like) of operation instruction.Being included in the container that has label of these materials or reagent and so on.Suitable containers comprises bottle, pipe-type bottles (vials) and test tube.Said container can make from multiple material, for example glass or plastics.
As one embodiment of the invention, when said material or reagent were the probe to CSTF2mRNA, said material or reagent can be immobilized onto solid substrate such as on the porous bar, to form at least one detection site.The measurement of said porous bar or surveyed area can comprise a plurality of sites, and each all comprises nucleic acid (probe).Test strip also can comprise the site of feminine gender and/or positive control.Perhaps, control site can be positioned on the bar different with test strip.Randomly, the different detection site can comprise the immobilized nucleic acids of difference amount, that is, amount is measured less on ensuing site greatly on first detection site.After adding specimen, the quantity in the site of demonstration detectable signal provides the quantitative indication of the CSTF2mRNA amount that in sample, exists.Said detection site is configurable for having any detectable suitably shape, normally across the strip or the point-like of test strip width.
Test kit of the present invention can further comprise positive control or CSTF2 standard model.Positive control sample of the present invention can be measured its CSTF2 level and prepare through collecting CSTF2 positive blood sample subsequently.Perhaps, can the CSTF2 albumen or the polynucleotide of purifying be added in the serum that does not contain CSTF2 to form said positive or CSTF2 standard substance.
The screening of anti-lung cancer material
In background of the present invention, treat that the material of identifying through this screening method can be any material or the compsn that comprises number of substances.And to be exposed to cell or proteic test substances can be single combination of planting material or multiple material to screening method according to the present invention.When in method, using combinations of substances, each material can in order or contact simultaneously.
Any test substances; For example; Cell extract, cells and supernatant, organism of fermentation product, marine organism extract, plant milk extract, purifying or crude protein, peptide, non-peptide material, synthetic scintilla material (comprise nucleic acid construct; For example sense-rna, siRNA, ribozyme and fit or the like) and crude substance, all can be used in the screening method of the present invention.Also can use any approach in a lot of combinatorial library method well known in the art to obtain test substances of the present invention; Comprise (1) biological library; (2) parallel solid phase of space addressable or liquid phase library (spatially addressable parallel solid phase or solution phase libraries); (3) need the synthetic library method of deconvolution (deconvolution); (4) " pearl one material " (" one-bead one-substance ") library method, and the synthetic library method of affinity chromatography selection is used in (5).The biological library method of using affinity chromatography to select is limited to peptide library, and other four kinds of approach are applicable to peptide, non-peptide oligomer or material small molecules library (Lam (1997) Anticancer Drug Des.12:145-67).The example of synthetic molecules library method can find (DeWitt et al., Proc Natl Acad Sci USA 1993,90:6909-13 in the prior art; Erb et al., Proc Natl Acad Sci USA 1994,91:11422-6; Zuckermann et al., J Med Chem 37:2678-85,1994; Cho et al., Science 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 et al., J Med Chem 1994,37:1233-51).The material library can be provided in the solution that (referring to Houghten, Bio/Techniques 1992, (Lam 13:412-21) or on the pearl; Nature 1991,354:82-4), on the chip (Fodor, Nature 1993; 364:555-6), (USP 5 on (USP 5,223,409), the spore on the bacterium; 571,698; 5,403,484 and 5,223,409), (Cull et al., Proc Natl Acad Sci USA 1992, (Scott and Smith, Science1990,249:386-90 89:1865-9) or on the phage on the plasmid; Devlin, Science 1990,249:404-6; 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).
A part of structure of the material that screens through any screening method of the present invention through add, deletion and/or substitute mode be by the material that conversion obtains, be included within the material of identifying through screening method of the present invention.
In addition, when the test substances of being screened is protein, in order to obtain this proteic DNA of coding; Can confirm proteic whole aminoacid sequence; Infer the nucleotide sequence of this proteins encoded with this, perhaps can analyze the proteic partial amino-acid series of gained, prepare few DNA as probe according to this sequence; And with this probe screening cDNA library, to obtain this proteic DNA of coding.DNA to gained confirms its availability in the material standed for test substances of preparation treatment or preventing cancer.
This paper is described screening combines CSTF2 albumen or its to lack the antibody of the partial peptide of BA in the former proteic body for useful specimen also can be specificity.
Although the structure in test agent library is well-known in the art, the guidance of characterization test material with the library that makes up these test substances that are used for this screening method is provided further hereinafter.
In the present invention, disclose the expression level and/or the BA of preventing CSTF2 and caused preventing of growth of cancer cells.Therefore, prevent the expression of CSTF2 and/or when active, this prevents the potential result of treatment among the indication experimenter when certain material.In the present invention, potential result of treatment refers to have the clinical benefit of rational expectation.In the present invention, this type of clinical benefit comprises:
(a) minimizing of CSTF2 genetic expression,
(b) reduction of the size of cancer, morbidity (prevalence) or metastatic potential among the experimenter,
(c) preventing cancer takes place, or
(d) clinical symptom of prevention or alleviation cancer.
(i) molecule modeling:
Provide convenience for people make up the test agent library to the molecular structure of material and/or to the knowledge of the molecular structure of CSTF2 with destination properties.It is that microcomputer modelling is carried out in the interaction that receives reagent agent and its target that prescreen is suitable for one of method that receives the reagent agent of further assessment.
Microcomputer modelling technology is for the visual of the three-dimensional atomic structure of selected molecule and can provide with the appropriate design of the novel substance of this interaction of molecules maybe.The three-dimensional structure typically depends on the x-ray crystal analysis that derives from selected molecule or the data of NMR imaging.Molecular dynamics needs field of force data.How computer graphics system connects target molecule for the prediction novel substance, and the structure of experimental implementation material and target molecule provides possibility to improve binding specificity.In order to predict which type of molecule-matter interaction is when tiny change all takes place for molecule and material one or both of; Need molecular mechanics software and computation-intensive computingmachine, their common couplings are joining the interface of user-friendly, the menu-drive between molecular designing program and the user.
An instance of the general molecule modeling of describing of preceding text comprises CHARMm and QUANTA program, Polygen Corporation, Waltham, Mass.CHARMm carries out energy minimization and molecular dynamics function.QUANTA carries out structure, graphical modeling and analysis of the molecular structure.Utilize QUANTA can carry out interactive structure, modification, the visual and analysis of molecule interbehavior.
Many pieces of literature reviews are arranged with the microcomputer modelling of the interactional medicine of differential protein, Rotivinen et al.Acta Pharmaceutica Fennica 1988 for example, 97:159-66; Ripka, New Scientist 1988,54-8; McKinlay&Rossmann, Annu Rev Pharmacol Toxiciol1989,29:111-22; Perry&Davies, Prog Clin Biol Res 1989,291:189-93; Lewis&Dean, Proc R Soc Lond 1989,236:125-40,141-62; And about the Askew et al. of nucleic acid component model acceptor, JAm Chem Soc 1989,111:1082-90.
Other can screen and the computer program of pattern description chemical substance can be from for example Mississauga, Ontario, the BioDesign of Canada, Inc.; Pasadena, Calif., Allelix; Inc company, Cambridge, the Hypercube of Ontario, companies such as Inc. obtain.See for example DesJarlais et al., J Med Chem1988,31:722-9; Meng et al., J Computer Chem 1992,13:505-24; Meng et al., Proteins 1993,17:266-78; Shoichet et al., Science 1993,259:1445-50.
In case identify the suppressor factor of inferring, can use combinatorial chemistry technique to make up any amount of variant based on the chemical structure of inferring suppressor factor that identifies, be described below.Gained infer suppressor factor, method screening of the present invention can be used, to identify the test agent of treatment or prevention lung cancer in or " test substances " library.
(ii) combinatorial chemistry is synthetic:
The combinatorial library of test substances can be used as the part that rational drug designs program and prepares, and rational drug is designed program and related to the knowledge of the core texture that exists in the relevant known suppressor factor.This strategy makes the library can keep rational scale, is convenient to carry out high flux screening.Perhaps, can make up simply molecular library that is particularly short, the polymerization rerum natura through synthetic all arrangements that constitute the molecule family in library simply.An instance of a kind of method in back is 6 peptide libraries that amino acid is formed by all length.This peptide library comprises all six amino acid series arrangement.Such library is called linear combination chemistry library.
