CN101133167A

CN101133167A - GITR antibodies for the diagnosis of NSCLC

Info

Publication number: CN101133167A
Application number: CNA2006800068915A
Authority: CN
Inventors: B·泰歇; S·尚卡拉; B·罗伯茨
Original assignee: Ilex Products Inc
Current assignee: Ilex Products Inc; Genzyme Corp
Priority date: 2005-01-19
Filing date: 2006-01-19
Publication date: 2008-02-27
Also published as: BRPI0606685A2; IL184343A0; EP1856281A2; WO2006078911A2; WO2006078911A3; JP2008527998A; AU2006206343A1; CA2592330A1

Abstract

The present invention provides methods and compositions for detecting, diagnosing, prognosing and monitoring the progress of cancers, e.g., NSCLC, of epithelial origin, e.g., lung, ovarian, breast, prostate and colon cancers and malignancies and kits for use in said methods. Further provided are methods for screening to identify agonists and antagonists of antigens associated with these cancers and malignancies.

Description

The GITR antibody that is used for the diagnosis of NSCLC

Cross

The application is according to the rights and interests of 35U.S.C. § 119 (e) requirement from the U.S. Provisional Patent Application series number of submitting on January 19th, 2,005 60/645,349, and this application is incorporated herein by reference in full with it.

Technical field

The present invention relates to be used for the composition and the method for treatment, diagnosis and the screening method of people's cancer and associated malignancies.

Background

Although obtained numerous progress in medical research, cancer remains the second largest cause of death in the U.S..In industrialized country, roughly 5 philtrums have one will die from cancer.The traditional mode of clinical care, for example excision, radiation and chemotherapy have quite high mortality, especially for solid tumor.Failure be because initial tumour is a unresponsiveness, or because the recurrence that causes owing to the regrowth on the original position and/or transfer.

Lung cancer is one of modal malignant tumour in the world wide, and is the second largest cancer mortality reason among the male sex.Referring to, American Cancer Society, Cancer facts andfigures, 1996, Atlanta.Diagnose about 178,100 routine lung cancer new cases in 1997, accounted for 13% of cancer diagnosis.160,400 examples have according to estimates taken place owing to the death that lung cancer causes in 1997, accounted for 29% of all cancer mortalities, this makes lung cancer more more fatal than the summation of mammary cancer, prostate cancer and colorectal carcinoma.Jemal, people such as A. (2004) CancerStatistics 2004, CA:A Cancer Journal for Clinicians 53:5-26.One annual survival rate of lung cancer from 1973 32% increase to 1993 41%, this is mainly owing to the raising of surgical technic.All stages 5 annual survival rates together have only 14%.For still being limited to the detected case of local time when disease, survival rate is 48%, but has only 15% lung cancer to be found so early.

In the various forms of lung cancer, nonsmall-cell lung cancer (NSCLC) accounts for about 80% of annual all new lung cancer cases.Small cell lung cancer is the most pernicious and the fastest form of growing in the lung cancer.Primary tumor has responsiveness to chemotherapy usually, but next is to shift widely.Mean survival time during diagnosis is about 1 year, and 5 annual survival rates are 5%.For the patient who is diagnosed as NSCLC, excision provides the unique opportunity of significant survival.

5 types nonsmall-cell lung cancer is arranged: squamous cell carcinoma, gland cancer, large cell carcinoma, adenosquamous carcinoma and undifferentiated carcinoma.Adenosquamous carcinoma starts from microscopic examination following time and seems flat cell.Undifferentiated cancer cells look with normal cell different and uncontrollably propagation.Squamous cell carcinoma is modal lung cancer type.It takes place from the cell of lining in air flue.Gland cancer takes place from glandular cell or the secretory cell that produces mucus (phlegm).So name maxicell lung cancer is because it seems huge and for circular when examining under a microscope these cells.

The feature of nonsmall-cell lung cancer also is four clinical stages.The I phase is the cancer that localizes very much, does not have cancer in lymphoglandula.II phase cancer has diffused to the lymphoglandula on the top of affected lung.III phase cancer diffused to position that cancer begins near.It can be the wall of the chest, middle part (mediastinum) or other lymphoglandula of the overcover of lung (pleura), chest.IV phase cancer has diffused to the other position of health.

Developing several antibody therapies with treatment lung cancer.Cetuximab and Gefitinib (gifitinib) are used for these cancers through FDA Food and Drug Administration's approval.The combination of Cetuximab and chemotherapy provides some benefits for NSCLC patient but still has needed further test.Kelly, people such as K. (2003) Proc.Am.Soc.Clin.Oncol.22:644.

Therefore, the effective methods of treatment that still needs NSCLC.The present invention has satisfied this needs, and relevant advantage is provided.

Disclosure of the present invention

The invention provides method, it is used to help the diagnosis of cell situation, be used for identifying and/or distinguish normal cell and neoplastic cell, and be used for identifying the potential therapeutical agent that can reverse tumorigenesis and/or the improvement symptom relevant the experimenter with the neoplastic cell existence.

Therefore, embodiment of the present invention are intended to come by the existence of the gene of the differential expression of evaluation in the screening table 1 method of diagnosis cell situation.In one aspect, the differential expression of described gene is for example indication of nonsmall-cell lung cancer (NSCLC), ovarian cancer, mammary cancer, prostate cancer and colorectal carcinoma of superfluous natural disposition state of the cell of epithelial origin.Can detect expression by any suitable method, for example comprise the amount by detecting the cDNA that produces from the amount of the mRNA of described genetic transcription or by the reverse transcription of transcribing from the mRNA of described gene or by the polypeptide or the proteinic amount of described genes encoding.Can implement these methods to sample by sampling principle (sample basis), maybe can revise these methods to carry out high throughput analysis.In addition, can retrieve and analytical database with regard to the existence and the amount of transcript or expressed genes product, described database comprises a certain amount of (quantitative) from isolating complete or transcript or the protein sequence partly of cell sample.

Another aspect of the present invention is screening, reverses or treatment tumorigenesis and tumor treatment agent to identify, wherein said cell and/or tumour are characterised in that polypeptide or the proteinic differential expression of identifying in the table 1.Described method comprises having this genotypic cell and contact with the potential reagent of significant quantity before being accredited as, and the reverse of the superfluous natural disposition situation of calibrating.

The polynucleotide of the protein shown in the coding schedule 1, its fragment or polypeptide (being also referred to as gene expression product herein) also are provided, have comprised the gene delivery vector of these polynucleotide and comprise the host cell of these polynucleotide.Described protein, polypeptide or its fragment also can be used for producing specifically identification and in conjunction with the antibody of these molecules.Described antibody can be polyclonal or monoclonal.These antibody can be used for separating the protein or the polypeptide that go out from the genetic expression of coding said polypeptide, and are used to detect neoplastic cell or tumour.

The present invention also provides isolating host cell and recombinant host cell, and it comprises peptide and/or its segmental polynucleotide of being identified in the coding schedule 1.Described cell can be protokaryon or eucaryon, and only as an example, can be for example any or multiple as in dendritic cell (DC) or the T cell of stem cell, antigen presenting cell (APC) of bacterium, yeast, animal, Mammals, people's cell and its specific hypotype.

Table 1

Gene	Unigene﹠ GenBank numbering	The chain ID of locus ^*	Normal cell is expressed	Cancer cells is expressed	Seq.ID Nos.
Gene	Unigene﹠ GenBank numbering	The chain ID of locus ^*	Normal cell is expressed	Cancer cells is expressed	Seq.ID Nos.	GITR (a/k/a. “TNFRSF18”)	Hs.212680 AF117297.1 AF241229.1 AF125304.1 AY358877.1 NM_148901.1 NM_148902.1 NM_004195.2 NP_004186 NP_683699 NT_077913	8784		Gland cancer and squama cancer; NSCLC; Ovarian cancer; Mammary cancer; Prostate cancer and colorectal carcinoma	1，2

* network address=ncbi.nlm.nih.gov/LocusLink/list.cgi

The present invention also provides the method for cancer that is used for monitoring the experimenter, and its expression level by measuring goal gene at different time and more described expression of gene level to be to determine whether expression increases or reduce, and has this to monitor cancer among the experimenter.The test kit that is used for diagnostic method or drug screening also is provided herein.Described test kit comprises the reagent (for example, probe, primer or antibody) and the working instructions of at least a detection genetic expression.

The sequence table general introduction

As used herein, term " GITR gene " is meant the ORF of continuous polynucleotide sequence at least, and its coding has the protein or the polypeptide of biologic activity shown here.LocusLink (on seeing) has reported, and is the member of TNF receptor superfamily by the protein of this genes encoding.Reported this receptor has increase when the T-cell-stimulating expression, and it is considered to play crucial effects in the dominance immunity self tolerance of being kept by CD25 (+) CD4 (+) control T cell (dominantimmunological self-tolerance).The gene knockout research of mouse shows that also the effect of this receptor is T-cell-stimulating and the apoptosis that CD3 drives.Three kinds of transcript variants of this gene have been reported, the isotype that its coding is different through selectively montage.

SEQ ID NO:1 is an example of GITR polynucleotide sequence, and other sequences are known in this area, and the example includes but not limited to sequence shown in the table 1 and the coding sequence of the GITR gene expression product of definition herein.The sequence that biologically is equal to is also included within the scope of this definition, described sequence for example be coding SEQ ID NO:2 polypeptide sequence and have the sequence of at least 90% or selectively at least 95% sequence homology with exemplary sequence such as SEQ ID NO:1, this can determine by the identity per-cent sequential analysis of carrying out under default parameter.By the gene that biologically is equal under high stringent condition, identified or polynucleotide with the ability of minus strand hybridization also in this range of definition.Can wish to use non-people's gene, its polynucleotide sequence is well known in the art.Referring to for example, UniGene ClusterHs.212680.Polynucleotide passage also is known in the art, and it includes but not limited to GenBank accession number: BI911657.1; AI499936.1; AI214481.1; And AI923712.1.These polynucleotide are useful especially as probe or primer.

As used herein; term " GITR gene expression product, protein or polypeptide " comprises the aminoacid sequence of SEQ ID NO:2 and the GITR genetic transcription of identifying from above and the aminoacid sequence of translation; and do not consider gene expression system, for example bacterium or other prokaryotic cell prokaryocytes, yeast cell, mammalian cell such as ape, ox or people's cell.This term comprises polypeptide isolated from tissue sample, isolating, natural generation, and the reorganization protein and the polypeptide that produce.This term also comprises having and SEQ ID NO:2 at least 90% or selectively at least 95% homologous aminoacid sequence and polypeptide with biologic activity described herein.The example of homologous amino acid sequence includes, but not limited to have the polypeptide of aminoacid sequence of SEQ ID NO:2 or other GITR gene expression products of having modified by the conservative amino acid displacement.

Be used to implement pattern of the present invention

In whole present disclosure, quote (identifying citation) as proof with reference to multiple publication, patent and disclosed patent specification by distinctive.The disclosure of quoting these publications, patent and disclosed patent specification in this disclosure is as a reference more fully to describe the state in the field under the present invention.

Definition

Unless otherwise noted, enforcement of the present invention will be adopted the routine techniques of immunology, molecular biology, microbiology, cytobiology and recombinant DNA, and described technology is within the skill of this area.Referring to, Sambrook for example, Fritsch and Maniatis, MOLECULARCLONING:A LABORATORY MANUAL, the 2nd edition (1989); CURRENT PROTOCOLSIN MOLECULAR BIOLOGY (people such as F.M.Ausubel, editor, (1987)); Book series METHODS IN ENZYMOLOGY (Academic Press, Inc.): PCR 2:A PRACTICALAPPROACH (M.J.MacPherson, B.D.Hames and G.R.Taylor, editor (1995)), Harlow and Lane, editor (1988) ANTIBODIES, A LABORATORYMANUAL and ANIMAL CELL CULTURE (R.I.Freshney,, editor (1987)).

As used herein, some term has the implication that defines below.

As used in this specification sheets and claim, singulative " a ", " an " and " the " comprise plural connotation, unless context is clearly stipulated in addition.For example, term " a cell " comprises a plurality of cells, comprises its mixture.

All digital nominals, for example, pH, temperature, time, concentration and molecular weight comprise scope, all are the approximations that changes by 0.1 increment (+) or (-).Although be not appreciated that and always mention clearly, all digital nominal fronts are all titled with term " approximately ".Although be not appreciated that also and always mention clearly that reagent described herein is exemplary, and the equivalent of these reagent is well known in the art.

Term " polynucleotide " and " oligonucleotide " are used interchangeably, and are meant the polymer form of the Nucleotide (deoxyribonucleotide or ribonucleotide or its analogue) of any length.Polynucleotide can have any three-dimensional structure and can exercise any known or unknown function.Following is the limiting examples of polynucleotide: isolating RNA, nucleic acid probe and the primer of gene or gene fragment (for example, probe, primer, EST or SAGE label), exon, intron, messenger RNA(mRNA) (mRNA), transfer RNA (tRNA), ribosome-RNA(rRNA), ribozyme, cDNA, recombination of polynucleotide, branched polynucleotide, plasmid, carrier, the separated DNA of any sequence, any sequence.Polynucleotide can comprise for example methylated Nucleotide of modified Nucleotide and nucleotide analog.If exist, can before or after the polymkeric substance assembling, modify nucleotide structure.Available non-nucleotide component is interrupted the sequence of Nucleotide.After polymerization, can further modify polynucleotide by for example puting together mutually with marker components.This term also refers to two strands and single chain molecule.Unless specify in addition or requirement, form each single stranded form in two complementary single stranded form of double chain form for of the present invention any embodiment of polynucleotide comprises double chain form and known or prediction.

Polynucleotide are made up of the particular sequence of 4 kinds of nucleotide bases, and described 4 kinds of nucleotide bases are: VITAMIN B4 (A), cytosine(Cyt) (C), guanine (G), thymus pyrimidine (T); Uridylic when polynucleotide are RNA (U) replaces guanine.Therefore, term " polynucleotide sequence " is the letter arrangement expression of polynucleotide molecule.Can will should arrange in the database of expression input in having the computer of central processing unit by letter, and be used for information biology application examples such as functional genome and homology search.

" gene " is meant the polynucleotide that comprise at least one can encode specific polypeptide or proteinic open reading frame (ORF) after being transcribed and translating.Arbitrary sequence in the polynucleotide sequence described herein can be used for identifying bigger fragment or the complete encoding sequence with they associated genes.The method of separating bigger fragment sequence is known to those skilled in the art.

" gene product " or selectively " gene expression product " be meant when gene is transcribed and translated the amino acid (for example, peptide or polypeptide) that produces.

Term " polypeptide " can exchange with term " protein " and use, and it is meant the compound of two or more subunit's amino acid, amino acid analogue or peptide mimics (peptidomimetic) on the widest meaning.Described subunit can connect by peptide bond.In another embodiment, described subunit can pass through for example connection such as ester bond, ehter bond of other keys.As used herein, term " amino acid " is meant natural and/or non-natural or synthetic amino acid, (comprising glycine and D and L optically active isomer), amino acid analogue and peptide mimics.If peptide chain is short, then usually three or more amino acid whose peptides are called oligopeptides.If the peptide chain length then is called polypeptide or protein with described peptide usually.

" transcribing under the control " is the term that fully understands in the art, and the transcribing of its expression polynucleotide sequence (being generally dna sequence dna) depends on it and be connected to effectively to facilitate and transcribe the element that opens the beginning or promote to transcribe." effectively connect " be meant wherein said element be in the arrangement that allows their performance functions and put.

As used herein, term " comprises " or " comprising " is intended to expression, and described composition and method comprise the key element of being stated, but do not get rid of other key elements." basically by ... form ", when being used for definitions section compound and method, expression is not comprised other key elements that have any substantive meaning for this combination.Therefore, basically from here the composition formed of the key element of definition will not get rid of to come the contaminant trace species of self-separation and purification process and pharmaceutically acceptable carrier for example phosphate buffered saline(PBS), sanitas etc." by ... form " the important method step that will represent not comprise other compositions that the eliminating minute element is outer and be used to use the present composition.By the defined embodiment of each term in these transition type terms (transition term) within the scope of the present invention.

