CN101208431A

CN101208431A - Compositions and methods for the diagnosis and treatment of tumor

Info

Publication number: CN101208431A
Application number: CNA2006800198001A
Authority: CN
Inventors: 帕特里克·多德; 格雷奇恩·弗朗兹; 保罗·波拉基斯; 维多利亚·史密斯; 苏珊·斯潘塞; 吴棣; 张泽民
Original assignee: Genentech Inc
Current assignee: Genentech Inc
Priority date: 2005-04-08
Filing date: 2006-04-05
Publication date: 2008-06-25
Also published as: CR9486A; MX2007012216A; CA2604377A1; BRPI0609729A2; RU2007141407A; AU2006235436A2; MA29587B1; NO20075698L; JP2008535498A; AU2006235436A1; IL185900A0; EP1871886A2; ZA200707967B; KR20080003405A

Abstract

The present invention is directed to compositions of matter useful for the diagnosis and treatment of tumor in mammals and to methods of using those compositions of matter for the same.

Description

Composition and method for tumor diagnosis and therapy

Related application

The application according to 35 U.S.C. § 120 be submit on June 19th, 2002, serial number 10/177, the part continuation application of 488 U.S. Patent application simultaneously requires its priority, the latter requires that on June 20th, 2001 is submitting, serial number 60/299 according to 35 U.S.C. § 119 (e), the priority of 500 U.S. Provisional Patent Application, takes in the disclosure of which and is used as reference herein.

Invention field

This invention address that diagnosing and treating the method for the tumour in mammal available for the composition for diagnosing and treating the tumour in mammal and using these compositions.

Background of invention

Malignant tumour (cancer) is that the second cause of death (Boring et al., the CA Cancel J.Clin.43 after heart disease are occupied in the U.S.：7(1993)).The feature of cancer is the exception derived from normal structure or the increase of the quantity of neoplastic cell, and the cell breeds to form tumor mass；These neoplasm cells invade adjacent tissue；And malignant cell is produced, these malignant cells are final to be traveled to regional nodes through blood or lymphatic system and is traveled at a distance by the process referred to as " shifted ".In cancerous condition, cell is bred under conditions of normal cell does not grow.Cancer manifests itself by the extremely diversified forms being characterized with different degrees of invasion and offensiveness.

During attempting to find the effective cell target of cancer diagnosis and treatment, researcher attempts identification specific expressed transmembrane polypeptide or polypeptide otherwise related to film compared with one or more normal non-cancerous cells on the surface of one or more certain types of cancer cells.Generally, amount of such membrane-associated polypeptide than being expressed on the surface of non-cancerous cell on the surface of cancer cell is bigger.The identification of such Tumor-associated cell surface antigen polypeptide to become possibility by the selectively targeted destruction cancer cell of the therapy based on antibody.In this regard it is noted that it is very effective in the treatment of some cancers that the therapy based on antibody is verified.For example, HERCEPTIN  and RITUXAN  (both being from GenentechInc., South San Francisco, California) are the antibody for being used successfully to treat breast cancer and non-Hodgkin's (Hodgkin) lymthoma respectively.In particular, HERCEPTIN  are the extracellular domains of its selective binding human epidermal growth factor receptor 2 (HER2) proto-oncogene from Humanized monoclonal antibodies derived from recombinant DNA.HER2 protein overexpressions are observed in 25-30% primary breast cancer.RITUXAN  are genetic engineering Chi-meric mice/human monoclonal antibodies, and it is directed in CD20 antigens present on the surface of normal and pernicious bone-marrow-derived lymphocyte.Both antibody are all the recombinant productions in Chinese hamster ovary celI.

In other trials of effective cell target of cancer diagnosis and treatment are found, researcher attempts to identify (1) by one or more certain types of cancer cells non-membrane-associated polypeptide that specificity is produced compared with one or more certain types of non-cancerous cells, (2) to be significantly higher than the polypeptide that the expression of one or more normal non-cancerous cells is produced by cancer cell, or the special polypeptide for being limited to single (or difference of the very limited number) organization type (such as normal prostate and prostate tumor tissue) in carcinous and non-cancerous state of (3) its expression.Such polypeptide can keep intracellular targeting, or can be by cancer cells secrete.In addition, such polypeptide can not be by cancer cell oneself expression, but the cell secretion that there is the polypeptide strengthened (potentiating) or grow enhancing effect to cancer cell by producing and/or secreting.Such secrete polypeptide typically provides the protein of the growth vigor more than normal cell, including such as material such as angiogenesis factor, CAF, growth factor for cancer cell.The identification of the antagonist of such non-membrane-associated polypeptide is expected to serve as effective therapeutic agent of such treatment of cancer.In addition, the particular cancers that the identification of the expression pattern of such polypeptide can be used in diagnosis mammal.

Although there is above-mentioned progress in mammalian cancer therapy, for can detect respectively in mammal the presence of tumour and the other diagnosis of effective inhibition of enoplastic cell growth and therapeutic agent still have it is very big the need for.Therefore, an object of the invention is identification：(1) a greater amount of cell membrane-associated polypeptides are expressed compared with normal cell or on other different cancer cells on the cancer cell of one or more types, (2) by one or more certain types of cancer cell (or by producing other cells of polypeptide of the growth to cancer cell with reinforcing effect) non-membrane-associated polypeptides that specificity is produced compared with one or more certain types of non-cancerous normal cells, (3) to be significantly higher than the non-membrane-associated polypeptide that the expression of one or more normal non-cancerous cells is produced by cancer cell, or (4) its expression specificity is limited to the polypeptide of carcinous single (or only a few the is different) organization type (such as normal prostate and prostate tumor tissue) with non-cancerous state, and the composition of the therapeutic treatment and diagnostic assays available for the cancer in mammal is generated using those polypeptides and its code nucleic acid.Another target of the present invention is to identify that its expression is limited to cell membrane-associated polypeptide, secrete polypeptide or the intracellular polypeptide of single or only a few tissue, and generates using those polypeptides and its code nucleic acid the composition of matter of the therapeutic treatment and diagnostic assays available for the cancer in mammal.

Summary of the invention

A. embodiment

In this manual, applicant describes the identification of various kinds of cell polypeptide (and its code nucleic acid or its fragment) first, and the degree that these cell polypeptides are expressed on the surface of the cancer cell of one or more types or expressed by the cancer cell of one or more types is higher than the degree that they express on the surface of the normal noncancerous cells of one or more types or expressed by the normal noncancerous cells of one or more types.Or, such polypeptide is expressed by producing and/or secreting the cell to cancer cell with the polypeptide strengthened or grow enhancing effect.Or, such polypeptide may be overexpressed compared with the normal cell of identical organization type by tumour cell, but may both normal cells and tumour cell by the organization type (for preferred pair life not required tissue, such as prostate) of single or very limited number it is specific expressed.All aforementioned polypeptides are referred to herein as tumor associated antigen target polypeptide (Tumor-associatedAntigenic Target polypeptides, " TAT " polypeptide), and they are expected to serve as effective target for the treatment of of cancer and diagnosis in mammal.

Therefore, in one embodiment of the invention, the invention provides the nucleic acid molecules of separation, it has the nucleotide sequence of codes for tumor associated antigenic target polypeptide or its fragment (" TAT " polypeptide).

In some aspects, the nucleic acid molecules of the separation are included and following nucleotide sequences of any one with least about 80% nucleic acid sequence identity, or the nucleic acid sequence identity of at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%：

(a) encode

Total length TAT polypeptides with amino acid sequence disclosed herein,

The TAT polypeptid acid sequences disclosed herein for lacking signal peptide,

The extracellular domain of the cross-film TAT polypeptides disclosed herein for being with or without signal peptide or

The DNA molecular of any other fragment being specifically defined of total length TAT polypeptid acid sequences disclosed herein；Or

(b) complementary series (complement) of the DNA molecular of (a).

In other side, the nucleic acid molecules of the separation are included and following nucleotide sequences of any one with least about 80% nucleic acid sequence identity, or the nucleic acid sequence identity of at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%：

(a) include

Total length TAT polypeptides cDNA disclosed herein coded sequence,

The coded sequence of the TAT polypeptides disclosed herein for lacking signal peptide,

The coded sequence of the extracellular domain of the cross-film TAT polypeptides disclosed herein for being with or without signal peptide or

The coded sequence of any other fragment being specifically defined of total length TAT polypeptid acid sequences disclosed herein

DNA molecular；Or

(b) complementary series of the DNA molecular of (a).

In other side, the present invention relates to the nucleic acid molecules of separation, it is included and following nucleotide sequences of any one with least about 80% nucleic acid sequence identity, or the nucleic acid sequence identity of at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%：

(a) DNA molecular of mature polypeptide identical with the full length coding region coding of any human protein cDNA disclosed herein for being preserved in ATCC；Or

(b) complementary series of the DNA molecular of (a).

This paper another aspect provides the nucleic acid molecules of separation, it includes following nucleotide sequence, TAT polypeptides that the nucleotide sequence coded membrane-spanning domain is deleted or membrane-spanning domain inactivation, or it is complementary with such coding nucleotide sequence, wherein there is disclosed herein the membrane-spanning domain of such polypeptide.It is therefore contemplated that the soluble ectodomains of TAT polypeptides described herein.

In other side, the present invention relates to the nucleic acid molecules of the separation with following any one hybridization：

(a) encode

TAT polypeptides with full length amino acid sequence disclosed herein,

The TAT polypeptid acid sequences disclosed herein for lacking signal peptide,

The nucleotide sequence of any other fragment being specifically defined of total length TAT polypeptid acid sequences disclosed herein, or

(b) complementary series of the nucleotide sequence of (a).At this point, one embodiment of the invention is related to the fragment or its complementary series of total length TAT polypeptid coding sequences disclosed herein, they can be used as such as hybridization probe, the hybridization probe can be used as such as diagnostic probe, PCR primer, antisense oligonucleotide probe, or the fragment for encoding full leng TAT polypeptides, it can optionally encode the polypeptide of the binding site of the organic molecule comprising anti-TAT polypeptide antibodies, TAT combinations oligopeptides or other combination TAT polypeptides.The length of such nucleic acid fragment is generally at least about 5 nucleotides,Or length is at least about 6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,35,40,45,50,55,60,65,70,75,80,85,90,95,100,105,110,115,120,125,130,135,140,145,150,155,160,165,170,175,180,185,190,195,200,210,220,230,240,250,260,270,280,290,300,310,320,330,340,350,360,370,380,390,400,410,420,430,440,450,460,470,480,490,500,510,520,530,540,550,560,570,580,590,600,610,620,630,640,650,660,670,680,690,700,710,720,730,740,750,760,770,780,790,800,810,820,830,840,850,860,870,880,890,900,910,920,930,940,950,960,970,980,990 or 1000 nucleotides,Wherein in this linguistic context,Term " about " means the 10% of the nucleotide sequence length plus or minus the length.In addition, such nucleic acid fragment is generally made up of the continuous nucleotide derived from TAT polypeptides complete encoding sequence or its complementary series.It should be noted that the new segment or its complementary series of TAT polypeptide-coding nucleotide sequences can be determined in a usual manner, i.e. using any of a variety of well-known sequence alignment programmes by TAT polypeptide-coding nucleotides sequence and other known nucleotide alignments, and determine which (a little) TAT polypeptide-coding nucleotides sequence or its complementary series are new.All such new segments or its complementary series of TAT polypeptide-coding nucleotide sequences are contemplated herein.Also contemplate the TAT polypeptide fragments of the TAT polypeptide fragments encoded by these nucleotide molecule fragments, the preferably binding site of those organic molecules comprising anti-TAT polypeptide antibodies, TAT combinations oligopeptides or other combination TAT polypeptides.

In another embodiment, the invention provides the TAT polypeptides of separation, it is by the nucleic acid sequence encoding for any separation identified above.

In terms of some, the present invention relates to the TAT polypeptides of separation, its include with

TAT polypeptides with full length amino acid sequence disclosed herein；

The TAT polypeptid acid sequences disclosed herein for lacking signal peptide；

The extracellular domain of the cross-film TAT polypeptides disclosed herein for being with or without signal peptide；

By the amino acid sequence of any nucleic acid sequence encoding disclosed herein；

Amino acid sequence of any other fragment being specifically defined of total length TAT polypeptid acid sequences disclosed herein with least about 80% amino acid sequence identity, or the amino acid sequence identity of at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

In another aspect, the present invention relates to the TAT polypeptides of separation, it is included and amino acid sequence of any amino acid sequence disclosed herein being preserved in coded by ATCC human protein cDNA with least about 80% amino acid sequence identity, or the amino acid sequence identity of at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

In yet another aspect, the present invention relates to the TAT polypeptides of separation, it is included by following nucleotide sequence coded amino acid sequence, the nucleotide sequence and coding

(a) with full length amino acid sequence disclosed herein TAT polypeptides,

(b) the TAT polypeptid acid sequences disclosed herein for lacking signal peptide,

(c) extracellular domain of the cross-film TAT polypeptides disclosed herein for being with or without signal peptide,

(d) by any nucleic acid sequence encoding disclosed herein amino acid sequence or

(e) complementary sequence hybridization of the DNA molecular of any other fragment being specifically defined of total length TAT polypeptid acid sequences disclosed herein.

In a specific aspect, the invention provides the TAT polypeptides of separation, it does not have N- terminus signal sequences and/or no initial methionine, and as coded by encoding the nucleotide sequence of amino acid sequence described above.There is also described herein the method for producing it, wherein methods described includes：Culture includes the host cell of following carrier under conditions of suitable for expression TAT polypeptides, and the carrier includes suitable coding nucleic acid molecule, and reclaims TAT polypeptides from cell culture.

Another aspect of the present invention provides the TAT polypeptides of separation, and it is that membrane-spanning domain is deleted or membrane-spanning domain inactivation.There is also described herein the method for producing it, wherein methods described includes：Culture includes the host cell of following carrier under conditions of suitable for expression TAT polypeptides, and the carrier includes suitable coding nucleic acid molecule, and reclaims TAT polypeptides from cell culture.

In other embodiments of the present invention, the invention provides the carrier for including the DNA for encoding any polypeptide described herein.Additionally provide the host cell for including any examples of such carriers.For example, the host cell can be Chinese hamster ovary celI, Escherichia coli (E.coli) cell or yeast cells.The method for producing any polypeptide described herein is additionally provided, is included in suitable for culture host cell under conditions of expression expectation polypeptide and expects polypeptide from cell culture recovery.

In other embodiments, the invention provides the chimeric polyeptides of separation, it includes any TAT polypeptides described herein with heterologous (non-TAT) peptide fusion.The example of such chimeric molecule includes any TAT polypeptides described herein merged with heterologous polypeptide such as epitope tag sequence or immunoglobulin fc region.

In another embodiment, the invention provides antibody, it is combined, and preferably specifically binds any foregoing or aftermentioned polypeptide.It is optional that, the antibody is monoclonal antibody, antibody fragment, chimeric antibody, humanized antibody, single-chain antibody or the anti-TAT polypeptide antibodies of Reverse transcriptase and its antibody that each antigenic epitopes are combined.The antibody of the present invention, which can optionally be coupled growth inhibitor or cytotoxic agent such as toxin, includes such as maytansinoid (maytansinoid) or Calicheamicin (calicheamicin), antibiotic, radio isotope, nucleolytic enzyme (nucleolytic enzyme) or the like.The antibody of the present invention can be produced optionally in Chinese hamster ovary celI or bacterial cell, and preferably suppress the growth or propagation or the cell death that induces it to be combined of its cell combined.For diagnostic purpose, antibody of the invention can be attached to solid support, etc. with detectably labeled.

In other embodiments of the present invention, the invention provides the carrier for including the DNA for encoding any antibody described herein.Additionally provide the host cell for including any examples of such carriers.For example, the host cell can be Chinese hamster ovary celI, Bacillus coli cells or yeast cells.The method for producing any antibody described herein is additionally provided, is included in suitable for culture host cell under conditions of expression expectation antibody and reclaims expectation antibody from cell culture.

In another embodiment, the invention provides oligopeptides (" TAT combinations oligopeptides "), it is combined, and preferably specifically binds any foregoing or aftermentioned TAT polypeptides.It is optional that, TAT combinations oligopeptides of the invention can be coupled growth inhibitor or cytotoxic agent, such as toxin, including such as maytansinoid or Calicheamicin, antibiotic, radio isotope, nucleolytic enzyme or the like.The TAT combinations oligopeptides of the present invention can be produced optionally in Chinese hamster ovary celI or bacterial cell, and preferably suppress the growth or propagation or the cell death that induces it to be combined of its cell combined.For diagnostic purpose, TAT combinations oligopeptides of the invention can be attached to solid support, etc. with detectably labeled.

In other embodiments of the present invention, the invention provides the carrier for including the DNA for encoding any TAT combinations oligopeptides described herein.Additionally provide the host cell for including any examples of such carriers.For example, the host cell can be Chinese hamster ovary celI, Bacillus coli cells or yeast cells.The method for producing any TAT combinations oligopeptides described herein is additionally provided, is included in suitable for culture host cell under conditions of expression expectation TAT combination oligopeptides and expects TAT Binding peptides from cell culture recovery.

In another embodiment, the invention provides organic molecule (" TAT combinations organic molecule "), it is combined, and preferably specifically binds any foregoing or aftermentioned TAT polypeptides.It is optional that, TAT combinations organic molecule of the invention can be coupled growth inhibitor or cytotoxic agent, such as toxin, including such as maytansinoid or Calicheamicin, antibiotic, radio isotope, nucleolytic enzyme or the like.The TAT combinations organic molecule of the present invention preferably suppresses the growth or propagation or the cell death that induces it to be combined of its cell combined.For diagnostic purpose, TAT combinations organic molecule of the invention can be attached to solid support, etc. with detectably labeled.

In still another embodiment, the present invention relates to composition, it includes the following substances with carrier combinations：TAT polypeptides as described herein, as described herein chimeric TAT polypeptides, as described herein anti-TAT antibody, TAT combinations oligopeptides as described herein or TAT combination organic molecules as described herein.It is optional that, the carrier is pharmaceutically acceptable carrier.

In still another embodiment, the present invention relates to product, it includes the composition accommodated in container and the container, wherein the composition can include TAT polypeptides as described herein, as described herein chimeric TAT polypeptides as described herein, anti-TAT antibody, TAT combinations oligopeptides as described herein or TAT combination organic molecules as described herein.The product optionally can also include investing the package insert that the label or the container of the container include, and it is related to purposes of the composition in the therapeutic treatment or diagnostic assays of tumour.

Another embodiment of the invention is related to TAT polypeptides as described herein, is fitted together to TAT polypeptides, the as described herein purposes of anti-TAT polypeptide antibodies, TAT combinations oligopeptides as described herein or TAT combinations organic molecule as described herein for preparing medicine as described herein, and the medicine can be used for treating the illness for having response to the TAT polypeptides, chimeric TAT polypeptides, anti-TAT polypeptide antibodies, TAT combinations oligopeptides or TAT combination organic molecules.

B. other embodiments

Another embodiment of the invention is related to the method for the cell growth for suppressing expression TAT polypeptides, wherein this method includes making antibody, oligopeptides or the organic molecule that cells contacting is combined with the TAT polypeptides, and wherein described antibody, oligopeptides or organic molecule and the TAT polypeptides combination cause the expression TAT polypeptides cell growth inhibition.In preferred embodiments, the cell is cancer cell, and the combination of the antibody, oligopeptides or organic molecule and the TAT polypeptides causes the cell death for expressing the TAT polypeptides.It is optional that, the antibody is monoclonal antibody, antibody fragment, chimeric antibody, humanized antibody or single-chain antibody.Antibody, TAT combinations oligopeptides and TAT combinations organic molecule employed in the inventive method can optionally be coupled growth inhibitor or cytotoxic agent, such as toxin, including such as maytansinoid or Calicheamicin, antibiotic, radio isotope, nucleolytic enzyme or the like.Antibody and TAT combinations oligopeptides employed in the inventive method can be produced optionally in Chinese hamster ovary celI or bacterial cell.

Yet another embodiment of the present invention is related to the method that therapeutic treatment has the mammal of cancerous tumour, the cancerous tumour includes the cell of expression TAT polypeptides, wherein this method includes applying the antibody of TAT polypeptides, oligopeptides or the organic molecule with reference to described in of therapeutically effective amount to the mammal, thus obtaining the effective treatment property of the tumour processing.It is optional that, the antibody is monoclonal antibody, antibody fragment, chimeric antibody, humanized antibody or single-chain antibody.Antibody, TAT combinations oligopeptides and TAT combinations organic molecule employed in the inventive method can optionally be coupled growth inhibitor or cytotoxic agent, such as toxin, including such as maytansinoid or Calicheamicin, antibiotic, radio isotope, nucleolytic enzyme or the like.Antibody and oligopeptides employed in the inventive method can be produced optionally in Chinese hamster ovary celI or bacterial cell.

Yet another embodiment of the present invention is related to the method for determining and suspecting the presence of the TAT polypeptides in the sample containing certain TAT polypeptide, wherein this method include make the sample exposed to can with reference to the TAT polypeptides antibody, oligopeptides or organic molecule, and the combination of antibody, oligopeptides or organic molecule and TAT polypeptides described in the sample is determined, wherein the presence of such combination shows there is the TAT polypeptides in the sample.It is optional that, the sample may include the cell (can be cancer cell) for suspecting expression TAT polypeptides.Antibody, TAT combinations oligopeptides or TAT combinations organic molecule employed in the inventive method can it is optionally detectably labeled, be attached to solid support, or the like.

Yet another embodiment of the present invention is related to the method for the presence of tumour in diagnosis mammal, wherein this method include detection (a) histiocytic test sample for being obtained from the mammal neutralize that (b) identical tissue-derived or control sample of known normal non-cancerous cell of type in coding TAT polypeptides gene expression, wherein the expression of the TAT polypeptides in the test sample shows to obtain in the mammal of the test sample higher than control sample has tumour.

Yet another embodiment of the present invention is related to the method for the presence of tumour in diagnosis mammal, wherein this method, which includes (a), makes that comprising the histiocytic test sample contact obtained from the mammal antibody, oligopeptides or the organic molecule of TAT polypeptides can be combined, and (b) detects the formation of compound between antibody, oligopeptides or organic molecule and the TAT polypeptides described in the test sample, the formation of wherein compound shows there is tumour in the mammal.It is optional that, antibody, TAT combinations oligopeptides or the TAT combination organic molecules used is detectably labeled, is attached to solid support, or the like, and/or the histiocytic test sample is obtained from individual of the suspection with cancerous tumour.

Yet another embodiment of the present invention is related to what is treated or prevented and change, the method for preferably enhanced TAT expression of polypeptides or the relevant cell proliferative disorders of activity, and this method includes applying the antagonist of the TAT polypeptides of effective dose to the subject of the such treatment of needs.Preferably, the cell proliferative disorders are cancers, and the antagonist of the TAT polypeptides is anti-TAT polypeptide antibodies, TAT combinations oligopeptides, TAT combinations organic molecule or ASON.Effective treatment or prevention of cell proliferative disorders can be grown by directly killing the cell of expression TAT polypeptides or suppressing it, or be realized by the cell growth reinforcing activity of antagonism TAT polypeptides.

Yet another embodiment of the present invention, which is related to, makes antibody, oligopeptides or organic molecule with expressing the method that the cell of TAT polypeptides is combined, wherein this method is included in suitable for the antibody, oligopeptides or organic molecule makes antibody, oligopeptides or organic molecule described in the cells contacting of expression TAT polypeptides with the TAT polypeptides with reference under conditions of, and allows the combination between them.In preferred embodiments, antibody, which is used, can be used for qualitative and/or quantitative determination antibody, oligopeptides or organic molecule and cell the position of combination and/or the molecule of amount or compound label.

Other embodiments of the present invention is related to (a) TAT polypeptides, the nucleic acid of (b) coding TAT polypeptides or the purposes of the anti-TAT polypeptide antibodies of carrier or host cell, (c), (d) TAT combinations oligopeptides or (e) TAT combinations organic molecule in the therapeutic treatment or diagnostic assays or the therapeutic treatment of (ii) cell proliferative disorders or the medicine of prevention available for (i) cancer or tumour is prepared comprising the nucleic acid.