The preparation in combinatorial chemistry library is well-known to those skilled in the art, can produce through chemistry or biosynthesizing.The combinatorial chemistry library include but not limited to, and peptide library (is seen for example USP 5,010,175; Furka, Int J Pept Prot Res 1991,37:487-93; Houghten et al., Nature1991,354:84-6).Also can use other to be used to produce the chemistry in Chemical Diversity library.These chemistry comprise, but are not limited only to peptide (for example PCT announces WO 91/19735), the peptide that is encoded (for example WO93/20242); Biological at random oligomer (for example WO 92/00091), (for example USP 5,288 for BENZODIAZEPINE (benzodiazepines); 514), diversomer such as NSC 9226, BENZODIAZEPINE and dipeptides (DeWitt et al., Proc Natl Acad Sci USA 1993; 90:6909-13), vinylogyization (vinylogous) polypeptide (Hagihara et al., J Amer Chem Soc 1992; 114:6568), the non-peptide class peptide mimics (Hirschmann et al., the J Amer Chem Soc 1992 that have glucose skeleton (scaffolding); 114:9217-8), the stand-in organic synthesis of little library of compounds (analogous organic synthese) (Chen et al., J.Amer Chem Soc 1994; 116:2661), oligomerization carbaminate (Cho et al., Science1993; 261:1303), and/or peptide acyl phosphonic acid ester (peptidylphosphonates) (Campbell et al., J Org Chem 1994; 59:658), nucleic acid library (is seen Ausubel, Current Protocols in Molecular Biology 1995 supplementary issues; Sambrook et al., Molecular Cloning:A Laboratory Manual, 1989, Cold Spring Harbor Laboratory; New York, USA), for example USP 5,539 (is seen in the PNAG3 library; 083), antibody library (see for example Vaughan et al., Nature Biotechnology 1996,14 (3): 309-14 and PCT/US96/10287); For example Liang et al. (is seen, Science1996,274:1520-22 in the glucide library; USP 5,593,853) and the organic molecule library (see for example BENZODIAZEPINE, Gordon EM.Curr Opin Biotechnol.1995 Dec 1; 6 (6): 624-31; Isoprenoid (isoprenoids), USP 5,569,588; Thiazolidone (thiazolidinones) and inclined to one side thia piperidine (metathiazanone), USP 5,549,974; Tetramethyleneimine (pyrrolidines), USP 5,525,735 and 5,519,134; Morpholino compounds, USP 5,506,337; BENZODIAZEPINE, 5,288,514; Deng).
The equipment that is used to prepare combinatorial library is commercially availablely (to see for example 357MPS, 390MPS, Advanced Chem Tech, Louisville KY; Symphony, Rainin, Woburn, MA; 433AApplied Biosystems, Foster City, CA, 9050 Plus; Millipore, Bedford, MA).In addition, have multiple combinatorial library itself also be commerce can get (see for example ComGenex, Princeton, N.J., Tripos, Inc., St.Louis, MO, 3D Pharmaceuticals, Exton, PA, Martek Biosciences, Columbia, MD, or the like).
(iii) other material standed for
Another kind of means are to use the recombinant bacteria phage to produce the library.Use " phage method " (Scott&Smith, Science 1990,249:386-90; Cwirla et al., Proc Natl Acad Sci USA 1990,87:6378-82; Devlin et al., Science 1990,249:404-6), can make up very large library (for example 106-108 chemical entities).Another means is mainly used chemical process, and the example comprises Geysen method (Geysen et al., Molecular Immunology 1986,23:709-15; Geysen et al., J Immunologic Method 1987,102:259-74) (Science 1991,251:767-73) with the method for Fodor etc.(14th International Congress of Biochemistry 1988, Volume#5, Abstract FR:013 such as Furka; Furka, Int J Peptide Protein Res1991,37:487-93); (USPs 5 such as Houghten (USP 4,631,211) and Rutter; 010,175) put down in writing the method that produces peptide mixt, can these peptides have been tested as agonist or antagonist.
Fit is the macromole that the nucleic acid by the specific molecular target of can combining closely constitutes.Tuerk and Gold (Science.249:505-510 (1990)) have disclosed SELEX (the Fas lignand system property evolution of carrying out through exponential enrichment) method and have selected fit.In the SELEX method, the large-scale library of nucleic acid molecule (for example 10 15Plant differing mol) can be used for screening.
The screening of CSTF2 binding substance
In the present invention, in lung cancer, detect crossing of CSTF2 gene and express, and in normal organ, do not express (Fig. 1 and 2).In addition, the siRNA to the CSTF2 gene induces prevent (Fig. 3) to growth of cancer cells to preventing of CSTF2 genetic expression.These results indicate the CSTF2 gene in cancer cells, to bring into play crucial effects.Therefore, the invention provides the albumen that uses CSTF2 gene, this genes encoding, screen the method for the material that combines the CSTF2 polypeptide.Because CSTF2 expression of gene in the lung cancer, be expected to prevent the propagation of lung carcinoma cell with CSTF2 polypeptide bonded material, and therefore useful to treatment or prevention lung cancer.Therefore, the present invention also provides and has used the screening of CSTF2 polypeptide to prevent the method for the candidate substances of proliferation of lung cancer cells, and the method for the candidate substances of screening treatment or prevention lung cancer.Particularly, be used for treating or an embodiment of the candidate substances method of preventing cancer or anticancer growth in screening, this method may further comprise the steps:
(a) test substances is contacted with CSTF2 polypeptide or its fragment;
(b) detect the activity that combines between said polypeptide or its fragment and the said test substances; With
(c) select to combine said polypeptide or its segmental test substances as being used to treat or the candidate substances of preventing cancer.
In another embodiment, the present invention also provides to use CSTF2 polypeptide or its fragment to screen to be used to and has treated or the method for the candidate substances of preventing cancer or anticancer growth, and it may further comprise the steps:
(a) test substances is contacted with CSTF2 polypeptide or its functional fragment;
(b) detect the activity that combines between polypeptide described in the step (a) or its fragment and the said test substances; And
(c) the active result of treatment with said test substances of the combination of (b) is associated.
Perhaps, according to the present invention, also can assess or estimate test substances or compound potential result of treatment to treatment or preventing cancer.In some embodiments, the present invention is provided for assessing or the evaluation test material is crossed the method for expressing relevant treatment for cancer effect to treatment or preventing cancer or inhibition with CSTF2, and this method comprises the steps:
(a) test substances is contacted with polypeptide by the CSTF2 polynucleotide encoding;
(b) combination that detects between said polypeptide and the said test substances is active; And
(c) potential result of treatment and test substances are associated, wherein when this material combines said polypeptide, show potential result of treatment.
In the present invention, can result of treatment be associated with the activity that combines of CSTF2 polypeptide or its functional fragment.For example, when certain test substances combines CSTF2 polypeptide or its functional fragment, can the candidate substances with result of treatment identified or be chosen as to said test substances.Perhaps, when certain test substances debond CSTF2 polypeptide or its functional fragment, can be the material that does not have remarkable result of treatment with said test medicament or compound identification.
Method of the present invention is more detailed the description below.
The CSTF2 polypeptide that need be used to screen can be recombinant polypeptide, or is derived from natural protein or its partial peptide.The polypeptide that contacts with test substances can be, for example, the polypeptide of purifying, soluble protein, with carrier-bound form, or the fusion rotein that merges with other polypeptide.
As using the CSTF2 polypeptide to screen protein, for example, can use to well known to a person skilled in the art several different methods with CSTF2 polypeptide bonded method of protein.This type of screening can be passed through, and for example, immunoprecipitation method is that mode described as follows is carried out specifically.Gene through the CSTF2 polypeptide of will encoding inserts the exogenous gene expression carrier, and for example pSV2neo, pcDNAI, pcDNA3.1, pCAGGS and pCD8 express this gene in host (for example, animal) cell etc.
Express used promotor for this reason and can be any common spendable promotor, comprise, for example; SV40 early promoter (Rigby in Williamson (ed.); Genetic Engineering, vol.3.Academic Press, London; 83-141 (1982)), EF-alpha promotor (Kim et al.; Gene 91:217-23 (1990)), CAG promotor (Niwa et al., Gene 108:193 (1991)), RSV LTR promotor (Cullen, Methods in Enzymology 152:684-704 (1987)), SR alpha promotor (Takebe et al.; Mol Cell Biol 8:466 (1988)), CMV immediate early promoter (Seed and Aruffo; Proc Natl Acad Sci USA 84:3365-9 (1987)), SV40 late promoter (Gheysen and Fiers, J Mol Appl Genet 1:385-94 (1982)), gland virus stage starting (Kaufman et al., Mol Cell Biol 9:946 (1989)), HSV TK promotor or the like.
Said importing host cell can be implemented according to any method with expression alien gene; Electroporation method ((Chu et al. for example; Nucleic Acids Res 15:1311-26 (1987)), calcium phosphate method (Chen and 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 Biol4:1641-3 (1984)), Lipofectin method (Derijard B., Cell 76:1025-37 (1994); Lamb et al., Nature Genetics 5:22-30 (1993): Rabindran et al., Science 259:230-4 (1993)) or the like.