Term " isolating " is meant with cell and otherwise component and separates mutually that wherein polynucleotide, peptide, polypeptide, protein, antibody or its fragment combine with it usually natively.In one aspect of the invention, isolating polynucleotide and 3 ' and 5 ' continuous nucleotide separate, described isolating polynucleotide at it born or natural surroundings for example common and described 3 on karyomit(e) ' be connected with 5 ' continuous nucleotide.As being significantly to those skilled in the art, polynucleotide, peptide, polypeptide, protein, antibody or its fragment that non-natural takes place do not need " separation " thereby the counterpart (counterpart) of its generation natural with it distinguished.In addition, " spissated ", " separating " or " dilution " polynucleotide, peptide, polypeptide, protein, antibody or its fragment can generation natural with it counterpart distinguish, wherein the molecular conecentration of every volume or number are than situation height (" spissated ") or low (" separating ") of its natural generation.Be its primary sequence or for example be that polynucleotide, peptide, polypeptide, protein, antibody or its fragment of its glycosylation pattern need not exist with its isolating form with the difference of the counterpart of natural generation because its can by its primary sequence or selectively by another feature for example the counterpart of glycosylation pattern generation natural with it distinguish.Therefore, the polynucleotide that provide non-natural to take place are as the embodiment that differentiates mutually with the polynucleotide of isolating natural generation.Provide the protein that in bacterial cell, produces, the embodiment that differentiates mutually as protein with isolated natural generation from wherein produce described proteinic eukaryotic cell natively.

As used herein, " gene delivery ", " transgenosis " etc. are such terms, and it refers to exogenous polynucleotide (being sometimes referred to as " transgenosis ") is imported in the host cell, and regardless of the method which kind of adopts be used to import.These methods comprise for example carrier mediated transgenosis of the multiple technology of knowing (by for example virus infection/transfection or various other based on protein or based on the gene delivery mixture of lipid) and the technology of sending (for example electroporation, " particle gun " are sent and the various other technologies that are used to import polynucleotide) that helps " exposed " polynucleotide.The polynucleotide that import can stably or momently remain in the host cell.The common requirement of stable maintenance, the polynucleotide that imported comprise can be compatible with host cell replication orgin, the replicon that perhaps is integrated into host cell is for example in extrachromosomal replication (for example plasmid) or nucleus or the m-chromosome.Many carriers known in the art can mediated gene to the transfer of mammalian cell.

" gene delivery vector " is defined as polynucleotide that portability inserts and enters any molecule in the host cell.The example of gene delivery vector is a liposome, and biocompatible polymer comprises natural polymer and synthetic polymer; Lipoprotein; Polypeptide; Polysaccharide; Lipopolysaccharides; Artificial viral envelope; Recombinant yeast cell, metallic particles; And bacterium or virus, baculovirus for example, adenovirus and retrovirus, phage, clay, plasmid, fungi carrier and other recombinant vectorss that are generally used for this area, described recombinant vectors has been described in multiple eucaryon and the prokaryotic hosts and has expressed, and can be used for gene therapy and be used for simple protein expression.

" virus vector " is defined as virus or the virion that reorganization produces, its comprise with in the body, exsomatize or external mode is sent polynucleotide into host cell.The example of virus vector comprises retroviral vector, adenovirus carrier, adeno-associated virus vector, Alphavirus carrier etc.The Alphavirus carrier for example based on the carrier of Semliki Forest virus with based on the carrier of sindbis virus, also has been developed and has been used for gene therapy and immunotherapy.Referring to, Schlesinger and Dubensky (1999) Curr.Opin.Biotechnol.5:434-439; With people (1999) Nat.Med.5 (7): 823-827 such as Ying.Transgenosis therein is by the aspect of retrovirus-mediated method, and the vector construction body is meant the polynucleotide that comprise reverse transcription virus gene group or its part and therapeutic gene.As used herein, " transgenosis of retrovirus-mediated method " or " retrovirus transduction " has the identical meaning, and be meant such method, promptly by this method, rely on virus to enter cell and its genome conformity gone in the host genome gene or nucleotide sequence stably are transferred in the host cell.Virus can enter host cell by its normal infection mechanism, or is transformed so that it can be in conjunction with different host cell surface acceptors or part to enter described cell.As used herein, " retroviral vector " is meant and can enters machine-processed virion with the exogenous nucleic acid transfered cell by virus or viral sample.

Retrovirus carries its genetic information with the form of RNA; Yet in case this virus infected cell, described RNA just is reversed and records into dna form, and this DNA is integrated in the genomic dna of infected cells.Described dna form through integrating is called provirus.

Transgenosis therein is by the dna viral vector aspect of adenovirus (Ad) or adeno associated virus (AAV) mediation for example, and the vector construction body is meant and comprises viral genome or its part and genetically modified polynucleotide.Adenovirus (Ad) comprises surpassing 50 kinds serotype through relative sign, homologous one papova fully.Referring to for example WO95/27071.Ad grows easily and does not require and is integrated in the host cell gene group.Also make up the carrier that derives from reorganization Ad, particularly reduced those carriers of the potential of the reorganization of wild-type virus and propagation.Referring to for example, WO95/00655 and WO95/11984.Wild-type AAV is integrated in the genome of host cell with highly infective and specificity.Referring to, Hermonat and Muzyczka (1984) Proc.Natl.Acad.Sci.USA81:6466-6470; People such as Lebkowski (1988) Mol.Cell.Biol.8:3988-3996.

Comprising promotor knows in the art with the carrier that polynucleotide effectively can be connected to cloning site wherein.Such carrier can be in external or body transcribe rna, and can be from (La Jolla, CA) (Madison, source WI) is commercially available with Promega Biotech such as Stratagene.For optimization expression and/or in-vitro transcription, may must remove, add or change the clone 5 ' and/or 3 ' untranslated part is extra to eliminate, potential, inappropriate, selectable translation initiation codon or can transcribe or translation skill on disturb or reduce other sequences of expression.Selectively, can be right after the total ribosome bind site of 5 of initiator codon ' insertion to strengthen expression.

Gene delivery vector also comprises several non-virus carriers, comprises DNA/ liposome complex, recombinant yeast cell and by the viral protein of target-DNA mixture.Also comprise target antibody or its segmental liposome can be used for method of the present invention.In order to increase cytotropic sending, nucleic acid of the present invention or protein can be conjugated in conjunction with cell-surface antigens for example antibody or its associativity fragment of TCR, CD3 or CD4.

" probe " when using under the situation in polynucleotide operations, is meant oligonucleotide, thereby it provides by hybridizing with target with the reagent form and detects the target that may be present in the purpose sample.Usually, probe can comprise mark or means, can adhere to mark by these means before or after hybridization.Suitable mark includes, but are not limited to, radio isotope, fluorochrome, chemiluminescence compound, dyestuff and albumen (comprising enzyme).

" primer " be have usually free 3 '-the short polynucleotide of OH group, it is by combine target or " template " that may be present in the purpose sample with target hybridization, then, promotes and the polymerization of target complementary polynucleotide." polymerase chain reaction " (" PCR ") is such reaction, in this reaction, the catalyzer that uses " primer to " be made up of " upstream " and " downstream " primer or " primer sets " and polymerization for example archaeal dna polymerase (being generally heat-staple polysaccharase) prepares the duplicate copy of target polynucleotide.The method that is used for PCR is known in the art, and instructs in for example " PCR:A PRACTICAL APPROACH " (people such as M.MacPherson, IRL Press at Oxford University Press (1991)).Produce all processes of the duplicate copy of polynucleotide, for example, PCR or gene clone are referred to as " duplicating " herein.Also can be with primer as the hybridization probe in Southern or the Northern engram analysis for example.People such as Sambrook are on seeing.

Statement " database " is meant the data of one group of storage having represented arrangement set, and described arrangement set has been represented the set of biology reference material conversely.

Term " cDNA " is meant complementary DNA, its be with enzyme for example reversed transcriptive enzyme be prepared into the mRNA molecule in cell or the organism of being present in of cDNA." cDNA library " is the set that is present in all the mRNA molecules in cell or the organism, with the reversed transcriptive enzyme this kind of enzyme all mRNA is transformed into the cDNA molecule, inserts then in " carrier " (other dna moleculars that can continue to duplicate after adding foreign DNA).The exemplary carrier that is used for the library comprises phage, i.e. the virus of bacterial infection, for example lambda particles phage.Can survey the library with regard to specific cDNA (thereby mRNA) then.

As used herein, " expression " be meant by it polynucleotide be transcribed into the process of mRNA and/or subsequently the mRNA that transcribes translated into peptide, polypeptide or proteinic process by it.If described polynucleotide derive from genomic dna, express the montage that can be included in mRNA in the eukaryotic cell so.When being used for gene, " differential expression " is meant from the mRNA of described genetic transcription and/or translation or by the difference of the protein of described genes encoding to produce.Compare with the expression level of normal or control cells, the gene of differential expression can be crossed and express or low the expression.Yet as used herein, crossing expression is than at least 1.25 times of detected expression height in normal or healthy corresponding cell or tissue, or selectively, at least 1.5 times, or selectively, at least 2 times expression.Term " differential expression " also refers to the nucleotide sequence in cell or tissue, and it is expressed in described cell or tissue and reticent in control cells, and perhaps it is not expressed in described cell or tissue and expresses in control cells.

As used herein, " solid support " or " solid support " (being used interchangeably) is not limited to the upholder of particular type.On the contrary, many upholders are obtainable and are known for those skilled in the art.Solid support comprises silica gel, resin, deutero-plastics film, granulated glass sphere, cotton, plastic beads, alumina gel, microarray and chip.As used herein, " solid support " also comprises synthetic antigen presentation matrix, cell and liposome.Can select suitable solid support based on the suitability of terminal use of wanting and various schemes.For example, synthetic for peptide, solid support can be that for example polystyrene is (for example for resin, available from Bachem Inc., the PAM-resin of Peninsula Laboratories, or the like), POLYHIPE  resin is (available from Aminotech, Canada), polyamide resin (available from Peninsula Laboratories), be grafted with polystyrene resin (TentaGel , the Rapp Polymere of polyoxyethylene glycol, Tubingen, Germany) or the polydimethylacrylamiin resin (available from Milligen/Biosearch, California).

Also polynucleotide can be attached to solid support to be used for the high flux screening assay method.For example PCT WO97/10365 discloses the structure of high density oligonucleotide chip.Also can referring to, United States Patent (USP) 5,405,783,5,412,087 and 5,445,934.Use these methods, can be also referred to as synthesising probing needle on the derivatize glass surface of chip array.The nucleoside phosphoramidites of light protection is coupled to glass surface, optionally goes protection, then itself and second kind of shielded nucleoside phosphoramidites are reacted by using the photodissociation that mask carries out.Repeat this coupling/go the protection process until finishing the probe of wanting.

" hybridization " is meant such reaction, one or more polynucleotide reactions in this reaction, thus form the mixture of stabilization by the hydrogen bond between the base of nucleotide residue.Described hydrogen bond can according to Watson-Crick base pairing, Hoogstein in conjunction with or produce in any other sequence-specific mode.Described mixture can comprise two chains that form the duplex structure, forms three or more chains of multichain mixture, and wall scroll is from hybridizing chain or these any combination.Hybridization can constitute a for example step in the enzymatic cutting of PCR reaction initial or the polynucleotide that undertaken by ribozyme of more complicated process.

Can under different " tight degree " conditions, carry out hybridization.Usually, under about 40 ℃ in 10 * SSC or have in the solution of identical ionic strength/temperature and carry out the low stringency hybridization.Usually, in 6 * SSC, carrying out medium tight degree hybridization under about 50 ℃ and under about 60 ℃, in 1 * SSC, carrying out high tight degree hybridization.

When hybridization took place with antiparallel configuration between two strand polynucleotide, described reaction was called " annealing ", and these polynucleotide are described to " complementary ".If hybridize between the chain in the chain of the chain that can be in the chain of first polynucleotide and second polynucleotide, so double-stranded polynucleotide can be " complementary " or " homologous " for another polynucleotide." complementarity " or " homology " (polynucleotide with another polynucleotide complementary degree) can come quantitative according to the expection in relative chain forms the base of hydrogen bond each other according to generally accepted base pairing rules ratio.

Polynucleotide or polynucleotide region (perhaps polypeptide or polypeptide zone) (for example have certain percentage with another sequence, 80%, " sequence identity " 85%, 90% or 95%) is expression, when comparing, the per-cent of identical base (or amino acid) in comparing two sequences.Can use software program as known in the art, for example CURRENT PROTOCOLS INMOLECULAR BIOLOGY (people such as F.M.Ausubel, the editor, 1987) addendum 30,7.7.18 joint, described in the table 7.7.1 those are compared and definite homology or sequence identity per-cent.Preferably, use default parameter to compare.Preferred comparison program is to use the BLAST of default parameter.Especially, preferred program is BLASTN and BLASTP, uses following default parameter: genetic code=standard; Strainer (filter)=nothing; Chain=two; Cutoff (cutoff)=60; Desired value=10; Matrix (Matrix)=BLOSUM62; Kind (Descriptions)=50 sequence: sort by (sortby)=high score; Database=nonredundant), GenBank+EMBL+DDBJ+PDB+GenBank CDStranslations+SwissProtein+SPupdate+PIR.Can in following IP address, find the detailed content of these programs: www.ncbi.nlm.nih.gov/cgi-bin/BLAST.

Hyperplasia is the form of in check cell proliferation, and it relates to the increase of the cell number in tissue or the organ, and the remarkable change of non-structure or function.Metaplasia (metaplasia) is a kind of in check cell growth forms, wherein the cell of the differentiation of the another kind of type of cell replacement of one type differentiation fully.Metaplasia can take place in epithelium or phoirocyte.The atypia metaplasia relates to some metaplastic disorderly epithelium.

As used herein, term " neoplastic cell ", " tumorigenesis ", " tumour ", " tumour cell ", " cancer " and " cancer cells " (being used interchangeably) are meant such cell, described cell shows autonomous relatively growth, thereby they show the misgrowth phenotype (that is, losing the cell fission of regulation and control) of the obvious forfeiture of control that is characterised in that cell proliferation.Neoplastic cell can be a virulent or benign.Metastatic cell or tissue is expression, and adjacent body structure can be invaded and destroy to described cell.

" inhibition " tumor growth represent when with do not have growth phase that treatment intervenes than the time growth conditions that weakened.Can assess growth of tumour cell by any method known in the art, described method includes, but not limited to measure tumour size, use ³The H-thymidine is integrated assay method and is determined whether tumour cell is being bred or counted tumour cell." inhibition " growth of tumour cell is meant any state or all states of following state: slow down, postpone or stop tumor growth and tumour and shrink.

Term " antigen " has obtained clear and definite understanding in this area, it comprises having immunogenic material.As used herein, this term also comprises the material that causes immunologic unresponsiveness or anergy.

" inborn " or " natural " or " wild-type " antigen are to comprise epi-position and isolated polypeptide, protein or fragment from natural biological source.It is conjugated antigen acceptor specifically also.

As used herein, " antibody " comprises whole antibody or its any Fab or strand.Therefore, term " antibody " comprises any protein or the peptide that comprises such molecule, described molecule comprise immunoglobulin molecules to small part.These example comprises, but be not limited to, the complementarity-determining region of heavy chain or light chain or its ligand binding moiety (CDR), heavy chain or variable region of light chain, heavy chain or constant region of light chain, framework region (FR) or its any part or protein-bonded at least one part, any of foregoing can be integrated in the antibody of the present invention.

Antibody can be polyclonal or monoclonal, and can for example separate muroid, rat, sheep and the dog from any suitable biogenetic derivation.Other sources have been described hereinafter.