Another embodiment of the invention is related to the method for suppressing growth of cancer cells, the growth of wherein described cancer cell rely at least partially upon TAT polypeptides growth reinforcing effect (wherein described TAT polypeptides can be by cancer cell oneself expression, either expressed by the cell for producing the polypeptide to cancer cell with growth reinforcing effect), wherein this method include making TAT polypeptides contact can with reference to the TAT polypeptides antibody, oligopeptides or organic molecule, thus the growth reinforcing activity of TAT polypeptides described in antagonism, then suppresses the growth of the cancer cell.Preferably completely suppress the growth of cancer cell.It is even furthermore preferable that the death of the antibody, oligopeptides or organic molecule and cancer cell described in the zygotic induction of the TAT polypeptides.It is optional that, the antibody is monoclonal antibody, antibody fragment, chimeric antibody, humanized antibody or single-chain antibody.Antibody, TAT combinations oligopeptides and TAT combinations organic molecule employed in the inventive method can optionally be coupled growth inhibitor or cytotoxic agent, such as toxin, including such as maytansinoid or Calicheamicin, antibiotic, radio isotope, nucleolytic enzyme or the like.Antibody and TAT combinations oligopeptides used in the inventive method can be produced optionally in Chinese hamster ovary celI or bacterial cell.

Yet another embodiment of the present invention is related to the method for the tumour in therapeutic treatment mammal, the growth of wherein described tumour relies at least partially upon the growth reinforcing effect of TAT polypeptides, wherein methods described include to the mammal apply therapeutically effective amount can with reference to the TAT polypeptides antibody, oligopeptides or organic molecule, thus the growth reinforcing activity of TAT polypeptides described in antagonism and handle the obtaining the effective treatment property of tumour.It is optional that, the antibody is monoclonal antibody, antibody fragment, chimeric antibody, humanized antibody or single-chain antibody.Antibody, TAT combinations oligopeptides and TAT combinations organic molecule employed in the inventive method can optionally be coupled growth inhibitor or cytotoxic agent, such as toxin, including such as maytansinoid or Calicheamicin, antibiotic, radio isotope, nucleolytic enzyme or the like.Antibody and oligopeptides employed in the inventive method can be produced optionally in Chinese hamster ovary celI or bacterial cell.

C. more other embodiments

In other embodiments, the present invention relates to the possible claim of following this set the application：

1. the nucleic acid of separation, it has the nucleotide sequence for having at least 80% nucleic acid sequence identity with following any one：

(a) DNA molecular, it encodes SEQ ID NO：Amino acid sequence shown in 2；

(b) DNA molecular, it encodes SEQ ID NO：Amino acid sequence shown in 2, lacks its associated signal peptide；

(c) DNA molecular, it encodes SEQ ID NO：The extracellular domain of polypeptide shown in 2, with its associated signal peptide；

(d) DNA molecular, it encodes SEQ ID NO：The extracellular domain of polypeptide shown in 2, lacks its associated signal peptide；

(e)SEQ ID NO：Nucleotide sequence shown in 1；

(f)SEQ ID NO：The full length coding region of nucleotide sequence shown in 1；Or

(g) complementary series of (a), (b), (c), (d), (e) or (f).

2. the nucleic acid of separation, it has：

(a) nucleotide sequence, it encodes SEQ ID NO：Amino acid sequence shown in 2；

(b) nucleotide sequence, it encodes SEQ ID NO：Amino acid sequence shown in 2, lacks its associated signal peptide；

(c) nucleotide sequence, it encodes SEQ ID NO：The extracellular domain of polypeptide shown in 2, with its associated signal peptide；

(d) nucleotide sequence, it encodes SEQ ID NO：The extracellular domain of polypeptide shown in 2, lacks its associated signal peptide；

(e)SEQ ID NO：Nucleotide sequence shown in 1；

(g) complementary series of (a), (b), (c), (d), (e) or (f).

3. the nucleic acid of separation, it hybridizes with following any one：

(a) nucleic acid, it encodes SEQ ID NO：Amino acid sequence shown in 2；

(b) nucleic acid, it encodes SEQ ID NO：Amino acid sequence shown in 2, lacks its associated signal peptide；

(c) nucleic acid, it encodes SEQ ID NO：The extracellular domain of polypeptide shown in 2, with its associated signal peptide；

(d) nucleic acid, it encodes SEQ ID NO：The extracellular domain of polypeptide shown in 2, lacks its associated signal peptide；

(e)SEQ ID NO：Nucleotide sequence shown in 1；

(g) complementary series of (a), (b), (c), (d), (e) or (f).

4. the nucleic acid of claim 3, wherein the hybridization occurs under strict conditions.

5. the nucleic acid of claim 3, its length is at least about 5 nucleotides.

6. expression vector, it includes the nucleic acid of claim 1,2 or 3.

7. the expression vector of claim 6, wherein the nucleic acid is operatively connected with control sequence, the control sequence is recognized by the host cell converted with the carrier.

8. host cell, it includes the expression vector of claim 7.

9. the host cell of claim 8, it is Chinese hamster ovary celI, Bacillus coli cells or yeast cells.

10. the method for producing polypeptide, is included in the host cell suitable for cultivating claim 8 under conditions of the expression polypeptide, and reclaim the polypeptide from cell culture.

11. the polypeptide of separation, it has at least 80% amino acid sequence identity with following any one：

(a)SEQ ID NO：Polypeptide shown in 2；

(b)SEQ ID NO：Polypeptide shown in 2, lacks its associated signal peptide；

(c)SEQ ID NO：The extracellular domain of polypeptide shown in 2, with its associated signal peptide；

(d)SEQ ID NO：The extracellular domain of polypeptide shown in 2, lacks its associated signal peptide；

(e)SEQ ID NO：Polypeptide coded by nucleotide sequence shown in 1；Or

(f)SEQ ID NO：Polypeptide coded by the full length coding region of nucleotide sequence shown in 1.

12. the polypeptide of separation, it has：

(a)SEQ ID NO：Amino acid sequence shown in 2；

(b)SEQ ID NO：Amino acid sequence shown in 2, lacks its associated signal peptide sequence；

(c)SEQ ID NO：The amino acid sequence of the extracellular domain of polypeptide shown in 2, with its associated signal peptide sequence；

(d)SEQ ID NO：The amino acid sequence of the extracellular domain of polypeptide shown in 2, lacks its associated signal peptide sequence；

(e)SEQ ID NO：Amino acid sequence coded by nucleotide sequence shown in 1；Or

(f)SEQ ID NO：Amino acid sequence coded by the full length coding region of nucleotide sequence shown in 1.

13. chimeric polyeptides, it includes the polypeptide with the claim 11 or 12 of heterologous polypeptide.

14. the chimeric polyeptides of claim 13, wherein the heterologous polypeptide is the Fc areas of epitope tag sequence or immunoglobulin.

15. the antibody of separation, it combines the polypeptide for having at least 80% amino acid sequence identity with following any one：

(a)SEQ ID NO：Polypeptide shown in 2；

(b)SEQ ID NO：Polypeptide shown in 2, lacks its associated signal peptide；

(e)SEQ ID NO：Polypeptide coded by nucleotide sequence shown in 1；Or

16. the antibody of separation, it is combined with any one of following polypeptides：

(a)SEQ ID NO：Amino acid sequence shown in 2；

(e)SEQ ID NO：Amino acid sequence coded by nucleotide sequence shown in 1；Or

17. the antibody of claim 15 or 16, it is monoclonal antibody.

18. the antibody of claim 15 or 16, it is antibody fragment.

19. the antibody of claim 15 or 16, it is chimeric or humanized antibody.

20. the antibody of claim 15 or 16, it is coupled with growth inhibitor.

21. the antibody of claim 15 or 16, itself and cytotoxic agent couplings.

22. the antibody of claim 21, wherein the cytotoxic agent is selected from toxin, antibiotic, radio isotope and nucleolytic enzyme.

23. the antibody of claim 21, wherein the cytotoxic agent is toxin.

24. the antibody of claim 23, wherein the toxin is selected from maytansinoid and Calicheamicin.

25. the antibody of claim 23, wherein the toxin is maytansinoid.

26. the antibody of claim 15 or 16, it is the antibody produced in bacterium.

27. the antibody of claim 15 or 16, it is the antibody produced in Chinese hamster ovary celI.

28. the antibody of claim 15 or 16, it induces the cell death that it is combined.

29. the antibody of claim 15 or 16, its is detectably labeled.

30. the nucleic acid of separation, it has the nucleotide sequence of the antibody of coding claim 15 or 16.

31. expression vector, it includes the nucleic acid for the claim 30 being operatively connected with control sequence, and the control sequence is recognized by the host cell converted with the carrier.

32. host cell, it includes the expression vector of claim 31.

33. the host cell of claim 32, it is Chinese hamster ovary celI, Bacillus coli cells or yeast cells.

34. the method for producing antibody, is included in the host cell suitable for cultivating claim 32 under conditions of the expression antibody, and reclaim the antibody from cell culture.

35. the oligopeptides of separation, it combines the polypeptide for having at least 80% amino acid sequence identity with following any one：

(a)SEQ ID NO：Polypeptide shown in 2；

(b)SEQ ID NO：Polypeptide shown in 2, lacks its associated signal peptide；

(e)SEQ ID NO：Polypeptide coded by nucleotide sequence shown in 1；Or

36. the oligopeptides of separation, it is combined with any one of following polypeptides：

(a)SEQ ID NO：Amino acid sequence shown in 2；

(e)SEQ ID NO：Amino acid sequence coded by nucleotide sequence shown in 1；Or

37. the oligopeptides of claim 35 or 36, it is coupled with growth inhibitor.

38. the oligopeptides of claim 35 or 36, itself and cytotoxic agent couplings.

39. the oligopeptides of claim 38, wherein the cytotoxic agent is selected from toxin, antibiotic, radio isotope and nucleolytic enzyme.

40. the oligopeptides of claim 38, wherein the cytotoxic agent is toxin.

41. the oligopeptides of claim 40, wherein the toxin is selected from maytansinoid and Calicheamicin.

42. the oligopeptides of claim 40, wherein the toxin is maytansinoid.

43. the oligopeptides of claim 35 or 36, it induces the cell death that it is combined.

44. the oligopeptides of claim 35 or 36, its is detectably labeled.

45.TAT combination organic molecules, it combines the polypeptide for having at least 80% amino acid sequence identity with following any one：

(a)SEQ ID NO：Polypeptide shown in 2；

(b)SEQ ID NO：Polypeptide shown in 2, lacks its associated signal peptide；

(e)SEQ ID NO：Polypeptide coded by nucleotide sequence shown in 1；Or

46. the organic molecule of claim 45, it is combined with any one of following polypeptides：

(a)SEQ ID NO：Amino acid sequence shown in 2；

(e)SEQ ID NO：Amino acid sequence coded by nucleotide sequence shown in 1；Or

47. the organic molecule of claim 45 or 46, it is coupled with growth inhibitor.

48. the oligopeptides of claim 45 or 46, itself and cytotoxic agent couplings.

49. the oligopeptides of claim 48, wherein the cytotoxic agent is selected from toxin, antibiotic, radio isotope and nucleolytic enzyme.

50. the oligopeptides of claim 48, wherein the cytotoxic agent is toxin.

51. the oligopeptides of claim 50, wherein the toxin is selected from maytansinoid and Calicheamicin.

52. the oligopeptides of claim 50, wherein the toxin is maytansinoid.

53. the oligopeptides of claim 45 or 46, it induces the cell death that it is combined.

54. the oligopeptides of claim 45 or 46, its is detectably labeled.

55. composition, it is included and carrier combinations：

(a) polypeptide of claim 11；

(b) polypeptide of claim 12；

(c) chimeric polyeptides of claim 13；

(d) antibody of claim 15；

(e) antibody of claim 16；

(f) oligopeptides of claim 35；

(g) oligopeptides of claim 36；

(h) the TAT combination organic molecules of claim 45；Or

(i) the TAT combination organic molecules of claim 46.

56. the composition of claim 55, wherein the carrier is pharmaceutically acceptable carrier.

57. product, it includes：

(a) container；And

(b) composition of the claim 55 accommodated in the container.

58. the product of claim 57, in addition to the package insert that the label or the container of the container include is invested, it, which is related to the composition, is used for the therapeutic treatment of cancer or the purposes of diagnostic assays.

59. cytostatic method, the cell expression has the protein of at least 80% amino acid sequence identity with following any one：

(a)SEQ ID NO：Polypeptide shown in 2；

(b)SEQ ID NO：Polypeptide shown in 2, lacks its associated signal peptide；

(e)SEQ ID NO：Polypeptide coded by nucleotide sequence shown in 1；Or

(f)SEQ ID NO：Polypeptide coded by the full length coding region of nucleotide sequence shown in 1, methods described include making the cells contacting can with reference to the protein antibody, oligopeptides or organic molecule, the thus combination of the antibody, oligopeptides or organic molecule and the protein causes the growth inhibition of the cell.

60. the method for claim 59, wherein the antibody is monoclonal antibody.

61. the method for claim 59, wherein the antibody is antibody fragment.

62. the method for claim 59, wherein the antibody is chimeric or humanized antibody.

63. the method for claim 59, wherein the antibody, oligopeptides or organic molecule and growth inhibitor are coupled.

64. the method for claim 59, wherein the antibody, oligopeptides or organic molecule and cytotoxic agent couplings.

65. the method for claim 64, wherein the cytotoxic agent is selected from toxin, antibiotic, radio isotope and nucleolytic enzyme.

66. the method for claim 64, wherein the cytotoxic agent is toxin.

67. the method for claim 66, wherein the toxin is selected from maytansinoid and Calicheamicin.

68. the method for claim 66, wherein the toxin is maytansinoid.

69. the method for claim 59, wherein the antibody is the antibody produced in bacterium.

70. the method for claim 59, wherein the antibody is the antibody produced in Chinese hamster ovary celI.

71. the method for claim 59, wherein the cell is cancer cell.

72. the method for claim 71, wherein making the cancer cell be further exposed to radiation treatment or chemotherapeutics.

73. the method for claim 71, wherein the cancer cell is selected from breast cancer cell, colorectal cancer cell, lung carcinoma cell, ovarian cancer cell, central nervous system cancer cell, liver cancer cells, transitional cell bladder carcinoma cell line, pancreatic cancer cell, cervical cancer cell, melanoma cells and leukaemia.

74. the method for claim 71, wherein the cancer cell expresses the protein in a larger amount compared with identical tissue-derived normal cell.

75. the method for claim 59, it causes the cell death.

76. the method for claim 59, wherein the protein has：

(a)SEQ ID NO：Amino acid sequence shown in 2；

(e)SEQ ID NO：Amino acid sequence coded by nucleotide sequence shown in 1；Or

77. the method that therapeutic treatment has the mammal of cancerous tumour, the cancerous tumour has the cell of at least protein of 80% amino acid sequence identity comprising expression with following any one：

(a)SEQ ID NO：Polypeptide shown in 2；

(b)SEQ ID NO：Polypeptide shown in 2, lacks its associated signal peptide；

(e)SEQ ID NO：Polypeptide coded by nucleotide sequence shown in 1；Or

(f)SEQ ID NO：Polypeptide coded by the full length coding region of nucleotide sequence shown in 1,

Methods described include to the mammal apply therapeutically effective amount can with reference to the protein antibody, oligopeptides or organic molecule, thus effectively treat the mammal.

78. the method for claim 77, wherein the antibody is monoclonal antibody.

79. the method for claim 77, wherein the antibody is antibody fragment.

80. the method for claim 77, wherein the antibody is chimeric or humanized antibody.

81. the method for claim 77, wherein the antibody, oligopeptides or organic molecule and growth inhibitor are coupled.

82. the method for claim 77, wherein the antibody, oligopeptides or organic molecule and cytotoxic agent couplings.

83. the method for claim 82, wherein the cytotoxic agent is selected from toxin, antibiotic, radio isotope and nucleolytic enzyme.

84. the method for claim 82, wherein the cytotoxic agent is toxin.

85. the method for claim 84, wherein the toxin is selected from maytansinoid and Calicheamicin.

86. the method for claim 84, wherein the toxin is maytansinoid.

87. the method for claim 77, wherein the antibody is the antibody produced in bacterium.

88. the method for claim 77, wherein the antibody is the antibody produced in Chinese hamster ovary celI.

89. the method for claim 77, wherein making the tumour be further exposed to radiation treatment or chemotherapeutics.

90. the method for claim 77, wherein the tumour is mammary tumor, colorectal tumours, lung neoplasm, ovarian neoplasm, central nerve neuroma, liver tumour, tumor of bladder, pancreatic neoplasm or cervix neoplasmses.

91. the method for claim 77, wherein the cancerous cells of the tumour express the protein in a larger amount compared with identical tissue-derived normal cell.

92. the method for claim 77, wherein the protein has：

(a)SEQ ID NO：Amino acid sequence shown in 2；

(e)SEQ ID NO：Amino acid sequence coded by nucleotide sequence shown in 1；Or

93. the method for suspecting the presence of the protein in the sample containing certain protein is determined, wherein the protein has at least 80% amino acid sequence identity with following any one：

(a)SEQ ID NO：Polypeptide shown in 2；

(b)SEQ ID NO：Polypeptide shown in 2, lacks its associated signal peptide；

(e)SEQ ID NO：Polypeptide coded by nucleotide sequence shown in 1；Or

(f)SEQ ID NO：Polypeptide coded by the full length coding region of nucleotide sequence shown in 1, methods described include make the sample exposed to can with reference to the protein antibody, oligopeptides or organic molecule, and the combination of antibody, oligopeptides or organic molecule and the protein described in the sample is determined, wherein the combination of the antibody, oligopeptides or organic molecule and the protein shows there is the protein in the sample.

94. the method for claim 93, wherein the sample includes the cell for suspecting the expression protein.

95. the method for claim 94, wherein the cell is cancer cell.

96. the method for claim 93, wherein the antibody, oligopeptides or organic molecule are detectably labeled.

97. the method for claim 93, wherein the protein has：

(a)SEQ ID NO：Amino acid sequence shown in 2；

(e)SEQ ID NO：Amino acid sequence coded by nucleotide sequence shown in 1；Or

98. the method for the presence of tumour in diagnosis mammal, methods described includes determining the expression that coding from the histiocytic test sample that the mammal obtains and in the control sample of identical tissue-derived known normal cell has at least gene of the protein of 80% amino acid sequence identity with following any one：

(a)SEQ ID NO：Polypeptide shown in 2；

(b)SEQ ID NO：Polypeptide shown in 2, lacks its associated signal peptide；

(e)SEQ ID NO：Polypeptide coded by nucleotide sequence shown in 1；Or

(f)SEQ ID NO：Polypeptide coded by the full length coding region of nucleotide sequence shown in 1, wherein the expression of the protein in the test sample shows there is tumour in the mammal of acquisition test sample higher than control sample.

99. the method for claim 98, wherein the step of determining the expression of the gene of code for said proteins, which is included in in situ hybridization or RT-PCR analyses, uses oligonucleotides.

100. the method for claim 98, wherein the step of determining the expression of the gene of code for said proteins, which is included in SABC or Western blot analysis, uses antibody.

101. the method for claim 98, wherein the protein has：

(a)SEQ ID NO：Amino acid sequence shown in 2；

(e)SEQ ID NO：Amino acid sequence coded by nucleotide sequence shown in 1；Or

102. diagnosing the method for the presence of tumour in mammal, methods described includes antibody, oligopeptides or the organic molecule for the protein for making the histiocytic test sample contact obtained from the mammal to combine the amino acid sequence identity for having at least 80% with following any one：

(a)SEQ ID NO：Polypeptide shown in 2；

(b)SEQ ID NO：Polypeptide shown in 2, lacks its associated signal peptide；

(e)SEQ ID NO：Polypeptide coded by nucleotide sequence shown in 1；Or

(f)SEQ ID NO：Polypeptide coded by the full length coding region of nucleotide sequence shown in 1, and the formation of compound between antibody described in test sample, oligopeptides or organic molecule and the protein is detected, the formation of wherein compound shows there is tumour in the mammal.

103. the method for claim 102, wherein the antibody, oligopeptides or organic molecule are detectably labeled.

104. the method for claim 102, wherein the histiocytic test sample is obtained from individual of the suspection with cancerous tumour.

105. the method for claim 102, wherein the protein has：

(a)SEQ ID NO：Amino acid sequence shown in 2；

(e)SEQ ID NO：Amino acid sequence coded by nucleotide sequence shown in 1；Or

106. treating or preventing the method for the cell proliferative disorders relevant with certain protein expression or increased activity, the protein has at least 80% amino acid sequence identity with following any one：

(a)SEQ ID NO：Polypeptide shown in 2；

(b)SEQ ID NO：Polypeptide shown in 2, lacks its associated signal peptide；

(e)SEQ ID NO：Polypeptide coded by nucleotide sequence shown in 1；Or

(f)SEQ ID NO：Polypeptide coded by the full length coding region of nucleotide sequence shown in 1, methods described is included to the subject for needing such processing using the antagonist of the protein of effective dose, thus effectively treats or prevents the cell proliferative disorders.

107. the method for claim 106, wherein the cell proliferative disorders are cancers.

108. the method for claim 106, wherein the antagonist is anti-TAT polypeptide antibodies, TAT combinations oligopeptides, TAT combinations organic molecule or ASON.

109. the method for making antibody, oligopeptides or organic molecule be combined with cell, the cell expression has the protein of at least 80% amino acid sequence identity with following any one：

(a)SEQ ID NO：Polypeptide shown in 2；

(b)SEQ ID NO：Polypeptide shown in 2, lacks its associated signal peptide；

(e)SEQ ID NO：Polypeptide coded by nucleotide sequence shown in 1；Or

(f)SEQ ID NO：Polypeptide coded by the full length coding region of nucleotide sequence shown in 1, methods described include make the cells contacting can with reference to the protein antibody, oligopeptides or organic molecule, and allow the antibody, oligopeptides or organic molecule to be combined with the protein, so that the antibody, oligopeptides or organic molecule are with reference to the cell.

110. the method for claim 109, wherein described is monoclonal antibody.

111. the method for claim 109, wherein the antibody is antibody fragment.

112. the method for claim 109, wherein the antibody is chimeric or humanized antibody.

113. the method for claim 109, wherein the antibody, oligopeptides or organic molecule and growth inhibitor are coupled.

114. the method for claim 109, wherein the antibody, oligopeptides or organic molecule and cytotoxic agent couplings.

115. the method for claim 114, wherein the cytotoxic agent is selected from toxin, antibiotic, radio isotope and nucleolytic enzyme.

116. the method for claim 114, wherein the cytotoxic agent is toxin.

117. the method for claim 116, wherein the toxin is selected from maytansinoid and Calicheamicin.

118. the method for claim 116, wherein the toxin is maytansinoid.

119. the method for claim 109, wherein the antibody is the antibody produced in bacterium.

120. the method for claim 109, wherein the antibody is the antibody produced in Chinese hamster ovary celI.

121. the method for claim 109, wherein the cell is cancer cell.

122. the method for claim 121, wherein making the cancer cell be further exposed to radiation treatment or chemotherapeutics.

123. the method for claim 121, wherein the cancer cell is selected from breast cancer cell, colorectal cancer cell, lung carcinoma cell, ovarian cancer cell, central nervous system cancer cell, liver cancer cells, transitional cell bladder carcinoma cell line, pancreatic cancer cell, cervical cancer cell, melanoma cells and leukaemia.

124. the method for claim 123, wherein the cancer cell expresses the protein in a larger amount compared with identical tissue-derived normal cell.

125. the method for claim 109, it causes the cell death.

126. any one of claim 1-5 or 30 nucleic acid is being prepared for the purposes in the therapeutic treatment of cancer or the medicine of diagnostic assays.

127. any one of claim 1-5 or 30 nucleic acid is preparing the purposes in being used to treat the medicine of tumour.

128. any one of claim 1-5 or 30 nucleic acid is preparing the purposes in being used to treat or prevent the medicine of cell proliferative disorders.

129. the expression vector of any one of claim 6,7 or 31 is being prepared for the purposes in the therapeutic treatment of cancer or the medicine of diagnostic assays.

130. the expression vector of any one of claim 6,7 or 31 is preparing the purposes in being used to treat the medicine of tumour.

131. the expression vector of any one of claim 6,7 or 31 is preparing the purposes in being used to treat or prevent the medicine of cell proliferative disorders.

132. the host cell of any one of claim 8,9,32 or 33 is being prepared for the purposes in the therapeutic treatment of cancer or the medicine of diagnostic assays.

133. the host cell of any one of claim 8,9,32 or 33 is preparing the purposes in being used to treat the medicine of tumour.