For polypeptide, can import N or the C end of this polypeptide to the recognition site of the known monoclonal antibody of specificity (epi-position), thereby be the fusion rotein that comprises this epi-position this expression of polypeptides by the CSTF2 genes encoding.Epitope-antibody system (Experimental Medicine 13:85-90 (1995)) that can commodity in useization.The carrier that can utilize its MCS to express the fusion rotein that forms with for example beta-galactosidase enzymes, maltose binding protein, glutathione s-transferase and green fluorescent protein (GFP) is commercially available.In addition, also reported following fusion rotein, it is prepared to the small-sized epi-position that 12 (a dozen) amino acid constitute by several through only importing, and makes fusion can not change the character of CSTF2 polypeptide.The monoclonal antibody that can use for example polyhistidine (His-label), influenza lectin HA, people c-myc, FLAG, vesicular stomatitis virus gp (VSV-GP), T7 gene 10 albumen (T7-label), human herpes simplex vicus's gp (HSV-label), E-label epi-positions such as (epi-positions on the mono-clonal phage) and discern them is as screening and the proteinic epitope-antibody of CSTF2 polypeptide bonded system (Experimental Medicine 13:85-90 (1995))
In immunoprecipitation, these antibody are added to in the cell lysate of suitable washing agent preparation and form immunocomplex.Immunocomplex by the CSTF2 polypeptide, comprise with the polypeptide and the antibody of this polypeptide bonded ability and form.Except using to the antibody of above-mentioned epi-position, can also use antibody to carry out immunoprecipitation to the CSTF2 polypeptide, such antibody can as indicated abovely prepare.Immunocomplex can be precipitated, and for example when antibody is mouse IgG antibody, can it be precipitated through albumin A sepharose or Protein G sepharose.If will be prepared into fusion rotein by the polypeptide of CSTF2 genes encoding with epi-position (for example GST); Then can use the material of these epi-positions of specific combination; Gsh-sepharose4B for example is according to forming immunocomplex with using the identical mode to the antibody of CSTF2 polypeptide.
Can follow or according to, for example, the method in the document is implemented immunoprecipitation (Harlow and Lane, Antibodies, 511-52, Cold Spring Harbor Laboratory publications, New York (1988)).
Generally use SDS-PAGE to analyze albumen, use the gel of suitable concn, can utilize the proteic molecular weight of bonded to analyze this albumen through immunoprecipitation.Owing to be difficult to detect with CSTF2 polypeptide bonded albumen, can improve proteic detection sensitivity through following method: containing ri through common dyeing processs such as blue dyeing of coomassie or silver dyeing 35The S-methionine(Met) or 35Culturing cell in the substratum of S-halfcystine, the albumen in the labeled cell, and detect this albumen.When proteic molecular weight is known, can directly be purified into target protein and measures its sequence from the SDS-polyacrylamide gel.
As utilizing the CSTF2 polypeptide to screen the proteic method that combines this polypeptide, can use for example West-Western engram analysis method (Skolnik et al., Cell 65:83-90 (1991)).Particularly; Can obtain through following method with CSTF2 polypeptide bonded albumen; Express the proteinic culturing cell (for example 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 SW1573) that combines the CSTF2 polypeptide from expection and utilize phage vector (for example ZAP) preparation cDNA library; Expressing protein on the LB agarose; The proteopexy that gives expression on filter membrane, is made the CSTF2 polypeptide and the reaction of above-mentioned filter membrane of purifying and mark, and detect expression and the proteic plaque of CSTF2 polypeptide bonded according to affinity tag.Polypeptide of the present invention can utilize combining between vitamin H and avidin, perhaps utilizes specific combination CSTF2 polypeptide or the peptide that merges with the CSTF2 polypeptide or the antibody of polypeptide (for example GST), carries out mark.Also can use the method for utilizing ri or fluorescence etc.
Perhaps; In another embodiment of screening method of the present invention; Can use the two-hybrid system (" MATCHMAKER Two-Hybrid system " that utilizes cell; " Mammalian MATCHMAKER Two-Hybrid Assay Kit ", " MATCHMAKER one-Hybrid system " are (Clontech); " HybriZAP Two-Hybrid Vector System " (Stratagene); Reference is seen " Dalton and Treisman, Cell 68:597-612 (1992) ", " Fields and Sternglanz, Trends Genet 10:286-92 (1994) ").
In two-hybrid system, polypeptide of the present invention and SRF land or GAL4 land are merged, and in yeast cell, express.Express and the proteic cell preparation cDNA of polypeptide bonded of the present invention library from expection, make this library when being expressed with VP16 or the fusion of GAL4 transcriptional activation domain.Then; The cDNA library is imported in the above-mentioned yeast cell; And from the cDNA of detected positive colony (when giving expression to the yeast cell can be with polypeptide bonded albumen of the present invention the time, both combinations activate reporter gene, and positive colony can be detected) separation source from this library.Through will above isolating cDNA import in the intestinal bacteria and express this albumen, can prepare by this cDNA encoded protein.As reporter gene, except the HIS3 gene, can also use for example Ade2 gene, lacZ gene, CAT gene, luciferase genes etc.
Also can screen the polypeptide bonded material with the CSTF2 genes encoding with affinity chromatography.For example, can be fixed on polypeptide of the present invention on the carrier of affinity column, can combine the proteic test substances of polypeptide of the present invention to be applied on this post and will contain.The test substances here can be for example cell extract, cell lysate etc.After loading test substances, the flushing pillar, thus can prepare the material that is incorporated into polypeptide of the present invention.When test substances is protein, the proteinic aminoacid sequence of gained is analyzed, synthesize few DNA according to this sequence, and screen the cDNA library as probe, thereby obtain this proteic DNA of coding with this widow DNA.
Utilize the biosensor of surface plasma resonance can be in the present invention as detecting or the quantitative device of binding substance.When using this biosensor, trace can only be used and not have the polypeptide of mark that (for example BIAcore Pharmacia), carries out real-time observation with the form of surface plasma body resonant vibration signal to the interaction between polypeptide of the present invention and test substances.Therefore, use biosensor, BIAcore for example, people just might assess combining between polypeptide of the present invention and test substances.
Be used to screen the method that the bonded molecule takes place when fixed CSTF2 polypeptide is exposed to synthetic chemical or crude substance library or random phage peptide display libraries; And use is based on high-throughput screening method (Wrighton et al., the Science 273:458-64 (1996) of combinatorial chemistry technique; Verdine, Nature 384:11-13 (1996); Hogan, Nature 384:17-9 (1996)), with not only separation and the protein bound protein of CSTF2, and the method for separation and the protein bound material of CSTF2 (comprising agonist and antagonist), be that those skilled in the art are well-known.
Except the CSTF2 polypeptide, the fragment of this polypeptide also can be used for screening of the present invention, as long as it keeps the natural at least a BA that has the CSTF2 polypeptide.
Said polypeptide or its fragment can further connect other materials, as long as this polypeptide and fragment keep its at least a BA.Available material comprises: peptide, lipid, sugar and sugar chain, ethanoyl, natural and synthetic polymer etc.The modification that can carry out these types is to give other function of this polypeptide and fragment or to make it stable.
The polypeptide or the fragment that are used for the inventive method can be used as naturally occurring protein and obtain from nature through conventional purification process, or obtain through chemosynthesis based on selected aminoacid sequence.For example, can be used for the conventional method of peptide synthesis of synthetic comprises:
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 Experiment of Peptide Synthesis (Japanese), Maruzen Co., 1985;
5) Development of Pharmaceuticals (second volume) (Japanese), Vol.14 (peptide synthesis), Hirokawa, 1991;
6) WO99/67288; With
7)Barany?G.&Merrifield?R.B.，Peptides?Vol.2，“Solid?Phase?Peptide?Synthesis”，Academic?Press，New?York，1980，100-118。
Perhaps, can obtain this protein (Morrison J. for example, J Bacteriology 1977,132:349-51 through any known gene engineering method that is used to produce polypeptide; Clark-Curtiss&Curtiss, Methods in Enzymology (editor Wu et al.) 1983,101:347-62).For example, but at first prepare the carrier that is fit to that comprises the polynucleotide of the target protein of encoding with expression-form (for example being positioned at the downstream of the adjusting sequence that comprises promotor), be transformed into proper host cell then, then cultivate host cell to produce protein.In particular, the gene through the CSTF2 polypeptide of will encoding inserts carrier such as pSV2neo, pcDNAI, pcDNA3.1, pCAGGS or the pCD8 that is used to express alien gene, this gene of expression in host (for example animal) cell etc.Promotor can be used for expressing.Can use any promotor commonly used, comprise for example SV40 early promoter (Rigby in Williamson (volume), Genetic Engineering; Vol.3.Academic Press, London, 1982; 83-141), EF-α promotor (Kim et al., Gene 1990,91:217-23), CAG promotor (Niwa et al.; Gene 1991,108:193), RSV LTR promotor (Cullen, Methods in Enzymology 1987; 152:684-704), SR α promotor (Takebe et al., Mol Cell Biol 1988,8:466), CMV immediate early promoter (Seed et al.; Proc Natl Acad Sci USA 1987,84:3365-9), SV40 late promoter (Gheysen et al., J Mol Appl Genet 1982; 1:385-94), gland virus stage starting (Kaufman et al., Mol Cell Biol 1989,9:946), HSV TK promotor etc.Can carrier be imported host cell to express the CSTF2 gene according to any method; For example electroporation (Chu et al., NucleicAcids Res 1987,15:1311-26), calcium phosphate method (Chen et al.; Mol Cell Biol 1987; 7:2745-52), DEAE DEXTRAN 500.000 method (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; Rabindran et al., Science 1993,259:230-4) etc.