Its digestion fragment, specific part, derivative and variant also wished to comprise in term " antibody ", comprising antibody analog or comprise analog antibody or its specific fragment or the structure of part and/or the antibody moiety of function, comprises single-chain antibody and its fragment.The example of the binding fragment that " antigen-binding portion thereof " of term antibody is included comprises the Fab fragment, the unit price fragment of being made up of VL, VH, CL and CH structural domain; F (ab ') 2 fragments are included in the segmental divalence fragment of two Fab that hinge area connects by disulfide linkage; The Fd fragment of forming by VH and CH structural domain; The Fv fragment of forming by the VL and the VH structural domain of antibody single armed, the dAb fragment of forming by the VH structural domain (people (1989) Nature 341:544-546 such as Ward); With isolating complementarity-determining region (CDR).In addition, although segmental two structural domain VL of Fv and VH are by the genes encoding that separates, but can use recombination method they to be coupled together by the synthetic linker, described linker makes them can be formed into one protein chain, thereby wherein match and form monovalent molecule (being called strand Fv (scFv)) in VL and VH district.People (1988) Proc.Natl.Acad.Sci.USA 85:5879-5883 such as people such as Bird (1988) Science 242:423-426 and Huston.Single-chain antibody also is hoped to be included in the term " fragment of antibody ".Use routine techniques well known by persons skilled in the art to obtain in the above-mentioned antibody fragment any, and with screen described fragment for the identical method of complete antibody with regard to binding specificity and neutralization activity.

Term " epi-position " is meant the protein determinant of binding antibody specifically.Epi-position usually by the chemically reactive surface group of molecule for example amino acid or sugared side chain form, and have specific Three Dimensions Structure and specific charge characteristic usually.The difference of conformational epitope and non-conformational epitope is the former but non-being combined under the situation that the sex change solvent exists of the latter is lost.

Such molecule wished to comprise in term " antibody derivatives ", and promptly described molecule is in conjunction with the molecule of epi-position defined above, and be the modifier or the derivative of natural monoclonal antibody of the present invention.Derivative includes, but not limited to for example antibody, double antibody, chimeric antibody, recombinant antibodies and the humanized antibody of dual specific, polyspecific, heterospecific, tri-specific, four specificitys, polyspecific.

Any reagent with two kinds of different binding specificities, for example protein, peptide or protein or peptide complex wished to comprise in term " bispecific molecule ".Term " polyspecific molecule " or " heterospecific molecule " wish to comprise any reagent that has more than two kinds of different binding specificities, for example protein, peptide or protein or peptide complex.

Term " heteroantibody " is meant two or more antibody that link together, its antibodies fragment (for example, Fab), derivative, or antigen binding domain, wherein at least two kinds have different specificitys.

As used herein, the variable region of the immunoglobulin sequences with the ethnic group of deriving from system and the antibody of constant region wished to comprise in term " people's antibody ".People's antibody of the present invention can comprise that to can't help ethnic group be immunoglobulin sequences amino acids coding residue (for example, by external random mutagenesis or site-directed mutagenesis or the sudden change that imports by somatic mutation in the body).Yet such antibody do not wished to comprise in term used herein " people's antibody ", and promptly in this antibody, for example the CDR sequence of the kind system of mouse is transplanted to people's frame sequence to derive from another kind of mammalian species.Therefore, as used herein, term " people's antibody " is meant such antibody, i.e. proteinic each part basically (for example, CDR, framework, C in described antibody _L, C _HStructural domain (for example, C _H1, C _H2, C _H3), hinge, (V _L, V _H)) right and wrong are immunogenic basically in the people, wherein have only the minority sequence to change or variation.Similarly, specified primate (monkey, baboon, chimpanzee etc.), rodent (mouse, rat, rabbit, cavy, logical sequence mouse etc.) and other Mammalss of antibody have been specified such kind, subgenus, genus, subfamily, section's specific antibody.In addition, chimeric antibody comprises any combination of above-mentioned antibody.With respect to the antibody of unmodified, these changes or change randomly and preferably keep or to have reduced immunogenicity in people or other species.Therefore, people's antibody is different with chimeric antibody or humanized antibody.It is to be noted that the non-human animal of human normal immunoglobulin (for example, heavy chain and/or light chain) gene that can be by can the expressive function rearrangement or protokaryon or eukaryotic cell produce people's antibody.In addition, when people's antibody was single-chain antibody, it can comprise the linker peptide of not finding in natural people's antibody.For example, Fv can comprise the linker peptide, for example 2 or about 8 glycine or other amino-acid residues, and it connects the variable region of heavy chain and the variable region of light chain.Such linker peptide is considered to the people source.

As used herein, if end user's immunoglobulin sequences, for example pass through the transgenic mice of immune carrier's immunoglobulin gene or pass through screening human immunoglobulin gene library, and obtain people's antibody from a system, it is sequence that so described people's antibody " derives from " specific kind.Like this, the aminoacid sequence that can be immunoglobulin (Ig) by aminoacid sequence and the ethnic group with people's antibody compares and identifies that " deriving from " ethnic group is people's antibody of immunoglobulin sequences.Selected people's antibody usually aspect aminoacid sequence be that the coded aminoacid sequence of immunoglobulin gene has at least 90% identity by ethnic group, and comprise and work as the amino-acid residue that when comparing described people's antibody is accredited as human antibodies with the racial immunity sphaeroprotein aminoacid sequence (for example, the muroid kind is a sequence) of other species.In some cases, people's antibody can have at least 95% aspect aminoacid sequence Yu by the coded aminoacid sequence of racial immunity globulin gene, or even at least 96%, 97%, 98% or 99% identity.Usually, deriving from specific ethnic group is that people's antibody of sequence will show and be that the coded aminoacid sequence of immunoglobulin gene is compared and is no more than 10 amino acid whose differences by ethnic group.In some cases, people's antibody can show with being compared by the coded aminoacid sequence of racial immunity globulin gene and be no more than 5, or does not even surpass 4,3,2 or 1 amino acid whose differences.

As used herein, term " monoclonal antibody " or " monoclonal antibody combination " are meant and have single molecular antibody molecule preparation of planting.Monoclonal antibody combination shows single binding specificity and the avidity for defined epitope.

" human monoclonal antibodies " is meant that showing single binding specificity, having the ethnic group of deriving from is the variable region of immunoglobulin sequences and the antibody of constant region.

Term " recombinant human antibody ", as used herein, comprise and pass through recombinant methods, express, form or isolating everyone antibody, for example (for example from animal, mouse) (it is genetically modified for the human immunoglobulin gene or transfection chromosome (transchromosomal)) or hybridoma isolated antibody prepared therefrom, thereby from host cell through transforming expressing antibodies for example from the transfectoma isolated antibody, from what recombinate, isolated antibody in people's antibody library of combination, and become any other method of other dna sequence dnas to prepare the montage of human immunoglobulin gene's sequence by involving, express, form or isolated antibody.Such recombinant human antibody has variable region and the constant region that the ethnic group of deriving from is an immunoglobulin sequences.Yet, in certain embodiments, such recombinant human antibody can be accepted vitro mutagenesis (perhaps, when using for people Ig sequence genetically modified animal, body endosome cell mutation), thereby the aminoacid sequence in the VH of described recombinant antibodies and VL district is such sequence, is VH and VL sequence and associated though promptly described sequence derives from ethnic group, may not be that to be present in people's antibody kind natively be in the storehouse in vivo.

As used herein, " isotype " is meant the antibody type (for example, IgM or IgG1) by the weight chain constant area gene coding.

Term " genetically modified non-human animal " is meant to have genome that comprises one or more people's heavy chains and/or light chain transgenosis or transfection chromosome (be integrated into or nonconformity is gone among the natural gene group DNA of animal) and the non-human animal that can express fully human antibodies.For example, transgenic rat can have people's light chain transgenosis and people's heavy chain transgenosis or people's heavy chain transfection chromosome, and described like this rat produces the anti-INF-Alpha antibodies of people.People's heavy chain transgenosis can be integrated in the chromosomal DNA of rat, and perhaps people's heavy chain transgenosis can keep in extrachromosomal mode.The animal of transgenosis and transfection chromosome can produce multiple isotype (for example, IgG, IgA and/or IgE) for the human monoclonal antibodies of α V by carrying out the conversions of V-D-J reorganization and isotype.

" composition " also wishes to comprise the combination of promoting agent and another kind of carrier, described carrier for example be inertia (for example, detectable reagent or mark) or active compound or composition, for example adjuvant, thinner, tackiness agent, stablizer, buffer reagent, salt, lipophilic solvent, sanitas, adjuvant etc.Carrier also comprises drug excipient and additive, and (for example, sugar comprises monose, disaccharides, trisaccharide, tetrose and oligosaccharides for protein, peptide, amino acid, lipid and carbohydrate; Deutero-sugar is the sugar etc. of sugar alcohol, glyconic acid, esterification for example; With polysaccharide or glycopolymers), described material can be individually or is existed in combination, comprises these materials alone or in combination of 1-99.99% (by weight or volumeter).Exemplary protein vehicle comprises serum albumin such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein etc.Representative amino acid/the antibody component that also can work in buffer capacity comprises L-Ala, glycine, arginine, trimethyl-glycine, Histidine, L-glutamic acid, aspartic acid, halfcystine, Methionin, leucine, Isoleucine, Xie Ansuan, methionine(Met), phenylalanine, aspartame etc.The carbohydrate vehicle wishes also that within the scope of the present invention the example includes but not limited to monose for example fructose, maltose, semi-lactosi, glucose, D-seminose, sorbose etc.; Disaccharides is lactose, sucrose, trehalose, cellobiose etc. for example; Polysaccharide, for example raffinose, melizitose, Star Dri 5, dextran, starch etc.; With sugar alcohol for example N.F,USP MANNITOL, Xylitol, maltose alcohol, Saccharum lactis, Xylitol, sorbyl alcohol (sorbitol) and inositol.

Term " carrier " also comprises buffer reagent or pH regulator agent; Usually, buffer reagent is the salt from organic acid or alkali preparation.Representational buffer reagent comprises for example salt of citric acid, xitix, glyconic acid, carbonic acid, tartrate, succsinic acid, acetate or phthalandione of organic acid salt; Tris, Tromethamine hydrochloride (tromethamine hydrochloride), or phosphate buffer.Other carriers comprise polymeric excipient/additive, and for example polyvinylpyrrolidone, Fick (polymerization sugar), dextrates be (for example, cyclodextrin, for example the 2-hydroxypropyl-. quadrature (quadrature) .-cyclodextrin), polyoxyethylene glycol, seasonings, biocide, sweetener, antioxidant, static inhibitor, tensio-active agent (for example, polysorbate is " TWEEN20 " and " TWEEN 80 " for example), lipid (for example, phosphatide, lipid acid), steroid (for example, cholesterol) and sequestrant (for example, EDTA).

As used herein, term " pharmaceutically acceptable carrier " comprises any in the pharmaceutical carrier of standard, for example phosphate buffered saline(PBS), water and emulsion such as oil/water or water/oil-type emulsion, and various types of wetting agent.Composition also can comprise stablizer and sanitas and any carrier of pointing out above, and supplementary condition are that they are acceptable for using in the body.The example of carrier, stablizer and adjuvant, referring to Martin REMINGTON ' S PHARM.SCI., the 15th edition (Mack Publ.Co., Easton (1975) and Williams﹠amp; Williams, (1995)) and at " PHYSICIAN ' S DESK REFERENCE ", the 52nd edition, MedicalEconomics, Montvale is among the N.J. (1998).

" significant quantity " is the amount that is enough to realize useful or desirable result.Can use at one or many, use significant quantity in application or the dosage.

The invention provides and discern specifically and be combined in by the expressed polypeptide of genes identified in the table 1 or antibody or its variant, derivative or the fragment of the epi-position on the protein.In one aspect, separate described antibody.In yet another aspect, they and suitable carriers are combined.Described antibody can be polyclonal or monoclonal, and can be from any species, and muroid, rat, ape separate, and perhaps reorganization produces and separates.The present invention also provides the hybridoma cell line that produces these monoclonal antibodies, described monoclonal antibody individually or with carrier combinedly in culture.

The present invention also provides the polypeptide that comprises antibody, its variant, derivative or fragment (including but not limited to immunoglobulin chain and CDR).

The present invention further provides antiidiotypic antibody.Antiidiotypic antibody comprises any protein or the peptide that comprises such molecule, described molecule comprise immunoglobulin molecules to small part, such as but not limited at least one complementarity-determining region, heavy chain or variable region of light chain, heavy chain or constant region of light chain, framework region or its any part of heavy chain or light chain or its ligand binding domain, it can be integrated in the antibody of the present invention.Antiidiotypic antibody of the present invention can comprise or derive from any Mammals, such as but not limited to people, mouse, rabbit, rat, rodent, primate etc.

The present invention further provides the isolating polynucleotide of the antibody of the present invention of encoding, it individually or combined with carrier, vehicle, pharmaceutically acceptable vehicle, thinner and host cell.

The present invention further provides isolated nucleic acid molecule in one aspect, described nucleic acid molecule comprises the polynucleotide of at least a antibody of the present invention of coding or antiidiotypic antibody, complementary with it, or hybridization with it, its at least one specific sequence, structural domain, part or variant comprised.The present invention further provides the recombinant vectors that comprises described Nucleotide, comprised the host cell of described nucleic acid and/or recombinant vectors, and the method for preparing and/or use these nucleic acid, carrier and/or host cell.The method that is used to separate, duplicate and expresses polynucleotide is well known in the art and is described hereinafter.

One or more above-mentioned antibody also can be with carrier, pharmaceutically acceptable carrier be adapted at diagnosing or methods of treatment in use antibody or compositions related medical apparatus combined.

Described carrier can be liquid phase carrier or solid phase carrier for example globule, gel or such as the carrier molecule of liposome.Described composition randomly also can comprise at least a other compounds, protein or composition.

Other examples of " carrier " comprise for example another kind of peptide of therapeutic activity agent or protein (for example, Fab ' fragment).For example, antibody of the present invention, its variant, derivative or fragment (for example can functionally connect, by chemical coupling, gene fusion, non-covalent combination or other modes) to one or more other molecular entities, for example another kind of antibody (for example, to produce dual specific or multi-specificity antibody), cytotoxin, cell ligand or antigen.Therefore, the present invention includes many kinds of antibody conjugates, two and polyspecific molecule and fusion rotein, no matter its target epi-position identical whether with antibody of the present invention.

Other examples of carrier be directly or indirectly covalent attachment to the organic molecule (being also referred to as modifier) or the activator of antibody of the present invention.Described molecular adhesion can improve pharmacokinetic property (for example, serum half-life in the body of increase).The example of organic molecule includes, but not limited to hydrophilic polymer group, fatty acid group or fatty acid ester group.As used herein, term " fatty acid " " comprise monocarboxylic acid and di-carboxylic acid." hydrophilic polymer group ", the term as use herein is meant easier dissolved organic polymer in water than in octane.

The hydrophilic polymer that is suitable for modifying antibody of the present invention can be linear or branched, it comprises, for example, the polyalkane glycol (for example, PEG, mono methoxy-polyoxyethylene glycol (mPEG), PPG etc.), carbohydrate (for example, dextran, Mierocrystalline cellulose, oligosaccharides, polysaccharide etc.), polymkeric substance (for example, polylysine, poly arginine, poly aspartic acid etc.), polyalkane oxide compound (for example, polyethylene oxide, poly(propylene oxide) etc.) and the polyvinylpyrrolidone of hydrophilic amino acid.The suitable hydrophilic polymer of modifying antibody of the present invention has about 800 to about 150,000 daltonian molecular weight as the molecular entity that separates.Available 1 to about 6 alkyl, lipid acid or fatty acid ester group replacement hydrophilic polymer group.Can prepare the hydrophilic polymer that replaces with lipid acid or fatty acid ester group by using suitable method.For example, the polymkeric substance that comprises amine groups can be coupled to the carboxylate radical of lipid acid or fatty acid ester and the activated carboxylate radical on lipid acid or the fatty acid ester (for example, using N, N-carbonyl dimidazoles activated) can be coupled to the hydroxyl on the polymkeric substance.