134. the host cell of any one of claim 8,9,32 or 33 is preparing the purposes in being used to treat or prevent the medicine of cell proliferative disorders.

135. any one of claim 11-14 polypeptide is being prepared for the purposes in the therapeutic treatment of cancer or the medicine of diagnostic assays.

136. any one of claim 11-14 polypeptide is preparing the purposes in being used to treat the medicine of tumour.

137. any one of claim 11-14 polypeptide is preparing the purposes in being used to treat or prevent the medicine of cell proliferative disorders.

138. any one of claim 15-29 antibody is being prepared for the purposes in the therapeutic treatment of cancer or the medicine of diagnostic assays.

139. any one of claim 15-29 antibody is preparing the purposes in being used to treat the medicine of tumour.

140. any one of claim 15-29 antibody is preparing the purposes in being used to treat or prevent the medicine of cell proliferative disorders.

Any one of 141. claim 35-44 oligopeptides is being prepared for the purposes in the therapeutic treatment of cancer or the medicine of diagnostic assays.

Any one of 142. claim 35-44 oligopeptides is preparing the purposes in being used to treat the medicine of tumour.

Any one of 143. claim 35-44 oligopeptides is preparing the purposes in being used to treat or prevent the medicine of cell proliferative disorders.

Any one of 144. claim 45-54 TAT combinations organic molecule is being prepared for the purposes in the therapeutic treatment of cancer or the medicine of diagnostic assays.

Any one of 145. claim 45-54 TAT combinations organic molecule is preparing the purposes in being used to treat the medicine of tumour.

Any one of 146. claim 45-54 TAT combinations organic molecule is preparing the purposes in being used to treat or prevent the medicine of cell proliferative disorders.

The composition of any one of 147. claim 55 or 56 is being prepared for the purposes in the therapeutic treatment of cancer or the medicine of diagnostic assays.

The composition of any one of 148. claim 55 or 56 is preparing the purposes in being used to treat the medicine of tumour.

The composition of any one of 149. claim 55 or 56 is preparing the purposes in being used to treat or prevent the medicine of cell proliferative disorders.

The product of any one of 150. claim 57 or 58 is being prepared for the purposes in the therapeutic treatment of cancer or the medicine of diagnostic assays.

The product of any one of 151. claim 57 or 58 is preparing the purposes in being used to treat the medicine of tumour.

The product of any one of 152. claim 57 or 58 is preparing the purposes in being used to treat or prevent the medicine of cell proliferative disorders.

153. for cytostatic method, wherein the growth of the cell at least partly relies on the growth reinforcing effect for having at least protein of 80% amino acid sequence identity with following any one：

(a)SEQ ID NO：Polypeptide shown in 2；

(b)SEQ ID NO：Polypeptide shown in 2, lacks its associated signal peptide；

(e)SEQ ID NO：Polypeptide coded by nucleotide sequence shown in 1；Or

(f)SEQ ID NO：Polypeptide coded by the full length coding region of nucleotide sequence shown in 1, methods described includes making antibody, oligopeptides or the organic molecule that the protein contacts can be combined with the protein, so as to suppress the growth of the cell.

The method of 154. claims 153, wherein the cell is cancer cell.

The method of 155. claims 153, wherein the protein is expressed by the cell.

The method of 156. claims 153, wherein the cell growth reinforcing activity of protein described in the combination antagonism of the antibody, oligopeptides or organic molecule and the protein.

The method of 157. claims 153, wherein the antibody, oligopeptides or organic molecule and cell death described in the zygotic induction of the protein.

The method of 158. claims 153, wherein the antibody is monoclonal antibody.

The method of 159. claims 153, wherein the antibody is antibody fragment.

The method of 160. claims 153, wherein the antibody is chimeric or humanized antibody.

The method of 161. claims 153, wherein the antibody, oligopeptides or organic molecule and growth inhibitor are coupled.

The method of 162. claims 153, wherein the antibody, oligopeptides or organic molecule and cytotoxic agent couplings.

The method of 163. claims 162, wherein the cytotoxic agent is selected from toxin, antibiotic, radio isotope and nucleolytic enzyme.

The method of 164. claims 162, wherein the cytotoxic agent is toxin.

The method of 165. claims 164, wherein the toxin is selected from maytansinoid and Calicheamicin.

The method of 166. claims 164, wherein the toxin is maytansinoid.

The method of 167. claims 153, wherein the antibody is the antibody produced in bacterium.

The method of 168. claims 153, wherein the antibody is the antibody produced in Chinese hamster ovary celI.

The method of 169. claims 153, wherein the protein has：

(a)SEQ ID NO：Amino acid sequence shown in 2；

(e)SEQ ID NO：Amino acid sequence coded by nucleotide sequence shown in 1；Or

The method of tumour in 170. therapeutic treatment mammals, wherein the growth of the tumour at least partly relies on the growth reinforcing effect for having at least protein of 80% amino acid sequence identity with following any one：

(a)SEQ ID NO：Polypeptide shown in 2；

(b)SEQ ID NO：Polypeptide shown in 2, lacks its associated signal peptide；

(e)SEQ ID NO：Polypeptide coded by nucleotide sequence shown in 1；Or

(f)SEQ ID NO：Polypeptide coded by the full length coding region of nucleotide sequence shown in 1, methods described includes making antibody, oligopeptides or the organic molecule that the protein contacts are combined with the protein, thus effectively treats the tumour.

The method of 171. claims 170, wherein the protein is expressed by the cell of the tumour.

The method of 172. claims 170, wherein the cell growth reinforcing activity of protein described in the combination antagonism of the antibody, oligopeptides or organic molecule and the protein.

The method of 173. claims 170, wherein the antibody is monoclonal antibody.

The method of 174. claims 170, wherein the antibody is antibody fragment.

The method of 175. claims 170, wherein the antibody is chimeric or humanized antibody.

The method of 176. claims 170, wherein the antibody, oligopeptides or organic molecule and growth inhibitor are coupled.

The method of 177. claims 170, wherein the antibody, oligopeptides or organic molecule and cytotoxic agent couplings.

The method of 178. claims 177, wherein the cytotoxic agent is selected from toxin, antibiotic, radio isotope and nucleolytic enzyme.

The method of 179. claims 177, wherein the cytotoxic agent is toxin.

The method of 180. claims 179, wherein the toxin is selected from maytansinoid and Calicheamicin.

The method of 181. claims 179, wherein the toxin is maytansinoid.

The method of 182. claims 170, wherein the antibody is the antibody produced in bacterium.

The method of 183. claims 170, wherein the antibody is the antibody produced in Chinese hamster ovary celI.

The method of 184. claims 170, wherein the protein has：

(a)SEQ ID NO：Amino acid sequence shown in 2；

(e)SEQ ID NO：Amino acid sequence coded by nucleotide sequence shown in 1；Or

The antibody of 186. separation, the epitope that its antibody for combining any hybridoma cell line generation as shown in Table 7 is combined.

The antibody of 187. claims 186, it is monoclonal antibody.

The antibody of 188. claims 186, it is antibody fragment.

The antibody of 189. claims 186, it is chimeric or humanized antibody.

The antibody of 190. claims 186, it is coupled with growth inhibitor.

The antibody of 191. claims 186, itself and cytotoxic agent couplings.

The antibody of 192. claims 191, wherein the cytotoxic agent is selected from toxin, antibiotic, radio isotope and nucleolytic enzyme.

The antibody of 193. claims 191, wherein the cytotoxic agent is toxin.

The antibody of 194. claims 193, wherein the toxin is selected from maytansinoid and Calicheamicin.

The antibody of 195. claims 193, wherein the toxin is maytansinoid.

The antibody of 196. claims 186, it is the antibody produced in bacterium.

The antibody of 197. claims 186, it is the antibody produced in Chinese hamster ovary celI.

The antibody of 198. claims 186, it induces the cell death that it is combined.

The antibody of 199. claims 186, it is detectably labeled antibody.

The antibody of 200. claims 186, it includes the complementary determining region of at least one any antibody of any hybridoma cell line generation as shown in Table 7.

201. monoclonal antibodies, it is the monoclonal antibody of any hybridoma generation as shown in Table 7.

202. hybridomas, it generates the monoclonal antibody for combining TAT113 polypeptides.

The method of the antibody for the epitope that the antibody that 203. identifications can combine any hybridoma cell line generation as shown in Table 7 is combined, methods described includes determining the ability that second of antibody of any hybridoma cell line generation of the first antibody blocking as shown in Table 7 is combined with TAT113 polypeptides, wherein the ability that the combination of second of the antibody and the TAT113 polypeptides is blocked at least 40% by the first described antibody under the conditions of equal antibody concentrations shows the epitope that the first described antibody can be combined with reference to second of the antibody.

Read after this specification, other embodiments of the present invention will be apparent to those skilled in the art.

Brief description

Fig. 1 shows TAT113 cDNA nucleotide sequence (SEQ ID NO：1), wherein SEQ IDNO：1 is the clone of referred herein as " DNA215609 ".

Fig. 2 is shown derived from the NO of SEQ ID shown in Fig. 1：Amino acid sequence (the SEQ ID NO of 1 complete encoding sequence：2).

The detailed description of preferred embodiment

I. define

Refer to various polypeptides when term " TAT polypeptides " and " TAT " are as used herein and followed by numerical designations, wherein complete name (i.e. TAT/ numerical value) refers to particular polypeptide sequence described herein.Term " TAT/ numerical value polypeptide " and " TAT/ numerical value ", wherein term " numerical value " is provided as actual numerical value title, as used herein, the fragment and polypeptide variants (having further definition herein) of natural sequence polypeptide, polypeptide variants and natural sequence polypeptide are covered.TAT polypeptides described herein can be from a variety of source separation, such as people's organization type or other sources, or is prepared by recombinantly or synthetically method.Term " TAT polypeptides " refers to each other TAT/ numerical value polypeptide disclosed herein.All disclosures of " TAT polypeptides " are addressed in this specification not only individually but also can collectively refer to each polypeptide.For example, on the preparation of polypeptide, purifying, derivative, for the polypeptide antibody formation, for the polypeptide TAT combination oligopeptides formation, for the polypeptide TAT combination organic molecules formation, using, the composition containing the polypeptide, with the polypeptide therapeutic disease, etc. description individually refer to each polypeptide of the present invention.Term " TAT polypeptides " also includes the variant of TAT/ numerical value polypeptide disclosed herein.

" native sequences TAT polypeptides " includes the polypeptide that TAT polypeptides corresponding to what it is derived from nature have same amino acid sequence.Such native sequences TAT polypeptides can be separated from nature, or can be generated by recombinantly or synthetically means.Term " native sequences TAT polypeptides " especially covers specific T AT polypeptides (such as ectodomain sequence), the naturally occurring variant form (such as alternative splice forms) and naturally occurring allelic variant of the polypeptide of naturally occurring truncation or secreted form.In certain embodiments of the invention, native sequences TAT polypeptides disclosed herein are maturation or total length natural sequence polypeptide comprising full length amino acid sequence shown in accompanying drawing.Starting and terminator codon (if showing) are shown in figure with boldface letter and underscore.With " N " or " X " nucleic acid represented it is any nucleic acid in accompanying drawing.But, although the TAT polypeptides disclosed in accompanying drawing are illustrated as what is started to be designated as the methionine residues of the 1st amino acids in scheming, it is contemplated that and it is possible that the starting amino acid residue for being located at other methionine residues in the 1st amino acids upstream or downstream in figure as TAT polypeptides can be used.

TAT polypeptides " extracellular domain " or " ECD " refer to the TAT polypeptide forms substantially free of membrane-spanning domain and cytoplasm domain.Generally, TAT polypeptides ECD is having less than 1% membrane-spanning domain and/or cytoplasm domain, preferably having less than 0.5% domain.It is appreciated that the standard that any membrane-spanning domain of TAT polypeptides of the present invention is the hydrophobic domain conventionally used for identifying this type according to this area is identified.The exact boundary of membrane-spanning domain may be varied from, but be most likely to be the either end in the domain initially determined that herein and be no more than about 5 amino acid.Therefore, it is optional that, the extracellular domain of TAT polypeptides can be containing the membrane-spanning domain mentioned in embodiment or specification/extracellular domain border either side about 5 or less amino acid, and present invention contemplates such polypeptide with and without associated signal peptide and encode their nucleic acid.

The approximate location of " signal peptide " of various TAT polypeptides disclosed herein is found in this specification and/or accompanying drawing.But, it should be noted that, the C- end boundaries of signal peptide can change, it is most likely that the signal peptide C- end boundaries either sides initially determined that herein are no more than about 5 amino acid, wherein the C- end boundaries of signal peptide can be according to this area conventionally used for identifying that the standard of this type amino acid sequence element identifies (such as Nielsen et al., Prot.Eng.10：1-6 (1997) and von Heinje et al., Nucl.Acids.Res.14：4683-4690(1986)).Moreover, it is to be understood that from the excision on secrete polypeptide not being complete and homogeneous in signal sequence in some cases, cause secretion type more than one.Present invention contemplates these mature polypeptides, the signal peptide C- end boundaries either sides that wherein signal peptide is identified herein are no more than about excision in 5 amino acid, and encode their polynucleotides.

" TAT polypeptide variants " mean with any other fragment of total length native sequences TAT peptide sequences disclosed herein, the TAT peptide sequences for lacking signal peptide disclosed herein, the TAT polypeptides extracellular domain with and without signal peptide disclosed herein or total length TAT peptide sequences disclosed herein (such as those be only total length TAT polypeptides complete encoding sequence a part encoded by nucleic acid fragment) have at least about 80% amino acid sequence identity TAT polypeptides as defined herein, preferred activity TAT polypeptides.Such TAT polypeptide variants include N- the or C- ends addition of such as wherein total length natural acid sequence or delete the TAT polypeptides of one or more amino acid residues.Generally, TAT polypeptide variants and total length native sequences TAT peptide sequences disclosed herein, lack the TAT peptide sequences of signal peptide disclosed herein, with and without the TAT polypeptide extracellular domains of signal peptide disclosed herein, or any other fragment being specifically defined of total length TAT peptide sequences disclosed herein has at least about 80% amino acid sequence identity, or at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity.Generally, the length of TAT variant polypeptides is at least about 10 amino acid, or length is at least about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600 amino acid or more.Be optional that, TAT variant polypeptides have compared with natural TAT peptide sequences to be substituted no more than conserved amino acid, or have with natural TAT peptide sequences compared with no more than 2,3,4,5,6,7,8,9 or 10 conserved amino acid replacements.

" percentage (%) amino acid sequence identity " for the TAT peptide sequences addressed in this article relatively is defined as in aligned sequences and introduces breach when necessary, to obtain after largest percentage sequence identity, and any conservative replacement be not considered as under conditions of a part for sequence identity, with the percentage of the amino acid residue identical amino acid residue in specific T AT peptide sequences in candidate sequence.Any algorithm needed for maximum contrast can be obtained with art technology comparative sequences total length.However, for the present invention, % amino acid sequence identity values are to compare computer program ALIGN-2 using sequence to obtain, and the complete source code of ALIGN-2 programs is provided wherein in table 1 below.ALIGN-2 sequences compare computer program and write by Genentech companies, source code shown in table 1 below submits to U.S. Copyright Office (US Copyright Office together with customer documentation, Washington D.C., 20559), and with U.S. Copyright Registration TXU510087 register.The public can obtain ALIGN-2 programs, or the compilation of source code that can be provided from table 1 below by Genentech companies (South San Francisco, Caiifornia).ALIGN2 programs should be compiled into UNIX operating system, used on preferably number UNIX V4.0D.All sequences compare parameter by ALIGN-2 program settings and constant.

In the case where being compared for amino acid sequence using ALIGN-2, given amino acid sequence A relative to (to), with (with) or for (against) given amino acid sequence B % amino acid sequence identities (or can be expressed as having or comprising relative to, with or for give amino acid sequence B a certain % amino acid sequence identities given amino acid sequence A) be calculated as below：

Fraction X/Y multiplies 100

Wherein X is the total number of atnino acid that alignment programs ALIGN-2 is assessed as identical match in comparison of the program to A and B, and Y is the total amino acid residues in B.It is understood that if amino acid sequence A length and amino acid sequence B length are unequal, % amino acid sequence identities of the A relative to B will be equal to % amino acid sequence identities of the B relative to A.The example calculated as the % amino acid sequence identities made in this way, how table 2 and 3 calculates % amino acid sequence identity of the amino acid sequence for being assigned as " comparison protein " relative to the amino acid sequence for being assigned as " TAT " if being demonstrated, wherein " TAT " represents the amino acid sequence that purpose assumes TAT polypeptides, " comparison protein " represents the amino acid sequence that purpose " TAT " polypeptide is directed to its polypeptide being compared, and " X ", " Y " and " Z " each represents different hypothesis amino acid residues.Unless otherwise expressly specified, all % amino acid sequence identities values used herein are all, according to described in the preceding paragraph, to be obtained using ALIGN-2 computer programs.

" TAT variant polynucleotides " or " TAT variant nucleic acid sequences " mean to encode TAT polypeptides as defined herein, it is preferred that activity TAT polypeptides and the total length native sequences TAT peptide sequence disclosed herein with coding, the total length native sequences TAT peptide sequences of shortage signal peptide disclosed herein, TAT polypeptide extracellular domains with and without signal peptide disclosed herein, or total length TAT peptide sequences disclosed herein any other fragment (such as those by be only total length TAT polypeptides complete encoding sequence a part nucleic acid coding fragment) nucleotide sequence have at least about 80% nucleic acid sequence identity nucleic acid molecules.Generally, TAT variant polynucleotides are with encoding total length native sequences TAT peptide sequences disclosed herein, the total length native sequences TAT peptide sequences of shortage signal peptide disclosed herein, TAT polypeptide extracellular domains with and without signal sequence disclosed herein, or the nucleotide sequence of any other fragment of total length TAT peptide sequences disclosed herein has at least about 80% nucleic acid sequence identity, or at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% nucleic acid sequence identity.The variant does not cover native nucleotide sequence.

Generally,The length of TAT variant polynucleotides is at least about 5 nucleotides,Or length is at least about 6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,35,40,45,50,55,60,65,70,75,80,85,90,95,100,105,110,115,120,125,130,135,140,145,150,155,160,165,170,175,180,185,190,195,200,210,220,230,240,250,260,270,280,290,300,310,320,330,340,350,360,370,380,390,400,410,420,430,440,450,460,470,480,490,500,510,520,530,540,550,560,570,580,590,600,610,620,630,640,650,660,670,680,690,700,710,720,730,740,750,760,770,780,790,800,810,820,830,840,850,860,870,880,890,900,910,920,930,940,950,960,970,980,990 or 1000 nucleotides,Wherein in this linguistic context,Term " about " means the 10% of the nucleotide sequence length plus or minus the length.

" percentage (%) nucleic acid sequence identity " of TAT nucleic acid sequence encodings on being identified herein is defined as contrast sequence and introduces breach when necessary to obtain after largest percentage sequence identity, with the percentage of the nucleotides identical nucleotides in TAT purpose nucleic acid sequences in candidate sequence.The alignment of percentage nucleic acid sequence identity purpose can be measured with the various ways in the range of art technology, for example using publicly available computer software, such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software.However, for the present invention, % nucleic acid sequence identity values are to compare computer program ALIGN-2 using sequence to obtain, and the complete source code of ALIGN-2 programs is provided wherein in table 1 below.ALIGN-2 sequences compare computer program and write by Genentech companies, source code shown in table 1 below submits to U.S. Copyright Office (US Copyright Office together with customer documentation, Washington D.C., 20559), and with U.S. Copyright Registration TXU510087 register.The public can obtain ALIGN-2 programs, or the compilation of source code that can be provided from table 1 below by Genentech companies (South San Francisco, California).ALIGN2 programs should be compiled into UNIX operating system, used on preferably number UNIX V4.0D.All sequences compare parameter by ALIGN-2 program settings and constant.

In the case where comparing nucleotide sequence with ALIGN-2, given nucleotide sequence C relative to (to), with (with) or for (against) give amino acid sequence D % nucleic acid sequence identities (or can be expressed as having or comprising relative to, with or for give nucleotide sequence D a certain % nucleic acid sequence identities given nucleotide sequence C) be calculated as below：

Fraction W/Z multiplies 100

Wherein W is the few nucleotide that sequence alignment programme ALIGN-2 is assessed as identical match in C and the D contrast of the program, and wherein Z is the total nucleotide number in D.It is appreciated that if nucleotide sequence C length and nucleotide sequence D length are unequal, % nucleic acid sequence identities of the C relative to D will be equal to % nucleic acid sequence identities of the D relative to C.The example calculated as % nucleic acid sequence identities, how table 4 and 5 calculates % nucleic acid sequence identity of the nucleotide sequence for being designated as " comparison dna " relative to the nucleotide sequence for being appointed as " TAT-DNA " if being demonstrated, wherein " TAT-DNA " represents purpose hypothesis TAT nucleic acid sequence encodings, " comparison dna " represents the nucleotide sequence that purpose " TAT-DNA " nucleic acid molecules are directed to its nucleic acid molecules being compared, and " N ", " L " and " V " each represents different hypothesis nucleotides.Unless otherwise expressly specified, all % nucleic acid sequence identities values used herein described in the preceding paragraph using ALIGN-2 computer programs all according to being obtained.

In other embodiments, TAT variant polynucleotides are to encode TAT polypeptides and can be with encoding the nucleotide sequence hybridizations of total length TAT polypeptides disclosed herein, the nucleic acid molecules preferably hybridized under stingent hybridization and wash conditions.TAT variant polypeptides can be the variant polypeptide that those are encoded by TAT variant polynucleotides.

Term " full length coding region " refers to the nucleotide sequence (usually showing in the accompanying drawings between starting and terminator codon (containing)) for encoding total length TAT polypeptides of the present invention in the nucleic acid for encoding TAT polypeptides.Term " full length coding region " refers to the TAT peptide codings part (usually being shown in the accompanying drawings between starting and terminator codon (containing)) for the cDNA that insertion is deposited in ATCC carrier when for ATCC preservation nucleic acid.

" separation ", when for describing various TAT polypeptides disclosed herein, it is intended that identify to come from some part of its natural surroundings, and separate and/or reclaim obtained polypeptide.The contaminant component of its natural surroundings refers to the material of the diagnosis that would generally disturb the polypeptide or therapeutical uses, it may include the solute of enzyme, hormone and other oroteins property or non-proteinaceous.In preferred embodiments, peptide purification to (1) is enough the N- ends by using spinning cup sequenator at least 15 residues of acquisition or the degree of internal amino acid sequence, or (2) reach homogeneity according to the SDS-PAGE under the non-reduced or reducing condition using Coomassie blue or preferred Silver stain.The polypeptide of separation includes the polypeptides in situ in recombinant cell, because at least one composition of TAT polypeptide natural surroundingses is not in.However, the polypeptide of separation is generally prepared by least one purification step.

" separation " TAT polypeptide encoding nucleic acids or other polypeptide encoding nucleic acids refer to from being generally accredited and disconnected nucleic acid molecules in adjoint at least one contaminative nucleic acid molecules therewith in the natural origin of the polypeptide encoding nucleic acid.The polypeptide encoding nucleic acid molecule of separation is present at the form of nature or background different from it.Therefore the polypeptide encoding nucleic acid molecule of separation has any different with the particular polypeptide coding nucleic acid molecule that is present in n cell.However, the polypeptide encoding nucleic acid molecule of separation includes the polypeptide encoding nucleic acid molecule included by the cell for being often expressed as the polypeptide, such as when the chromosome mapping when the nucleic acid molecules in the cell is different from its chromosome mapping in n cell.

Term " control sequence " refers to DNA sequence dna necessary to the coded sequence expressed and be operatively connected in specific host organism.For example, the control sequence suitable for prokaryotes includes promoter, optional operator sequence and ribosome bind site.Known eukaryotic utilizes promoter, polyadenylation signal and enhancer.

If a nucleic acid is in functional interrelationship with another nucleotide sequence, it is " being operatively connected ".If for example, presequence (presequence) or secreting the DNA of leading (secretory leader) and being expressed as participating in the preceding protein (preprotein) of polypeptide secretion, the DNA of it and polypeptide is operatively connected；If promoter or enhancer influence the transcription of coded sequence, it is operatively connected with the sequence；Or, if the position of ribosome bind site promotes translation, it is operatively connected with coded sequence.Generally, " being operatively connected " means that connected DNA sequence dna is adjacent, and means adjacent in the case of secretion is leading and be in read state.However, enhancer need not be adjacent.Connection can be realized by the coupled reaction at convenient restriction site.If without such site, then according to oligonucleotides adapter or joint of the conventional practice using synthesis.