Also can adopt external translating system at external generation CSTF2 albumen.
The CSTF2 polypeptide that will contact with test substances can be polypeptide, the soluble protein of for example purifying or the fusion rotein that merges with other polypeptide.
In the present invention, disclosed and prevent CSTF2 genetic expression to reduce the cell growth.Therefore, combine the candidate substances of CSTF2 polypeptide, can identify and can be used for treating or the candidate substances of preventing cancer through screening.These candidate substances or medicament can through secondary and/or further screening assess treatment or the potentiality of preventing cancer are used for treatment for cancer property material with evaluation.
Prevent the screening of the material of CSTF2 BA
The invention provides screening and can prevent the method for the material of cancer cell multiplication, and screening is used for treatment or preventing cancer, comprise the method for the material of lung cancer.Therefore, the invention provides the method that use is screened the material of treatment or preventing cancer or anticancer growth by the polypeptide of CSTF2 genes encoding, it comprises the following steps:
(a) test substances is contacted with the CSTF2 polypeptide;
(b) BA of the said polypeptide of detection step (a); And
(c) select the BA of CSTF2 polypeptide when test substances does not exist, prevent the test substances of the BA of this polypeptide.
In another embodiment, the present invention also provides the polypeptide screening of using by the CSTF2 genes encoding to be used to treat or preventing cancer or be used for the method for the material of anticancer growth, and it may further comprise the steps:
(a) test substances is contacted with the CSTF2 polypeptide; And
(b) BA of the said polypeptide of detection step (a); And
(c) BA of (b) and the result of treatment of said test substances are associated.
Perhaps, in some embodiments, the present invention provides a kind of and is used to assess or the treatment of evaluation test material or preventing cancer or inhibition are crossed the method for expressing relevant treatment for cancer effect with CSTF2, and this method may further comprise the steps:
(a) test substances is contacted with polypeptide by the polynucleotide encoding of CSTF2 gene;
(b) BA of the said polypeptide of detection step (a); And
(c) potential result of treatment and test substances are associated; Wherein when the BA of the polypeptide that does not have down detected polynucleotide encoding by the CSTF2 gene than test substances, this material shows potential result of treatment when preventing the BA of said polypeptide.
In the present invention, can the BA of result of treatment and CSTF2 polypeptide be associated.For example, when not existing down detected level to compare that this test substances is prevented or when suppressing the BA of CSTF2 polypeptide, can or be chosen as the candidate substances with result of treatment with said test substances evaluation with certain material.Perhaps, when not existing down detected level to compare that this test medicament or compound are not prevented or when suppressing the BA of CSTF2 polypeptide, can said test substances be accredited as the material that does not have remarkable result of treatment with certain test substances.
The method of the invention is more detailed the description below.
Any CSTF2 polypeptide all can be used for screening, as long as they comprise the proteic BA of CSTF2.Such BA comprises that the proteic cell-proliferation activity of CSTF2, RNA combine activity, mRNA nicking activity and mRNA polyadenylation active.For example, can use CSTF2 albumen, also can use with the CSTF2 protein function on polypeptide of equal value.Aforementioned polypeptides can the endogenous or external source ground expression by cell.
Material through this screening and separating is the candidate of antagonist of the polypeptide of CSTF2 genes encoding.Term " antagonist " is meant through combining polypeptide to suppress the molecule of polypeptide function.This term also refers to reduce or to suppress the molecule of the expression of gene of coding CSTF2.And the material through this screening and separating is the material standed for of following material, and said material suppresses interaction in the body of CSTF2 polypeptide and molecule (comprising DNA, RNA and albumen).
When the BA that will detect in present method is cell proliferation; Can detect through for example following method: the cell of CSTF2 polypeptide is expressed in preparation; Culturing cell in the presence of test substances is confirmed cell proliferation rate, measures the cell cycle etc.; And through measurement survivaling cell or colony formation ability, for example shown in Fig. 3 or 4.The material of the rate of propagation of the cell of selection reduction expression CSTF2 comprises the candidate substances of lung cancer as treatment or preventing cancer.
In the present invention, disclosed and prevent CSTF2 genetic expression to reduce the cell growth.So, reduce the candidate substances of CSTF2 polypeptide BA, can identify and can be used for treating or the candidate substances of preventing cancer through screening.The potentiality of treatment of these candidate substances or preventing cancer can through secondary and/or further screening assess with evaluation and be used for treatment for cancer property material.
More specifically, this method may further comprise the steps:
(a) make test substances and express the cells contacting of CSTF2 gene excessively;
(b) measure cell-proliferation activity; And
(c) selection is compared the test substances that reduces cell-proliferation activity with the cell-proliferation activity under test substances does not exist.
In preferred embodiments, method of the present invention can further may further comprise the steps:
(d) selection does not have the test substances of influence to the cell of seldom or not expressing CSTF2.
" prevent BA " and be defined herein as when not having said material, to preferred at least 10% prevent of CSTF2 BA, more preferably at least 25%, 50% or 75% prevent, most preferably at least 90% prevent.
In preferred embodiments, the control cells of CSTF2 polypeptide is not expressed in use.Thereby the present invention also provides and has used CSTF2 polypeptide or its fragment to screen the method that is used for cytostatic candidate substances or is used to treat or prevent the candidate substances of CSTF2 relative disease, comprises the following steps:
(a) cell of culture expression CSTF2 polypeptide or its functional fragment and do not express the control cells of CSTF2 polypeptide or its functional fragment in the presence of test substances;
(b) detect the BA of expressing said proteic cell and control cells; And
(c) select with control cells in and said test substances do not exist the propagation that detects down to compare the test substances that suppresses to express the BA in the said proteic cell.
In some embodiments, can use RNA to combine activity, mRNA nicking activity or mRNA polyadenylation active as the CSTF2 polypeptide BA that will in screening method of the present invention, detect.It is well known in the art being used to detect these active methods.During in screening, detecting these activity any, can preferably use the function equivalent of the polypeptide of the RNA identification motif that contains the CSTF2 polypeptide as the CSTF2 polypeptide.For example, the RNA that has the CSTF2 polypeptide of aminoacid sequence SEQ ID NO:2 discerns the zone that motif is made up of the amino acid/11 7 to 90 of SEQ ID NO:2.
Change the screening of the material of CSTF2 expression
The invention provides the method that screening suppresses the material of CSTF2 genetic expression.The material that suppresses CSTF2 genetic expression is expected to prevent proliferation of lung cancer cells, and therefore useful to treatment or prevention lung cancer.Therefore, the method that the present invention also provides screening to prevent the candidate substances of proliferation of lung cancer cells, and the method for the candidate substances of screening treatment or prevention lung cancer.In background of the present invention, above-mentioned screening can comprise, for example, and the following step:
(a) with test substances and the cells contacting of expressing the CSTF2 gene;
(b) detect CSTF2 expression of gene level; And
(c) test substances that reduces the CSTF2 gene expression dose is compared in selection with detected expression level when test substances does not exist.
In another embodiment, the present invention also provides the method for screening the candidate substances of preventing cancer cell multiplication and has screened the method that is used to treat or prevent the candidate substances of CSTF2 relative disease.
In background of the present invention, this type of screening can comprise for example following steps:
(a) make test substances and the cells contacting of expressing the CSTF2 gene;
(b) detect the CSTF2 gene expression dose; And
(c) expression level of (b) and the result of treatment of said test substances are associated.
Perhaps, in some embodiments, the present invention also provides a kind of and is used to assess or the treatment of evaluation test material or preventing cancer or inhibition are crossed the method for expressing relevant treatment for cancer effect with CSTF2, and this method may further comprise the steps:
(a) make test substances and the cells contacting of expressing CSTF2;
(b) potential result of treatment and test substances are associated, wherein when test substances is compared the expression level that reduces CSTF2 with contrast, show potential result of treatment.
In the present invention, result of treatment can associate with the CSTF2 gene expression dose.For example, when not existing following detected level to compare this test substances reduction CSTF2 expression of gene level, can the candidate substances with result of treatment identified or be chosen as to said test substances with certain test substances.Perhaps, when when certain test substances does not exist down detected level to compare this test substances not reduce the CSTF2 gene expression dose, can said test substances be accredited as the material that does not have remarkable result of treatment.
Below method of the present invention will be described in further detail.