The lipid acid and the fatty acid ester that are suitable for modifying antibody of the present invention can maybe can be comprised one or more unsaturated units by saturated.The example comprises, but be not limited to n-dodecane hydrochlorate (ester), n-tetradecane hydrochlorate (ester), Octadecane hydrochlorate (ester), NSC 62789 hydrochlorate (ester), n-docosane hydrochlorate (ester), positive melissate (ester), positive tetracontane hydrochlorate (ester), suitable-Δ-9-octadecane hydrochlorate (ester), all suitable-Δ-5,8,11,14-arachidonate (ester), suberic acid, tetradecane diacid, octadecane diacid, docosandioic acid.The suitable fatty acids ester comprises the monoesters of the dicarboxylic acid that comprises linear or branched low alkyl group.Described low alkyl group can comprise 1 to about 12, and preferably 1 to about 6 carbon atoms.

In yet another aspect, the invention provides genetically modified non-human animal, transgenic mice (being also referred to as " HuMAb mouse " herein) for example, it expresses complete human monoclonal antibodies, in the described antibody with the similar protein subtype of antibody of the present invention at least a and defined above.In specific embodiment, described genetically modified non-human animal has the people's of comprising heavy chain transgenosis and a genetically modified genomic transgenic mice of people's light chain, described people's heavy chain and light chain transgenes encoding anti-α V antibody of the present invention all or part of.Preferably, genetically modified non-human animal, transgenic mice for example can produce multiple isotype at the human monoclonal antibodies of purpose epi-position by carrying out V-D-J reorganization and isotype conversion.Isotype conversion can be undertaken by for example traditional or unconventional isotype conversion.

Therefore, in another embodiment, the invention provides isolated cells, it derives from or separates from the transgenic nonhuman animal of above-mentioned expressing human antibody transgenic mice for example.Make isolating B-cell immortalityization by merging to immortalized cells then, thereby the source (for example, hybridoma) of people's antibody is provided.These hybridomas are also included within the scope of the present invention.

At least a antibody method or composition have been the present invention further provides, it is used for cell, tissue, organ, animal or patient, and/or known in the art and/or described herein conditions associated before, afterwards or during, cancer for example nonsmall-cell lung cancer (NSCLC), ovarian cancer, mammary cancer, prostate cancer or the colorectal carcinoma of diagnosis epithelial origin.They also are used for prognosis or monitoring disease progress.

Composition also is provided, described composition comprises at least a antibody of the present invention, its variant, derivative or fragment, and they are fit to for the cancer of adjusting or improvement and the epithelial origin symptom that for example nonsmall-cell lung cancer (NSCLC), ovarian cancer, mammary cancer, prostate cancer are relevant with colorectal carcinoma or treat at least a such cancer effective measuring and use.Described composition comprises, for example medicine and diagnosis composition/test kit, and it comprises pharmaceutically acceptable carrier and at least a antibody of the present invention, its variant, derivative or fragment.As noted above, described composition can also comprise other antibody or therapeutical agent, and they provide through tailor-make so that the multiple therapy of maximum therapy benefit to be provided combinedly.

Whether selectively, composition of the present invention can be used altogether with other treatment agent and cytotoxic agent, no matter be connected with them or use in same administration according to dosage.They can use simultaneously altogether with such reagent (for example, in single composition or the mode to separate), perhaps can use before or after using such reagent.Such reagent can comprise reflunomide, on-steroidal immunosuppressor, antimalarial drug and nonsteroidal antiinflammatory drug.It is combined described composition and selectable therapy for example can be used reflunomide, on-steroidal immunosuppressor, antimalarial drug and nonsteroidal antiinflammatory drug.

Can in external or body, implement method of the present invention.When in external enforcement, described method need be with contacts (for example, use or send to cell) such as cell and one or more antibody and/or the relevant therapeutic composition that comprises above-mentioned antibody, derivatives.

Can send described antibody and composition by any suitable method with any appropriate formulation.Therefore, further provide the preparation that comprises antibody of the present invention herein.Described preparation also can comprise one or more sanitass or stablizer for example phenol, meta-cresol, p-cresol, ortho-cresol, parachlorometacresol, phenylcarbinol, nitrous acid benzene mercury, phenoxyethyl alcohol, formaldehyde, trichloro-butyl alcohol, magnesium chloride (for example, hexahydrate), alkyl paraben (methyl esters, ethyl ester, propyl ester, butyl ester etc.), benzalkonium chloride, benzethonium chloride, Sodium dehydroacetate and Thiomersalate or its mixture in aqueous diluent.As known in the art, can use any suitable concentration or mixture, 0.001-5% for example, or therein any scope or value, such as but not limited to 0.001,0.003,0.005,0.009,0.01,0.02,0.03,0.05,0.09,0.1,0.2,0.3,0.4,0.5,0.6,0.7,0.8,0.9,1.0,1.1,1.2,1.3,1.4,1.5,1.6,1.7,1.8,1.9,2.0,2.1,2.2,2.3,2.4,2.5,2.6,2.7,2.8,2.9,3.0,3.1,3.2,3.3,3.4,3.5,3.6,3.7,3.8,3.9,4.0,4.3,4.5,4.6,4.7,4.8,4.9, or therein any scope or value.Non-limiting example comprises, preservative-free, the meta-cresol of 0.1-2% (for example, 0.2,0.3,0.4,0.5,0.9,1.0%), the phenylcarbinol of 0.1-3% (for example, 0.5,0.9,1.1,1.5,1.9,2.0,2.5%), the Thiomersalate of 0.001-0.5% (for example, 0.005,0.01), the phenol of 0.001-2.0% (for example, 0.05,0.25,0.28,0.5,0.9,1.0%), the alkyl paraben of 0.0005-1.0% (for example, 0.00075,0.0009,0.001,0.002,0.005,0.0075,0.009,0.01,0.02,0.05,0.075,0.09,0.1,0.2,0.3,0.5,0.75,0.9 and 1.0%).

As noted above, the invention provides goods, it comprises wrapping material and at least one bottle, this bottle is equipped with the antibody of the present invention at least randomly prepared and the specified buffer reagent and/or the solution of sanitas in aqueous diluent, wherein said wrapping material comprise and indicate that such solution can keep 1,2,3,4,5,6,9,12,18,20,24,30,36,40,48,54,60,66,72 hour or the label of longer time.The present invention further comprises goods, second bottle that it comprises wrapping material, first bottle of at least a freeze dried antibody of the present invention is housed and the aqueous diluent of specified buffer reagent or sanitas is housed, wherein said wrapping material comprise and instruct patient's reshaped antibody in aqueous diluent can keep 24 hours or the label of the solution of longer time to form.

After described antibody scope is included in reconstruct, if in wet/system of doing, produce the amount of about 1.0 μ g/ml to the concentration of about 1000mg/ml, although lower and higher concentration is exercisable, and depend on think the delivery vector of usefulness, for example, pharmaceutical solutions will be with transdermal patch method, lung's method, through mucous membrane method or infiltration or micro pump method and difference.

Can prepare preparation of the present invention by being included in the method for mixing at least a antibody of the present invention and sanitas in the aqueous diluent, described sanitas is selected from phenol, meta-cresol, p-cresol, ortho-cresol, parachlorometacresol, phenylcarbinol, alkyl paraben (methyl esters, ethyl ester, propyl ester, butyl ester etc.), benzalkonium chloride, benzethonium chloride, Sodium dehydroacetate and Thiomersalate or its mixture.Use conventional dissolving and blending means in aqueous diluent, to mix described antibody and sanitas.For example, with the amount of the antibody and the sanitas that are enough to provide desired concn, will be at least a antibody of the measuring vol in solutions buffered with combined at the required sanitas in solutions buffered.It will be recognized by those skilled in the art the variation of this method, the order, temperature and the pH when whether using other additives, preparation preparation that add of component for example is the factor that can be optimized for the application concentration that is adopted and method.

Can provide described composition and preparation to the patient with clear soln or two doleiform formula, described two bottles comprise the one bottle of freeze dried antibody that is reconstructed with second bottle that aqueous diluent is housed.Require single solution bottle or two bottle of reconstruct repeatedly reusable, and can satisfy the single or multiple circulations of patient treatment, thereby provide than present obtainable treatment plan treatment plan more easily.The authorized device that comprises these single bottle systems comprises the pen-type injector that is used to send solution, BD Pens for example, BD Autojectore, Humaject.RTM. ' NovoPen.RTM., B-D.RTM.Pen, AutoPen.RTM., and OptiPen.RTM., GenotropinPen.RTM., Genotromorm Pen.RTM., Humatro Pen.RTM., Reco-Pen.RTM., Roferon Pen.RTM., Biojector.RTM., iject.RTM., J-tip Needle-Free Injector.RTM., Intraject.RTM., Medi-Ject.RTM., for example, by Becton Dickensen (Franklin Lakes, N.J. can obtain on bectondickenson.com), Disetronic (Burgdorf, Switzerland can obtain on disetronic.com; Bioject, Portland, Oregon (can on bioject.com, obtain); National Medical Products, Weston Medical (Peterborough, UK can obtain on weston-medical.com), Medi-Ject Corp (Minneapolis, Minn. can obtain on mediject.com) make or develop.

Antibody

Antibody of the present invention is monoclonal antibody, although in some aspects, can use polyclonal antibody.They can also be functional fragment, antibody derivatives or antibody variants.They can be chimeric, humanized or be people's antibody fully.The functional fragment of antibody includes but not limited to Fab, Fab ', Fab2, Fab ' 2 and strand variable region.Can include but not limited to produce in ox, rabbit, goat, mouse, rat, logical sequence mouse, cavy, sheep, dog, cat, monkey, chimpanzee, the ape etc. antibody cell culture, phage or various animal.As long as described fragment or derivative keep the binding specificity or the neutralising capacity of antibody of the present invention, just can use it.Can by under given one-tenth set condition will to suitable antigenic combination with combining of incoherent antigen or antigen mixture compared, come test antibody with regard to the bonded specificity.If antibody surpasses combination at least 2,5 to incoherent antigen or antigen mixture, 7 and preferably 10 times the time to suitable antigenic combination, can think that so it is specific.Be used for determining that specific specific assay rule such as ELISA are well known in the art.

The feature of described antibody is that also it discerns the ability with the binding purposes epi-position specifically.

Can use conventional hybridization knurl technology known in the art and that describe in detail in the literature to produce monoclonal antibody of the present invention.For example, by (for example with suitable immortal cell line, the myeloma cell, such as but not limited to Sp2/0, Sp2/0-AG14, NS0, NS1, NS2, AE-1, L.5,＞243, P3X63Ag8.653, Sp2 SA3, Sp2 MAI, Sp2 SS1, Sp2 SA5, U397, MLA 144, ACT IV, MOLT4, DA-1, JURKAT, WEHI, K-562, COS, RAJI, NIH 3T3, HL-60, MLA 144, NAMAIWA, NEURO 2A, CHO, PerC.6, YB2/O) or like that, perhaps allos myelomatosis (heteromyelomas), its fusion product or derive from its any cell or fused cell, any other suitable clone perhaps known in the art (referring to, for example, www.atcc.org, www.lifetech.com. etc.), producing sexual cell with antibody merges and produces hybridoma, described antibody produces sexual cell for for example, but be not limited to, isolating or clone splenocyte, peripheral blood cells, lymphocyte, tonsilla cell or other immunocytes or comprise the cell of B cell, perhaps any other cell, described other cells are to express heavy chain or constant region of light chain or variable region or framework region or CDR sequence as the mode of endogenous or heterologous nucleic acids, described nucleic acid is as reorganization or endogenous virus, bacterium, algae, prokaryotic organism, Amphibians, insect, Reptilia, fish, Mammals, rodent, horse, sheep, goat, sheep, primate, Eukaryotic genomic dna, cDNA, rDNA, Mitochondrial DNA or RNA, chloroplast DNA or RNA, hnRNA, mRNA, tRNA (strand, double-stranded or three chains, heterozygosis) etc. or its any combination.Antibody produce sexual cell also can from peripheral blood or preferably spleen or the lymphoglandula of people or other suitable animals obtain, described human or animal has used the purpose antigen immune.Any other proper host cell also can be used for the nucleic acid of expressing heterologous or endogenous coding antibody of the present invention, its specific fragment or variant.Can be by using selectivity culture condition or other suitable currently known methodss to separate the cell (hybridoma) of fusion or reconstitution cell and cloning by limiting dilution or cell sorting or other known methods.

Can use and be used to produce or separate other appropriate method with required specific antibody, include but not limited to, by using the method known in the art (for example, but to be not limited to display libraries such as phage, rrna, oligonucleotide, RNA, cDNA from peptide or protein library; For example, can be from various commercial vendor Cambridge AntibodyTechnologies (Cambridgeshire for example, UK), MorphoSys (Martinsreid/Planegg, Del.), Biovation (Aberdeen, Scotland, UK), select the method for recombinant antibodies among the BioInvent (Lund Sweden) obtains).Referring to U.S. Patent number 4,704,692,5,723,323,5,763,192,5,814,476,5,817,483,5,824,514,5,976,862.Alternative methods depends on known in the art and/or transgenic animal (for example, SCID mouse, people such as Nguyen (1977) Microbiol.Immunol.41:901-907 (1997) that can produce people's antibody library described herein; People such as Sandhu, (1996) Crit.Rev.Biotechnol.16:95-118; People such as Eren (1998) Immunol.93:154-161) immunity.Such technology includes, but not limited to ribosomal display (people (1997) Proc.Natl.Acad.Sci.USA such as Hanes, 94:4937-4942; People such as Hanes, (1998) Proc.Natl.Acad.Sci.USA, 95:14130-14135); Unicellular antibody generating technique (for example, the lymphocyte antibody method of selection (selected lymphocyte antibody method) (" SLAM ") (U.S. Patent number 5,627,052, people such as Wen (1987) J.Immunol.17:887-892; People such as Babcook, Proc.Natl.Acad.Sci.USA (1996) 93:7843-7848); Gel microdrop (gel microdroplet) and flow cytometry (people (1990) Biotechnol.8:333-337 such as Powell; One Cell Systems, (Cambridge, Mass).; People such as Gray (1995) J.Imm.Meth.182:155-163; People such as Kenny (1995) Bio/Technol.13:787-790); The B cell is selected people (1994) Molec.Biol.Reports 19:125-134 (1994) such as () Steenbakkers.

Also can by send the coding antibody of the present invention polynucleotide to appropriate host, thereby for example provide transgenic animal or Mammals for example (they produce these antibody in its milk) such as goat, ox, horse, sheep prepare antibody variants of the present invention.These methods are well known in the art, and for example are described in the U.S. Patent number 5,827,690,5,849,992,4,873,316,5,849,992,5,994,616,5,565,362 and 5,304,489.

Term " antibody variants " comprises the posttranslational modification of antagonist or segmental linear polypeptide sequence.For example, U.S. Patent number 6,602,684B1 has described the method for the antibody that is used to produce modified glycosylation form and the glycoprotein that is produced, described antibody comprises complete antibody molecule, antibody fragment or comprises the fusion rotein in the zone in the Fc district that is equal to immunoglobulin (Ig) to have the cytotoxicity of enhanced Fc mediation.