" stringency " of hybridization reaction can be determined readily by those of ordinary skill in the art, and be calculated by rule of thumb generally according to probe length, wash temperature and salinity.Generally, the higher temperature of longer probes call is correctly to anneal, and shorter probe needs relatively low temperature.Hybridization is often relied on when complementary strand is present in the ability that time variation DNA anneals again in the environment less than its melting temperature.Probe and expectation degree of homology that can be between hybridization sequences are higher, and workable relative temperature is also higher.As can be seen here, higher relative temperature would tend to make reaction condition more strict, and lower temperature is also just less stringent.On the other details of hybridization reaction stringency and explanation, referring to Ausubel et al.,《CurrentProtocols in Molecular Biology》, Wiley Interscience Publishers, 1995.

" stringent condition " defined herein or " high stringency " can be determined by following condition：(1) washed using low ionic strength and high temperature, the lauryl sodium sulfate of such as 0.015M sodium chloride/0.0015M sodium citrates/0.1%, 50 DEG C；(2) denaturant is used in crossover process, such as formamide, such as 50% (v/v) formamide and 0.1% bovine serum albumin/0.1%Ficoll/0.1% polyvinylpyrrolidones/50mM sodium phosphate buffers pH 6.5, sodium chloride containing 750mM, 75mM sodium citrates, 42 DEG C；Or (3) are using 50% formamide, 5x SSC (0.75M NaCl, 0.075M sodium citrates), 50mM sodium phosphates (pH 6.8), 0.1% sodium pyrophosphate, 5x DenhardtShi solution, the salmon sperm dna (50 μ g/ml) of ultrasonication, 0.1%SDS, with in the solution of 10% dextran glucosides in 42 DEG C of hybridized overnights, and in 42 DEG C of washing 10 minutes in 0.2x SSC (sodium chloride/sodium citrate), then carry out 10 minutes high stringency wash in 55 DEG C in the 0.1x SSC containing EDTA.

" medium stringency condition " can such as Sambrook et al.,《Molecular Cloning：ALaboratory Manual》, New York, Cold Spring Harbor Press, determination described in 1989, including the use of than less stringent wash solution described above and hybridization conditions (such as temperature, ionic strength and %SDS).One example of medium stringency condition be in 37 DEG C containing：20% formamide, 5x SSC (150mM NaCl, 15mM trisodium citrates), 50mM sodium phosphates (pH 7.6), 5x DenhardtShi solution, 10% dextran glucosides, and 20mg/ml are denatured in the solution of the salmon sperm dna of shearing and are incubated overnight, and then wash filter membrane in about 37-50 DEG C in 1x SSC.Technical staff will appreciate how to adjust temperature, ionic strength etc. as needed to adapt to the factors such as probe length.

Term " epitope tag " refers to comprising the TAT polypeptides merged with " tag polypeptide " or the chimeric polyeptides of anti-TAT antibody as used herein.Tag polypeptide has enough residues can prepare the epitope of antibody to provide for it, but its activity for not disturbing the polypeptide merged with it that causes short enough.Tag polypeptide preferably also has suitable uniqueness so that substantially with other epitopes cross reaction does not occur for the antibody.Suitable tag polypeptide generally has at least six amino acid residue and generally between about 8 to about 50 amino acid residues (preferably between about 10 to about 20 amino acid residues).

" active " or " activity " retains natural or naturally occurring TAT biology and/or the TAT polypeptide forms of immunologic competence in order to which the present invention refers to, wherein " biology " activity refers to as caused by natural or naturally occurring TAT, induction is for the biological function (inhibition or irritating) beyond the ability of the antibody tormation of the natural or naturally occurring TAT antigenic epitopes having, and " immunology " activity refers to ability of the induction for the antibody tormation of the natural or naturally occurring TAT antigenic epitopes having.

Term " antagonist " is used with broadest, including partially or completely blocks, suppresses or neutralize any molecule of the biological activity of natural TAT polypeptides disclosed herein.Similarly, term " activator " is used with broadest, includes any molecule of the biological activity of simulation natural TAT polypeptides disclosed herein.Suitable activator or antagonist molecules clearly include excitability or antagonistic antibodies or antibody fragment, the fragment of natural TAT polypeptides or amino acid sequence variation, peptide, ASON, organic molecule etc..For identifying that the activator of TAT polypeptides or the method for antagonist molecules may include to make TAT polypeptides contact potential agonist or antagonist molecules and measure the detectable change of one or more biological activities generally relevant with TAT polypeptides.

" processing " or " treatment " or " mitigation " refer to therapeutic treatment and preventative or precaution measure both, wherein target is prevention or slows down (mitigation) targeted pathological condition or illness.Needing the subject for the treatment of includes subject already with illness and tends to suffer from the subject of illness or to prevent the subject of illness.If after the anti-TAT antibody, TAT combinations oligopeptides or TAT combination organic molecules of therapeutic dose is received according to the method for the present invention, patient shows observable and/or measurable reduction or disappearance in following one or more, then the cancer of TAT polypeptides is expressed in subject or mammal success " treatment "：Cancer cell number is reduced or cancer-free cell；Tumor mass reduction；Cancer cell is to the infiltration of peripheral organs, including propagation of the cancer into soft tissue and bone be suppressed (i.e. a certain degree of to slow down, preferably stop)；Metastases are suppressed (i.e. a certain degree of to slow down, preferably to stop)；Tumour growth is suppressed by a certain degree of；And/or the one or more symptoms relevant with particular cancers obtain a certain degree of mitigation；Morbidity and mortality are reduced；And quality of life is improved.Anti- TAT antibody or TAT combinations oligopeptides can be cell inhibiting and/or cytotoxicities, can prevent growth of cancer cells with it and/or kill existing cancer cell to be limited.The mitigation of these signs or symptom can also by patient perceptions to.

Above-mentioned parameter for assessing the successful treatment of disease and improving can be measured readily by routine protocols known to internist.For treatment of cancer, effect can be measured for example, by time (time to disease progression, TTP) before assessment progression of disease and/or measure responsiveness (response rate, RR).Transfer can be determined by testing (staging test) by stages, and by the test of bone scanning and calcium level and other enzymes to determine whether to travel to bone.CT scan can be also carried out to ascertain whether the lymph node traveled in pelvis and the region.The liver enzyme level measurement that is carried out respectively using chest X-ray and by known method ascertains whether to be transferred to lung and liver.Other conventional methods for monitoring of diseases include transrectal ultrasonography (TRUS) and per rectum needle biopsy (TRNB).

For this cancer more localized of carcinoma of urinary bladder, the urinary cytology that determining the method for progression of disease includes carrying out by cystoscopy art is assessed, monitors band blood situation in urine, shows urothelium road (urothelial tract) or intravenous injection pyelogram (intravenous pyelogram), computerized tomography (computed tomography) (CT) and magnetic resonance imaging (magneticresonance imaging) (MRI) by Ultrasonography.The presence remotely shifted can be assessed by abdominal CT, chest X-ray or bone radionuclide imaging.

" long-term " administration refers to applies medicament with the continuous mode opposite with short term patterns, so that initial treatment effect (activity) is maintained into long period of time." interval ", which is applied, refers to that processing is not continuously to carry out without interruption, but circulative.

For treatment, mitigation symptom or diagnosis for cancer, " mammal " aim enters mammiferous any animal, including people, domestic animal and livestock, and zoo, motion or pet animals, dog, cat, ox, horse, sheep, pig, goat, rabbit etc..Preferably, mammal refers to people.

The administration of " joint " one or more other therapeutic agents includes simultaneously (common) continuous administration applied with any order.

" carrier " includes pharmaceutically acceptable carrier, excipient or stabilizer as used herein, and they are nontoxic to the cell exposed to it or mammal in the dosage and concentration used.Generally, the acceptable carrier of physiology is pH aqueous buffer solutions.The example of physiology acceptable carriers includes buffer, such as phosphate, citrate and other organic acids；Antioxidant, including ascorbic acid；Low molecule amount (less than about 10 residues) polypeptide；Protein, such as serum albumin, gelatin or immunoglobulin；Hydrophilic polymer, such as polyvinylpyrrolidone；Amino acid, such as glycine, glutamine, asparagine, arginine or lysine；Monose, disaccharides and other sugar, including glucose, mannose or dextrin；Chelating agent, such as EDTA；Sugar alcohol, such as mannitol or sorbierite；Into salt gegenion, such as sodium；And/or nonionic surfactant, such as TWEEN , polyethylene glycol (PEG) and PLURONICS .

" solid phase " or " solid support " means that antibody, TAT combinations oligopeptides or the TAT combinations organic molecule of the present invention can adhere or adhere to non-aqueous base thereon.The example of the solid phase covered herein includes the solid phase that those are partially or completely made up of glass (such as controlled pore glass), polysaccharide (such as agarose), polyacrylamide, polystyrene, polyvinyl alcohol and polysiloxanes (silicone).In certain embodiments, according to actual conditions, solid phase may include the hole of assay plate；In other embodiments, it refers to purification column (such as affinity column).This term also includes the discontinuous solid phase of discrete particle, such as United States Patent (USP) 4, described in 275,149.

" liposome " refers to what is be made up of all kinds lipid, phosphatide and/or surfactant, available for the vesicles that medicine (such as TAT polypeptides, the antibody for it or TAT combinations oligopeptides) is delivered to mammal.Similar to the lipid arrangement of biomembrane, the composition of liposome is typically arranged to bilayer formation.

" small " molecule or organic " small " molecule are defined herein as molecular weight less than about 500 dalton.

Polypeptide disclosed herein, antibody, TAT combinations oligopeptides, " effective dose " of TAT combinations organic molecule or its activator or antagonist refer to the amount for being enough the purpose for realizing clear stipulaties." effective dose " can by rule of thumb and in a usual manner, and contact described purpose is determined.

Term " therapeutically effective amount " refer to can in subject or mammal the antibody of effective " treatment " disease or illness, polypeptide, TAT combinations oligopeptides, TAT combinations organic molecule or other medicines quantity.In the case of cancer, the therapeutically effective amount of medicine can reduce cancer cell number；Reduce gross tumor volume；Suppress (i.e. a certain degree of to slow down, preferably to stop) cancer cell infiltration into peripheral organs；Suppress (i.e. a certain degree of to slow down, preferably to stop) metastases；Suppress tumour growth to a certain extent；And/or mitigate the one or more symptoms relevant with cancer to a certain extent.Referring to the definition of " treatment " herein.With it growth of cancer cells and/or the existing cancer cell of kill can be prevented to be limited, medicine can be cell inhibiting and/or cytotoxicity.

Anti- TAT antibody, TAT polypeptides, " the growth inhibition amount " of TAT combinations oligopeptides or TAT combination organic molecules refer to suppress cell in vitro or in vivo, especially tumour, the amount of the growth of such as cancer cell.Anti- TAT antibody, TAT polypeptides, TAT combinations oligopeptides or TAT combinations organic molecule can be determined by rule of thumb and in a usual manner for " the growth inhibition amount " of inhibition of enoplastic cell growth.

Anti- TAT antibody, TAT polypeptides, " the cytotoxicity amount " of TAT combinations oligopeptides or TAT combination organic molecules refer to cause cell, especially tumour in vitro or in vivo, the amount of such as cancer cell destruction.Anti- TAT antibody, TAT polypeptides, TAT combinations oligopeptides or TAT combinations organic molecule can be determined by rule of thumb and in a usual manner for " the cytotoxicity amount " of inhibition of enoplastic cell growth.

Term " antibody " is used with broadest, for example single anti-TAT monoclonal antibodies (including excitability, Antagonism and neutrality antibody), the fragment (seeing below) with the specific anti-TAT antibody compositions of multi-epitope, polyclonal antibody, single-stranded anti-TAT antibody and anti-TAT antibody are clearly covered, as long as they show expectation biology or immunologic competence.Term " immunoglobulin " (Ig) is used interchangeably with antibody herein.

" antibody of separation " refers to be identified from some part of its natural surroundings, and separates and/or reclaim obtained antibody." contaminant component " of its natural surroundings refers to diagnosis or the material of therapeutical uses that can disturb the antibody, it may include the solute of enzyme, hormone and other oroteins property or non-proteinaceous.In preferred embodiments, by antibody purification to the measure of (1) according to Lowry methods, antibody weight is more than 95%, most preferably weight is more than 99%, (2) the N- ends by using spinning cup sequenator at least 15 residues of acquisition or the degree of internal amino acid sequence are enough, or (3) reach homogeneity according to the SDS-PAGE under the reproducibility or non-reducing conditions using Coomassie blue or preferred Silver stain.The antibody of separation includes the antibody iM situ in recombinant cell, because at least one composition of antibody natural surroundings is not in.However, the antibody of separation is generally prepared by least one purification step.

(IgM antibody is made up of the heterotetrameric glycoproteins that 4 basic chain antibody units are made up of two identical light chains (L) and two identical heavy chains (H) the other polypeptide of 5 different tetramer units and referred to as J chains substantially, therefore includes 10 antigen binding sites；And the polymerizable multivalence assemblage formed comprising the individual 4 basic chain elements of 2-5 and J chains of secretory IgA antibody).In the case of IgG, 4 chain elements typically about 150,000 dalton.Every light chain is connected by a covalent disulfide bonds with heavy chain, and two heavy chains are connected with each other by one or more disulfide bond, and the number of disulfide bond depends on the isotype of heavy chain.Every heavy chain and light chain also have the intrachain disulfide bond of regular interval.Every heavy chain has a variable region (V in N- ends_H), followed by three (for α and γ chains) or four (for μ and ε isotypes) constant region (C_H).Every light chain has a variable region (V in N- ends_L), followed by a constant region (C of its other end_L)。V_LWith V_HIt is arranged together, and C_LWith the first constant region (C of heavy chain_H1) it is arranged together.Think that specific amino acid residue forms interface between light chain and weight chain variable district.A paired V_HWith a V_LAn antigen binding site is formed together.On the structure and property of different classes of antibody, see, for example,《Basic and Clinical Immunology》, the 8th edition, Daniel P.Stites, Abba I.Terr and Tristram G.Parslow are compiled, Appleton ＆ Lange, Norwalk, CT, 1994, page 71 and the 6th chapter.

Light chain from any invertebrate species, according to its amino acid constant region sequence, can be included into one of two kinds of completely different types, both types are referred to as Kappa (κ) and lambda (λ).According to its heavy chain (C_H) amino acid constant region sequence, immunoglobulin can be included into different classification or isotype.Immunoglobulin has five classes：IgA, IgD, IgE, IgG and IgM, the heavy chain respectively with referred to as α, δ, ε, γ and μ.According to C_HThe secondary difference of sequence and function, γ and α classes can be further divided into multiple subclass, and such as mankind express following subclass：IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2.

It is very big that term " variable " refers to sequence difference of some of variable region section between antibody.V domains mediate antigen combines and determines specificity of the specific antibodies to its specific antigen.However, variability is not uniformly distributed in 110 amino acid of variable region leap.Conversely, V areas by 15-30 amino acid relative consistency section (be referred to as framework region (KR)), and each length for distinguishing framework is 9-12 amino acid, the shorter region of variability (be referred to as " hypervariable region ") composition.Each self-contained four FR in variable region of native heavy and light chain, they take beta sheet conformation mostly, are connected by three hypervariable regions for forming loop connecting and formed in some cases a beta sheet structure part.Hypervariable region in every chain is very closely kept together by FR, and participate in together with the hypervariable region of another chain the antigen binding site of antibody formation (referring to Kabat et al.,《Sequences of Proteins of ImmunologicalInterest》, the 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991).Constant region does not participate in the combination of antibody and antigen directly, but shows the participation of antibody in a variety of effector functions, the cytotoxicity (ADCC) of such as antibody dependent cellular mediation.

Term " hypervariable region " refers to the amino acid residue for being responsible for antigen binding in antibody as used herein.Hypervariable region generally comprises amino acid residue (such as V from " complementary determining region " or " CDR "_LIn about residue 24-34 (L1), 50-56 (L2) and 89-97 (L3) nearby and V_HIn near about residue 1-35 (H1), 50-65 (H2) and 95-102 (H3)；Kabat et al.,《Sequences of Proteins of ImmunologicalInterest》, the 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991) and/or those residue (such as V from " hypervariable loop "_LIn residue 26-32 (L1), 50-52 (L2) and 91-96 (L3) and V_HIn residue 26-32 (H1), 53-55 (H2) and 96-101 (H3)；Chothia and Lesk, J.Mol.Biol.196：901-917(1987)).

Term " monoclonal antibody " refers to the antibody obtained from the antibody of a group substantially homogeneity as used herein, that is, each antibody for constituting colony is identical, in addition to may be with the possible naturally occurring mutation of indivisible presence.Monoclonal antibody is high special, for single antigenic site.In addition, different from the polyclonal antibody preparations for generally comprising the different antibodies for being directed to different determinants (epitope), every kind of monoclonal antibody is for the single determinant on antigen.Except their specificity, the superiority of monoclonal antibody is embodied in them can be not affected by the pollution of other antibody in synthesis.Modifier " monoclonal " can not be construed to require to generate antibody by any ad hoc approach.For example, the monoclonal antibody available for the present invention can be by initially by Kohler et al., Nature, 256：Prepared by the hybridoma method of 495 (1975) description, or can be prepared by recombinant DNA method in bacterium, eucaryon animal or plant cell (see, for example, United States Patent (USP) 4,816,567)." monoclonal antibody " it is also possible to use such as Clackson et al., Nature, 352：624-628 (1991) and Marks et al., J.Mol.Biol., 222：Technology described in 581-597 (1991) is separated from phage antibody library.

Monoclonal antibody includes " chimeric " antibody herein, a wherein part for heavy chain and/or light chain is identical or homologous with derived from particular species or the corresponding sequence belonged in the antibody of specific antibodies classification or subclass, and the remainder of chain with derived from another species or the corresponding sequence belonged in the antibody of another antibody isotype or subclass, and the fragment of this antibody-like is identical or homologous, as long as they show required biological activity (referring to United States Patent (USP) 4,816,567 and Morrison et al., Proc.Natl.Acad.Sci.USA, 81：6851-6855(1984)).Chimeric antibody interested includes including variable region antigen-binding subsequences and " primatized (primatized) " antibody of human constant region sequence derived from non-human primate (such as Old World monkey class (Old World Monkey), ape) herein.

" complete antibody " refers to comprising antigen binding site and C_LAt least heavy-chain constant domains C_H1、C_H2 and C_H3 antibody.Constant region can be native sequences constant domain (such as naive sequence constant domain) or its amino acid sequence variation.Preferably, complete antibody has one or more of effector function.

" antibody fragment " includes the antigen binding domain or variable region of a part for complete antibody, preferably complete antibody.The example of antibody fragment includes Fab, Fab ', F (ab ')₂With Fv fragments；Double antibody；Linear antibodies are (referring to United States Patent (USP) 5,641,870, embodiment 2；Zapata et al., Protein Eng.8 (10)：1057-1062(1995))；Single-chain antibody molecules；And the multi-specificity antibody formed by antibody fragment.

Two identical antigen-binding fragments are produced with Papain digestion of antibodies, are referred to as " Fab " fragment, and remaining " Fc " fragment, its title reflects the ability that it is easy to crystallization.Fab fragments are by a Whole light chains and the variable domain (V of a heavy chain_H) and the first constant domain (C_H1) constitute.Each Fab fragments are monovalent for antigen binding, i.e., it has an antigen binding site.Pepsin antibody produces a larger F (ab ')₂Fragment, it is roughly equivalent to two Fab fragments being connected by disulfide bond, with bivalent antigen binding activity and still is able to crosslinking antigen.Fab ' fragments are because in C_HThe carboxyl terminal in 1 domain adds a small number of residues, including one or more cysteines from antibody hinge region, and different with Fab fragments.Fab '-SH are the appellations for the Fab ' that herein wherein constant region cysteine residues are carried with a free sulphur alcohol radical.F(ab′)₂Antibody fragment is generated as paired Fab ' fragments, has hinge cysteine between Fab ' fragments.Also know other chemical couplings of antibody fragment.

Fc fragments include the carboxy-terminal sections of two heavy chains kept together by disulfide bond.What the sequence in the effector function Shi You Fc areas of antibody was determined, the region is still present in the part that the Fc acceptors (FcR) on some cell types are recognized.

" Fv " is that the minimum antibody fragment with binding site is recognized comprising intact antigen.This fragment is made up of the dimer of the non-covalent heavy chain variable domain combined closely and a light chain variable domain.Six hypervariable loops (heavy chain and each 3 rings of light chain) are produced by the folding in the two domains, they contribute the amino acid residue with reference to antigen and assign antibody with antigen-binding specificity.Even however, single variable domain (or only including half of Fv of three CDR to antigen-specific) also has the ability for recognizing and combining antigen, although affinity is less than entire binding site.

" scFv ", can also be abbreviated as " sFv " or " scFv ", be comprising the antibody V for connecting into a polypeptide chain_HAnd V_LThe antibody fragment in domain.Preferably, sFv polypeptides are in V_HAnd V_LPeptide linker is also included between domain so that the sFv formation desired structures of antigen binding.Summary on sFv referring to Pl ü ckthun,《The Pharmacology of Monoclonal Antibodies》, vol.113, Rosenburg and Moore compile, Springer-Verlag, New York, pp.269-315,1994；Borrebaeck 1995, sees below.

Term " double antibody " refers to by V_HAnd V_LPrepared by building sFv fragments (see the preceding paragraph) using short circuit head (about 5-10 residue) between domain small antibody fragments, because joint is short, matched so that V domains are taken in interchain rather than chain, so as to generate bivalent fragment, i.e. the fragment with two antigen binding sites.Bispecific double antibody is the heterodimer of two " intersection " sFv fragments, the V of two of which antibody_HAnd V_LDomain is present on different polypeptide chains.Double antibody it is more complete be described in such as EP 404,097；WO93/11161；Hollinger et al., Proc.Natl.Acad.Sci.USA, 90：6444-6448(1993).

" humanization " form of inhuman (such as rodent) antibody refers to the chimeric antibody that bottom line includes the sequence derived from non-human antibody.Generally, some hypervariable region residues that humanized antibody refers in human immunoglobulin(HIg) (receptor antibody) immunoglobulin that some hypervariable region residues of non-human species' (donor antibody) such as mouse, rat, rabbit or non-human primates with expectation antibody specificity, affinity and ability are replaced.In some cases, framework region (FR) residue of human immunoglobulin(HIg) is replaced with into corresponding non-human residues.In addition, humanized antibody can be included in the residue for not having to find in receptor antibody or donor antibody.It is to further improve the performance of antibody to carry out these modifications.Generally, humanized antibody comprising at least one, be usually two variable domains completely or generally all, the wherein hypervariable loop for completely or generally all corresponding to non-human immunoglobulin of hypervariable loop, and entirely or substantially upper whole FR is the FR of human immunoglobulin sequence.Humanized antibody optionally also includes at least part constant region for immunoglobulin (Fc), the typically constant region of human immunoglobulin(HIg).More details are referring to Jones et al., Nature 321：522-525(1986)；Riechmann et al., Nature 332：323-329(1988)；Presta, Curr.Op.Struct.Biol.2：593-596(1992).

" species-dependent antibody ", such as mammalian anti-human's IgE antibody, refer to the antibody for having the binding affinity for being better than homologue of the antigen from the second mammalian species to the antigen from the first mammalian species.Generally, species-dependent antibody " specific binding " human antigen is (i.e. with no more than about 1 × 10^-7M, preferably more than about 1 × 10^-8M, is most preferably not more than about 1 × 10^-9M binding affinity (Kd)), but have homologue of the antigen from the second non-human mammal species at least about 50 times weak to the binding affinity of human antigen than it, or at least about 500 times, or at least about 1000 times of binding affinity.Species-dependent antibody can be all kinds antibody defined above, it is preferred that humanized antibody or human antibody.