The cell of expressing the CSTF2 gene comprises, for example, and the clone of setting up from lung cancer; Such cell can be used for screening (A427 for example, A549, LC319, PC14, PC3, the PC9 of the invention described above; 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, LU61).The well-known method of available those skilled in the art, for example, RT-PCR, Northern engram analysis, Western engram analysis, immunostaining and flow cytometry are estimated expression level." reduction expression level " in this definition is preferably than the expression level when said material does not exist, and makes the CSTF2 gene expression dose reduce at least 10% at least, more preferably reduces at least 25%, 50% or 75%, most preferably reduces by 95% level.Material among this paper comprises chemical substance, double chain nucleotide or the like.The preparation of double chain nucleotide has been described in front.In screening method, the material that can select to reduce the CSTF2 gene expression dose is as the candidate substances that is used for treating or preventing lung cancer.
In the present invention, disclosed and prevent CSTF2 genetic expression to reduce the cell growth.Therefore, reduce the material of CSTF2 gene expression dose, can identify and can be used for treating or the candidate substances of preventing cancer through screening.These candidate substances can through secondary and/or further screening assess treatment or the potentiality of preventing cancer are used for treatment for cancer property material with evaluation.
Perhaps, said screening method of the present invention can comprise the following steps:
(a) with the cells contacting of test substances with the carrier that has imported the reporter gene that comprises CSTF2 gene transcription regulation zone and under this transcriptional control zone control, express;
(b) expression level or the activity of measurement reporter gene; And
(c) select to reduce reporter gene expression level or active test substances.
In another embodiment, the present invention's method that the candidate substances that screening is used to prevent cancer cell multiplication also is provided and screening are used to treat or prevent the method for the candidate substances of CSTF2 relative disease.
According to another aspect, the invention provides a kind of method, it may further comprise the steps:
(a) make test substances and the cells contacting that has wherein imported carrier, the reporter gene that this carrier comprises CSTF2 gene transcription regulatory region and under this transcriptional regulatory district control, expresses;
(b) expression or the activity of the said reporter gene of detection; And
(c) expression level of (b) and the result of treatment of test substances are associated.
Perhaps, in some embodiments, the present invention also provides a kind of and is used to assess or the treatment of evaluation test material or preventing cancer or inhibition are crossed the method for expressing relevant treatment for cancer effect with CSTF2, and this method may further comprise the steps:
A) make test substances and the cells contacting that has wherein imported carrier, the reporter gene that this carrier comprises CSTF2 gene transcription regulatory region and under this transcriptional regulatory district control, expresses
B) expression or the activity of the said reporter gene of measurement; And
C) potential result of treatment and test substances are associated, wherein reduce the expression of said reporter gene or show potential result of treatment when active when test substances.
In the present invention, result of treatment can associate with the expression level or the activity of said reporter gene.For example, detected level is compared this material and is reduced the expression level of said reporter gene or when active, can the candidate substances with result of treatment identified or be chosen as to said test substances when not existing with certain test substances.Perhaps, detected level is compared this material and is not reduced the expression level of said reporter gene or when active, can said test substances be accredited as the material that does not have remarkable result of treatment when not existing with certain test substances.
Suitable reporter gene and host cell are well known in the art.For example; Reporter gene is luciferase, green fluorescent protein (GFP), mushroom coral (Discosoma sp.) red fluorescent protein (DsRed), chloramphenicol acetyltransferase (CAT), Laz and beta-glucuronic acid Glycosylase (GUS), and host cell is COS7, HEK293, HeLa etc.The required report construct of said screening can prepare through reporter gene sequence is connected with CSTF2 gene transcription regulation zone.CSTF2 transcriptional control as herein described zone is from initiator codon to the zone at the 500bp upper reaches at least, and is preferred 1,000bp, more preferably 5000 or 10, the 000bp upper reaches.The nucleotide fragments that contains said transcriptional control zone can be separated from genomic library, maybe can pass through pcr amplification.The required report construct of said screening can prepare through arbitrary transcriptional control zone in reporter gene sequence and these genes is connected.Differentiate the method in transcriptional control zone, with and the assay method rules all be well-known (Molecular Cloning, the third edition, the 17th chapter, 2001, Cold Springs Harbor Laboratory Press).
Use and contain the carrier host cells infected of reporting construct, and detect the expression or the activity (for example, using luxmeter (luminometer), absorption spectrometer, flow cytometer or the like) of reporter gene with method well-known in the art." reduce and express or activity " in this definition is that the expression of reporter gene or activity preferably are lowered at least 10% than when said material does not exist, and more preferably, reduces by 25%, 50% or 75%, most preferably, reduces at least 95%.
In the present invention, disclosed and prevent CSTF2 genetic expression to reduce the cell growth.Therefore, reduce reporter gene through screening and express or active candidate substances, can identify the candidate substances of potential treatment or preventing cancer.These candidate substances can through secondary and/or further screening assess treatment or the potentiality of preventing cancer are used for treatment for cancer property material with evaluation.
Each side of the present invention is described in following embodiment, and they also limit the scope of the invention of describing in the claim unintentionally.
Only if definition separately, all technology of this use and scientific terminology all with the present invention under the same meaning of field those skilled in the art's common sense.Suitable method and material are described below, though also can be used for practice or test the present invention with method and material similar or that be equal to described here.
The present invention will further describe in the following example, and it does not limit the scope of the invention of describing in the claim.
Embodiment
Material and method
Clone and tissue sample.
15 kinds of human lung cancer cell lines that use in this research comprise 5 kinds of gland cancer (NCI-H1781, NCI-H1373, LC319, A549 and PC-14), 5 kinds of squamous cell carcinomas (SK-MES-1, NCI-H520, NCI-H1703, NCI-H2170 and LU61), a kind of large cell carcinoma (LX1) and 4 kinds of small cell lung cancers (SBC-3, SBC-5, DMS114 and DMS273).All cells in being supplemented with the appropriate media of 10%FCS with monolayer culture and containing in the humidification air of 5%CO2 and keep in 37 ℃.The stingy tract epithelial cell of people (SAEC) that is used as normal control is substratum (Cambrex Bioscience, Inc) the middle cultivation through optimizing.In the early time under informed consent, from before tumor resection, obtaining primary NS CLC tissue sample and closing on corresponding healthy tissues (Kikuchi T, Daigo Y, Katagiri T, the et al.Oncogene 2003 of margins of excision through the patient of anticancer therapy; 22:2192-205, Taniwaki M, 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).To the pathologic tumour-lymphoglandula-transfer classification (table 1 of all tumours based on international cancer federation; Sobin L, Wittekind CH.TNM classification of malignant tumors.6th ed.New York:Wiley-Liss; 2002) come by stages.Be used for the primary tumors of lung of formalin fixed that tissue Microarray lists immunostaining with to close on normal lung tissue's sample be 327 patients (196 routine gland cancer, 98 routine squamous cell carcinomas, 23 routine large cell carcinomas and 10 routine gland squamous cell carcinomas from undergoing surgery in Saitama Cancer center; 99 women and 228 male patients; Median age 64.7 years old, scope 29-85 year) obtain.These patients that accept the excision of its preinvasive cancer do not accept treatment before any art, and only have the patient that positive lymph nodes shifts among them and after its operation, accepted the NACT treatment based on platinum.The use of this research and all said clinical materials has all obtained each scientific research Ethics Committee approval.
[table 1]
Related (n=327) between the positive characteristic with the patient of CSTF2 in the NSCLC tissue
Figure BDA0000156064560000771
ADC, gland cancer
Non-ADC, squamous cell carcinoma add large cell carcinoma and glandular scale shape cell carcinoma
*P＜0.05 (FisherShi checks without fail)
Semiquantitative reverse transcription-PCR.
Use random primer (Roche Diagnostics) will become strand cDNA from the 3 microgram mRNA sample aliquot rts altogether of every duplicate samples with Superscript II (Invitrogen).Semiquantitative reverse transcription-PCR (RT-PCR) experiment with following each organize people CSTF2 (gene accession number NM_001325) specific synthetic primer or beta-actin (ACTB) Auele Specific Primer that is used as internal contrast carried out: CSTF2,5 '-GTCATGCAGGGAACAGGAAT-3 ' (SEQ ID NO:3) and 5 '-TGAGTCATTCAAGGGTTAGGATG-3 ' (SEQ ID NO:4); ACTB, 5 '-GAGGTGATAGCATTGCTTTCG-3 ' (SEQ ID NO:5) and 5 '-CAAGTCAGTGTACAGGTAAGC-3 ' (SEQ ID NO:6).The cycle number of optimizing the PCR reaction is to guarantee that product intensity is in the linear phase of amplification.
The Northern engram analysis.
The people that to contain 23 kinds of tissues organizes the trace (BD Bioscience) and the 521-bp PCR product (it uses primer 5 '-CGAGGCTTGTTAGGAGATGC-3 ' (SEQ ID NO:7) and 5 '-CCCCCATGTTAAGGACTG-3 ' (SEQ ID NO:8) to be prepared into probe) of the 32P mark of CSTF2 to hybridize more.Prehybridization, hybridization and cleaning are followed the recommendation of manufacturers and are carried out.Trace is shielded radioautograph 14 days in-80 ℃ with strengthening.