Also can be by (for example sending polynucleotide of the present invention with vegetable cell that transgenic plant and cultivation are provided, but be not limited to, tobacco, corn and duckweed) prepare antibody variants, the vegetable cell of described transgenic plant or cultivation produces these antibody, specific part or variant in the part of plant or in by its cultured cells.For example, people such as Cramer (1999) Curr.Top.Microbol.Immunol.240:95-118 and the bibliography of wherein being quoted have been described and have for example been used inducible promoter to express the generation of the transgene tobacco leaf of a large amount of recombinant proteins.Used transgenic corns on the commercial production level, to express mammalian proteins matter, described mammalian proteins matter have with in other recombination systems, produce or from the identical biologic activity of those mammalian proteins matter of natural origin purifying.Referring to, for example, people such as Hood, Adv.Exp.Med.Biol. (1999) 464:127-147 and the bibliography of wherein being quoted.Also produce antibody variants in large quantities from transgenic plant seed, comprise antibody fragment, for example (scFv ' s), described transgenic plant seed comprises tobacco seed and potato tuber to single-chain antibody.Referring to, for example, people such as Conrad (1998) Plant Mol.Biol.38:101-109 and the bibliography of wherein being quoted.Therefore, also can use transgenic plant to produce antibody of the present invention in accordance with known methods.

Antibody derivatives can produce in the following way: add exogenous array to modify immunogenicity or minimizing, enhancing or to modify combination, avidity, combination rate (on-rate), the rate of dissociation (off-rate), avidity (avidity), specificity, transformation period or any other suitable feature.Common retained part or all inhuman or people CDR sequences, and personnel selection or other amino acid replace the non-human sequence of variable regions and constant region.

Usually, the CDR residue directly and the overwhelming majority participate in influencing the antigen combination.Can use any known method to carry out the humanization of antibody of the present invention or engineered, described method is such as but not limited to U.S. Patent number 5,723,323,5,976,862,5,824,514,5,817,483,5,814,476,5,763,192,5,723,323,5,766,886,5,714,352,6,204,023,6,180,370,5,693,762,5,530,101,5,585,089, the method for describing in 5,225,539 and 4,816,567.

Be used to prepare part to the technology of fully human antibodies and be well known in the art, and can use any such technology.According to an embodiment, thereby can in the transgenic mice of transforming expressing human heavy chain and light chain antibody gene, prepare the fully human antibodies sequence.Prepared a plurality of strains of such transgenic mice, they can produce different classes of antibody.Can merge the hybridoma cell line that is used for producing continuously required antibody from the B cell of the transgenic mice that produces required antibody with preparation.(referring to, for example, Russel, people such as N.D. (2000) Infection and Immunity April 2000:1820-1826; Gallo, people such as M.L. (2000) European J.of Immun.30:534-540; Green, L.L. (1999) J.of Immun.Methods 231:11-23; Yang, people such as X-D (1999A) J.ofLeukocyte Biology 66:401-410; Yang, X-D (1999B) Cancer Research59 (6): 1236-1243; Jakobovits, A. (1998) Advanced Drug DeliveryReviews 31:33-42; Green, L. and Jakobovits, A. (1998) J.Exp.Med.188 (3): 483-495; Jakobovits, A. (1998) Exp.Opin.Invest.Drugs7 (4): 607-614; Tsuda, people such as H. (1997) Genomics 42:413-421; Sherman-Gold, R. (1997) Genetic Engineering News 17 (14); Mendez, people such as M. (1997) Nature Genetics 15:146-156; Jakobovits, A. (1996) WEIR ' S HANDBOOK OF EXPERIMENTAL IMMUNOLOGY, THE INTEGRATEDIMMUNE SYSTEM IV volume, 194.1-194.7; Jakobovits, A. (1995) Current Opinion in Biotechnology 6:561-566; Mendez, people such as M. (1995) Genomics 26:294-307; Jakobovits, A. (1994) CurrentBiology 4 (8): 761-763; Arbones, people such as M. (1994) Immunity 1 (4): 247-260; Jakobovits, A. (1993) Nature 362 (6417): 255-258; Jakobovits, people such as A. (1993) Proc.Natl.Acad.Sci.USA 90 (6): 2551-2555; People such as Kucherlapati, U.S. Patent number 6,075,181).

Also can produce human monoclonal antibodies by hybridoma, described hybridoma comprise merge to immortalized cells from the transgenic nonhuman animal B cell that obtains of transgenic mice for example, described transgenic nonhuman animal has the people's of comprising heavy chain transgenosis and the genetically modified genome of light chain.

Also can modify antibody of the present invention to produce chimeric antibody.Chimeric antibody is that the multiple structural domain of the heavy chain of wherein antibody and light chain is by from those antibody more than the dna encoding of species.Referring to, for example, U.S. Patent number 4,816,567.

Term " antibody derivatives " also comprises " double antibody ", and it is the little antibody fragment with two antigen binding sites, and wherein fragment is at same polypeptide chain (V _HV _L) in comprise and be connected to light chain variable structural domain (V _L) weight chain variable structural domain (V _H).(referring to for example, EP404,097; WO93/11161; With people such as Hollinger, (1993) Proc.Natl.Acad.Sci.USA 90:6444-6448).Do not allow two structural domains on same chain to carry out the paired linker by using too short, force the complementary structure territory pairing of described structural domain and another chain, thereby produce two antigen binding sites.(referring to, for example, the U.S. Patent number 6 that belongs to people such as Chen, 632,926, it discloses antibody variants, and described antibody variants has the amino acid in the hypervariable region of one or more insertion parental antibodies, and its for the binding affinity of target antigen than parental antibody for this antigenic binding affinity twice at least by force).

Term " antibody derivatives " further comprises " linear antibody ".The method that produces it is well known in the art, and is described in people (1995) Protein Eng.8 (10) such as Zapata: among the 1057-1062.In brief, these antibody comprise the paired series connection Fd section (V that forms paired antigen binding domain territory _H-C _H1-V _H-C _H1).Linear antibody can be dual specific or monospecific.

Can from the reconstitution cell culture, reclaim and purifying antibody of the present invention by known method, described method includes but not limited to, A protein purification, ammonium sulfate or ethanol sedimentation, acid extraction, negatively charged ion or cation-exchange chromatography, phosphorylated cotton chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite and lectin chromatography.Also can use high performance liquid chromatography (" HPLC ") to carry out purifying.

Antibody of the present invention comprise natural purifying product, chemical synthesis process product and as mentioned above by recombinant technology from eukaryote host or the product that alternatively produces from prokaryotic cell prokaryocyte, described eukaryote host comprises, for example, yeast, higher plant, insect and mammalian cell.

Aspect more of the present invention, can detect ground or therapeutic ground mark antibody be useful.Being used for that antibody is conjugated to these compositions and methods is well known in the art.Just for illustration purpose, can for example radioactive atom, chromophore, fluorophore wait traget antibody with detectable part.These antibody through mark can be used in vivo or the diagnostic techniques in isolating given the test agent.Also antibody can be conjugated to for example pharmacology reagent, for example chemotherapeutics or toxin.Other can be connected to cytokine, part, another kind of antibody.Be used to be coupled to antibody and comprise cytokine, for example interleukin II (IL-2) and tumour necrosis factor (TNF) with the suitable reagent that obtains antitumous effect; The photosensitizers that is used for photodynamic therapy comprises ALPCS4 (III), haematoporphyrin and phthalocyanine; Radionuclide, for example iodine-131 ( ¹³¹I), Yttrium-90 ( ⁹⁰Y), bismuth-212 ( ²¹²Bi), bismuth-213 ( ²¹³Bi), technetium-99m ( ^99mTc), rhenium-186 ( ¹⁸⁶Re) and rhenium-188 ( ¹⁸⁸Re); Microbiotic, for example Dx, Zorubicin, daunorubicin, methotrexate, daunomycin, neocarzinostatin and carboplatin; Bacteriotoxin, plant poison and other toxin, for example diphtheria toxin, ETA, staphylococcal enterotoxin A, toxalbumin-A toxin, ricin A (deglycosylated ricin A and natural ricin A), TGF-alpha toxin, from the cytotoxin of Chinese Naja (naja naja atra) with spend more white tree toxalbumin (plant poison); From the ribosome inactivating protein of plant, bacterium and fungi, for example restrictocin (ribosome inactivating protein that produces by Aspergillus restrictus (Aspergillus restrictus)), Saponaria officinalis toxalbumin (from the ribosome inactivating protein of Saponaria officinalis (Saponaria officinalis)) and RNA enzyme; Tyrosine kinase inhibitor; Ly207702 (bifluoride purine nucleoside); The liposome that comprises anti-wafer (anti cystic agents) (for example, the plasmid of antisense oligonucleotide, toxin-encoding, methotrexate etc.); With other antibody or antibody fragment, F (ab) for example.

About comprising the preparation of the antibody that covalently is connected to organic molecule, can use suitable method for example to prepare them by reacting with one or more modifiers.The example of these reagent comprises the group of modification property and reactivity.Term used herein " modifier " is meant the suitable organic group (for example, hydrophilic polymer, lipid acid, fatty acid ester) that comprises activating group.The front provides the particular instance of these groups." activating group " thus be can be under appropriate condition and the chemical part or the functional group of second chemical group reaction formation covalent linkage between modifier and second chemical group.These activity examples of groups are for example tosylate, methylsulfonic acid, halo (chloro, bromo, fluoro, iodo), N-hydroxy-succinamide base esters (NHS) etc. of electrophilic group.Can comprise with the activating group of sulfydryl reaction, for example, maleimide, iodoacetyl, acryl, pyridyl disulfide, 5-sulfydryl-2-nitrobenzoic acid mercaptan (5-thiol-2-nitrobenzoic acid thiol, TNB-thiol) etc.Aldehyde functional group can be coupled to the molecule that comprises amine or hydrazides, and azido-and three valent phosphors radical reaction can be formed phosphoramidate or phosphinylidyne imine linkage.The suitable method that is used for activating group is introduced molecule is well known in the art (referring to for example, Hermanson, G.T., BIOCONJUGATETECHNIQUES, Academic Press:San Diego, Calif. (1996)).Can be directly or by linker part and organic group (for example, hydrophilic polymer, lipid acid, fatty acid ester) combination, described linker part for example is divalence C with activating group ₁-C ₁₂Group, wherein one or more carbon atoms can for example oxygen, nitrogen and sulphur substitute by heteroatoms.Suitable linker partly comprises, for example, and TEG.Can produce the modifier that comprises the linker part by following manner: for example, under the situation that 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) exists with list-Boc-alkyl diamine (for example, list-Boc-quadrol, list-Boc-diamino hexane) thereby and fatty acid response between unhindered amina and fatty acid carboxylate, form amido linkage.Can remove the Boc blocking group to expose primary amine by handling with trifluoroacetic acid (TFA) from product; this primary amine can be coupled to another carboxylic acid as described; perhaps can react with maleic anhydride; and with the product cyclisation of gained, thereby produce the activatory maleimide aminoderivative of lipid acid.

Can produce modified antibody of the present invention by people's antibody or Fab and modifier are reacted.For example, can in non-locus specificity mode organic moiety be bonded to antibody by using for example NHS ester of PEG of the reactive modifier of amine.Also can prepare modified people's antibody or Fab by the disulfide linkage (for example, intrachain disulfide bond) of going back original antibody or Fab.Then, can be with through reductive antibody or Fab and the reactive modifier reaction of sulfydryl, thus produce modified antibody of the present invention.Can use for example reverse proteolysis (reverse proteolysis) of suitable method to prepare modified people's antibody and the Fab that comprises organic moiety, described organic moiety is connected to the specific site of antibody of the present invention.Usually referring to, Hermanson, G.T., BIOCONJUGATE TECHNIQUES, Academic Press:San Diego, Calif. (1996).

Polynucleotide and polypeptide

By the sequence information that provides in the table 1 (with sequence table hereinafter) being provided and being purchased obtainable automated peptide synthesizer (for example by Perkin Elmer/AppliedBiosystems through employing, Inc., 430A or 431A type, Foster City, the synthesizer that CA, USA produce) chemosynthesis of being carried out can obtain polynucleotide, polypeptide and its fragment.Precipitable synthetic protein or polypeptide, and further carry out purifying by for example high performance liquid chromatography (HPLC).Therefore, the present invention also provides and has been used for chemosynthesis method of protein of the present invention, and described method is by providing proteinic sequence and reagent for example amino acid and enzyme, and with correct direction and linear precedence amino acid is linked together synthetic protein.

Alternatively, can use host cell described herein and carrier system to obtain protein and polypeptide by the recombination method of knowing described herein.Host cell can be protokaryon or eucaryon.Host cell systems has above been described.

Diagnostic method

As noted above, the invention provides the several different methods that is used for the assisted diagnosis cell state, described cell state is characterised in that the abnormal cell growth that exists with for example malignant tumour, hyperplasia or metaplastic form.For example nonsmall-cell lung cancer (NSCLC), ovarian cancer, mammary cancer, prostate cancer and colorectal carcinoma are useful especially to described method for the cancer of assisted diagnosis epithelial origin.Can whether not be subjected to the restriction of common normal growth to control to determine the superfluous natural disposition state of cell by the growth of noting cell.For the purposes of the present invention, described term comprises that also genotype changes, and described genotype changes and occurs in before this growth that detection carries out with the tumour form, and is the reason of these phenotypic alternations.The phenotypic alternation relevant with the superfluous natural disposition state of cell (in groups with body in the relevant external feature of tumorigenesis ability) comprise that round cellular form, looser matrix are adhered to, for example release, the sugar transport of increase, the serum needs of minimizing, the expression of fetal antigen etc. of plasminogen activator of the forfeiture of the forfeiture of contact inhibition, anchorage dependence, proteolytic enzyme.(referring to, people such as Luria (1978) GENERAL VIROLOGY, the 3rd edition, 436-446 (John Wiley﹠amp; Sons, New York)).

Therefore, an embodiment is the method for the situation of diagnosis cell, it is diagnosed from the polynucleotide of the differential expression of sample separation or the existence of polypeptide by screening, described sample comprises or the cell that comprises expressing said gene under a cloud, and the differential expression of wherein said gene is the sign of the superfluous natural disposition state of cell.As follows, to compare with corresponding normal or healthy cell or tissue, described gene is expressed in cancer or tumour cell more, and wherein said cell is one or more cells in lung, ovary or the prostate gland.

Can detect by any suitable method, comprise the amount that for example detects the cDNA that produces from the amount of the mRNA of described genetic transcription or from reverse transcription or by the polypeptide or the proteinic amount of described genes encoding by the mRNA of described genetic transcription.Be provided for the probe of each method in these methods by peptide of identifying in reverse translation (reversetranslating) table 1 and the polynucleotide that use the described peptide of coding.Can implement these methods to sample by the sampling principle, maybe can revise these methods to carry out high throughput analysis.In addition, can retrieve and analytical database with regard to the existence and the amount of transcript or expressed genes product, described database comprises a certain amount of (quantitative) from isolating complete or transcript or the protein sequence partly of cell sample.In one aspect, described database comprises the sequence shown at least one table 1 and/or its polynucleotide of encoding.

Only presented for purposes of illustration, by measuring genetic expression in the above expression of gene amount of level of the mRNA that transcribes from goal gene (if having, for example, change) in the record test macro.In the embodiment of separating, indicated the existence of the superfluous natural disposition state of cell by the raising of goal gene encoded polypeptides or proteinic level.Described method can be used for helping the diagnosis of lung cancer, ovarian cancer or prostate cancer.Therefore, by before tumor growth, detecting this genotype, the measurable susceptibility of people (predisposition) and/or early diagnosis and therapy is provided to cancer.

Be used for cell or tissue sample of the present invention comprise body fluid, solid tissue's sample, from tissue culture or cell and its offspring in its source, and the section or the smear of any preparation from these sources, maybe can comprise any other sample of the cell with gene described herein.In one embodiment, described sample comprises from organizing of experimenter and for example may comprise the lung of metastasis or the cell of tissue preparation.

In the mensuration of the change that is used for the mRNA level, the nucleic acid that is extracted in the aforementioned sample to be comprised according to the standard method of this area at first.For example, can use various lytic enzymes or chemical solution to separate or extract mRNA with the nucleic acid binding resin according to the specification sheets of enclosing that provides by manufacturer according to the method shown in the people such as Sambrook (1989) (on seeing).Then, according to method well-known in the art or based on the method that exemplifies, detect the mRNA of the gene that in the nucleic acid samples that is extracted, comprises by hybridization (for example, Northern engram analysis) and/or amplification program herein.