" TAT combinations oligopeptides " refers to combination, preferably specifically binds the oligopeptides of TAT polypeptides described herein.Known oligopeptides synthetic method can be used and chemical synthesis in TAT combinations oligopeptides, or recombinant technique can be used to prepare and purify.The length of TAT combination oligopeptides is typically at least about 5 amino acid, or length is at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 amino acid or more, it can wherein combine, it is preferred that widely-known technique can be used just to be identified without excessively testing for the such TAT combinations oligopeptides for specifically binding TAT polypeptides described herein.Thus, it should be noted that the technology of the oligopeptides for being capable of specific binding polypeptide target to few peptide library selection is well-known in the art (see, for example, United States Patent (USP) 5,556,762,5,750,373,4,708,871,4,833,092,5,223,409,5,403,484,5,571,689,5,663,143；PCT Publication WO 84/03506 and WO 84/03564；Geysen et al., Proc.Natl.Acad.Sci.U.S.A., 81：3998-4002(1984)；Geysen et al., Proc.Natl.Acad.Sci.U.S.A., 82：178-182(1985)；Geysen et al., in Synthetic Peptides asAntigens, 130-149 (1986)；Geysen et al., J.Immunol.Meth., 102：259-274(1987)；Schoofs et al., J.Immunol., 140：611-616 (1988), Cwirla, S.E.et al. (1990) Proc.Natl.Acad.Sci.USA, 87：6378；Lowman, H.B.et al. (1991) Biochemistry, 30：10832；Clackson, T.et al. (1991) Nature, 352：624；Marks, J.D.et al. (1991) J.Mol.Biol., 222：581；Kang, A.S.et al. (1991) Proc.Natl.Acad.Sci.USA, 88：8363；Smith, G.P. (1991) Current Opin.Biotechnol., 2：668.

" TAT combinations organic molecule " refers in addition to oligopeptides as defined herein or antibody, with reference to preferably specifically binding the organic molecule of TAT polypeptides described herein.TAT combination organic molecules can be used known method to identify and chemical synthesis (see, for example, PCT Publication WO 00/00823 and WO00/39585).TAT is typically less than about 2000 dalton with reference to organic bulk of molecule, or size is less than about 1500,750,500,250 or 200 dalton, it can wherein combine, widely-known technique can be used just to be identified without excessively testing for the such organic molecule for preferably specifically binding TAT polypeptides described herein.Thus, it should be noted that the technology for screening the molecule for being capable of Binding peptide target from organic molecule library is (see, for example, PCT Publication WO 00/00823 and WO00/39585) well-known in the art.

" with reference to " purpose antigen, such as antibody of tumor relative polypeptide antigen target, oligopeptides, siRNA or other organic molecules, refer to that affinity the antigen can be combined enough, so that the antibody, oligopeptides or other organic molecules can be used for the cell or tissue of the targeted expression antigen as diagnosticum and/or therapeutic agent, and do not occur the molecule of cross reaction with other oroteins significantly.In such embodiment, analyzed according to fluorescence-activated cell sorting (FACS) or radioimmunoprecipitation (RIA) measure, antibody, oligopeptides, siRNA or other organic molecules combine the degree of " non-target " protein by less than the antibody, oligopeptides, siRNA or other organic molecules to about the 10% of the combination of its specific target protein.Combination for antibody, oligopeptides or other organic molecules to target molecule, term " specific bond " or " specific binding " particular polypeptide or epitope in particular polypeptide target mean measurable combination different from non-specific interaction to its " special ".Specific bond can be for example, by determining the combination of molecule and being compared and measured with the combination of control molecule, and the control molecule is typically that structure is similar but be not bound with the molecule of activity.For example, specific bond can be determined by the competition with control molecule, the control molecule is similar to target, such as excessive unmarked target material.In this case, if the combination of labeled target and probe by excessive unmarked target material Reverse transcriptase, it indicates that specific bond.Term " specific bond " or " specific binding " particular polypeptide or epitope in particular polypeptide target or to its " special ", can be at least about 10 by the Kd for example to target as used herein^-4M, or at least about 10^-5M, or at least about 10^-6M, or at least about 10^-7M, or at least about 10^-8M, or at least about 10^-9M, or at least about 10^-10M, or at least about 10^-11M, or at least about 10^-12M or bigger (greater) molecule is showed.In one embodiment, term " specific bond " refers to such combination, the wherein epitope in molecule combination particular polypeptide or particular polypeptide, and does not combine any other polypeptide or polypeptide epitope substantially.

Antibody, oligopeptides or other organic molecules or " growth inhibiting " antibody, oligopeptides or other organic molecules of the growth of tumour cell of TAT polypeptides " suppress expression ", which show expression or be overexpressed the cancer cells of suitable TAT polypeptides, causes measurable growth inhibiting molecule.TAT polypeptides can be the transmembrane polypeptide expressed on cancer cell surfaces, or can be the polypeptide for being generated and being secreted by cancer cell.Compared with appropriate controls, it is preferred that the anti-TAT antibody of growth inhibiting, oligopeptides or other organic molecules will express TAT tumour cell growth inhibition more than 20%, preferably from about 20% to about 50%, even more preferably more than 50% (e.g., from about 50% to about 100%), described to compare the tumour cell that typically unused institute's test antibody, oligopeptides or other organic molecules are handled.In one embodiment, growth inhibition effect can be measured under about 0.1 to 30 μ g/ml or about 0.5nM to 200nM antibody concentration in cell culture, wherein growth inhibition effect is determined within 1-10 days after tumour cell is exposed to antibody.Tumour cell tumor growth inhibitory action can be determined in many ways, described in such as Examples below part.If applying anti-TAT antibody with about 1 μ g/kg to about 100mg/kg body weight causes in about 5 days to 3 months away from administration of antibodies first, tumour body tumour cell is exposed to what is determined within 1-10 days after antibody in preferably from about 5 to 30 days.Tumour cell tumor growth inhibitory action can be determined in many ways, described in such as Examples below part.If applying anti-TAT antibody with about 1 μ g/kg to about 100mg/kg body weight causes in about 5 days to 3 months away from administration of antibodies first, tumor mass reduction or tumor cell proliferation reduction in preferably from about 5 to 30 days, then the antibody is growth inhibiting in vivo.

Antibody, oligopeptides, the siRNA or other organic molecules of " apoptosis-induced ", refer to the molecule of inducement of apoptosis, wherein apoptosis is combined by annexin V, DNA break, cellular contraction, endoplasmic reticulum expand, cell rupture, and/or membrane vesicle form and (are referred to as apoptotic body) to determine.The cell is typically the cell for being overexpressed TAT polypeptides.Preferably, the cell is tumour cell, for example prostate, breast, ovary, stomach, endometrium, lung, kidney, colon, bladder cells.There are a variety of methods to can be used for assessing the cell event relevant with apoptosis.For example, can combine to measure phosphatidylserine (PS) transposition by annexin；DNA break can be assessed by DNA ladder (laddering)；Core/Chromatin condensation along with DNA break can be assessed by any increase of hypodiploid cells.Preferably, apoptosis-induced antibody, oligopeptides or other organic molecules are such molecules：In annexin binding assay, it induces annexin to combine strengthens about 2 to 50 times, most preferably from about preferably from about 5 to 50 times, 10 to 50 times relative to untreated cell.

Antibody " effector function " refers to those and is attributable to the biological activity in antibody Fc district (native sequences Fc areas or amino acid sequence variation Fc areas), and changes with antibody isotype.The example of antibody mediated effect thing function includes：Clq is combined and complement-dependent cytotoxicity；Fc acceptors are combined；The cytotoxicity (ADCC) of antibody dependent cellular mediation；Phagocytosis；The downward of cell surface receptor (such as B-cell receptor)；With B cell activation.

" cytotoxicity of antibody dependent cellular mediation " or " ADCC " refer to the secreting type Ig being wherein attached to present on some cytotoxic cells (such as natural killer (NK) cell, neutrophil cell and macrophage) on Fc acceptors (FcR) and enable these cytotoxic effect cells to specifically bind the target cell for carrying antigen, and the cytotoxic form of target cell is then killed with cytotoxin.Antibody " arms " (arm) cytotoxic cell, and be that such lethal effect is absolutely required.Mediation ADCC main cell, NK cells, an expression Fc γ RIII, and monocytes Fc γ RI, Fc γ RII and Fc γ RIII.Ravetch and Kinet, Annu.Rev.Immunol., 9：The 464th page table 3 summarizes the FcR expression on hematopoietic cell in 457-92 (1991).For the ADCC activity of purpose of appraisals molecule, external ADCC determination methods, such as United States Patent (USP) 5,500,362 or 5, described in 821,337 can be carried out.Effector cell available for such determination method includes PMBC (PBMC) and natural killer (NK) cell.Or can purpose of appraisals molecule in vivo ADCC activity, such as in animal model, the acceptor of such as Clynes etFc γ RII and Fc γ RIII subclass includes the allelic variant and alternative splice forms of these acceptors.Fc γ RII acceptors include Fc γ RIIA (" activated receptor ") and Fc γ RIIB (" suppression acceptor "), and they have similar amino acid sequence, and difference essentially consists in its cytoplasm domain.Activated receptor Fc γ RIIA are in its cytoplasm domain comprising immunity receptor the activation motifs (ITAM) based on tyrosine.Suppress acceptor Fc γ RIIB and suppression motif (ITIM) of the immunity receptor based on tyrosine is included in its cytoplasm domain (referring to summary

, Annu.Rev.Immunol.15：203-234(1997)).FcR summary is referring to Ravetch and Kinet, Annu.Rev.Immunol.9：457-492(1991)；Capel et al., Immunomethods 4：25-34(1994)；De Haas et al., J.Lab.Clin.Med.126：330-341(1995)).Term " FcR " covers other FcR, including the following FcR that will be identified herein.The term also include neonatal receptor, FcRn, it is responsible for the IgG of parent being transferred to fetus (Guyer et al., J.Immunol.117：587 (1976) and Kim et al., J.Immunol.24：249(1994)).

" human effector cell " refers to expression one or more FcR, and performs the leucocyte of effector function.Preferably, the cell at least expresses Fc γ RIII and performs ADCC effector functions.Mediating the example of ADCC human leukocytes includes PMBC (PBMC), natural killer (NK) cell, monocyte, cytotoxic T cell and neutrophil cell, preferably PBMC and NK cells.Effector cell can separate from natural origin, such as blood.

" complement-dependent cytotoxicity " or " CDC " refers to the dissolving of the target cell in the presence of complement.The activation of classic complement approach is to be associated with antigen binding (suitable hypotype) antibody by the component of complement system first (Clq) combination to trigger.In order to assess complement activation, CDC determination methods, such as Gazzano-Santoro et al., J.Immunol.Methods 202 can be carried out：Described in 163 (1996).

Term " cancer " and " carcinous " refer to or described in mammal generally with the not modulated physiological status being characterized of cell growth.The example of cancer includes but is not limited to cancer, lymthoma, blastoma, sarcoma and leukaemia or lymphoid malignancies.The more specific example of such cancer includes squamous cell carcinoma (such as epithelial squamous cell cancer), lung cancer includes ED-SCLC, non-small cell lung cancer, the gland cancer of lung and the carcinoma squamosum of lung, peritoneal cancer, hepatocellular carcinoma, stomach cancer includes human primary gastrointestinal cancers, cancer of pancreas, spongioblastoma, cervical carcinoma, oophoroma, liver cancer, carcinoma of urinary bladder, carcinoma of urethra, hepatoma (hepatoma), breast cancer, colon cancer, the carcinoma of the rectum, colorectal cancer, endometrium or uterine cancer, salivary-gland carcinoma, kidney, prostate cancer, carcinoma of vulva, thyroid cancer, liver cancer, cancer of anus, carcinoma of penis, melanoma, Huppert's disease and B cell lymphoma, the cancer of the brain, and head and neck cancer, and associated transitions.

Term " cell proliferative disorders " and " proliferative disorders " refer to the illness relevant with a certain degree of abnormal cell proliferation.In one embodiment, the cell proliferative disorders refer to cancer.

B cell lymphoma, the cancer of the brain and head and neck cancer, and associated transitions.

" tumour " refers to the growth of all neoplastic cells and bred as used herein, either pernicious or benign, and (pre-cancerous) and cancerous cells and tissue before all cancers.

Antibody, oligopeptides or other organic molecules of " inducing cell death " instruct the molecule for causing the cell that can be survived to become unable to survival.The cell is the cell for expressing TAT polypeptides, and the cell of TAT polypeptides is overexpressed preferably compared with the normal cell of same organization type.TAT polypeptides can be the transmembrane polypeptide expressed on cancer cell surfaces, or can be the polypeptide for being generated and being secreted by cancer cell.Preferably, the cell is cancer cell, for example breast, ovary, stomach, endometrium, salivary gland, lung, kidney, colon, thyroid gland, pancreas or bladder cells.Cell death in vitro can be determined under conditions of in the absence of complement or immune effector cell, to distinguish by the cell death of cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) induction of antibody dependent cellular mediation.Therefore, hot inactivated serum (i.e. without complement) can be used to be carried out when in the absence of immune effector cell for the determination method for cell death.In order to determine whether antibody, oligopeptides or other organic molecules being capable of inducing cell deaths, the forfeiture of film integrality can be assessed relative to untreated cell, it is (referring to Moore et al.Cytotechnology 17 by propidium iodide (propidium iodide) (PI), trypan blue：1-11 (1995)) or 7AAD intake assess.It is preferred that inducing cell death antibody, oligopeptides or other organic molecules be PI absorb determination method in BT474 cells induce PI intake molecule.

" expression TAT cell " refers to or expressed on cell surface or with secreted form the cell of endogenous or transfection TAT polypeptides." expression TAT cancer " refers to the cancer for the cell that there is TAT polypeptides or generation on cell surface and secrete TAT polypeptides." expression TAT cancer " optionally generates the TAT polypeptides of enough levels on its cell surface so that anti-TAT antibody, oligopeptides or other organic molecules can be in connection and produce therapeutic effect to the cancer.In another embodiment, " expression TAT cancer " optionally generates and secreted the TAT polypeptides of enough levels so that anti-TAT antibody, oligopeptides or other organic molecule antagonists can be in connection and produce therapeutic effect to the cancer.For the latter, the antagonist can be reduction, suppress or prevent tumour cell from generating and secrete the ASON of secretion-type T AT polypeptides.The cancer of " overexpression " TAT polypeptides refers to compared with the non-cancerous cell of same organization type, the cancer of the TAT polypeptides of TAT polypeptides or generation and the significantly higher level of secretion on its cell surface with significantly higher level.Such overexpression can be either by transcribing or translating caused by enhancing by gene magnification.Can in diagnosis or prognostic assays by assess present on cell surface or TAT protein matter level that cell is secreted rise nucleic acid or mRNA level, for example by FISH, the nucleic acid or the probe (FISH based on nucleic acid of its complementary strand corresponding to coding TAT are used；Referring to WO 98/45479, in October, 1998 is disclosed in)；Southern traces；Northern traces；Or PCR (PCR) technology, such as real-time quantitative PCR (RT-PCR).The determination method based on antibody is it is also possible to use, TAT polypeptides overexpression is studied by measuring the released antigen in biological fluid such as serum and (referring also to such as United States Patent (USP) 4,933,294, is issued to June nineteen ninety 12；WO 91/05264, is disclosed on April 18th, 1991；United States Patent (USP) 5,401,638, is issued to March nineteen ninety-five 28；Sias et al., J.Immunol.Methods 132：73-80(1990)).Except said determination method, skilled practitioner is also using a variety of in vivoassay methods.For example, can be by cell in patient's body exposed to optionally with the antibody of detectable such as labelled with radioisotope, and the combination of cell in antibody and patient's body can be assessed, such as the biopsy being derived from by external scan radioactivity or by analysis in advance exposed to the patient of the antibody is cut into slices.

As used herein, term " immunoadhesin " refers to the antibody sample molecule that the effector function of the binding specificity of heterologous protein (" adhesin ") and immunoglobulin constant domain is joined together.In structure, immunoadhesin includes the fusions of antigen recognizing and binding site (being " heterologous ") different from antibody, the amino acid sequence with expectation binding specificity and immunoglobulin constant domain sequence.The adhesin part of immunoadhesin molecule is typically continuous (contiguous) amino acid sequence of the binding site including at least acceptor or part.Constant region for immunoglobulin sequence in immunoadhesin can be obtained from any immunoglobulin, such as IgG-1, IgG-2, IgG-3 or IgG-4 hypotype, IgA (including IgA-1 and IgA-2), IgE, IgD or IgM.

Word " label " refers to as used herein to be directly or indirectly coupled to produce the detectable compounds or composition of " labeled " antibody, oligopeptides or other organic molecules with antibody, oligopeptides or other organic molecules.Label can be itself just detectable (such as radioisotopic tracer or fluorescent marker), or, for enzyme marker, substrate compounds or composition can be catalyzed and occur detectable chemical modification.

Term " cytotoxic agent " refers to suppression or prevents the function of cell and/or cause the material of cytoclasis as used herein.The term is intended to include：Radio isotope (such as At²¹¹、I¹³¹、I¹²⁵、Y⁹⁰、Re¹⁸⁶、Re¹⁸⁸、Sm¹⁵³、Bi²¹²、P³²With Lu radio isotope)；Chemotherapeutics；Enzyme and its fragment, such as nucleolytic enzyme；Antibiotic；And toxin, such as small molecule toxins or bacterium, fungi, the enzyme activity toxin of plant or animal origin, including its fragment and/or variant；And the various antineoplastics or anticarcinogen being disclosed below.Hereafter describe other cytotoxic agents.Kill the destruction that tumour efficacy-enhancing ingredient plays tumour cell.

" chemotherapeutics " refers to the chemical compound available for treating cancer.The example of chemotherapeutics includes alkylating agents (alkylating agents), such as phosphinothioylidynetrisaziridine (thiotepa) and CYTOXAN  endoxan (cyclophosphamide)；Alkylsulfonates (alkyl sulfonates), such as busulfan (busulfan), Improsulfan (improsulfan) and piposulfan (piposulfan)；Aziridines (aziridines), such as Benzodepa (benzodepa), carboquone (carboquone), meturedepa (meturedepa) and uredepa (uredepa)；Ethylenimines (ethylenimines) and methylamelamines (methylamelamines), including hemel (altretamine), triethylenemelamine (triethylenemelamine), triethylphosphoramide (triethylenephosphoramide), triethylene thiophosphamide (triethylenethiophosphoramide) and trimethylolmelamine (trimethylolomelamine)；Annonaceousacetogenicompounds (acetogenins) (especially bullatacin (bullatacin) and bullatacinone (bullatacinone))；Delta-9-Tetrahydrocannabinol (tetrahydrocannabinol) (Dronabinol (dronabinol), MARINOL )；β-lapachol (lapachone)；Lapachol (lapachol)；Colchicines (colchicines)；Betulic acid (betulinicacid)；Camptothecine (camptothecin) (including synthetic analogues Hycamtin (topotecan) (HYCAMTIN ), CPT-11 (Irinotecan (irinotecan), CAMPTOSAR ), acetyl camptothecine, scopoletin (scopoletin) and 9-aminocamptothecin (9-amino camptothecin)；Bryostatin (bryostatin)；callystatin；CC-1065 (including its Adozelesin (adozelesin), Carzelesin (carzelesin) and Bizelesin (bizelesin) synthetic analogues)；Podophyllotoxin (podophyllotoxin)；Podophyllic acid (podophyllinic acid)；Teniposide (teniposide)；Cryptophycins (cryptophycins) (particularly cryptophycin 1 and cryptophycin 8)；Dolastatin (dolastatin)；Duocarmycin (including synthetic analogues, KW-2189 and CB1-TM1)；Eleutherobin (eleutherobin)；pancratistatin；sarcodictyin；Spongistatin (spongistatin)；Nitrogen mustards (nitrogen mustards), such as Chlorambucil (chlorambucil), Chlornaphazine (chlornaphazine), cholophosphamide (cholophosphamide), Estramustine (estramustine), ifosfamide (ifosfamide), chlormethine (mechlorethamine), mustron (mechlorethamine oxide hydrochloride), melphalan (melphalan), novoembichin (novembichin), phenesterin (phenesterine), prednimustine (prednimustine), Trofosfamide (trofosfamide), uracil mastard (uracil mustard)；Nitrosourea (nitrosoureas), such as BCNU (carmustine), chlorozotocin (chlorozotocin), Fotemustine (fotemustine), lomustine (lomustine), Nimustine (nimustine) and Ranimustine (ranimustine)；Antibioticses, such as Enediyne Antibiotic (enediyne) (such as Calicheamicin (calicheamicin), especially Calicheamicin γ 1I and Calicheamicin ω I1 are (see, for example, Agnew, Chem.Intl.Ed.Engl.33：183-186(1994))；Anthracycline antibiotic (dynemicin), including dynemicinA；Ai sibo mycin (esperamicin)；And Neocarzinostatin (neocarzinostatin) chromophore and related chromoprotein Enediyne Antibiotic chromophore),Aclacinomycin (aclacinomycin),D actinomycin D (actinomycin),Anthramycin (authramycin),Azaserine (azaserine),Bleomycin (bleomycin),Act-C (cactinomycin),carabicin,Carminomycin (carminomycin),Cardinophyllin (carzinophilin),Chromomycin (chromomycin),Actinomycin D (dactinomycin),Daunorubicin (daunorubicin),Detorubicin (detorubicin),6- phenodiazine -5- oxygen-L- nor-leucines,ADRIAMYCIN  Doxorubicins (doxorubicin) (including morpholino Doxorubicin,Cyanomorpholino Doxorubicin,2- pyrroles is for Doxorubicin and deoxydoxorubicin),Epirubicin (epirubicin),Esorubicin (esorubicin),Idarubicin (idarubicin),Marcellomycin (marcellomycin),Mitomycin (mitomycins) such as mitomycin C,Mycophenolic acid (mycophenolic acid),Nogalamycin (nogalamycin),Olivomycin (olivomycin),Peplomycin (peplomycin),potfiromycin,Puromycin (puromycin),Triferricdoxorubicin (quelamycin),Rodorubicin (rodorubicin),Streptonigrin (streptonigrin),Streptozotocin (streptozocin),Tubercidin (tubercidin),Ubenimex (ubenimex),Zinostatin (zinostatin),Zorubicin (zorubicin)；Antimetabolic species, such as methopterin (methotrexate) and 5 FU 5 fluorouracil (5-FU)；Folacin, such as denopterin (denopterin), methopterin (methotrexate), pteroyltriglutamic acid (pteropterin), Trimetrexate (trimetrexate)；Purine analogue, such as fludarabine (fludarabine), Ismipur (mercaptopurine), thiapurine (thiamiprine), thioguanine (thioguanine)；Pyrimidine analogue, such as ancitabine (ancitabine), azacitidine (azacitidine), 6- azauridines (azauridine), Carmofur (carmofur), cytarabine (cytarabine), dideoxyuridine (dideoxyuridine), doxifluridine (doxifluridine), enocitabine (enocitabine), floxuridine (floxuridine)；Androgens, such as calusterone (calusterone), dromostanolone propionate (dromostanolone propionate), epitiostanol (epitiostanol), Mepitiostane (mepitiostane), Testolactone (testolactone)；Anti- adrenal gland class, such as aminoglutethimide (aminoglutethimide), mitotane (mitotane), Trilostane (trilostane)；Folic acid supplement, such as folinic acid (folinic acid)；Aceglatone (aceglatone)；Aldophosphamideglycoside (aldophosphamide glycoside)；Amino-laevulic acid (aminolevulinic acid)；Eniluracil (eniluracil)；Amsacrine (amsacrine)；bestrabucil；Bisantrene (bisantrene)；Edatrexate (edatraxate)；Defosfamide (defosfamide)；Demecolcine (demecolcine)；Diaziquone (diaziquone)；elfomithine；Elliptinium Acetate (elliptinium acetate)；Epothilones (epothilone)；Ethoglucid (etoglucid)；Gallium nitrate；Hydroxyurea (hydroxyurea)；Lentinan (lentinan)；Lonidamine (lonidamine)；Maytansinoids (maytansinoids), such as maytansine (maytansine) and ansamitocin (ansamitocin)；Mitoguazone (mitoguazone)；Mitoxantrone (mitoxantrone)；Mopidamol (mopidamol)；C-283 (nitracrine)；Pentostatin (pentostatin)；Phenamet (phenamet)；THP (pirarubicin)；Losoxantrone (losoxantrone)；2- ethylhydrazides (ethylhydrazide)；Procarbazine (procarbazine)；PSK  polysaccharide compounds (JHS NaturalProducts, Eugene, OR)；Razoxane (razoxane)；Rhizomycin (rhizoxin)；Sizofiran (sizofiran)；Spirogermanium (spirogermanium)；Tenuazonic acid (tenuazonic acid)；Triethyleneiminobenzoquinone (triaziquone)；2,2 ', 2 "-trichlorotriethylamines；Trichothecin class (trichothecenes) (especially T-2 toxin, verrucarine (verrucarin) A, roridin (roridin) A and snake rhzomorph (anguidin))；Urethane (urethan)；Eldisine (vindesine) (ELDISINE , FILDESIN )；Dacarbazine (dacarbazine)；Mannomustine (mannomustine)；Dibromannitol (mitobronitol)；Mitolactol (mitolactol)；Pipobroman (pipobroman)；gacytosine；Cytarabine (arabinoside) (" Ara-C ")；Phosphinothioylidynetrisaziridine (thiotepa)；Taxoids (taxoids), such as TAXOL  taxols (paclitaxel) (Bristol-Myers Squibb Oncology, Princeton, NJ.), the ABRAXANE without cremophor (Cremophor)^TM, albumin transformation nano particle formulation taxol (paclitaxel) (American Pharmaceutical Partners, Schautoberg, Illinois) and TAXOTERE  Taxoteres (doxetaxel) (

- Poulenc Rorer, Antony, France)；Chlorambucil (chlorambucil)；Gemcitabine (gemcitabine) (GEMZAR )；6- sulphur crow purine (thioguanine)；Purinethol (mercaptopurine)；Methopterin (methotrexate)；Platinum analogs, such as cis-platinum (cisplatin) and carboplatin (carboplatin)；Vincaleukoblastinum (vinblastine) (VELBAN )；Platinum (platinum)；Etoposide (etoposide) (VP-16)；Ifosfamide (ifosfamide)；Mitoxantrone (mitoxantrone)；Vincristine (vincristine) (ONCOVIN )；Oxaliplatin (oxaliplatin)；Folinic acid (leucovorin)；Vinorelbine (vinorelbine) (NAVELBINE )；NSC-279836 (novantrone)；Edatrexate (edatrexate)；Daunomycin (daunomycin)；Aminopterin (aminopterin)；Ibandronate (ibandronate)；Topoisomerase enzyme inhibitor RFS 2000；DFMO (DMFO)；Retinoic acid-like class (retinoids), such as retinoic acid (retinoic acid)；Capecitabine (capecitabine) (XELODA )；Pharmaceutically acceptable salt, acid or the derivative of any of above material；And the combination of two or more above-mentioned substances, such as CHOP (endoxan, Doxorubicin, the abbreviation of vincristine and prednisolone conjoint therapy) and FOLFOX (oxaliplatin (ELOXATIN^TM) joint 5-FU and folinic acid therapeutic scheme abbreviation).