The Western trace.
With tumour cell cracking in lysis buffer; 50mmol/L Tris-HCl (pH 8.0), 150mmol/L NaCl, 0.5%NP40,0.5% Sodium desoxycholate and protease inhibitor cocktail group III (Calbiochem).Measure the protein contnt of every part of lysate through Bio-Rad protein determination test kit, with bovine serum albumin as standard substance.Every part of lysate get 10 micrograms 7.5% to 12% denaturing polyacrylamide gel (being with 3% SEPIGEL 305 stacking gel) go up resolve and electrophoretic transfer to nitrocellulose filter (GE Healthcare Biosciences).With after 5% alipoidic milk power among the TBST (the Tris BS that the contains Tween 20) sealing, with film with tame rabbit polyclonal antibody in room temperature incubation 1 hour.The commercialization man anti-CSTF2 antibody of rabbit polyclonal is available from ATLAS company, and the Western engram analysis of the lysate through using lung cancer cell line, detects to specific to people CSTF2.Immunoreactive protein matter arised from the room temperature incubation 1 hour with two anti-(the GE Healthcare Bio-sciences) that are conjugated with horseradish peroxidase.After the TBST cleaning, reactant uses enhanced chemical luminescence reagent box (GE Healthcare Bio-sciences) colour developing.
Immunofluorescence analysis.
Cultured cells cleans twice with PBS (-), in 4% formaldehyde solution in 4 ℃ fixing 60 minutes, and made in 5 minutes and can pass through through handling with the PBS (-) that contains 0.1%Triton X-100.Cell covers 10 minutes with the sealing non-specific binding with CASBlock (Zymed), carries out an anti-reaction afterwards.Then, with the protein incubation of cell with antibody that is directed against people CSTF2 or band c-myc label.
Immunohistochemistry and micro-array tissue.
For formalin fixed and paraffin mass in the proteic clinicopathlogical significance of investigation CSTF2 in the clinical lung cancer sample of embedding, use Envision+ test kit/horseradish peroxidase (DakoCytomation) to dye in the following manner to section.For antigen retrieval, slide glass is soaked in target thing reparation pH value of solution 9 (DakoCytomation) and in high-pressure sterilizing pot, boiled 15 minutes in 108 ℃.Seal after endogenous px and the protein, every slide glass adds the anti-people CSTF2 of tame rabbit polyclonal antibody (0.06 microgram/ml; ATLAS), and will cut into slices with as the anti-tame rabbit igg [Histofine Simple Stain MAX PO (G), Nichirei] of two anti-horseradish peroxidase-labeled incubation together.Add substrate-chromogen, and sample is used the haematoxylin redyeing look.
Primary NS CLC with 327 parts of formalin fixed makes up the tumor tissues microarray; These primary NS CLC obtains (Chin S F, Daigo Y, Huang HE, et al.Mol Pathol 2003 by single institution (please see above) according to the rules of collecting after the identical excision, fixing and preservation being organized; 56:275-9, Callagy G, Cattaneo E, Daigo Y, et al.Diagn Mol Pathol 2003; 12:27-34, Callagy G, Pharoah P, Chin SF, et al.J Pathol 2005; 205:388-96).The histology of considering each tumour is heterogeneous, based on slide glass on the range estimation of corresponding H&E stained compare and select the tissue regions of sampling.To take from 3,4 or 5 tissue core (diameter, 0.6mm of a donor tumor mass with micro-array tissue appearance (Beecher Instruments); The degree of depth 3-4mm) is placed in the acceptor paraffin mass.Get 1 healthy tissues core from each case punching, and 5 microns sections of gained microarray piece are used for immunohistochemical analysis.By 3 independently the investigator second assesses the CSTF2 positive quantitatively in the condition of not knowing the clinical pathology data in advance.Because the staining power in each tumor tissues core is uniform mostly; So use criterion sxemiquantitative assessment CSTF2 staining power: strong positive (score 2+); Vandyke brown dyeing makes nucleus and tenuigenin fuzzy fully in＞50% tumour cell; Weak positive (1+), discernable any brown dyeing in neoplastic cell nuclei and the tenuigenin than low degree; Do not have (score 0), do not have perceptible dyeing in the tumour cell.Only they are defined as under the situation of strong positive independently the investigator, just accepting case is strong positive.
Statistical study.
Statistical study uses StatView statistics program (SaS) to carry out.Use Fisher to check the related of the strong CSTF2 immunoreactivity of assessment and clinical pathology variable such as age, sex, pathologic tumour-lymphoglandula-transition phase and histological type without fail.Calculate the data tumour-specific survivorship curve that ends during during to the NSCLC associated death or to last follow-up observation from from the operation.For each related variable be that CSTF2 expresses calculating K aplan-Meier curve; Use sequence check to analyze the difference of survival time between the inferior group of patient.Carry out single argument and multivariate analysis to confirm the association between clinical pathology variable and the cancer associated death with the Cox proportional hazards regression models.At first, analyze death and possible prognosis factor and comprise the association between age, sex, histology, pT classification and the pN classification, once consider a factor.Secondly, with reverse (progressively) program multivariate analysis, it always forces strong CSTF2 to express and the variable of the horizontal P of any satisfied entering＜0.05 gets into model together.Along with this model continues the interpolation factor, independent factor is above withdrawing from horizontal P＜0.05.
The RNA interferometry.
In order to estimate the biological function of CSTF2 in lung and esophageal cancer cell, use siRNA (siRNA) duplex (SIGMA) to target gene.The target sequence that is used for RNA interferential synthetic oligonucleotide is following: si-CSTF2-#1,5 '-GGCUUUAGUCCCGGGCAGA-3 ' (SEQ ID NO:9); Si-CSTF2-#2; 5 '-CACUUUACUUUCUGUAACU-3 ' (SEQ ID NO:10); Contrast 1: (EGFP; Enhanced green fluorescence protein [GFP] gene, a kind of two mutants of Victoria jellyfish (Aequorea gictoria) GFP), 5 '-GAAGCAGCACGACUUCUUC-3 ' (SEQ ID NO:11); Contrast 2 (LUC is from the luciferase genes of Lampyridea (Photinus pyralis)), 5 '-CGUACGCGGAAUACUUCGA-3 ' (SEQ ID NO:12).Lung cancer cell line A549 and LC319 are applied to (each coils 8.0x105) on the 10-cm dish, and use 30 microlitre Lipofectamine2000 (Invitrogen) according to the instruction of manufacturers with every kind of siRNA oligonucleotide (100nmol/L) transfection.Behind the incubation 7 days, form with the assessment colony through these cells of Giemsa solution-dyed, and through bromination 3-(4,5-dimethylthiazole-2-yl)-2,5-phenylbenzene tetrazolium (MTT) assay method assessment cell viability.
The result
The expression of CSTF2 in lung cancer and the healthy tissues.
Be used to develop the therapeutical agent of lung cancer and/or the novel target molecule of biomarker in order to identify; At first; Use the cDNA microarray of forming by 27,648 kinds of genes or EST to carry out genome range gene expression spectrum analysis (Kikuchi T, Daigo Y to 101 parts of lung cancer; Katagiri T, et al.Oncogene2003; 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.Hum Mol Genet 2004; 13:3029-43, Kikuchi T, Daigo Y, Ishikawa N, et al.Int J Oncol2006; 28:799-805, Taniwaki M, Daigo Y, Ishikawa N, et al.Int J Oncol2006; 29:567-75).Identify the CSTF2 transcript and in the lung cancer sample of great majority inspection, cross expression (3 times or higher), and seldom expression (except that testis) in all 29 kinds of healthy tissuess of CSTF2.As a result, think that CSTF2 is the better candidate gene of recruit's target.Through sxemiquantitative RT-PCR experimental verification CSTF2 in 12/15 cancerous lung tissue being checked with in 15/15 lung cancer cell line, cross and express (Figure 1A).In addition, confirmed the proteic expression of CSTF2 (Figure 1B) in 9 lung cancer cell lines through using anti-CSTF2 antibody to carry out the Western engram analysis.In order to confirm the Subcellular Localization of endogenous CSTF2 in the lung carcinoma cell, use anti-CSTF2 antibody to carry out immunofluorescence analysis, found its dyeing (Fig. 1 C) in the nucleus of SBC5 cell.
Carry out the Northern engram analysis with CSTF2cDNA as probe, in 16 kinds of health adult tissues that checked, only in testis, identify 2.6-kb transcript (Fig. 2 A).In addition, use anti-CSTF2 antibody to check the CSTF2 protein expression in 5 kinds of healthy tissuess (heart, lung, liver, kidney and testis) and the lung cancer.The CSTF2 positive staining is observed in discovery in the nucleus of testicular cell, but in other healthy tissues, does not observe (Fig. 2 B).
CSTF2 crosses and expresses related with NSCLC patient's poor prognosis.