Have at least 10 Nucleotide and show the nucleic acid molecule that polynucleotide to the peptide identified at least a coding schedule 1 have sequence complementarity or homology and can be used as hybridization probe.Be known in the art, needn't use " mating fully " probe for specific hybrid.The little change of the probe sequence that displacement, disappearance or the insertion by a small amount of base obtains does not influence the hybridization specificity.Usually, can tolerate the base-pair mismatch (when the time) of as many as 20% by best comparison.Preferably, the homology zone of the similar size that is comprised in the gene of the probe that is used for detecting mRNA and the peptide of identifying at coding schedule 1 or the polynucleotide has about at least 80% identity.In one aspect, after comparing in the homology zone, described probe has 85% identity with corresponding polynucleotide sequence, or alternatively, it shows 90% identity.These probes can be used for radiometry (for example, Southern and Northern engram analysis) with detection, prognosis, diagnosis or the monitoring various superfluous natural disposition state that differential expression was caused by polynucleotide of interest.Segmental total size and complementary segmental big young pathbreaker depend on the desirable purposes or the application of specific nucleic acid section.The littler fragment that derives from known array can be used for hybridizing embodiment usually, and wherein the length of complementary region can change according to the complementary sequence that hope detects, for example about 10 and about 100 Nucleotide or even total length between change.

In one aspect, use has stability and the selectivity of the nucleotide probe of complementary sequence with the increase hybrid in the sheet segment limit of length greater than about 10 Nucleotide, thereby improves the specificity of the specific cross molecule that obtains.Alternatively, when needs, can design and have length and surpass about 25 or selectively surpass about 50 Nucleotide or even the longer segmental nucleic acid molecule of gene complementation.Can for example easily prepare such fragment by following manner: use the directly synthetic described fragment of chemical process, use the nucleic acid replication technology, for example at U.S. Patent number 4,603, two PCR that cause oligonucleotide of employing that describe in 102 ^TMTechnology, or selected sequence imported in the recombinant vectors to carry out recombinant production.In one aspect, probe length is about 50 to about 75 Nucleotide, or selectively about 50 to about 100 Nucleotide.Can design these probes according to the sequence of full-length gene.

In certain embodiments, thus use nucleotide sequence described herein and suitable means for example the combination of mark to detect complementary sequence be very favorable to detect hybridization.The indicator means that many kinds are suitable are well known in the art, and comprise fluorescence, radioactive, enzymatic or other parts, avidin/biotin for example, and it can produce detectable signal.People can use fluorescent mark or enzyme label for example urase, alkaline phosphatase or peroxidase to replace radioreagent or other are at undesirable reagent aspect the environment.Under the situation of enzyme label, known such colorimetric indicator substrate, its can be used for providing for human eye or on spectrophotometry de termination the visible means, to identify and the specific hybrid that comprises the sample of complementary nucleic acid.

Can under different " tight degree " conditions, carry out hybridization.Relevant condition comprises temperature, ionic strength, the time of incubation, for example existence of methane amide of other solutes in the reaction mixture, and washing procedure.Higher tight degree condition is such condition, promptly for example higher temperature and lower Na ion concentration, and it requires, and higher minimum complementarity forms stable hybridization complex between the hybridization component.The condition that increases the tight degree of hybridization is well-known, and is disclosed in the art.Referring to, people (1989) such as Sambrook for example is on seeing.

Nucleotide probe of the present invention also can be used as primer and detects the gene or the genetic transcription thing of differential expression in some bodily tissue.In addition, the homology zone that can be used for detecting the similar size that is comprised in the sequence of the peptide of identifying in primer and the coding schedule of formerly identifying 1 of mRNA of aforementioned differential expression has about at least 80% identity.For the purposes of the present invention, " amplification " be meant that use can duplicate any method of the primer dependency polysaccharase of target sequence with suitable fidelity of reproduction.For example the Klenow fragment and the reversed transcriptive enzyme of T7DNA polysaccharase, e. coli dna polymerase increase available archaeal dna polymerase natural or reorganization.

Known amplification method is people such as PCR, MacPherson, PCR:A PRACTICALAPPROACH, (IRL Press at Oxford University Press (1991)).Yet the PCR condition that is used for each application response is rule of thumb to determine.The success of many parameter influence reactions.Annealing temperature and time, extension time, Mg are wherein arranged ²⁺The relative concentration of ATP concentration, pH and primer, template and deoxyribonucleotide.

After the amplification, can pass through agarose gel electrophoresis, manifest with ethidium bromide and ultraviolet lighting then, detect the dna fragmentation of gained.Can have by the dna fragmentation that proof amplifies prediction size, show expection the restrictive diges-tion pattern and/or with correct clone's dna sequence dna hybridization, verify the specific amplification of the goal gene of differential expression.

Also can use the method known in the art that probe is attached to solid support to be used for the high flux screening assay method.PCT WO97/10365 and U.S. Patent number 5,405,783,5,412,087 and 5,445,934 for example disclose the structure of the high density oligonucleotide chip that can comprise one or more sequences disclosed herein.Use disclosed method in the U.S. Patent number 5,405,783,5,412,087 and 5,445,934, synthetic probe of the present invention on the derivatize glass surface.The nucleoside phosphoramidites of light protection is coupled to glass surface, optionally goes protection, then itself and second kind of shielded nucleoside phosphoramidites are reacted by using the photodissociation that mask carries out.Repeat this coupling/go the protection process until finishing the probe of wanting.

Also can measure the expression of gene level by nucleic acid samples is exposed to through the chip of probe modification.For example, preferably during amplification step, use the nucleic acid of fluorescence labels marker extraction.Hybridization at the sample of the suitable enterprising mark of passing through of tight degree level.Use for example Laser Scanning Confocal Microscope degree of coming quantitative measurment probe-nucleic acid hybridization of proofing unit.Referring to, U.S. Patent number 5,578,832 and 5,631,734.The measuring result and the gene expression dose positive correlation that obtain.

Probe and high density oligonucleotide probe array also provide the effective means that is used for the monitoring purposes expression of gene.They also can be used for screening the composition that raises or reduce destination gene expression.

In another embodiment, method of the present invention be used to monitor for the stimulation of determining for example cell or experimenter be exposed to medicine and make the expression of goal gene when replying, described goal gene specifically with probe hybridization of the present invention.

In one embodiment, by detecting the nucleic acid that one or more marks that are connected to sample nucleic acid detect hybridization.Can integrate mark by any method in many methods known to those skilled in the art.Yet, in one aspect, in the preparation of sample nucleic acid, during amplification step, integrate mark simultaneously.Therefore, for example, use the amplified production that will provide through the primer of mark or through the polymerase chain reaction (PCR) of the Nucleotide of mark through mark.In the embodiment of separating, as mentioned above, use is integrated into mark in the nucleic acid of transcribing out through the transcription amplification of the Nucleotide (for example, through fluorescein-labeled UTP and/or CTP) of mark.

Alternatively, mark directly can be added in the original nucleic acid samples (for example, mRNA, polyA, mRNA, cDNA etc.) or after amplification is finished, add in the amplified production.The method that mark is connected to nucleic acid is known to those skilled in the art, and (for example for example comprise nick-translation method or end-labelling, use is through the RNA of mark), it is by the described nucleic acid of phosphorylation (kinasing), the nucleic acid linker that will connect sample nucleic acid then adheres to (connection) to mark (for example, fluorophore).

Be suitable for detectable label of the present invention and comprise any composition that can detect by spectroscope, photochemistry, biological chemistry, immunochemistry, electricity, optics or chemical means.Useful in the present invention mark comprises vitamin H (it uses the avidin conjugate through mark to dye), magnetic bead (for example, Dynabeads ^TM), fluorescence dye (for example, fluorescein, texas Red, rhodamine, green fluorescent protein etc.), radio-labeled thing (for example, ³H, ¹²⁵I, ³⁵S, ¹⁴C or ³²P), for example Radioactive colloidal gold or tinted shade pearl or plastic bead (for example, polystyrene, polypropylene, latex etc.) of enzyme (for example, horseradish peroxidase, alkaline phosphatase and normally used other enzymes in ELISA) and colorimetric marker.Instruct the patent of the purposes of these marks to comprise U.S. Patent number 3,817,837,3,850,752,3,939,350,3,996,345,4,277,437,4,275,149 and 4,366,241.

The method that is used to detect these marks is known to those skilled in the art.Therefore, for example, can use photographic film or scintillometer to come detection of radioactive labels, can detect fluorescent mark by using photodetector to detect the light of launching.Usually by substrate being provided to enzyme and detecting through enzyme the reaction product that the effect of substrate produces is detected enzyme labelling and has color marker to detect the colorimetric mark by manifesting simply.

As in WO97/10365, describing in more detail, can before or after hybridization, in target (sample) nucleic acid, add mark.These are the detectable marks that directly are attached to or are integrated into target (sample) nucleic acid before hybridization.On the contrary, after hybridization, " indirect labelling " is connected to the heteroduplex body.Usually, indirect labelling is attached to the bound fraction that before hybridization, is attached to target nucleic acid.Therefore, for example, can before hybridization, carry out biotinylation to target nucleic acid.After hybridization, the fluorophore that is conjugated with avidin will be in conjunction with the heteroduplex body that has vitamin H, thereby the mark of easy detection is provided.About nucleic acid being carried out mark and detection detailed summary through the method for the nucleic acid of the hybridization of mark, referring to, LABORATORYTECHNIQUES IN BIOCHEMISTRY AND MOLECULAR BIOLOGY, the 24th volume: Hybridization with Nucleic Acid Probes, P.Tijssen, ed.Elsevier, N.Y. (1993).

Also can use method as known in the art before hybridizing with high-density probe array, for example disclosed method among the WO97/10365 is come the modification of nucleic acids sample, with the complexity of minimizing sample, thereby reduces background signal and the sensitivity that improves measurement.

Usually the result of software program analysis from the chip assay method uses a computer.Referring to, for example, EP 0,717 113 A2 and WO95/20681.With the hybridization data read-in programme, this program is calculated by the i.e. expression level of genes identified in the table 1 of the gene of target.With the data set of the gene expression dose of these data and existing ill and healthy individual relatively.Association between the data of data that obtained and disease individuality in groups shows disease takes place in being tried the patient.

Also within the application's scope, it comprises the sequence of the polynucleotide of peptide listed in one or more coding schedules 1 or its part to the database that can be used for detecting superfluous natural disposition lung tissue, and the sequence of described peptide.

These polynucleotide sequences are stored in the digital storage media, thus the data handling system that the stdn of the gene of the evaluation lung carcinoma cell that collected out is described.Have superfluous natural disposition phenotype or genotypic cell by at first selecting to suspect, then from these cellular segregation polynucleotide, this data handling system can be used for analyzing two genetic expressions between the cell.Then isolating polynucleotide are checked order.As mentioned above, use the homology search technology to compare from the sequence that exists in the sequence of sample and the database.In one aspect, at least one sequence of in being tried sequence and table 1, identifying or encode its polynucleotide or its complementary sequence between select greater than 90%, or selectively select greater than 95%, or selectively select sequence identity more than or equal to 97%, be the positive indication of from lung cancer defined above, prostate cancer or ovarian cancer cell, having isolated described polynucleotide.

Alternatively, sample and database can be compared.In brief, use method as known in the art and that for example in people such as Sambrook (1989) (on seeing), describe from the cell or tissue sample, to separate multiple RNA.Randomly, the genetic transcription thing can be transformed into cDNA.With the sampling receiving sequence specificity analyses of genetic transcription thing and quantitatively.With the comparable data storehouse sequence relative abundance of these genetic transcription thing sequence abundance and the routine data collection that comprises ill and healthy individual.The patient has the disease with the tight association of patient's data collection, comprises that the crossing of transcript of identifying express herein.

Also can determine the differential expression of goal gene by checking protein.Can obtain to be used for the various technology of protein analysis in the art.It includes but not limited to radioimmunoassay, ELISA (enzyme linked immunological radiometric determination), " sandwich " immunoassay, immunoradiometric assay, in-site detecting method (for example using Radioactive colloidal gold, enzyme or labelled with radioisotope), Western engram analysis, immunoprecipitation assay, immunofluorescence assay and PAGE-SDS.Means measuring protein level comprise that (a) provides the biological sample that comprises polypeptide; (b) measure any immunologic opsonin bonded amount that between the expression product to goal gene has component in reactive antibody and the sample, takes place, the wherein bright expressed proteinic level of immunologic opsonin bonded scale.

For these immunoassays, need identification specifically and in conjunction with the antibody of the protein of these genes.These antibody can be buied from commercial vendor, or use the method for knowing in this area to produce and screen.Referring to, people (1989) (on seeing) such as Harlow and Lane (1988) (on seeing) and Sambrook.Alternatively, can use known method to prepare polyclone or monoclonal antibody with the protein that separates specifically identification and binding purposes gene.

Be in malignant tumour, hyperplasia or the metaplasia of the differential expression of gene that at diagnostic characteristic people carry out the comparative analysis of experimenter and suitable contrast usually.Preferably, diagnostic detection comprises the control sample (being called " positive control " hereinafter) that derives from the experimenter, and described experimenter shows expection variation and purpose malignant tumour or the metaplastic Clinical symptoms in the destination gene expression.Alternatively, diagnosis comprises that also the control sample (hereinafter being called " negative control ") that derives from the experimenter, described experimenter lack the Clinical symptoms of superfluous natural disposition state and its described expression of gene level within normal range.Be relevant to the change of being identified, the positive correlation between experimenter and the positive control shows the susceptibility of existence to described disease.Lack dependency between experimenter and the negative control and proved conclusively this diagnosis.In preferred embodiments, this method is used for diagnosing based on the differential expression of goal gene the cancer of epithelial origin, for example, and lung cancer, ovarian cancer or prostate cancer.

The screening assay method

The present invention also provides screening, and it is used to identify and is used to reverse the superfluous natural disposition situation of cell or optionally suppresses the growth of above-mentioned cell or the lead compound of propagation (lead), medicine, therapeutic biotechnological formulation and method.In one aspect, described Screening and Identification can be used for treating malignant tumour, hyperplasia or the metaplastic lead compound or the biological reagent of the differential expression that is characterised in that goal gene.

Therefore, in the described method of external enforcement, at first provide suitable cell culture or tissue culture.Described cell can be cultured cells or genetically modified cell, the described cell differential expression goal gene relevant with neoplastic cell.Alternatively, described cell can be from biopsy.With described cell at certain condition (temperature, growth or developing medium and gas (CO ₂)) under cultivate and carry out reasonable time to obtain the exponential growth that no density dependency limits.Also wish the cell culture that separates that maintenance is other; Thereby it is not accept to be had a try agent with the cell culture that compares.

As being obvious to those skilled in the art, can change this method to carry out high throughput analysis, and can in microtiter plate, cultivate suitable cell and can measure several reagent simultaneously by the variation and/or the necrocytosis of writing down genotypic variation, phenotype.

When reagent was composition except DNA or RNA nucleic acid molecule, appropriate condition comprised and directly is added in the cell culture or is added in the substratum to add.As being significantly to those skilled in the art, must adding can measure by " effectively " that experience is determined.

Described screening comprises the subject cell of described reagent with the differential expression that is characterised in that goal gene is contacted that the expression level with regard to goal gene detects described cell then.In some respects, may need to be determined at the expression level that detects preceding goal gene.This provides the baseline that is used for the expression of comparison after the pair cell culture is used described reagent.In another embodiment, subject cell is the culturing cell from the clone of having set up of differential expression goal gene.If genetic expression is replied (reduce or increase) to the level that exists in the cell that is in normal or non-superfluous natural disposition state, the perhaps dead or growth velocity of showing minimizing in cell selective ground, then this reagent is possible therapeutical agent.