This definition also includes acting as adjusting, reduce, block or suppressing that the antihormone agent of the hormone effect of cancer growth, and the often form of system or whole body therapeutic can be promoted.Their own can be hormone.Example includes anti-estrogens and SERM class (SERM), including such as TAM (tamoxifen) (including NOLVADEX  TAMs), EVISTA  Raloxifenes (raloxifene), Droloxifene (droloxifene), 4-hydroxytamoxifen, Trioxifene (trioxifene), that Lip river former times fragrant (keoxifene), LY117018, Onapristone (onapristone) and FARESTON  Toremifenes (toremifene)；Antiprogestin class；Estrogen receptor down agent class (ERD)；Function is suppression or the medicament of closing ovary, such as luteinizing hormone releasing hormone (LHRH) activator, such as LUPRON  and ELIGARD  leuprorelin acetates (leuprolide acetate), goserelin acetate (goserelinacetate), buserelin acetate (buserelin acetate) and Triptorelin (triptorelin)；Other anti-androgenses, such as Drogenil (flutamide), Nilutamide (nilutamide) and bicalutamide (bicalutamide)；And suppress to adjust the aromatase inhibitor of the aromatase enzyme of estrogen production, such as 4 (5)-imidazoles, aminoglutethimide (aminoglutethimide), MEGASE  megestrol acetates (megestrol acetate), AROMASIN  Exemestanes (exemestane), formestane (fbrmestane), Fadrozole (fadrozole), RIVISOR  Vorozoles (vorozole), FEMARA  Letrozoles (letrozole) and ARIMIDEX  Anastrozoles (anastrozole) in adrenal gland.In addition, this definition of chemotherapeutics includes diphosphonates (bisphosphonates), such as clodronate (clodronate) (such as BONEFOS  or OSTAC ), DIDROCAL  etidronates (etidronate), NE-58095, ZOMETA  zoledronic acids/zoledronate (zoledronic acid/zoledronate), FOSAMAX  alendronates (alendronate), AREDIA  Pamidronates (pamidronate), SKELID  Tiludronates (tiludronate) or ACTONEL  Risedronates (risedronate)；And troxacitabine (troxacitabine) (DOX nucleosides analogue of cytosine)；ASON, particularly those ASONs for suppressing to participate in gene expression of the signal of adherent cell proliferation in, such as PKC- α, Raf, H-Ras and EGF-R ELISA (EGF-R)；Vaccine, such as THERATOPE  vaccines and gene therapy vaccine, such as ALLOVECTIN  vaccines, LEUVECTIN  vaccines and VAXID  vaccines；The inhibitor of LURTOTECAN  topoisomerases 1；ABARELIX  rmRH；Lapatinib ditosylate (ErbB-2 and EGFR dual tyrosine kinase micromolecular inhibitors, also referred to as GW572016)；And pharmaceutically acceptable salt, acid or the derivative of any of above material.

" growth inhibitor " refers to suppress cell in vitro or in vivo as used herein, especially expresses the compound or composition of TAT growth of cancer cells.Therefore, growth inhibitor can be the medicament of the ratio for the TAT expressivity cells for significantly reducing the S phases.The example of growth inhibitor includes blocking the cell cycle to carry out the medicament (at the position beyond the S phases), such as induces the medicament that G1 is stagnated and the M phases stagnate.Classical M phases blocking agent includes long aphrodisiac class (vincas) (vincristine (vincristine) and vincaleukoblastinum (vinblastine)), taxanes (taxanes) and Topoisomerase II inhibitors such as Doxorubicin (doxorubicin), epirubicin (epirubicin), daunorubicin (daunorubicin), Etoposide (etoposide) and bleomycin (bleomycin).Those retardances G1 medicaments also extend to the S phases and stagnated, such as DNA alkylating agents such as TAM (tamoxifen), metacortandracin (prednisone), Dacarbazine (dacarbazine), chlormethine (mechlorethamine), cis-platinum (cisplatin), methotrexate (MTX) (methotrexate), 5 FU 5 fluorouracil (5-fluorouracil) and ara-C.More information can be found in《TheMolecular Basis of Cancer》, Mendelsohn and Israel compile, the 1st chapter, entitled " Cell cycleregulation, oncogenes, and antieioplastic drugs ", Murakaini et al., WB Saunders, Philadelphia, 1995, especially the 13rd page.Taxanes (taxol (paclitaxel) and docetaxel (docetaxel)) is all the anticarcinogen derived from yew tree.Docetaxel (TAXOTERE , Rhone-Poulenc Rorer) derived from European yew is taxol (TAXOL , Bristol-MyersSquibb) semi-synthetic analog.Taxol and docetaxel promote to be assembled into micro-pipe and by preventing depolymerization from making microtubule stabilization by tubulin dimer, cause the suppression to cell mitogen.

" Doxorubicin (Doxorubicin) " is anthracycline antibiotic.The full chemical name of Doxorubicin is (8S- is cis) -10- [(3- amino -2, 3, deoxidation-α-L- the lysols of 6- tri--pyranohexose base) epoxide] -7, 8, 9, 10- tetrahydrochysenes -6, 8, 11- trihydroxies -8- (hydroxyacetyl) -1- methoxyl groups -5, 12- naphthalenediones ((8S-cis) -10- [(3-amino-2, 3, 6-trideoxy- α-L-lyxo-hexapyranosyl) oxy] -7, 8, 9, 10-tetrahydro-6, 8, 11-trihydroxy-8- (hydroxyacetyl) -1-methoxy-5, 12-naphthacenedione).

Term " cytokine " " refers to a kind of common name of protein discharged by cell mass, that another cell is acted on as extracellular medium.The example of this type cytokines has lymphokine, monokine and traditional polypeptide hormone.Cell factor includes growth hormone, such as human growth hormone (HGH), N- methionyl human growth hormones and bovine growth hormone；Parathormone；Thyroxine；Insulin；Proinsulin；Relaxins；Relaxins is former；Glycoprotein hormone, such as follicle-stimulating hormone (FSH) (FSH), thyrotropic hormone (TSH) and metakentrin (LH)；Liver growth factor；Fibroblast growth factor；Prolactin；Human placental lactogen；Tumor necrosis factor-alpha and-β；Mu Leshi (Mullerian) inhibitory substance；Small mouse promoting sexual gland hormone related peptide；Inhibin；Activin；VEGF；Integrin；TPO (TPO)；Nerve growth factor, such as NGF- β；Platelet growth factor；TGF (TGF), such as TGF- α and TGF-β；Insulin like growth factor-1 and-II；Hematopoietin (EPO)；Bone-inducing factor；Interferon, such as interferon-' alpha ' ,-β and-γ；Colony stimulating factor (CSF), such as macrophage CSF (M-CSF), granulocytes-macrophages CSF (GM-CSF) and granulocyte CSF (G-CSF)；Interleukin (IL), such as IL-1, IL-1a, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12；TNF, such as TNF-α or TNF-β；And other polypeptide factors, including LIF and kit parts (KL).As used herein, term cell factor includes the biological activity equivalent of the protein and native sequence cytokines from natural origin or from recombinant cell culture thing.

Term " package insert ", which is used to refer to, is typically included in specification in the commodity packaging for the treatment of product, and they, which include, concerns and the indication of such treatment products application, usage, dosage, the information using, contraindication and/or warning.

Table 1

/*

* C-C increased from 12 to 15

* Z is average of EQ

* B is average of ND

* match with stop is_M；Stop-stop=0；J (joker) match=0

*/

#define_M -8 /* value of a match with a stop */

Int _ day [26] [26]=

/* A B C D E F G H I J K L M N O P Q R S T U V W X Y Z */

/ * A */{ 2,0, -2,0,0, -4,1, -1, -1,0, -1, -2, -1,0, _ M, 1,0, -2,1,1,0,0, -6,0, -3,0 },

/ * B */{ 0,3, -4,3,2, -5,0,1, -2,0,0, -3, -2,2, _ M, -1,1,0,0,0,0, -2, -5,0, -3,1 },

/ * C */{ -2, -4,15, -5, -5, -4, -3, -3, -2,0, -5, -6, -5, -4, _ M, -3, -5, -4,0, -2,0, -2, -8,0,0, -5 },

/ * D */{ 0,3, -5,4,3, -6,1,1, -2,0,0, -4, -3,2, _ M, -1,2, -1,0,0,0, -2, -7,0, -4,2 },

/ * E */{ 0,2, -5,3,4, -5,0,1, -2,0,0, -3, -2,1, _ M, -1,2, -1,0,0,0, -2, -7,0, -4,3 },

/ * F */{ -4, -5, -4, -6, -5,9, -5, -2,1,0, -5,2,0, -4, _ M, -5, -5, -4, -3, -3,0, -1,0,0,7, -5 },

/ * G */{ 1,0, -3,1,0, -5,5, -2, -3,0, -2, -4, -3,0, _ M, -1, -1, -3,1,0,0, -1, -7,0, -5,0 },

/ * H */{ -1,1, -3,1,1, -2, -2,6, -2,0,0, -2, -2,2, _ M, 0,3,2, -1, -1,0, -2, -3,0,0,2 },

/ * I */{ -1, -2, -2, -2, -2,1, -3, -2,5,0, -2,2,2, -2, _ M, -2, -2, -2, -1,0,0,4, -5,0, -1, -2 },

/ * J */{ 0,0,0,0,0,0,0,0,0,0,0,0,0,0, _ M, 0,0,0,0,0,0,0,0,0,0,0 },

/ * K */{ -1,0, -5,0,0, -5, -2,0, -2,0,5, -3,0,1, _ M, -1,1,3,0,0,0, -2, -3,0, -4,0 },

/ * L */{ -2, -3, -6, -4, -3,2, -4, -2,2,0, -3,6,4, -3, _ M, -3, -2, -3, -3, -1,0,2, -2,0, -1, -2 },

/ * M */{ -1, -2, -5, -3, -2,0, -3, -2,2,0,0,4,6, -2, _ M, -2, -1,0, -2, -1,0,2, -4,0, -2, -1 },

/ * N */{ 0,2, -4,2,1, -4,0,2, -2,0,1, -3, -2,2, _ M, -1,1,0,1,0,0, -2, -4,0, -2,1 },

/ * O */{ _ M, _ M, _ M, M, M, _ M, _ M, _ M, _ M, _ M, _ M, _ M, _ M, _ M, 0, _ M, _ M, _ M, _ M, _ M, _ M, _ M, _ M, _ M, _ M, _ M },

/ * P */{ 1, -1, -3, -1, -1, -5, -1,0, -2,0, -1, -3, -2, -1, _ M, 6,0,0,1,0,0, -1, -6,0, -5,0 },

/ * Q */{ 0,1, -5,2,2, -5, -1,3, -2,0,1, -2, -1,1, _ M, 0,4,1, -1, -1,0, -2, -5,0, -4,3 },

/ * R */{ -2,0, -4, -1, -1, -4, -3,2, -2,0,3, -3,0,0, _ M, 0,1,6,0, -1,0, -2,2,0, -4,0 },

/ * S */{ 1,0,0,0,0, -3,1, -1, -1,0,0, -3, -2,1, _ M, 1, -1,0,2,1,0, -1, -2,0, -3,0 },

/ * T */{ 1,0, -2,0,0, -3,0, -1,0,0,0, -1, -1,0, _ M, 0, -1, -1,1,3,0,0, -5,0, -3,0 },

/ * U */{ 0,0,0,0,0,0,0,0,0,0,0,0,0,0, _ M, 0,0,0,0,0,0,0,0,0,0,0 },

/ * V */{ 0, -2, -2, -2, -2, -1, -1, -2,4,0, -2,2,2, -2, _ M, -1, -2, -2, -1,0,0,4, -6,0, -2, -2 },

/ * W */{ -6, -5, -8, -7, -7,0, -7, -3, -5,0, -3, -2, -4, -4, _ M, -6, -5,2, -2, -5,0, -6,17,0,0, -6 },

/ * X */{ 0,0,0,0,0,0,0,0,0,0,0,0,0,0, _ M, 0,0,0,0,0,0,0,0,0,0,0 },

/ * Y */{ -3, -3,0, -4, -4,7, -5,0, -1,0, -4, -1, -2, -2, _ M, -5, -4, -4, -3, -3,0, -2,0,0,10, -4 },

/ * Z */{ 0,1, -5,2,3, -5,0,2, -2,0,0, -2, -1,1, _ M, 0,3,0,0,0,0, -2, -6,0, -4,4 }

}；

/*

*/

#include<stdio.h>

#include<ctype.h>

#define MAXJMP 16 /* max jumps in a diag */

#define MAXGAP 24 /* don′t continue to penalize gaps larger than this */

#define JMPS 1024 /* maxjmps in an path */

#define MX 4 /* save if there′s at least MX-1 bases since last jmp */

#define DMAT 3 /* value of matching bases */

#define DMIS 0 /* penalty for mismatched bases */

#define DINS0 8 /* penalty for a gap */

#define DINS1 1 /* penalty per base */

#define PINS0 8 /* penalty for a gap */

#define PINS1 4 /* penalty per residue */

struct jmp {

short n[MAXJMP]； /* size of jmp(neg for dely)*/

unsigned short x[MAXJMP]； /* base no. of jmp in seq x */

}； /* limits seq to 2^16-1 */

struct diag {

int score； /* score at last jmp */

long offset； /* offset of prey block */

short ijmp； /* current jmp index */

struct jmp jp； /* list of jmps */

}；

struct path {

int spc； /* number of leading spaces */

short n[JMPS]； /* size of jmp(gap)*/

int x[JMPS]； /* loc of jmp(last elem before gap) */

}；

char *ofile； /* output file name */

char *namex[2]； /* seq names:getseqs()*/

char *prog； /* prog name for err msgs */

char *seqx[2]； /* seqs:getseqs()*/

int dmax； /* best diag:nw()*/

int dmax0； /* final diag */

int dna； /* set if dna:main()*/

int endgaps； /* set if penalizing end gaps */

Int gapx, gapy； /* total gaps in seqs */

Int len0, len1； /* seq lens */

Int ngapx, ngapy； /* total size of gaps */

int smax； /* max score:nw()*/

int *xbm； /* bitmap for matching */

long offset； /* current offset in jmp file */

struct diag *dx； /* holds diagonals */

structpath pp[2]； /* holds path for seqs */

Char * calloc (), * malloc (), * index (), * strcpy ()；

Char * getseq (), * g_calloc ()；

/* Needleman-Wunsch alignment program

*

* usage: progs file1 file2

* where file1 and file2 are two dna or two protein sequences.

* The sequences can be in upper-or lower-case an may contain ambiguity

* Any lines beginning with ′；', ' ＞ ' or ' ＜ ' are ignored

* Max file length is 65535(limited by unsigned short x in the jmp struct)

* A sequence with 1/3 or more of its elements ACGTU is assumed to be DNA

* Output is in the file ″align.out″

*

* The program may create a tmp file in/tmp to hold info about traceback.

* Original version developed under BSD 4.3 on a vax 8650

*/

#include ″nw.h″

#include ″day.h″

Static_dbval [26]=

1,14,2,13,0,0,4,11,0,0,12,0,3,15,0,0,0,5,6,8,8,7,9,0,10,0

}；

Static_pbval [26]=

1,2 | (1 ＜＜ (' D '-' A ')) | (1 ＜＜ (' N '-' A ')), 4,8,16,32,64,

The ＜＜ 14 of 128,256,0xFFFFFFF, 1 ＜＜, 10,1 ＜＜, 11,1 ＜＜, 12,1 ＜＜ 13,1,

1 ＜＜ 15,1 ＜＜ 16,1 ＜＜ 17,1 ＜＜ 18, the ＜＜ 21 of 1 ＜＜, 19,1 ＜＜ 20,1,1 ＜＜ 22

1 ＜＜ 23, the ＜＜ 25 of 1 ＜＜ 24,1 | (1 ＜＜ (' E '-' A ')) | (1 ＜＜ (' Q '-' A '))

}；

Main (ac, ay) main

int ac；

char *av[]；

{

Prog=av [0]；

if(ac！=3)

Fprintf (stderr, " usage:%s file1 file2 n ", prog)；

Fprintf (stderr, " where file1 and file2 are two dna or two protein sequences. n ")；

Fprintf (stderr, " The sequences can be in upper-or lower-case n ")；

Fprintf (stderr, " Any lines beginning with '；' or ' ＜ ' are ignored n ")；

Fprintf (stderr, " Output is in the file " align.out " n ")；

exit(1)；

}

Namex [0]=av [1]；

Namex [1]=av [2]；

Seqx [0]=getseq (namex [0] , ＆len0)；

Seqx [1]=getseq (namex [1] , ＆len1)；

Xbm=(dna)_dbval:_pbval；

Endgaps=0； /* 1 to penalize endgaps */

Ofile=" align.out "；/* output file */

nw()；/ * fill in the matrix, get the possible jmps */

readjmps()； /* get the actual jmps */

print()；/ * print stats, alignment */

cleanup(0)； /* unlink any tmp files */

}

/ * do the alignment, return best score:main()

* dna:Values in Fitch and Smith, PNAS, 80,1382-1386,1983

* pro:PAM 250 values

* When scores are equal, we prefer mismatches to any gap, prefer

* a new gap to extending an ongoing gap, and prefer a gap in seqx

* to a gap in seq y.

*/

nw() nw

{

Char * px, * py； /* seqs and ptrs */

Int * ndely, * dely； /* keep track of dely */

Int ndelx, delx；/* keep track of delx */

int *tmp；/ * for swapping row0, row1 */

int mis； /* score for each type */

Int ins0, ins1； /* insertion penalties */

register id； /* diagonal index */

register ij； /* jmp index */

Register * col0, * col1；/ * score for curt, last row */

Register xx, yy； /* index into seqs */

Dx=(struct diag *) g_calloc (" to get diags ", len0+len1+1, sizeof (struct diag))；

Ndely=(int *) g_calloc (" to get ndely ", len1+1, sizeof (int))；

Dely=(int *) g_calloc (" to get dely ", len1+1, sizeof (int))；

Col0=(int *) g_calloc (" to get col0 ", len1+1, sizeof (int))；

Col1=(int *) g_calloc (" to get col1 ", len1+1, sizeof (int))；

Ins0=(dna)DINS0:PINS0；

Ins1=(dna)DINS1:PINS1；

Smax=-10000；

if(endgaps){

For (col0 [0]=dely [0]=- ins0, yy=1；Yy ＜=len1；yy++){

Col0 [yy]=dely [yy]=col0 [yy-1]-ins1；

Ndely [yy]=yy；

}

Col0 [0]=0；/* Waterman Bull Math Biol 84 */

}

else

For (yy=1；Yy ＜=len1；yy++)

Dely [yy]=- ins0；

/* fill in match matrix

*/

For (px=seqx [0], xx=1；Xx ＜=len0；Px++, xx++)

/* initialize first entry in col

*/

if(endgaps){

If (xx==1)

Col1 [0]=delx=- (ins0+ins1)；

else

Col1 [0]=delx=col0 [0]-ins1；

Ndelx=xx；

}

else{

Col1 [0]=0；

Delx=-ins0；

Ndelx=0；

}

...nw

For (py=seqx [1], yy=1；Yy ＜=len1；Py++, yy++)

Mis=col0 [yy-1]；

if(dna)

Mis+=(xbm [* px- ' A ']s ＆xbm [* py- ' A '])DMAT:DMIS；

else

Mis+=_day [* px- ' A '] [* py- ' A ']；

/* update penalty for del in x seq；

* favor new del over ongong del

* ignore MAXGAP if weighting endgaps

*/

If (endgaps | | ndely [yy] ＜ MAXGAP)

If (col0 [yy]-ins0 ＞=dely [yy])

Dely [yy]=col0 [yy]-(ins0+ins1)；

Ndely [yy]=1；

}else{

Dely [yy] -=ins1；

ndely[yy]++；

}

}else{

If (col0 [yy]-(ins0+ins1) ＞=dely [yy])

Dely [yy]=col0 [yy]-(ins0+ins1)；

Ndely [yy]=1；

}else

ndely[yy]++；

}

/* update penalty for del in y seq；

* favor new del over ongong del

*/

If (endgaps | | ndelx ＜ MAXGAP)

If (col1 [yy-1]-ins0 ＞=delx)

Delx=col1 [yy-1]-(ins0+ins1)；

Ndelx=1；

}else{

Delx-=ins1；

ndelx++；

}

}else{

If (col1 [yy-1]-(ins0+ins1) ＞=delx)

Delx=col1 [yy-1]-(ins0+ins1)；

Ndelx=1；

}else

ndelx++；

}

/* pick the maximum score；we′re favoring

* mis over any del and delx over dely

*/

...nw

Id=xx-yy+len1-1；

If (mis ＞=delx ＆＆ mis ＞=dely [yy])

Col1 [yy]=mis；

Else if (delx ＞=dely [yy])

Col1 [yy]=delx；

Ij=dx [id] .ijmp；

if(dx[id].jp.n[0]&&(！Dna | | (ndelx ＞=MAXJMP

＆＆ xx ＞ dx [id] .jp.x [ij]+MX) | | mis ＞ dx [id] .score+DINS0))

dx[id].ijmp++；

If (++ ij ＞=MAXJMP)

writejmps(id)；

Ij=dx [id] .ijmp=0；

Dx [id] .offset=offset；

Offset+=sizeof (struct jmp)+sizeof (offset)；

}

Dx [id] .jp.n [ij]=ndelx；

Dx [id] .jp.x [ij]=xx；

Dx [id] .score=delx；

}

else{

Col1 [yy]=dely [yy]；

Ij=dx [id] .ijmp；

if(dx[id].jp.n[0]&&(！Dna | | (ndely [yy] ＞=MAXJMP

＆＆ xx ＞ dx [id] .jp.x [ij]+MX) | | mis ＞ dx [id] .score+DINS0))

dx[id].ijmp++；

If (++ ij ＞=MAXJMP)

writejmps(id)；

Ij=dx [id] .ijmp=0；

Dx [id] .offset=offset；

Offset+=sizeof (struct jmp)+sizeof (offset)；

}

Dx [id] .jp.n [ij]=- ndely [yy]；

Dx [id] .jp.x [ij]=xx；

Dx [id] .score=dely [yy]；

}

If (xx==len0 ＆＆ yy ＜ len1)