In order to verify biology and the clinicopathlogical significance of CSTF2 in lung cancer takes place, on the micro-array tissue of the primary NS CLC tissue that contains the patient who accepts healing property excision from 327, carried out immunohistochemical staining.In the nucleus of lung carcinoma cell, observe the CSTF2 positive staining of anti-CSTF2 polyclonal antibody, but what contiguous normal lung cell in office or in the stroma cell of tumour cell dyeing be negative.With the classification of the CSTF2 expression level on the tissue array, scope is not from there being (score 0) to weak/strong positive (score 1+ to 2+) (Fig. 2 C).In 327 routine NSCLC, CSTF2 is dyeing (24% by force in 77 examples; Score 2+), weak dyeing (50% in 165 examples; Score 1+), dye-free (26%: score 0 and in 85 examples; Details are shown in table 1).Then, checked the related of CSTF2 expression level (strong positive is to the weak positive/nothing) and various clinical pathology variablees, and found that strong CSTF2 expresses and primary tumor excision NSCLC patient's poor prognosis relevant (P=0.0079, sequence check afterwards; Fig. 2 D), still irrelevant with any other clinical pathology variable.In addition, checked that univariate analysis (comprises age (＞=65 pairs＜65 years old), sex (male sex is to the women), histology (non-gland cancer is to gland cancer), smoking history (smoker is to the non-smoker), pT stage (tumour size with evaluate patient prognosis and several clinical pathological factors; T2-T3 is to T1), pN stage (nodus lymphoideus transferring rate; N1-N2 is to N0) and CSTF2 express (score 2+ to 0,1+)) between association.All these parameters except that smoking history all with poor prognosis significant correlation (table 2).Multivariate analysis indication pT stage, pN stage, age and the strong CSTF2 positive of using the Cox proportional hazard model to carry out are the independent prognostic factors (table 2) of NSCLC.
[table 2]
The CoxShi proportional hazard model of prognosis factor is analyzed among the NSCLC patient
ADC, gland cancer
Non-ADC, squamous cell carcinoma add large cell carcinoma and glandular scale shape cell carcinoma
To the siRNA of CSTF2 inhibition to the lung carcinoma cell growth.
For whether the rise of assessing CSTF2 plays a role in the growth of lung carcinoma cell or survival, will be transfected into endogenous mistake together with the synthetic oligonucleotide that contrasts siRNA (si-LUC and si-EGFP) to the siRNA (si-CSTF2-#1 and si-CSTF2-#2) of CSTF2 and express among the A549 and LC319 cell of CSTF2.With the mRNA level of CSTF2 in si-CSTF2-#1 and the si-CSTF2-#2 cells transfected with compare remarkable reduction (Fig. 3 A) with arbitrary contrast siRNA cells transfected.Cell viability and colony number through MTT assay method and the investigation of CFA method significantly reduce (Fig. 3 B and 3C) in si-CSTF2-#1 and si-CSTF2-#2 cells transfected.
CSTF2 is to the activation of mammalian cell proliferation.
In order to check the latent effect of CSTF2 in tumour takes place, made up the plasmid (pcDNA3.1/myc-His-CSTF2) that is designed to express CSTF2 and be transfected in the COS-7 cell.Confirmed that through the Western engram analysis external source CSTF2 expresses (Fig. 4 A).Carried out MTT and CFA method, found to be strengthened (Fig. 4 B and 4C) with comparing significantly with the COS-7 cell of analog carrier transfection with the growth of the COS-7 cell of CSTF2 transfection.
Discuss
In order to develop expection, set up a kind of strong target thing screening system and identified specificity activated protein and interaction protein thereof in lung carcinoma cell the molecular targeted anti-tumor agents malignant cell high degree of specificity, that the untoward reaction risk is minimum.At first, through containing the genome range cDNA microarray system of 27,648 kinds of genes, the coupling laser capture microdissection is dissected, and 101 parts of lung cancer samples have been analyzed the genome range express spectra.Through the cDNA microarray analysis with organize the Northern engram analysis verified that this genoid is expressed very low or do not had expression in normal organ after more; List the protein expression of logarithm in tissue Microarray in hundred clinical sample analysis candidate target; Use the RNA EVAC to investigate the afunction phenotype then, and further confirmed this proteinic biological function.Through these analyses; Identified the candidate gene that is used to develop new diagnostic biomarker, curative drug and/or immunotherapy; They raise in cancer cells but in the normal organ except that testis, placenta and/or fetal tissue, do not express (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, DaigoY, Ishikawa N, et al.Hum Mol Genet 2004; 13:3029-43, Kikuchi T, Daigo Y, Ishikawa N, et al.Int J Oncol 2006; 28:799-805, Taniwaki M, Daigo Y, Ishikawa N, et al.Int J Oncol 2006; 29:567-75, 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 Res2005; 65:5638-46, Furukawa C, Daigo Y, Ishikawa N, et al.Cancer Res2005; 65:7102-10, Ishikawa N, Daigo Y, Takano A, et al.Cancer Res2005; 65:9176-84, Suzuki C, Daigo Y, Ishikawa N, et al.Cancer Res2005; 65:11314-25, Ishikawa N, Daigo Y, Takano A, et al.Cancer Sci2006; 97:737-45, Takahashi K, Furukawa C, Takano A, et al.Cancer Res2006; 66:9408-19, Hayama S, Daigo Y, Kato T, et al.Cancer Res2006; 66:10339-48, Kato T, Hayama S, Yamabuki Y, et al.Clin Cancer Res2007; 13:434-42, Suzuki C, Takahashi K, Hayama S, et al.Mol Cancer Ther2007; 6:542-51, Yamabuki T, Takano A, Hayama S, et al.Cancer Res2007; 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 Res2007; 13:6624-31, Ishikawa N, Takano A, Yasui W, et al.Cancer Res2007; 67:11601-11, Mano Y, Takahashi, K, Ishikawa N, et al.Cancer Sci2007; 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, Ito T, et al.Clin Cancer Res 2009,15:256-66, Suda T, Tsunoda T, Daigo Y, Nakamura Y, Tahara H.Cancer Sci 2007; 98:1803-8, Mizukami Y, Kono K, Daigo Y, et al.Cancer Sci 2008; 99:1448-54).
As stated, CSTF2 (member of its coding cutting stimulating factor) crosses in lung cancer with higher frequency and expresses, and might in the growth of lung cancer, play a significant role.Strike low CSTF2 expression through siRNA and prevented the growth of lung carcinoma cell.In addition, the clinical pathology evidence that obtains through our micro-array tissue experiment shows, the cancer specific survival time with NSCLC patient that the CSTF2 strong positive expresses is than having NSCLC patient's weak point that the weak male/female of CSTF2 is expressed.The result who obtains through assay method in external and the body points out strongly, and CSTF2 might be important growth factor, and the virulent phenotype is relevant more with lung carcinoma cell.
In a word, the CSTF2 gene can play a significant role in the growth/progress of lung cancer.For the patients with lung cancer that possibly have poor prognosis, the CSTF2 in the excision sample crosses the useful indication that expression can be an adjuvant therapy.
Industrial applicability
The gene expression analysis of the cancer of the use genome range cDNA microarray of describing among this paper identifies the target of specific gene as cancer prevention and treatment.Based on difference expression gene---the expression of CSTF2, the present invention are provided for identifying and detecting the molecular diagnostic markers of cancer, particularly lung cancer.
The data that provide among this paper increase the comprehensive understanding to cancer, are convenient to develop novel diagnosis policy, and are that the molecule target of identifying curative and preventive is given a clue.This type of information helps more profoundly to understand tumour to be taken place, and is that exploitation is used to diagnose, treat and the New Policy of final preventing cancer provides indication.
Like what prove among this paper, cell growth receives the preventing of duplex molecule of selectively targeted CSTF2 gene.Therefore, these new duplex molecule useful as anti-cancer agents things.
The CSTF2 expression of gene is in cancer, particularly compares remarkable rising in the lung cancer with normal organ.Thereby this gene can be used as the diagnosis marker of cancer, particularly lung cancer easily, and can be used for the diagnostic assay method of cancer by its encoded protein.In addition, the prognosis of having found the cancer patients that CSTF2 expression of gene level is higher is tending towards relatively poor.Therefore, the CSTF2 gene can be used for assessment of cancer patient's prognosis.
In addition, the present invention is provided for treating cancer, comprises the new treatment approach of lung cancer.The CSTF2 gene is the useful target of cancer therapy drug exploitation.
Through addressing all patents, patented claim and the publication of quoting among the complete this paper of including.
In addition, though at length and with reference to its specific embodiments the present invention is described, be appreciated that being described in of front be in nature exemplary with indicative, and intention illustration the present invention and preferred embodiment thereof.Through normal experiment, those skilled in the art can easily recognize and can carry out various changes and modification therein, without departing from the spirit and scope of the present invention.So, the invention is intended to not receive preceding text to describe and limit, but limit accompanying claims and equivalent thereof.