In yet another aspect, subject cell or tissue sample separate from the experimenter who waits to be treated, and screen one or more potential reagent to determine the therapeutical agent and/or the course of treatment best for this individual patient.

For the purposes of the present invention, " reagent " includes, but not limited to for example simple or complicated organic or inorganic molecule, peptide, protein or the oligonucleotide of compound of biological or chemical.But the vast array of synthetic compound, described compound for example are oligomer, for example oligopeptides and oligonucleotide, and based on the anthropogenics of various core textures; These compounds are also included within the term " reagent ".In addition, the compound that various natural origins can be provided for screening, for example plant or animal extracts etc.Although always do not point out clearly, should be appreciated that to use described reagent individually or with another kind of reagent that combinedly described another kind of reagent has and the identical or different biologic activity of being identified by sieve method of the present invention of reagent.Also wish described reagent and method and other therapies combined.

As used herein, the term superfluous natural disposition state of cell " reverse " is intended to comprise apoptosis, necrosis or stops the reverse of forfeiture, maturation, differentiation or superfluous natural disposition phenotype described herein of tumour generating ability, the drug resistance of fissional any other means, minimizing.As noted above, handle the pneumonocyte of differential expression suitably with the goal gene that causes superfluous natural disposition state with this method.Can identify these cells by any method as known in the art, described method allows to identify the differential expression of described gene.

When described reagent is nucleic acid, can it be added in the cell culture by method as known in the art, described method includes, but not limited to calcium phosphate precipitation, microinjection or electroporation.Alternatively or additionally, nucleic acid can be integrated into and express or insert in the carrier to be integrated in the cell.Comprise promotor and know in the art, and be briefly described below with the carrier that polynucleotide effectively can be connected into the cloning site in it.

Use the method for knowing in this area that polynucleotide are inserted in the vector gene group.For example, can under appropriate condition inset and carrier DNA be contacted with restriction enzyme to produce complementary end on each molecule, it can match mutually, and couples together by ligase enzyme.Alternatively, synthetic nucleic acid linker can be connected to end through the polynucleotide of restriction enzyme digestion.These synthetic linkers comprise the nucleotide sequence corresponding to the specific limited site in the carrier DNA.In addition, can connect the oligonucleotide that comprises terminator codon and suitable restriction site to insert in the carrier, described carrier comprises, for example, some or all of following parts: selectable marker gene for example is used for the neomycin gene at the stable or temporary transient transfectant of mammalian cell selection; Enhancers/promoters sequence from the instant early gene of people CMV is used for high level and transcribes; From transcription termination signal and the RNA processing signal of SV40, be used for the stability of mRNA; SV40 polyoma replication orgin and ColE1 are used for correct episomal replication; The multi-usage multiple clone site; And T7 and SP6 RNA promotor, be used to have the in-vitro transcription of justice and sense-rna.Other means be in the art know and be obtainable.

Can determine whether to realize the purpose of present method by the minimizing of cell fission minimizing, cytodifferentiation or mensuration gene overexpression, promptly reverse the superfluous natural disposition state of cell.Can or lose and monitor cytodifferentiation by Histological method or the existence by monitoring some cell surface marker, described cell surface marker may be relevant with undifferentiated phenotype (for example expression of goal gene).

Also claimed test kit, it comprises and carries out screening described herein and necessary reagent of in vitro method and specification sheets.

When the experimenter is an animal for example when rat or mouse, described method provides animal model system easily, and described animal model system can use before the clinical trial of therapeutical agent, or selectively is used for lead optimization (lead optimization).In this system, if genetic expression returns back to normal level, improve if perhaps compare with untreated animal with pathological cells separately with the existence symptom relevant or that be associated of the cell of the differential expression that comprises goal gene, candidate agent is the potential medicine so.The negative control group of separating with cell or animal (it is healthy or is not subject to processing) also is useful, and it provides basis relatively.

Methods of treatment

Include, but not limited to small molecules, polynucleotide, peptide, antibody, antigen presenting cell and comprise identification specifically and the immune effector cell of the cell of cracking expression goal gene by therapeutical agent provided by the invention.Can determine to use reagent whether will treat experimenter or patient valuably by the reagent that screens one or more antitumor cells, described tumour cell is by using method as known in the art isolated cells from experimenter or patient.Additive method is provided hereinafter.

In one embodiment, with for the cancer of treatment epithelial origin for example lung cancer, ovarian cancer, mammary cancer, prostate cancer and the colorectal carcinoma effectively amount use described therapeutical agent.Therapeutical agent of the present invention also can be used for preventing before canceration or non-malignant state advances to superfluous natural disposition or malignant state.

Various delivery systems are known, and can be used for using therapeutical agent of the present invention, for example be encapsulated in liposome, particulate, the microcapsule, express by reconstitution cell, receptor mediated endocytosis is (referring to for example, Wu and Wu (1987) J.Biol.Chem.262:4429-4432), therapeutic nucleic acids is built into the part of retroviral vector or other carriers, or the like.The method of sending includes but not limited in intra-arterial, intramuscular, intravenously, the nose and oral route.In specific embodiment, can wish pharmaceutical composition of the present invention is applied to the zone that needs treatment partly; This can by for example (but indefiniteness) at intra-operative by injection or carry out local infusion by means of conduit and realize.

Can effective agents be administered to experimenter or individuality for its desirable purpose with being accredited as herein, described experimenter or individually be easy to the disease relevant with the differential expression of goal gene take place or be in such risk.When described agent administration being given the experimenter for example when mouse, rat or people patient, described reagent can be added to pharmaceutically acceptable carrier and general or be administered to the experimenter partly.In one aspect, be to determine to obtain the patient of useful treatment, take out tumor sample on one's body and measure with regard to the differential expression pair cell of goal gene from described patient.Therapeutic dose can be determined by experience, and will change with the pathologic state of being treated, the experimenter who is treated and the effect and the toxicity of reagent.When being delivered to animal, described method can be used for further verifying the effect of reagent.As the example of animal model, and nude mice in groups (Balb/c NCR nu/nu is female, Simonsen, and Gilroy, CA) each uses by oneself about 10 ⁵To about 10 ⁹The individual cancer or the target cell of the hyperplasia of definition herein carry out subcutaneous vaccination.When forming tumour, use this reagent by for example subcutaneous injection around tumour.Weekly twice, use vernier callipers on two dimension, to carry out measurement of tumor to determine the minimizing of tumour size.In the time of suitably, also can use other animal models.

In the whole course of treatment, can be in potion, carry out using in the body continuously or off and on.Be used for the most effective definite method of using means and dosage and know to those skilled in the art, and will change with the purpose of the composition that is used for the treatment of, treatment, the target cell of being treated and the experimenter who is treated.Selected dosage level of available treatment doctor and pattern are carried out single or multiple and are used.Can find suitable formulation below and use described compositions and methods.

Reagent of the present invention and composition can be used for preparing medicine and are used for the treatment of people or other animals by for example activeconstituents being formulated in to use in the pharmaceutical composition according to ordinary method.

Described pharmaceutical composition can by in oral, the nose, parenteral route or use by anapnotherapy, and can adopt tablet, lozenge, granule, capsule, pill, ampulla, suppository or aerosol form.They can also adopt suspension, solution and the emulsion of activeconstituents in water-based or non-aqueous thinner, syrup, the form of granula or pulvis.Except reagent of the present invention, pharmaceutical composition also can comprise other pharmaceutically active compounds or multiple composition of the present invention.

More particularly, can be by any suitable way, comprise the approach of oral, rectum, nose, part (comprising), vagina, parenteral (comprising subcutaneous, intramuscular, intravenously and intracutaneous) and lung, use reagent of the present invention (being also referred to as activeconstituents herein) to treat through skin, aerosol, cheek and hypogloeeis.Will recognize that also preferred approach can change with recipient's situation and age and the disease of being treated.

Ideally, should use reagent to obtain the peak concentration of active compound in the site of disease.This can for example realize by following manner: intravenous injection reagent (randomly in salt solution), or Orally administered reagent, and for example as the tablet, capsule or the syrup that comprise activeconstituents.The desired blood level that can keep reagent by the successive infusion is to provide the activeconstituents of therapeutic dose in illing tissue.Effectively combination (operative combinations) is considered to provide the therapeutic combination, the total dose of needed each the Anti-virus agent component of described therapeutic combination is compared lower with needed dosage when using each single therapeutic compound or medicine individually, thereby has reduced undesirable action.

Although can use described reagent individually, preferably the form with pharmaceutical preparation provides it, and described pharmaceutical preparation comprises at least a activeconstituents defined above and for this reason one or more pharmaceutically acceptable carriers and randomly other treatment agent.Each carrier with the meaning of other component compatibility of preparation on must be " acceptable ", and harmless to the patient.

Preparation comprises and is suitable for those preparations that oral, rectum, nose, part (comprising through skin, cheek and hypogloeeis), vagina, parenteral (comprising subcutaneous, intramuscular, intravenously and intracutaneous) and lung are used.Preparation can provide and can be by any method preparation of knowing in the pharmaceutical field easily with unit dosage.These methods comprise makes activeconstituents and the carrier step linked together that constitutes one or more Synergist S-421 95s.Usually, prepare described preparation in the following way: make activeconstituents evenly and closely linked together, then if desired, moulding to product with the solid carrier of liquid vehicle or segmentation or both.

Be suitable for Orally administered preparation of the present invention and can be provided as unit separately for example capsule, cachet or tablet, the activeconstituents of each self-contained predetermined amount; Pulvis or granule; Solution in water-based or non-aqueous liquid or suspension agent; Perhaps oil-in-water liq emulsion or water-in-oil-type liquid emulsion.Can also bolus, the form of electuary or paste provides described activeconstituents.

Can be by randomly suppressing or the molded tablet for preparing with one or more Synergist S-421 95s.Can prepare compressed tablets by compacting activeconstituents in suitable machine, for example powder or particulate form exist described activeconstituents with free-pouring form, and randomly with tackiness agent (for example, polyvinylpyrrolidone, gelatin, Vltra tears), lubricant, inert diluent, sanitas, disintegrating agent (for example, primojel, crosslinked polyvinylpyrrolidone, crosslinked Xylo-Mucine), tensio-active agent or dispersant.Can be by in suitable machine, the mixture with the moistening powder compound of inert liquid diluent being carried out the molded molded tablet for preparing.Described tablet randomly can carry out dressing or cut, and available for example Vltra tears prepares so that required release characteristics to be provided with different ratios, thereby the slowly-releasing or the controlled release of activeconstituents herein are provided.Randomly, can provide enteric coating with in intestines rather than release is provided under one's belt to tablet.

Be suitable for that the preparation of topical application comprises the lozenge that includes the activeconstituents in flavoured base (being generally sucrose and gum arabic or tragacanth gum) in mouth; Include at the inert base pastille of the activeconstituents in gelatin and glycerine or sucrose and the gum arabic for example; With the mouth wash shua that includes the activeconstituents in suitable liquid vehicle.

The pharmaceutical composition that is used for topical application of the present invention can be formulated as ointment, ointment, suspension, lotion, pulvis, solution, paste, gel, sprays, aerosol or oil.Alternatively, preparation can comprise and is impregnated with activeconstituents and for example bandage or adhesivity plaster of the patch of one or more vehicle or thinner or dressing randomly.

If want, the water of emulsifiable paste matrix for example can comprise at least approximately polyvalent alcohol of 30%w/w, promptly has the alcohol of two or more hydroxyls, for example propylene glycol, butane-1,3-glycol, N.F,USP MANNITOL, sorbyl alcohol, glycerine and polyoxyethylene glycol with and composition thereof.If wish, topical formulations can comprise the described reagent of enhancing by skin or the absorption of other involved areas or the compound of infiltration.The example of these skin penetration enhancers comprises methyl-sulphoxide and relevant analogue.

The oil phase of emulsion of the present invention can constitute from known composition in a known way.Although this can only comprise emulsifying agent mutually, if wish its can comprise at least a emulsifying agent and fat or oil or with fat and both mixture of oil.Preferably, hydrophilic emulsifier is included in the lipophilic emulsifier with used as stabilizers.Both also are preferred to comprise oil ﹠ fat.In a word, the emulsifying agent that has or do not have stablizer has been formed so-called emulsifying wax, and described wax and oil and/or fat have been formed so-called emulsification ointment base, and it has formed the oily disperse phase of cream formulation.

The emulsifying agent and the emulsion stabilizer that are suitable for using in preparation of the present invention comprise Tween60, Span 80, cetostearyl alcohol (cetostearyl alcohol), tetradecyl alcohol, glyceryl monostearate and Sodium Lauryl Sulphate BP/USP.

Be appropriate to the oil of described preparation or fatty selection based on the cosmetic property that obtains to want, because active compound is very low in the solubleness of most of oil that may be used for the pharmaceutical emulsion preparation.Therefore, emulsifiable paste preferably should right and wrong oleaginous (non-greasy), non-chromatic (non-staining) and rinsable product, and it has suitable denseness to avoid seepage from pipe or other containers.Can use straight chain or branching, one or bibasic alkyl ester for example two dissidents, two acid esters, the different hexadecanol ester of stearic acid, coconut fatty acid propylene glycol diesters, Isopropyl myristate, decyl oleate, Wickenol 111, butyl stearate, palmitinic acid 2-(ethyl hexyl) ester or be called the mixture of the branched chain ester of Crodamol CAP, last three kinds is preferred ester.Depend on needed characteristic, can be individually or use these esters in combination.Alternatively, can use the high-melting-point lipid, for example white soft wax and/or whiteruss or other mineral oil.

Be suitable for also comprising eye drops to the preparation of eyes topical application, wherein said activeconstituents is dissolved in or is suspended in the suitable carriers (especially for the aqueous solvent of described reagent).

Can be provided for the preparation of rectal administration with suppository form with suitable matrix (comprising for example theobroma oil or salicylate).

Can be that the form of suppository, tampon, emulsifiable paste, gel, paste, foaming agent or the sprays preparation of suitable carriers provides the preparation that is suitable for vaginal application except described reagent, also to comprise known in the art.

Be suitable for the preparation (wherein said carrier is a solid) that nose uses and comprise having for example about 20 dust bases to about 500 microns granular size, it is used by this way, promptly wherein adopt snuffing, just by from take the dust container of nearly nose, sucking fast through nasal meatus.Wherein carrier is liquid and the aqueous solution or oil solution that be suitable for using or comprising by the preparation that atomizer is used with aerosol with for example form of nasal mist, nasal drop described reagent.

The preparation that is suitable for parenteral administration comprises, water-based and non-aqueous isotonic sterile injection liquid, and it can comprise antioxidant, buffer reagent, bacteriostatic agent and make preparation and the isoosmotic solute of desirable recipient's blood; With water-based and non-aqueous sterile suspensions, it can comprise suspension agent, thickening material and liposome or be designed to other microparticulate systems with described targeting compounds blood constitutent or one or more organs.Described preparation can for example provide in ampoule and the bottle at the container of the sealing of unitary dose or multiple doses, and can store under cryodesiccated (freeze dried) condition, only needs for example water for injection of adding sterile liquid carrier before being about to use.Can be from the interim injection solution of sterile powder, granule and tablet preparation and the suspension of type noted earlier.

Preferred unit dose formulations is to comprise the per daily dose of reagent or the preparation of unit, day sub-doses (as recited above) or its suitable part herein.

Transgenic animal

In yet another aspect, goal gene can be used for producing transgenic animal model.In recent years, the geneticist successfully imports by the embryo's that growing of operation gene and with foreign gene and has produced for example mouse of transgenic animal among these embryos.In case these gene integrations are gone in recipient embryo's the genome, just can analyze the embryo of gained or adult animals to determine the function of described gene.Produce the mutant animal to understand known function in vivo and the animal model of setting up human diseases.(referring to for example, people such as Chisaka (1992) 355:516-520; People such as Joyner (1992), POSTIMPLANTATION DEVELOPMENT IN THE MOUSE (Chadwick and Marsh, editor, John Wiley﹠amp; Sons, United Kingdom) pp:277-297; People such as Dorin (1992) Nature 359:211-215).