/*last col

*/

if(endgaps)

Col1 [yy] -=ins0+ins1* (len1-yy)；

If (col1 [yy] ＞ smax)

Smax=col1 [yy]；

Dmax=id；

}

If (endgaps ＆＆ xx ＜ len0)

Col1 [yy-1] -=ins0+ins1* (len0-xx)；

If (col1 [yy-1] ＞ smax)

Smax=col1 [yy-1]；

Dmax=id；

}

Tmp=col0；Col0=col1；Col1=tmp；

}

(void)free((char *)ndely)；

(void)free((char *)dely)；

(void)free((char *)col0)；

(void)free((char *)col1)； }

/*

*

* print()--only routine visible outside this module

*

* static:

* getmat () -- trace back best path, count matches: print()

* pr_align()--print alignment of described in array p[]:print()

* dumpblock () -- dump a block of lines with numbers, stars: pr_align()

* nums()--put out a number line:dumpblock()

* putline () -- put out a line (name, [num], seq, [num]):dumpblock()

* stars()--put a line of stars:dump block()

* stripname()--strip any path and prefix from a seqname

*/

#include ″nw.h″

#define SPC 3

#define P_LINE 256 /* maximum output line */

#define P_SPC 3 /* space between name or num and seq */

extern _day[26][26]；

int olen； /*set output line length */

FILE *fx； /* output file */

print() print

{

Int lx, ly, firstgap, lastgap； /* overlap */

If ((fx=fopen (ofile, " w "))==0)

Fprintf (stderr, " %s:Can ' t write %s n ", prog, ofile)；

cleanup(1)；

}

Fprintf (fx, " ＜ first sequence:%s (length=%d) n ", namex [0], len0)；

Fprintf (fx, " ＜ second sequence:%s (length=%d) n ", namex [1], len1)；

Olen=60；

Lx=len0；

Ly=len1；

Firstgap=lastgap=0；

If (dmax ＜ len1-1)/* leading gap in x */

Pp [0] .spc=firstgap=len1-dmax-1；

Ly-=pp [0] .spc；

}

Else if (dmax ＞ len1-1)/* leading gap in y */

Pp [1] .spc=firstgap=dmax- (len1-1)；

Lx-=pp [1] .spc；

}

If (dmax0 ＜ len0-1)/* trailing gap in x */

Lastgap=len0-dmax0-1；

Lx-=lastgap；

}

Else if (dmax0 ＞ len0-1)/* trailing gap in y */

Lastgap=dmax0- (len0-1)；

Ly-=lastgap；

}

Getmat (lx, ly, firstgap, lastgap)；

pr_align()；

}

}}

/*

* trace back the best path, count matches

*/

static

Getmat (lx, ly, firstgap, lastgap) getmat

Int lx, ly； /* ″core″(minus endgaps) */

Int firstgap, lastgap； /* leading trailing overlap */

{

Int nm, i0, i1, siz0, siz1；

char outx[32]；

double pct；

Register n0, n1；

Register char * p0, * p1；

/ * get total matches, score

*/

I0=i1=siz0=siz1=0；

P0=seqx [0]+pp [1] .spc；

P1=seqx [1]+pp [0] .spc；

N0=pp [1] .spc+1；

N1=pp [0] .spc+1；

Nm=0；

while(*p0 && *p1){

if(siz0){

p1++；

n1++；

siz0--；

}

else if(siz1){

p0++；

n0++；

siz1--；

}

else{

if(xbm[*p0-′A′]&xbm[*p1-′A′])

nm++；

If (n0++==pp [0] .x [i0])

Siz0=pp [0] .n [i0++]；

If (n1++==pp [1] .x [i1])

Siz1=pp [1] .n [i1++]；

p0++；

p1++；

}

/* pct homology:

* if penalizing endgaps, base is the shorter seq

* else, knock off overhangs and take shorter core

*/

if(endgaps)

Lx=(len0 ＜ len1)len0:len1；

else

Lx=(lx ＜ ly)lx:ly；

Pct=100.* (double) nm/ (double) lx；

Fprintf (fx, " n ")；

Fprintf (fx, " ＜ %d match%s in an overlap of %d:%.2f percent similarity n ",

Nm, (nm==1)″″:" es ", lx, pct)；

Fprintf (fx, " ＜ gaps in first sequence:%d ", gapx)； ...getmat

if(gapx){

(void) sprintf (outx, " (%d %s%s) ",

Ngapx, (dna)″base″:" residue ", (ngapx==1)″″:″s″)；

Fprintf (fx, " %s ", outx)；

Fprintf (fx, ", gaps in second sequence:%d ", gapy)；

if(gapy){

(void) sprintf (outx, " (%d %s%s) ",

Ngapy, (dna)″base″:" residue ", (ngapy==1)″″:″s″)；

Fprintf (fx, " %s ", outx)；

}

if(dna)

Fprintf (fx,

" n ＜ score:%d (match=%d, mismatch=%d, gap penalty=%d+%d per base) n ",

Smax, DMAT, DMIS, DINS0, D1NS1)；

else

Fprintf (fx,

" n ＜ score:%d (Dayhoff PAM 250 matrix, gap penalty=%d+%d per residue) n ",

Smax, PINS0, PINS1)；

if(endgaps)

Fprintf (fx,

" ＜ endgaps penalized. left endgap:%d %s%s, right endgap:%d %s%s n ",

Firstgap, (dna)″base″:" residue ", (firstgap==1)″″:" s ",

Lastgap, (dna)″base″:" residue ", (lastgap==1)″″: ″s″)；

else

Fprintf (fx, " ＜ endgaps not penalized n ")；

}

static nm； /* matches in core--for checking */

static lmax； /* lengths of stripped file names */

static ij[2]； /* jmp index for a path */

static nc[2]； /* number at start of current line */

static ni[2]； /* current elem number--for gapping */

static siz[2]；

static char *ps[2]； /* ptr to current element */

static char *po[2]； /* ptr to next output char slot */

static char out[2][P_LINE]； /* output line */

static char star[P_LINE]； /* set by stars() */

/*

* print alignment of described in struct path pp[]

*/

static

pr_align() pr_align

{

int nn； /* char count */

int more；

register i；

For (i=0, lmax=0；I ＜ 2；i++){

Nn=stripname (namex [i])；

If (nn ＞ lmax)

Lmax=nn；

Nc [i]=1；

Hi [i]=1；

Siz [i]=ij [i]=0；

Ps [i]=seqx [i]；

Po [i]=out [i]； }

For (nn=nm=0, more=1；more；){ ...pr_align

For (i=more=0；I ＜ 2；i++){

/*

* do we have more of this sequence

*/

if(！*ps[i])

continue；

more++；

if(pp[i].spc){ /* leading space */

* po [i] ++="；

pp[i].spc--；

}

else if(siz[i]){ /* in a gap */

* po [i] ++='-'；

siz[i]--；

}

else{ /* we′re putting a seq element

*/

* po [i]=* ps [i]；

if(islower(*ps[i]))

* ps [i]=toupper (* ps [i])；

po[i]++；

ps[i]++；

/*

* are we at next gap for this seq

*/

If (ni [i]==pp [i] .x [ij [i]])

/*

* we need to merge all gaps

* at this location

*/

Siz [i]=pp [i] .n [ij [i] ++]；

While (ni [i]==pp [i] .x [ij [i]])

Siz [i] +=pp [i] .n [ij [i] ++]；

}

ni[i]++；

}

If (++ nn==olen | |！more && nn){

dumpblock()；

For (i=0；I ＜ 2；i++)

Po [i]=out [i]；

Nn=0；

}

/*

* dump a block of lines, including numbers, stars:pr_align()

*/

static

dumpblock() dumpblock

{

register i；

For (i=0；I ＜ 2；i++)

* po [i] --=' 0 ',

...dumpblock

(void) putc (' n ', fx)；

For (i=0；I ＜ 2；i++){

if(*out[i]&&(*out[i]！=" | | * (po [i])！="))

If (i==0)

nums(i)；

If (i==0 ＆＆ * out [1])

stars()；

putline(i)；

If (i==0 ＆＆ * out [1])

Fprintf (fx, star)；

If (i==1)

nums(i)；

}

/*

* put out a number line:dumpblock()

*/

static

nums(ix) nums

int ix；/* index in out[] holding seq line */

{

char nline[P_LINE]；

Register i, j；

Register char * pn, * px, * py；

For (pn=nline, i=0；I ＜ lmax+P_SPC；I++, pn++)

* pn="；

For (i=nc [ix], py=out [ix]；*py；Py++, pn++)

If (* py==" | | * Py=='-')

* pn="；

else{

If (i%10==0 | | (i==1 ＆＆ nc [ix]！=1))

J=(i ＜ 0)-i:i；

For (px=pn；j；J/=10, px--)

* px=j%10+ ' 0 '；

If (i ＜ 0)

* px='-'；

}

else

* pn="；

i++；

}

* pn=' 0 ',

Nc [ix]=i；

For (pn=nline；*pn；pn++)

(void) putc (* pn, fx)；

(void) putc (' n ', fx)；

}

/*

* put out a line (name, [num], seq, [num]):dumpblock()

*/

static

putline(ix) putline

int ix； {

...putline

int i；

register char *px；

For (px=namex [ix], i=0；*px && *px！=':′；Px++, i++)

(void) putc (* px, fx)；

for (；I ＜ lmax+P_SPC；i++)

(void) putc (", fx)；

/* these count from 1:

* ni[] is current element (from 1)

* nc[] is number at start of current line

*/

For (px=out [ix]；*px；px++)

(void) putc (* px＆0x7F, fx)；

(void) putc (' n ', fx)；

}

/*

* put a line of stars (seqs always in out [0], out [1]):dumpblock()

*/

static

stars() stars

{

int i；

Register char * p0, * p1, cx, * px；

if(！* out [0] | | (* out [0]==" ＆＆ * (po [0])==") | |

！* out [1] | | (* out [1]==" ＆＆ * (po [1])=="))

return；

Px=star；

For (i=lmax+P_SPC；i；i--)

* px++="；

For (p0=out [0], p1=out [1]；*p0 && *p1；P0++, p1++)

if(isalpha(*p0)&& isalpha(*p1)){

if(xbm[*p0-′A′]&xbm[*p1-′A′]){

Cx=' * '；

nm++；

}

else if(！Dna ＆＆_day [* p0- ' A '] [* p1- ' A '] ＞ 0)

Cx=' '；

else

Cx="；

}

else

Cx="；

* px++=cx；

}

* px++=' n '；

* px=' 0 '；

}

/*

* strip path or prefix from pn, return len:pr_align()

*/

static

stripname(pn) stripname

char *pn；/* file name(may be path)*/

{

Register char * px, * py；

Py=0；

For (px=pn；*px；px++)

If (* px=='/')

Py=px+1；

if(py)

(void) strcpy (pn, py)；

return(strlen(pn))；

/*

* cleanup()--cleanup any tmp file

* getseq () -- read in seq, set dna, len, maxlen

* g_calloc()--calloc()with error checkin

* readjmps () -- get the good jmps, from tmp file if necessary

* writejmps()--write a filled array of jmps to a tmp file:nw()

*/

#include ″nw.h″

#include<sys/file.h>

Char * jname="/tmp/homgXXXXXX "； /* tmp file for jmps */

FILE *fj；

int cleanup()； /* cleanup tmp file */

long lseek()；

/*

* remove any tmp file ifwe blow

*/

cleanup(i) cleanup

int i；

{

if(fj)

(void)unlink(jname)；

exit(i)；

}

/*

* read, return ptr to seq, set dna, len, maxlen

* skip lines starting with′；', ' ＜ ', or ' ＞ '

* seq in upper or lower case

*/

char *

Getseq (file, len) getseq

char *file；/* file name */

int *len； /* seq len */

{

Char line [1024], * pseq；

Register char * px, * py；

Int natgc, tlen；

FILE *fp；

If ((fp=fopen (file, " r "))==0)

Fprintf (stderr, " %s:Can ' t read %s n ", prog, file)；

exit(1)；

}

Tlen==natgc=0；

While (fgets (line, 1024, fp))

If (* line=='；' | | * line==' ＜ ' | | * line==' ＞ ')

continue；

For (px=line；*px！=' n '；px++)

if(isupper(*px)||islower(*px))

tlen++；

}

If ((pseq=malloc ((unsigned) (tlen+6)))==0)

Fprintf (stderr, " %s:Malloc () failed to get %d bytes for %s n ", prog, tlen+6, file)；

exit(1)；

}

Pseq [0]=pseq [1]=pseq [2]=pseq [3]=' 0 '；

...getseq

Py=pseq+4；

* len=tlen；

rewind(fp)；

While (fgets (line, 1024, fp))

If (* line=='；' | | * line==' ＜ ' | | * line==' ＞ ')

continue；

For (px=line；*px！=' n '；px++){

if(isupper(*px))

* py++=*px；

else if(islower(*px))

* py++=toupper (* px)；

If (index (" ATGCU ", * (py-1)))

natgc++；

}

* py++=' 0 '；

* py=' 0 '；

(void)fclose(fp)；

Dna=natgc ＞ (tlen/3)；

return(pseq+4)；

}

char *

G_calloc (msg, nx, sz) g_calloc

char *msg；/ * program, calling routine */

Int nx, sz； /* number and size of elements */

{

Char * px, * calloc ()；

If ((px=calloc ((unsigned) nx, (unsigned) sz))==0)

if(*msg){

Fprintf (stderr, " %s:G_calloc () failed %s (n=%d, sz=%d) n ", prog, msg, nx, sz)；

exit(1)；

}

return(px)；

}

/*

* get final jmps from dx [] or tmp file, set pp [], reset dmax:main()

*/

readjmps() readjmps

{

Int fd=-1；

Int siz, i0, i1；

Register i, j, xx；

if(fj){

(void)fclose(fj)；

If ((fd=open (jname, O_RDONLY, 0)) ＜ 0)

Fprintf (stderr, " %s:Can ' t open () %s n ", prog, jname)；

cleanup(1)；

}

For (i=i0=i1=0, dmax0=dmax, xx=len0；；i++){

while(1){

For (j=dx [dmax] .ijmp；＆＆ dx [dmax] .jp.x [j] ＞=xx of j ＞=0；j--)

；

...readjmps

If (＆＆ dx [dmax] the .offset ＆＆ fj of j ＜ 0)

(void) lseek (fd, dx [dmax] .offset, 0)；

(void) read (fd, (char*)s ＆dx [dmax] .jp, sizeof (struct jmp))；

(void) read (fd, (char*)s ＆dx [dmax] .offset, sizeof (dx [dmax] .offset))；

Dx [dmax] .ijmp=MAXJMP-1；

}

else

break；

}

If (i ＞=JMPS)

Fprintf (stderr, " %s:Too many gaps in alignment n ", prog)；

cleanup(1)；

}

If (j ＞=0)

Siz=dx [dmax] .jp.n [j]；

Xx=dx [dmax] .jp.x [j]；

Dmax+=siz；

If (siz ＜ 0)/* gap in second seq */

Pp [1] .n [i1]=- siz；

Xx+=siz；

/ * id=xx-yy+len1-1

*/

Pp [1] .x [i1]=xx-dmax+len1-1；

gapy++；

Ngapy-=siz；

/* ignore MAXGAP when doing endgaps */

Siz=(- siz ＜ MAXGAP | | endgaps)-siz: MAXGAP；

i1++；

}

Else if (siz ＞ 0)/* gap in first seq */

Pp [0] .n [i0]=siz；

Pp [0] .x [i0]=xx；

gapx++；

Ngapx+=siz；

/* ignore MAXGAP when doing endgaps */

Siz=(siz ＜ MAXGAP | | endgaps)siz:MAXGAP；

i0++；

}

else

break；

}

/* reverse the order of jmps

*/

For (j=0, i0--；J ＜ i0；J++, i0--)

I=pp [0] .n [j]；Pp [0] .n [j]=pp [0] .n [i0]；Pp [0] .n [i0]=i；

I=pp [0] .x [j]；Pp [0] .x [j]=pp [0] .x [i0]；Pp [0] .x [i0]=i；

}

For (j=0, i1--；J ＜ i1；J++, i1--)

I=pp [1] .n [j]；Pp [1] .n [j]=pp [1] .n [i1]；Pp [1] .n [i1]=i；

I=pp [1] .x [j]；Pp [1] .x [j]=pp [1] .x [i1]；Pp [1] .x [i1]=i；

}

If (fd ＞=0)

(void)close(fd)；

if(fj){

(void)unlink(jname)；

Fj=0；

Offset=0；

} }

/*

* write a filled jmp struct offset of the prev one(if any):nw()

*/

writejmps(ix) writejmps

int ix；

{

char *mktemp()；

if(！fj){

If (mktemp (jname) ＜ 0)

Fprintf (stderr, " %s:Can ' t mktemp () %s n ", prog, jname)；

cleanup(1)；

}

If ((fj=fopen (jname, " w "))==0)

Fprintf (stderr, " %s:Can ' t write %s n ", prog, jname)；

exit(1)；

}

(void) fwrite ((char *)s ＆dx [ix] .jp, sizeof (struct jmp), 1, fj)；

(void) fwrite ((char *)s ＆dx [ix] .offset, sizeof (dx [ix] .offset), 1, fj)；

}

Table 2

TAT XXXXXXXXXXXXXXX (length=15 amino acid)

Comparison protein XXXXXYYYYYYY (length=12 amino acid)

% amino acid sequence identities=(total number of atnino acid for being defined as identical match between two peptide sequences by ALIGN-2) ÷ (total amino acid residues of TAT polypeptides)=5 ÷ 15=33.3%

Table 3

TAT XXXXXXXXXX (length=10 amino acid)

Comparison protein XXXXXYYYYYYZZYZ (length=15 amino acid)

% amino acid sequence identities=(total number of atnino acid for being defined as identical match between two peptide sequences by ALIGN-2) ÷ (total amino acid residues of TAT polypeptides)=5 ÷ 10=50%

Table 4

TAT-DNA NNNNNNNNNNNNNN (length=14 nucleotides)

Comparison dna NNNNNNLLLLLLLLLL (length=16 nucleotides)

% nucleic acid sequence identities=(total number of atnino acid for being defined as identical match between two peptide sequences by ALIGN-2) ÷ (total nucleotide number of TAT-DNA nucleotide sequences)=6 ÷ 14=42.9%

Table 5

TAT-DNA NNNNNNNNNNNN (length=12 nucleotides)

Comparison dna NNNNLLLVV (length=9 nucleotides)

% nucleic acid sequence identities=((total number of atnino acid for being defined as identical match between two peptide sequences by ALIGN-2) ÷ (total nucleotide number of TAT-DNA nucleotide sequences)=4 ÷ 12=33.3%

IL. the compositions and methods of the invention

A. anti-TAT antibody

In one embodiment, the invention provides the anti-TAT antibody that can be used herein as therapeutic agent and/or diagnosticum.Exemplary antibody includes polyclonal, monoclonal, humanization, bispecific and Heteroconjugate antibody.

1. polyclonal antibody

Polyclonal antibody is preferably generated by animal multiple subcutaneous (sc) or intraperitoneal (ip) injection related antigen and adjuvant.With there is the protein molecule of immunogenicity in immune species are treated it is probably useful by related antigen when synthetic peptide (especially using).For example; difunctional or derivatization reagent, such as maleimidobenzoyl sulfosuccinimide ester (coupling through cysteine residues), n-hydroxysuccinimide (through lysine residue), glutaraldehyde, succinic anhydride, SOCl can be used₂Or R¹N=C=NR, wherein R and R¹It is different alkyl, antigen and keyhole  hemocyanin (KLH), serum albumin, bovine thyroglobulin or soybean trypsin inhibitor is coupled.

By the way that such as 100 μ g or 5 μ g proteins or conjugate (being respectively used to rabbit or mouse) and the Freund's complete adjuvant of 3 times of volumes are mixed, and thus solution intracutaneous injection animal is immunized for antigen, immunogenic conjugate or derivative in multiple positions.After one month, by the hypodermic injection at multiple positions, animal is strengthened with the 1/5-1/10 of peptide in Freund's complete adjuvant or conjugate primary quantity.After 7-14 days, the blood of animal is gathered, and determines the antibody titer of serum.Animal is strengthened until titre reaches stabilization.Conjugate can also be prepared as protein fusions in recombinant cell culture thing.Equally, suitably immune response is strengthened using flocculating agent such as alum.

2. monoclonal antibody

Monoclonal antibody can be by initially by Kohler et al., Nature 256：Prepared by the hybridoma method of 495 (1975) description, or can prepare (United States Patent (USP) 4,816,567) by recombinant DNA method.

In hybridoma method, immune mouse as described above or other suitable host animals, such as hamster, to trigger generation or can generate the lymphocyte of following antibody, the antibody will specifically bind the protein for being used for being immunized.Or, can immunological lymphocyte in vitro.After immune, lymphocyte is separated, is then merged lymphocyte with myeloma cell line using suitable fusion agent such as polyethylene glycol, hybridoma (Goding, Monoclonal Antibodies is formed：Principles and Practice, pp.59-103, Academic Press, 1986).

By thus prepared hybridoma in suitable inoculation of medium and culture, the culture medium preferably comprises the one or more materials for suppressing Parent Myeloma Cell (also referred to as merging spouse) the growth or survival do not merged.For example, if Parent Myeloma Cell lacks hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), then the selective medium for hybridoma will generally contain hypoxanthine, aminopterin-induced syndrome and thymidine (HAT culture mediums), and these materials prevent to lack HGPRT cell growth.

Preferably fusion spouse myeloma cell is that those the high-level of antibody-producting cell stabilization for efficiently merging, supporting selection generate antibody and to for not merging the myeloma cell that parental cell carries out the selective medium sensitivity of selection.It is preferred that myeloma cell line be mouse source myeloma system, such as can be from SalkInstitute Cell Distribution Center (San Diege, California, USA the Derivative line for the MOPC-21 and MPC-11 mouse tumors) bought, and can be from American Type Culture Collection (Manassas, Virginia, USA) SP-2 and derivative that buy, such as X63-Ag8-653 cells.Human myeloma and mouse-people's heteromyeloma cell lines for generating human monoclonal antibodies also have been described (Kozbor, J.Immunol.133：3001(1984)；Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, pp.51-63, Marcel Dekker, Inc., NewYork, 1987).

Generation of the culture based assays that hybridoma just can be wherein being grown for the monoclonal antibody of antigen.Preferably, by immunoprecipitation or by external binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA), the binding specificity of the monoclonal antibody generated by hybridoma is determined.

The binding affinity of monoclonal antibody can be for example, by Munson et al., Anal.Biochem.107：Scatchard described in 220 (1980) analyzes to determine.

Once identification obtains the hybridoma of antibody of the generation with required specificity, affinity and/or activity, the clone can be subcloned by limiting dilution flow, and be cultivated (Goding, Monoclonal Antibodies using standard method：Principles and Practice, pp.59-103, Academic Press, 1986).Culture medium suitable for this purpose includes such as D-MEM or RPMI-1640 culture mediums.In addition, hybridoma can be in animal as ascites tumor progress In vivo culture, such as by the way that cell i.p. is expelled in mouse.

Flow can be purified by conventional antibody, such as affinity chromatography (such as using albumin A or Protein G-Sepharose) or ion-exchange chromatography, hydroxyapatite, gel electrophoresis, dialysis, the monoclonal antibody for being subcloned secretion is suitably separated with culture medium, ascites or serum.

The DNA of coding monoclonal antibody is easy to separate and be sequenced by old process (such as using the oligonucleotide probe for the gene that can specifically bind coding mouse source heavy chain of antibody and light chain).Such DNA preferred source is used as using hybridoma.Once separation, DNA can be placed in expression vector, then the expression vector is transfected into the host cell for not producing antibody protein originally, in such as Bacillus coli cells, Simian COS cells, Chinese hamster ovary (CHO) cell or myeloma cell, to obtain the synthesis of monoclonal antibody in recombinant host cell.The summary paper of recombination expressions of the DNA in bacterium on encoding antibody includes Skerra et al., Curr.Opinion in Immunol.5：256-262 (1993) and Pl ü ckthun, Immunol.Revs.130：151-188(1992).