Figure IDA0000156064640000021
Figure IDA0000156064640000031
Figure IDA0000156064640000041
Figure IDA0000156064640000051

Claims (30)

1. one kind is used at experimenter's diagnosing cancer or the tendentious method of cancer takes place, and wherein this method comprises the steps:
(a) measure CSTF2 expression of gene level in the biological sample that is derived from the experimenter through any method that is selected from down group:
(i) mRNA of detection CSTF2 gene,
(ii) detect by the protein of CSTF2 genes encoding and
(iii) detect proteinic BA by the CSTF2 genes encoding; And
(b) existence of cancer associates among the rising of the expression level that is measured in the step (a) being compared with the normal control level of CSTF2 gene and this experimenter.
2. the process of claim 1 wherein that the expression level that is measured in the step (a) is than this normal control level height at least 10%.
3. the process of claim 1 wherein that this biological sample that is derived from the experimenter comprises biopsy samples, phlegm, blood, hydrothorax or urine.
4. method of prognosis that is used to assess or confirms to suffer from the experimenter of cancer, wherein this method comprises the steps:
(a) detection resources CSTF2 expression of gene level in experimenter's biological sample;
(b) detected expression level and control level are compared; And
(c) based on the prognosis of relatively confirming this experimenter of (b).
5. the method for claim 4, wherein this control level is the good prognosis control level, and the rising indication poor prognosis compared with this control level of this expression level.
6. the method for claim 5, wherein said rising is than this control level height at least 10%.
7. the method for claim 4, wherein this expression level is to measure through any method that is selected from down group:
(a) mRNA of detection CSTF2 gene;
(b) detection is by the protein of CSTF2 genes encoding; With
(c) detection is by the proteinic BA of CSTF2 genes encoding.
8. the method for claim 4, wherein this biological sample that is derived from the experimenter comprises biopsy samples, phlegm or blood, hydrothorax or urine.
9. one kind is used for experimenter's diagnosing cancer or the assessment of suffering from cancer or the test kit of measuring prognosis, and it comprises the reagent that is selected from down group:
(a) be used to detect the reagent of the mRNA of CSTF2 gene;
(b) be used to detect proteinic reagent by the CSTF2 genes encoding; With
(c) be used to detect reagent by the proteinic BA of CSTF2 genes encoding.
10. the test kit of claim 9, wherein this reagent comprises to the probe of the genetic transcription thing of CSTF2 gene or primer or to the antibody of the translation product of CSTF2 gene.
11. an isolating duplex molecule, it suppresses the expression in vivo and the cell proliferation of CSTF2 gene in transfered cell the time, and wherein this molecule comprises sense strand and complementary antisense strand thereof, and wherein these chains are hybridized to form this duplex molecule each other.
12. the duplex molecule of claim 11, wherein this sense strand comprises the sequence corresponding with the target sequence that is selected from SEQ ID NO:9 and 10.
13. the duplex molecule of claim 11 or 12, wherein to form this duplex molecule, the length of this duplex molecule is 19～25 base pairs in this target sequence place hybridization for this sense strand and antisense strand.
14. each duplex molecule of claim 11 to 13, it is made up of single polynucleotide, these single polynucleotide comprise through this that interleaves that strand connects have justice and antisense strand the two.
15. the duplex molecule of claim 14; It has general formula 5 '-[A]-[B]-[A ']-3 ', and wherein [A] is sense strand, and it comprises the sequence corresponding with the target sequence that is selected from SEQ ID NO:9 and 10; [B] is for interleaving strand; It is made up of 3 to 23 Nucleotide, and [A '] be antisense strand, it comprises the complementary sequence of selected target sequence in [A].
16. a carrier, its each duplex molecule of coding claim 11 to 15.
17. the treatment or the method for preventing cancer in an experimenter; Wherein this method comprise to the experimenter use pharmacy effective dose to the duplex molecule of CSTF2 gene or the carrier of this duplex molecule of encoding, suppress the CSTF2 expression of gene when wherein this duplex molecule is in importing the cell of expressing the CSTF2 gene.
18. the method for claim 17, wherein this duplex molecule is each a duplex molecule of claim 11 to 15.
19. the method for claim 17, wherein this carrier is the carrier of claim 16.
20. a compsn that is used to treat the cancer of expressing the CSTF2 gene, wherein said composition comprises
At least a isolating to the duplex molecule of CSTF2 gene or the carrier of this duplex molecule of encoding, suppress the CSTF2 expression of gene when wherein this duplex molecule is in importing the cell of expressing the CSTF2 gene; And
The pharmaceutically useful body that supports.
21. the compsn of claim 20, wherein this duplex molecule is each a duplex molecule of claim 11 to 15.
22. the compsn of claim 20, wherein this carrier is the carrier of claim 16.
23. a screening is used to treat or the method for the candidate substances of preventing cancer or anticancer growth, wherein this method comprises the steps:
(a) test substances is contacted with CSTF2 polypeptide or its fragment;
(b) combination that detects between this polypeptide or fragment and this test substances is active; And
(c) select to combine this polypeptide or segmental test substances as being used to treat or the candidate substances of preventing cancer.
24. a screening is used to treat or the method for the candidate substances of preventing cancer or anticancer growth, wherein this method comprises the steps:
(a) test substances is contacted with CSTF2 polypeptide or its fragment;
(b) detect this polypeptide or segmental BA;
(c) this polypeptide or segmental BA are compared with detected BA when this test substances does not exist; And
(d) test substances of BA of selecting to prevent this polypeptide is as being used to treat or the candidate substances of preventing cancer.
25. the method for claim 24, wherein this BA is that cell-proliferation activity, RNA combine activity, mRNA nicking activity or mRNA polyadenylation active.
26. a screening is used to treat or the method for the candidate substances of preventing cancer or anticancer growth, wherein this method comprises the steps:
(a) make test substances and the cells contacting of expressing the CSTF2 gene; And
(b) test substances that reduces CSTF2 expression of gene level is compared in selection with detected expression level when this test substances does not exist.
27. a screening is used to treat or the method for the candidate substances of preventing cancer or anticancer growth, wherein this method comprises the steps:
(a) make test substances and wherein import the cells contacting that carrier is arranged, this carrier comprises CSTF2 gene transcription regulatory region and the reporter gene of under this transcriptional regulatory district control, expressing;
(b) measure this report expression of gene or activity; And
(c) test substances that reduces this report expression of gene or activity level is compared in selection with detected expression or activity level when this test substances does not exist.
28. a method that is used in experimenter's treatment or preventing cancer comprises this experimenter is used anti-CSTF2 antibody or its immunologic competence fragment.
29. claim 1 to 8,17 to 19 and 23 to 28 each methods, each compsn of claim 9 or 10 test kit or claim 20 to 22, wherein this cancer is a lung cancer.
30. carrier; They comprise arbitrary in the polynucleotide combination that comprises sense strand nucleic acid and antisense strand nucleic acid; Wherein said sense strand nucleic acid comprise with SEQ ID NO:9 or 10 corresponding nucleotide sequences and said antisense strand nucleic acid by forming with this sense strand complementary sequence; The transcript of wherein said sense strand and said antisense strand is hybridized forming duplex molecule each other, and wherein said carrier suppresses cell proliferation in importing the cell of expressing the CSTF2 gene time.
CN2010800477754A 2009-08-21 2010-08-18 CSTF2 for target genes of lung cancer therapy and diagnosis Pending CN102575300A (en)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001036470A2 (en) * 1999-11-16 2001-05-25 Matritech, Inc. Methods and compositions for detection and treatment of breast cancer, based on breast cancer-associated polypeptides
US6753314B1 (en) * 1999-04-01 2004-06-22 Curagen Corporation Protein-protein complexes and methods of using same
US20050053985A1 (en) * 2003-07-02 2005-03-10 Ptc Therapeutics, Inc. RNA processing protein complexes and uses thereof
CN101283106A (en) * 2005-07-27 2008-10-08 肿瘤疗法科学股份有限公司 Method of diagnosing small cell lung cancer

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1226439A1 (en) * 1999-11-01 2002-07-31 Universiteit van Amsterdam Method for the detection of mammalian carcinomas
US20070224201A1 (en) * 2002-10-02 2007-09-27 Genentech, Inc. Compositions and methods for the diagnosis and treatment of tumor

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6753314B1 (en) * 1999-04-01 2004-06-22 Curagen Corporation Protein-protein complexes and methods of using same
WO2001036470A2 (en) * 1999-11-16 2001-05-25 Matritech, Inc. Methods and compositions for detection and treatment of breast cancer, based on breast cancer-associated polypeptides
US6936424B1 (en) * 1999-11-16 2005-08-30 Matritech, Inc. Materials and methods for detection and treatment of breast cancer
US20050053985A1 (en) * 2003-07-02 2005-03-10 Ptc Therapeutics, Inc. RNA processing protein complexes and uses thereof
CN101283106A (en) * 2005-07-27 2008-10-08 肿瘤疗法科学股份有限公司 Method of diagnosing small cell lung cancer

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