U.S. Patent number 5,464,764 and 5,487,992 have described one type transgenic animal, goal gene are enough to destroy the disappearance or the sudden change of its function in described transgenic animal.(also can referring to, U.S. Patent number 5,631,153 and 5,627,059).These can be used for studying specific gene sequence function in vivo by " knocking out " animal that the phenomenon of utilizing homologous recombination produces.Polynucleotide sequence described herein can be used for preparing the animal model of lung cancer.Experimental technique

Experiment 1: expression analysis

Nonsmall-cell lung cancer has been represented huge unsatisfied medical science needs generally.From the individual patient of non-communicable disease, obtain fresh not downright bad NSCLC tumor tissues and corresponding healthy tissues.The flesh tissue sample is the tissue of at least 1.5 grams, and it is no more than 24 hours from the back of performing the operation in wet transportation on ice.Before analyzing, with regard to per-cent tumour content, tumour stage and histological type this tissue is assessed by the pathologist.The tissue of all acceptance is I, II or IIIa phase primary lung cancer, and wherein operation is initial or primary treatment.

After receiving tissue, sample (tissue) is shredded with the cruciform scalpel.The tissue of handling chopping with collagenase and elastoser is up to obtaining single-cell suspension liquid.Washing single-cell suspension liquid several, and splitting erythrocyte.Remove leucocyte-removing by the antibody that cell suspending liquid is exposed to the anti-CD 64, CD45 and the CD14 that are connected to magnetic bead.The anti-BerEP4 antibody that use is connected to magnetic bead separates epithelial cell.The anti-CD31 antibody that use is connected to magnetic bead separates endotheliocyte.Immediately from epithelial cell and endotheliocyte specimen preparation RNA.

Estimate character generally by the expression of the specific marker for described cell type from the RNA of epithelium and endotheliocyte.Cytokeratin 18, Feng's von willebrand's factor, EF1, P1H12, hevin and cytokeratin 8 usefulness are marked to be used for determining whether collected RNA represents the relative pure epithelial cell or the cell colony of endotheliocyte.After the assessment of the character of RNA, 4 parts of squama cancer samples, 2 parts of gland cancer samples and 3 parts of normal lung tissue's samples are in quantity and be enough to carry out SAGE in nature and analyze.

Disclosed method is carried out LongSAGE to 9 RNA samples among use Nature Biotechnology (2002) 20:508-512 ^TMUntil the degree of depth for about 50,000 labels in each library.Carry out completely bioinformatic analysis characterizing the SAGE data, based on comparing increase with the expression of mRNA in squama cancer and gland cancer with the normal lung cell between tumor type.For potential Antybody therapy target, focus concentrates in plasma membrane on the expressed protein.

GITR, the TNFR associated protein of glucocorticoid inducible, known to activated T cells and Treg cell expressing.GITR is also referred to as activation-inducing type TNRF family receptors (AITR) and tumor necrosis factor receptor super family member 18 (TNFRSF-18) (Stephens, G.L. wait people (2004) J.Immunol.173:5008-5020 and Nocentini, people such as G. (1997) P.N.A.S.94:6216-6221).The GITR part causes NF-κ B activation in conjunction with GITR and by TRAF2.The GITR-GITR ligand interaction has destroyed the apoptosis of TCR-CD3 activation-inducing in the T cell, and may participate in cell survival.The GITR part is also referred to as AITRL, GITRL, TL6 and hGIRTL.GITR is 228 amino acid whose transmembrane proteins that are considered to similar with CD27 to 4-1BB.GITR albumen has that 19 amino acid whose signal sequences, 134 are amino acid whose to have 3 and be rich in the zone, extracellular of the primitive of halfcystine, 23 amino acid whose TMDs and 52 amino acid whose cytoplasmic structure territories.The GITR part is expressed (Stephens is on people such as G. (2004) see) by endotheliocyte (comprising HUVEC), B1 lymphocyte, maturation and immature dendritic cell and scavenger cell.The regulation and control of the necrocytosis of interaction between GITR participation T-lymphocyte and the endotheliocyte and T-cell receptors mediation.GITR is by the activation of TRAF2/NIK approach mediation NF-κ B.GITR in conjunction with TNF acceptor correlation factor-1 (TNF receptor-associated factor-1, TRAF1), TRAF2 and TRAF3, but debond TRAF5 and TRAF6 (Nocentini is on people such as G. (1997) see).

GITR expresses on the CD4+CD25+T cell, and reduces T modulability inhibition activity after interacting with GITRL.But GITR on the target tumor cell and the effect (Kohm that exhausts CD4+CD25+T cell enhanced activity tomour specific Sex therapy, A.P. wait people (2004) J.Immunol.172:4686-4690, and Shimizu, people such as J. (2002) Nature Immunol.3:135-142).

The RT-PCR that organizes RNA in a large number from 55 lung tumors and 18 normal lung tissues shows, compares with healthy tissues, and the level of GITR RNA increases 〉=2 times in 76% tumour.According to RT-PCR, GITR comprises having very MIN expression in mammary gland, prostate gland, brain, the heart, kidney, liver, sialisterium, spleen, stomach, thymus gland and the uterus in multiple healthy tissues.The RT-PCR that organizes RNA in a large number from kinds of tumors and corresponding healthy tissues shows, 〉=2 times of the level increases of GITR RNA in the ovarian cancer 50% (n=40), 25% melanoma (n=22), 50% prostate cancer (n=24), 20% colorectal carcinoma (n=26) and 66% the mammary cancer (n=23).

To carrying out immunohistochemical analysis through formalin fixed and through paraffin-embedded people's nonsmall-cell lung cancer sample.Used antibody is anti-GITR (Research Systems, BA689), anti-RDC1 (Lifescience, RDC1-LP1439), anti-CD31 (DAKO, M0823), anti-α-smooth muscle actin (DAKO, M0851), anti-EMA (DAKO, M0804) and anti-wide spectrum cytokeratin (DAKO, antibody Z0622).

Generally speaking, on the tumour cell of the gland cancer of lung and squama cancer, there is strong GITR reactivity.Also there is strong reactivity on the wetting property cell in the tumor stroma of the gland cancer of lung and squama cancer.On the tumour cell of the gland cancer of lung and squama cancer, there is strong RDC1 reactivity.The RDC1 reactivity that on the endotheliocyte that is associated with tumor vessels and pericyte/smooth muscle cell, has moderate.

By fluorescence activated cell metering art (FACS), NCI-H358 and NCI-H1436 clone have the expression of extraordinary GITR, and NCI-H1299, NCI-H522, NCI-H23 and NCI-H647 have the expression of good GITR.By FACS, the expression that the expression ratio of the GITR on activated PBMC and activated CD3+T cell is fastened at non-small cell lung cancer cell is much lower.

Experiment 2: functional living being is measured

After generation, use and to screen the antibody group, with by using method as known in the art to identify the function of the described target protein of neutralization and the antibody of reversion of malignant phenotype based on the assay method of cell.Explanation for example, these methods are described in: Stanton, people such as C.A. (2004) Blood103 (2): 601-606 and Malinda, people such as K.M. (1999) Exp.Cell Res.250:168-173 (migration assay); [0210] Duan Zhidi [0226] section (apoptosis) of U.S. Patent application 2004/0253708A1; [0183] Duan Zhidi [0194] section (inhibition, antiproliferative, blocking-up and epi-position binding assay) of U.S. Patent application 2004/0258685A1; Stanton, people such as C.A. (2004) (on seeing) (propagation and cytotoxicity assay); Manches, 0. people such as grade (2003) Blood 101 (3): 949-954 (apoptosis, engulf assay method) with ADCC; [0066] section (CDC assay method) with U.S. Patent application 2004/0228859A1.

Experiment 3: effect in the body

Then, use syngeneic tumor model or human tumor xenograft model, further screen for example to have with regard to effect in the body and testing the essential bioactive antibody of describing in 2.Such assay method and model are known to those skilled in the art.Explanation for example, these methods are described in Tumor Models in Cancer Research, Teicher, B.A., editor, book series Cancer Drug Discovery and Development, Humana Press, 2004; With people (2004) PNAS 101 (24): 9051-9056 such as Lev.A..

Although above-mentioned experiment and detailed description are to be described about the application at NSCLC, should be apparent that to those skilled in the art method and composition of the present invention is relevant with other cancers of evaluation in the table 1.Therefore, the present invention also provides the method and composition that is used to diagnose with these malignant tumours of prognosis.

Though be appreciated that and described the present invention in conjunction with above-mentioned embodiment, the description of front and the embodiment of back are intended to illustrate rather than limit the scope of the invention.Other aspects, advantage and change within the scope of the invention will be tangible the those of skill in the art under the present invention.

Sequence list

SEQ ID NO：1

1 atggcacagc acggggcgat gggcgcgttt cgggccctgt gcggcctggc gctgctgtgc

61 gcgctcagcc tgggtcagcg ccccaccggg ggtcccgggt gcggccctgg gcgcctcctg

121 cttgggacgg gaacggacgc gcgctgctgc cgggttcaca cgacgcgctg ctgccgcgat

181 tacccgggcg aggagtgctg ttccgagtgg gactgcatgt gtgtccagcc tgaattccac

241 tgcggagacc cttgctgcac gacctgccgg caccaccctt gtcccccagg ccagggggta

301 cagtcccagg ggaaattcag ttttggcttc cagtgtatcg actgtgcctc. ggggaccttc

361 tccgggggcc acgaaggcca ctgcaaacct tggacagact gcacccagtt cgggtttctc

421 actgtgttcc ctgggaacaa gacccacaac gctgtgtgcg tcccagggtc cccgccggca

481 gagccgcttg ggtggctgac cgtcgtcctc ctggccgtgg ccgcctgcgt cctcctcctg

541 acctcggccc agcttggact gcacatctgg cagctgagga agacccagct gctgctggag

601 gtgccgccgt cgaccgaaga cgccagaagc tgccagttcc ccgaggaaga gcggggcgag

661 cgatcggcag aggagaaggg gcggctggga gacctgtggg tgtga

SEQ ID NO：2

MAQHGAMGAFRALCGLALLCALSLGQRPTGGPGCGPGRLLLGTGTDARCCRVHTTRCCRDYPGEECCSE

WDCMCVQPEFHCGDPCCTTCRHHPCPPGQGVQSQGKFSFGFQCIDCASGTFSGGHEGHCKPWTDCTQFG

FLTVFPGNKTHNAVCVPGSPPAEPLGWLTVVLLAVAACVLLLTSAQLGLHIWQLRKTQLLLEVPPSTED

ARSCQFPEEERGERSAEEKGRLGDLWV

<110>Genzyme Corporation

Teicher，Beverly A.

Roberts，Bruce L.

Shankara，Srinivas

＜120〉be used for the GITR antibody of the diagnosis of NSCLC

<130>5297 PCT

<140>PCT

<141>2006-01-19

<150>US 60/645,349

<151>2005-01-19

<160>2

<170>PatentIn version 3.3

<210>1

<211>705

<212>DNA

＜213〉homo sapiens

<400>1

atggcacagc acggggcgat gggcgcgttt cgggccctgt gcggcctggc gctgctgtgc 60

gcgctcagcc tgggtcagcg ccccaccggg ggtcccgggt gcggccctgg gcgcctcctg 120

cttgggacgg gaacggacgc gcgctgctgc cgggttcaca cgacgcgctg ctgccgcgat 180

tacccgggcg aggagtgctg ttccgagtgg gactgcatgt gtgtccagcc tgaattccac 240

tgcggagacc cttgctgcac gacctgccgg caccaccctt gtcccccagg ccagggggta 300

cagtcccagg ggaaattcag ttttggcttc cagtgtatcg actgtgcctc ggggaccttc 360

tccgggggcc acgaaggcca ctgcaaacct tggacagact gcacccagtt cgggtttctc 420

actgtgttcc ctgggaacaa gacccacaac gctgtgtgcg tcccagggtc cccgccggca 480

gagccgcttg ggtggctgac cgtcgtcctc ctggccgtgg ccgcctgcgt cctcctcctg 540

acctcggccc agcttggact gcacatctgg cagctgagga agacccagct gctgctggag 600

gtgccgccgt cgaccgaaga cgccagaagc tgccagttcc ccgaggaaga gcggggcgag 660

cgatcggcag aggagaaggg gcggctggga gacctgtggg tgtga 705

<210>2

<211>234

<212>PRT

＜213〉homo sapiens

<400>2

Met Ala Gln His Gly Ala Met Gly Ala Phe Arg Ala Leu Cys Gly Leu

1 5 10 15

Ala Leu Leu Cys Ala Leu Ser Leu Gly Gln Arg Pro Thr Gly Gly Pro

20 25 30

Gly Cys Gly Pro Gly Arg Leu Leu Leu Gly Thr Gly Thr Asp Ala Arg

35 40 45

Cys Cys Arg Val His Thr Thr Arg Cys Cys Arg Asp Tyr Pro Gly Glu

50 55 60

Glu Cys Cys Ser Glu Trp Asp Cys Met Cys Val Gln Pro Glu Phe His

65 70 75 80

Cys Gly Asp Pro Cys Cys Thr Thr Cys Arg His His Pro Cys Pro Pro

85 90 95

Gly Gln Gly Val Gln Ser Gln Gly Lys Phe Ser Phe Gly Phe Gln Cys

100 105 110

Ile Asp Cys Ala Ser Gly Thr Phe Ser Gly Gly His Glu Gly His Cys

115 120 125

Lys Pro Trp Thr Asp Cys Thr Gln Phe Gly Phe Leu Thr Val Phe Pro

130 135 140

Gly Asn Lys Thr His Asn Ala Val Cys Val Pro Gly Ser Pro Pro Ala

145 150 155 160

Glu Pro Leu Gly Trp Leu Thr Val Val Leu Leu Ala Val Ala Ala Cys

165 170 175

Val Leu Leu Leu Thr Ser Ala Gln Leu Gly Leu His Ile Trp Gln Leu

180 185 190

Arg Lys Thr Gln Leu Leu Leu Glu Val Pro Pro Ser Thr Glu Asp Ala

195 200 205

Arg Ser Cys Gln Phe Pro Glu Glu Glu Arg Gly Glu Arg Ser Ala Glu

210 215 220

Glu Lys Gly Arg Leu Gly Asp Leu Trp Val

225 230

Claims

1. be used for diagnosis or prognosis method for cancer, described cancer is selected from nonsmall-cell lung cancer (NSCLC), ovarian cancer, mammary cancer, prostate cancer and colorectal carcinoma, described method comprises detection institute's genes identified or gene product or its segmental expression level, wherein positive diagnosis or prognosis of expressing the predictability that is cancer described in the experimenter excessively of gene or gene product in table 1 in the experimenter.

2. the process of claim 1 wherein and measure the segmental expression level of described gene or gene product or its by immuno-chemical method.

3. the method for claim 2 wherein uses antibody to measure the segmental expression level of described gene or gene product or its by immuno-chemical method.

4. the process of claim 1 wherein and use monoclonal antibody, its variant or derivative to measure the segmental expression level of described gene or gene product or its by immuno-chemical method.

5. the process of claim 1 wherein that described antibody is selected from monoclonal antibody, polyclonal antibody, antibody variants, antibody derivatives, humanized antibody and antibody fragment by using the described gene of TPPA or gene product or its segmental expression level.

6. the process of claim 1 wherein the expression level of measuring described gene or gene product by the amount that detects polynucleotide.

7. the process of claim 1 wherein to detecting from the isolating suitable sample of experimenter.

8. the method for claim 7, wherein said suitable sample are tissue sample, biopsy sample or the humoral samples of preserving.

9. the method for claim 7, wherein said suitable sample is the sample that is selected from the body fluid of urine, blood and serum.

10. the method for claim 6, wherein said polynucleotide are selected from mRNA and cDNA.