In another embodiment, can be from use McCafferty et al., Nature 348：Monoclonal antibody or antibody fragment are separated in the phage antibody library of 552-554 (1990) described technique construction.Clackson et al., Nature 352：624-628 (1991) and Marks et al., J.Mol.Biol.222：581-597 (1991) is respectively described separates mouse source and human antibody using phage library.Subsequent publications are described reorganizes (Marks et al., Bio/Technology 10 by chain：779-783 (1992)) generation high-affinity (nM scopes) human antibody, and combination infection and In vivo recombination are used as strategy (Waterhouse et al., the Nuc.Acids Res.21 for building very big phage library：2265-2266 (1993)),.Thus, these technologies are the feasible replacement methods for separating the conventional monoclonal antibody hybridoma technology of monoclonal antibody.

The DNA of encoding antibody can be modified to generate chimeric or fusion antibody polypeptide, for example, pass through employment heavy chain and light-chain constant domains (C_HAnd C_L) the homologous mouse source sequence (United States Patent (USP) 4,816,567 of sequence replacing；Morrison et al., Proc.Natl.Acad.Sci.USA 81：6851 (1984)), or by merging the coded sequence all or in part of immunoglobulin coding sequence and NIg polypeptide (heterologous polypeptide).The constant domain of the alternative antibody of NIg polypeptide sequence, or the variable domain of an antigen binding site of antibody is substituted with them, so as to producing chimeric bivalent antibody, it is comprising having a specific antigen binding site to a kind of antigen and have another specific antigen binding site to not synantigen.

3. human antibody and humanized antibody

The anti-TAT antibody of the present invention may also include humanized antibody or human antibody.The humanization form of inhuman (such as mouse source) antibody refers to the gomphosis immunoglobulin, immunoglobulin chain or its fragment (such as Fv, Fab, Fab ', F (ab ') that bottom line includes the sequence derived from non-human immunoglobulin₂Or other antigen binding sub-sequences of antibody).Complementary determining region (CDR) residue that humanized antibody is included in human immunoglobulin(HIg) (receptor antibody) immunoglobulin that the CDR residues with non-human species' (donor antibody) such as mouse, rat or rabbit for expecting specificity, affinity and ability are replaced.In some cases, the Fv Framework residues of human immunoglobulin(HIg) are replaced with corresponding non-human residues.Humanized antibody can be additionally included in receptor antibody or input CDR or Frame sequence and residue certainly be not present.Generally, humanized antibody includes at least one, usually two substantially whole following variable regions：Wherein entirely or substantially upper whole CDR region corresponds to the CDR region of non-human immunoglobulin, and entirely or substantially upper whole FR areas are the FR areas of human immunoglobulin(HIg) consensus sequence.Humanized antibody preferably also includes constant region (Jones the et al., Nature 321 of at least part constant region for immunoglobulin (Fc), typically human immunoglobulin(HIg)：522-525(1986)；Riechmann et al., Nature 332：323-329(1988)；Presta, Curr.Op.Struct.Biol.2：593-596(1992)).

For the method for non-human antibody's humanization to be well known in the art.Generally, humanized antibody has one or more amino acid residues introduced from non-people source.These non-human amino acid residues are often referred to as " inputting " residue, and they are normally taken from " inputting " variable region.Humanization can carry out (Jones et al., Nature 321 substantially according to the method for Winter and its colleague：522-525(1986)；Riechmann et al., Nature 332：323-327(1988)；Verhoeyen et al., Science 239：1534-1536 (1988)), substitute corresponding human antibody sequence by using rodent CDR sequence.Therefore, such " humanization " antibody is chimeric antibody (United States Patent (USP) 4,816,567), wherein will be substituted considerably less than whole people source variable domain with the corresponding sequence from non-human species.In practice, humanized antibody is typically the human antibody that some of CDR residues are substituted with some possible FR residues with the residue in the similar site in rodent antibodies.

When antibody is intended for human therapeutic use, people's variable domain for preparing humanized antibody includes the selection of light chain and heavy chain variable domain, is very important for reduction antigenicity and HAMA responses (human anti-mouse antibody).According to so-called " most suitable (best-fit) " method, the whole library of known people's variable region sequences is screened with rodent antibodies variable region sequences.Find out with the immediate people V domains sequence of rodent, and receive people's framework region (FR) therein be used for humanized antibody (Sims et al., J.Immunol.151：2296(1993)；Chothia et al., J.Mol.Biol.196：901(1987)).Another method uses the specific frame area derived from the consensus sequence of all human antibodies from specific light chain or heavy chain subgroup.Same framework can be used for several different humanized antibody (Carter et al., Proc.Natl.Acad.Sci.USA 89：4285(1992)；Presta et al., J.Immunol.151：2623(1993)).

In addition, it is important that antibody keeps the high binding affinity and other favourable biological characteristicses to antigen after humanization.In order to realize this purpose, according to a kind of preferred method, analyze the method for parental array and various conceptual humanized products to prepare humanized antibody by using the threedimensional model of parent and humanized sequence.Three dimensional immunoglobulin model is typically available, and is known to those skilled in the art.It can also be illustrated and be shown the computer program of the possibility three-dimensional conformation structure of selected candidate immunoglobulin sequences sequence.Check that these display images can analyze possibility effect of the residue in candidate immunoglobulin sequences sequence functions, i.e. analyzing influence candidate immunoglobulin sequences combine the residue of the ability of its antigen.So, FR residues can be selected from acceptor and list entries and are combined, so as to obtain required antibody characteristic, such as the affinity to target antigen is improved.Generally, the direct and most substantive influence being related to antigen binding of some hypervariable region residues.

Contemplate the various forms of the anti-TAT antibody of humanization.For example, humanized antibody can be antibody fragment, such as Fab, optionally coupling has one or more cytotoxic agents to generate immune conjugate.Or, humanized antibody can be complete antibody, such as complete IgG1 antibody.

As the alternative of humanization, human antibody can be generated.For example, it is now possible to which the transgenic animals (such as mouse) of human antibody full repertoire can be generated in the case where lacking endogenous immunoglobulin generation after immune by generating.For example, it has been described that antibody heavy chain joining region (J in chimeric and germ line mutant mice_H) gene homozygosis delete cause the complete inhibition of endogenous antibody tormation.A large amount of human germline immunoglobulin's gene transfers will be caused into such germ line mutant mice to generate human antibody after antigen is attacked.See, for example, Jakobovits et al., Proc.Natl.Acad.Sci.USA 90：2551(1993)；Jakobovits etal., Nature 362：255-258(1993)；Bruggemann et al., Year in Immuno.7：33(1993)；United States Patent (USP) 5,545,806,5,569,825,5,591,669 (being all GenPharm)；5,545,807；And WO 97/17852.

Or, display technique of bacteriophage (McCafferty et al., Nature 348 can be used：552-553 (1990)) it is used in vitro from immunoglobulin variable (V) area gene complete or collected works generation human antibody and antibody fragment from epidemic disease donor rather.According to this technology, antibody V domain genes are cloned into filobactivirus such as M13 or the fd reading frame of main or secondary coat protein gene, and functional antibody fragment is shown as on phage particle surface.Because filamentous particle includes the single-stranded DNA copy of phage genome, the selection carried out based on the functional characteristic of antibody also causes the selection of the gene of the antibody of those characteristics of coding displaying.Thus, bacteriophage simulates some characteristics of B cell.Phage display can be carried out in a variety of forms, be summarized see, for example, Johnson, Kevin S.and Chiswell, David J., Current Opinion inStructural Biology 3：564-571(1993).The Several sources of V constant gene segment Cs can be used for phage display.Clackson et al., Nature 352：624-628 (1991) is from the isolated a large amount of different anti- oxazolones antibody of the small-sized V genes random combinatorial libraries derived from immune mice spleen.Can be substantially according to Markset al., J.Mol.Biol.222：581-597 (1991) or Griffith et al., EMBO are J.12：725-734 (1993) described technology, V gene complete or collected works are built by people donor is not immunized, and Separated pin is to the largely not antibody of synantigen (including autoantigen).Referring also to United States Patent (USP) 5,565,332 and 5,573,905.

As described above, can also pass through vitro activated B cells (referring to United States Patent (USP) 5,567,610 and 5,229,275) next life human antibodies.

4. antibody fragment

In some cases, the use of antibody fragment rather than complete antibody is favourable.The small volume of fragment, allows quick removing, can cause to improve entering for solid tumor.

The multiple technologies for generating antibody fragment are developed.Traditionally, these fragments are derived by proteolytic digestion complete antibody (see, for example, Morimoto et al., Journal of Biochemicaland Biophysical Methods 24：107-117(1992)；Brennan et al., Science 229：81(1985)).However, these fragments directly can be generated by recombinant host cell now.Thus Fab, Fv and scFv antibody fragment can all allow readily to generate these substantial amounts of fragments in expression in escherichia coli and by E. coli secretion.Can be from antibody phage libraries isolated antibody fragment discussed above.Or, Fab '-SH fragments directly can be reclaimed from Escherichia coli, and be coupled to form F (ab ') by chemical method₂Fragment (Carteret al., Bio/Technology 10：163-167(1992))., can be directly from recombinant host cell culture separation F (ab ') according to another method₂Fragment.The Fab and F (ab ') of Half-life in vivo comprising salvage receptor binding epitope residue, with extension₂Fragment is described in United States Patent (USP) 5,869,046.Other technologies for generating antibody fragment are obvious to skilled practitioner.In other embodiments, the antibody of selection is Single-Chain Fv Fragment of Murine (scFv).Referring to WO 93/16185；United States Patent (USP) 5,571,894；With United States Patent (USP) 5,587,458.Fv and sFv are the unique class with entire binding site and shortage constant region；Therefore, they are suitable for reducing non-specific binding during use in vivo.SFv fusion proteins can be built to generate fusion of the effector albumen matter positioned at sFv amino terminals or carboxyl terminal.Referring to《AntibodyEngineering》, Borrebaeck compile, see above.Antibody fragment can also be " linear antibodies ", such as United States Patent (USP) 5, the antibody described in 641,870.Such linear antibody fragments can be monospecific or bispecific.

5. bispecific antibody

Bispecific antibody refers to the antibody for having binding specificity at least two different epitopes.Exemplary bispecific antibody can combine two kinds of different epitopes of TAT protein described herein.This other antibody-like can join together TAT binding sites with the binding site for another protein.Or, the arm of anti-TAT arms and triggering molecule such as φt cell receptor molecule (such as CD3) or IgG Fc acceptors (Fc γ R) such as Fc γ RI (CD64), Fc γ RII (CD32) and Fc γ RIII (CD16) on combination leucocyte can be joined together, so that cellular defence mechanisms are focused on and are positioned at expression TAT cell.Bispecific antibody can be additionally used in the cell that cytotoxic agent is positioned to expression TAT.These antibody possess TAT combination arms and combine the arm of cytotoxic agent (such as saporin, anti-interferon-α, vinca alkaloids, ricin A chains, methotrexate (MTX) or radioactive isotope hapten).Bispecific antibody can be prepared into full length antibody or antibody fragment (such as F (ab ')₂Bispecific antibody).

WO 96/16673 describes bispecific anti-ErbB/anti- Fc γ RIII antibody, and United States Patent (USP) 5,837,234 discloses bispecific anti-ErbB/anti- Fc γ RI antibody.Bispecific anti-ErbB/Fc Alpha antibodies are shown in WO 98/02463.United States Patent (USP) 5,821,337 teaches bispecific anti-ErbB/anti-cd 3 antibodies.

Method for preparing bispecific antibody is known in the art.The production of traditional total length bispecific antibody is the coexpression based on two kinds of heavy chain immunoglobulin-light chains pair, and two of which chain has different specificity (Millstein et al., Nature 305：537-539(1983)).Due to being randomly assigned for heavy chain immunoglobulin and light chain, these hybridomas (four source hybridomas (quadroma)) generate the potential mixture of 10 kinds of different antibodies molecules, wherein only a kind of have correct bispecific structure.The purifying of the correct molecule generally carried out by affinity chromatography step is fairly cumbersome, and Product yields are low.WO 93/08829 and Traunecker et al., EMBO are J.10：3655-3659 discloses similar flow in (1991).

According to a kind of different method, the antibody variable domains will with required binding specificity (antibody-antigen binding site) are merged with immunoglobulin constant domain sequence.Preferably, fusion uses and includes at least part hinge, C_H2 and C_HThe heavy chain immunoglobulin constant domain in 3rd area.Preferably, there is the first heavy chain constant region (C that necessary site is combined comprising light chain at least one fusions_H1).By encoding immune immunoglobulin heavy chain fusions thing and, if it is desired, the DNA of light chain immunoglobulin inserts separated expression vector, and cotransfection is into suitable host cell.There is provided when not waited for the three of structure kind polypeptide chain ratio in the embodiment for the optimum point of production for expecting bispecific antibody, this provides greater flexibility to adjust the mutual ratio of three kinds of polypeptide fragments.However, when at least two polypeptide chains cause high yield with same ratio expression or when the ratio is to it is expected that the yield of chain combination has no significant effect, it is possible to which the coded sequence of two kinds or all three polypeptide chains is inserted into same carrier.

In a preferred embodiment of this method, bispecific antibody has the hybrid immunoglobulin heavy chain of the first binding specificity on an arm, and hybrid immunoglobulin heavy chain-light chain on another arm is constituted to (providing the second binding specificity).Because presence of the light chain immunoglobulin only in half of bispecific molecule provides the facility approach of separation, consequently found that this dissymmetrical structure is easy to separate required bispecific compound (compound) and undesired immunoglobulin chain combinations.This method is disclosed in WO 94/04690.On generating other details of bispecific antibody see, for example, Suresh et al., Methods in Enzymology 121：210(1986).

According to United States Patent (USP) 5, another method described in 731,168 can transform the interface between a pair of antibody molecules, by the percent maximum of the heterodimer reclaimed from recombinant cell culture thing.It is preferred that interface include at least part C_H3 domains.In the method, one or more small amino acid side chains of first antibody molecular interface are replaced with larger side chain (such as tyrosine or tryptophan).Big amino acid side chains are replaced by using compared with small amino acid side chains (such as alanine or threonine), are produced on the interface of secondary antibody molecule compensatory " cavity " of the same or similar size for bulky side chain.This provides the mechanism that heterodimer yield is improved than other undesired end-products such as homodimer.

Bispecific antibody includes crosslinking or " Heteroconjugate " antibody.For example, a kind of antibody in Heteroconjugate thing can be coupled with avidin, another antibody is coupled with biotin.For example, this antibody-like is proposed to be used in targets undesired cell (United States Patent (USP) 4,676,980) by immune system cell, and for treating HIV (WO 91/00360, WO 92/200373 and EP 03089).Any easily cross-linking method can be used to prepare Heteroconjugate antibodies.Suitable crosslinking agent is well-known in the art, and is disclosed in United States Patent (USP) 4,676,980 together with many crosslinking technologicals.

The technology that bispecific antibody is generated by antibody fragment is also described in document.It is connected chemically to prepare bispecific antibody for example, can be used.Brennan et al., Science 229：81 (1985) are described cuts complete antibody to generate F (ab ') by proteolysis₂The method of fragment.These fragments are reduced in the case where there are two mercaptan complexing agent sodium arsenites, to stablize two neighbouring mercaptan and prevent the formation of intermolecular disulfide bond.Then Fab ' the fragments of generation are changed into thionitrobenzoate ester (TNB) derivative.Then one of Fab '-TNB derivatives are reverted into Fab '-mercaptan by the reduction of mercaptoethylmaine again, and mixed with another Fab '-TNB derivatives of equimolar amounts, to form bispecific antibody.The bispecific antibody of generation can be used as the selective immobilized reagent of enzyme.

Nearest progress make it that directly reclaiming Fab '-SH fragments from Escherichia coli becomes to be more prone to, and these fragments are chemically coupled to form bispecific antibody.Shalaby et al., J.Exp.Med.175：217-225 (1992) describes the bispecific antibody F (ab ') of generation full-length human₂Molecule.Each Fab ' fragments are separately secreted by Escherichia coli, and are oriented chemical coupling in vitro to form bispecific antibody.The bispecific antibody being thusly-formed can combine cell and the normal human T cells for being overexpressed ErbB2 acceptors, and triggering people's cell Cytotoxic Lymphocytes for the dissolving activity of human breast tumor target.

Also describe the multiple technologies for directly preparing and separating bispecific antibody fragment from recombinant cell culture thing.For example, generating bispecific antibody using leucine zipper.Kostelny et al., J.Immunol.148 (5)：1547-1553(1992).Leucine zipper peptide from Fos and Jun albumen is connected by Gene Fusion with the Fab ' parts of two kinds of different antibodies.Antibody homodimer reduces in hinge area and forms monomer, then reoxidized and form antibody heterodimer.This method can also be used for generating antibody homodimer.By Hollinger et al., Proc.Natl.Acad.Sci.USA 90：" double antibody " technology of 6444-6448 (1993) descriptions provides the replacement mechanism for preparing bispecific antibody fragment.The fragment includes the V being connected by joint_HAnd V_L, the joint too it is short cause same chain on two domains between can not match.Therefore, the V in a fragment is forced_HAnd V_LDomain and the complementary V in another fragment_LAnd V_HDomain is matched, and is consequently formed two antigen binding sites.It is also reported that preparing another strategy of bispecific antibody fragment by using scFv (sFv) dimer.Referring to Gruber et al., J.Immunol.152：5368(1994).

Contemplate the antibody with more than two potency.For example, three-specific antibody can be prepared.Tutt et al., J.Immunol.147：60(1991).

6. Heteroconjugate antibodies

Heteroconjugate antibodies are also within the scope of the invention.Heteroconjugate antibodies are made up of the antibody of two kinds of covalent attachments.It has been proposed that this antibody-like is used to immune system cell is targetted undesired cell (United States Patent (USP) 4,676,980) and for treating HIV (WO 91/00360；WO 92/200373；EP 03089).Contemplating can be in vitro using the known method of synthetic protein chemistry, including those are related to the method for crosslinking agent, to prepare these antibody.For example, disulfide exchange can be used to react or build immunotoxin by forming thioether bond.Example suitable for the reagent of this purpose is included disclosed in imino group mercaptan alcohol ester/salt (iminothiolate) and 4- sulfydryl fourth methyl ester imidates (methyl-4-mercaptobutyrimidate) and such as United States Patent (USP) 4,676,980.

7. multivalent antibody

Multivalent antibody can quickly be expressed the internalization (and/or alienation) of the cell of the antibody combination antigen than bivalent antibody.The antibody of the present invention can be easy to generate by the recombination expression of the nucleic acid of encoding antibody polypeptide chain, the multivalent antibody (being different from IgM classes) (such as tetravalent antibody) with three or more antigen binding sites.Multivalent antibody can include dimerization domain and three or more antigen binding sites.It is preferred that dimerization domain include (or being made from it) Fc areas or cross-linking zone.In this case, antibody is by three or more antigen binding sites comprising Fc areas and Fc areas amino terminal.Multivalent antibody preferred herein includes (or being made from it) three to about eight, but preferably four antigen binding sites.Multivalent antibody includes at least one polypeptide chain (and preferably two polypeptide chains), wherein the polypeptide chain includes two or more variable domains.For example, polypeptide chain can include VD1- (X1)_n-VD2-(X2)_n- Fc, wherein VD1 are the first variable domains, and VD2 is the second variable domain, and Fc is a polypeptide chain in Fc areas, X1 and X2 represented amino acids or polypeptide, and n is 0 or 1.For example, polypeptide chain can be included：VH-CH1- flexible joint-VH-CH1-Fc areas chain；Or VH-CH1-VH-CH1-Fc areas chain.Multivalent antibody herein is preferably also comprising at least two (and preferably four) light chain variable district polypeptides.Multivalent antibody herein can include e.g., from about two to about eight light chain variable district polypeptides.The light chain variable district polypeptide contemplated herein includes light chain variable district, and optionally also includes CL domains.

8. effector function is engineered

It may want to modify the antibody of the present invention in terms of effector function, such as in order to strengthen the cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC) that the antigen dependent cell of antibody is mediated.This can be realized by introducing one or more amino acid replacements in antibody Fc district.Or cysteine residues are introduced in Ke Fc areas, it is possibly realized so that forming interchain disulfide bond in this zone.The homodimer antibody so generated can have the cell killing and antibody dependent cellular toxicity (ADCC) of improved internalization ability and/or the complement-mediated of raising.Referring to Caron et al., J.Exp.Med.176：1191-1195 (1992) and Shopes, B., J.Immunol.148：2918-2922(1992).Homodimer antibody with enhanced antitumor activity it is also possible to use such as Wolff et al., Cancer Research 53：It is prepared by heterobifunctional crosslinker described in 2560-2565 (1993).Or, antibody can be transformed into dual Fc areas, thus can have enhanced complement lysis and ADCC abilities.Referring to Stevenson et al., Anti-Cancer Drug Design 3：219-230(1989).In order to improve the serum half-life of antibody, salvage receptor binding epitope can be mixed into antibody (especially antibody fragment) described in 739,277 such as such as United States Patent (USP) 5.As used herein, term " salvage receptor binding epitope " refers to IgG molecules (such as IgG₁、IgG₂、IgG₃Or IgG₄) it is responsible for improving the epitope of serum half-life in IgG molecule bodies in Fc areas.

9. immune conjugate

The invention further relates to have the immune conjugate of the antibody of cytotoxic agent, the cytotoxic agent such as chemotherapeutics, growth inhibitor, toxin (such as bacterium, fungi, plant or the enzyme activity toxin of animal origin or its fragment) or radio isotope (radiating conjugate) comprising coupling.

It is hereinbefore described available for the chemotherapeutics for generating such immune conjugate.Workable enzyme activity toxin and its fragment include diphtheria toxin A chains, the nonbinding active fragments of diphtheria toxin, exotoxin A chain (comes from pseudomonas aeruginosa Pseudomonas aeruginosa), ricin (ricin) A chains, abrin (abrin) A chains, capsule lotus root toxalbumin (modeccin) A chains, α-broom aspergillin (sarcin), tung oil tree (Aleutites fordii) toxalbumin, carnation (dianthin) toxalbumin, dyers' grapes (Phytolacaamericana) toxalbumin (PAPI, PAPII and PAP-S), balsam pear (Momordica charantia) mortifier, curcin (curcin), crotin (crotin), Saponaria officinalis (sapaonaria officinalis) mortifier, gelonin (gelonin), morphine (mitogellin), restrictocin (restrictocin), phenomycin (phenomycin), enomycin (enomycin) and trichothecin (trichothecenes).A variety of radionuclides can be used for generation radiation coupled antibody.Example includes²¹²Bi、¹³¹I、¹³¹In、⁹⁰Y and¹⁸⁶Re.A variety of bifunctional protein coupling agents can be used to prepare for the conjugate of antibody and cytotoxic agent, such as N- succinimides base -3- (2- pyridine radicals two is thio) propionic ester (SPDP), iminothiolane (IT), imino-ester (such as hydrochloric acid adipyl imido dimethyl phthalate), active esters (such as succinimide base ester of suberic acid two), aldehydes (such as glutaraldehyde), double azido compound (such as double (p- azido benzoyl base) hexamethylene diamines), dual azepine derivatives (such as double (p- diazoniumbenzoyl) hexamethylene diamines), diisocyanate (such as toluene 2, 6- diisocyanate), with double activated fluorine compounds (such as 1, 5- bis- fluoro- 2, 4- dinitro benzenes) dual-function derivative.For example, can be such as Vitetta et al., Science 238：Ricin immunotoxin is prepared described in 1098 (1987).The 1- isothiocyanic acid benzyl -3- methyl diethylene-triamine pentaacetic acids (MX-DTPA) of carbon-14 mark are for by the exemplary chelating agent of radioactive nucleotides and antibody coupling.Referring to WO 94/11026.

Antibody and one or more small molecule toxins such as Calicheamicin (calicheamicin), maytansinoids (maytansinoids), trichothecin (trichothecene) and CC1065 has been contemplated herein, and these toxin have the conjugate of the derivative of neurotoxin active.

Maytansine and maytansinoids

In a preferred embodiment, anti-TAT antibody (total length or fragment) of the invention and one or more maytansinoid molecule coupling labeleds.

Maytansinoids are the mitotic inhibitors played a role by suppressing tubulin polymerization.Maytansine is initially isolated (United States Patent (USP) 3,896,111) from East Africa shrub tingia Caulis Mayteni (Maytenus serrata).It is subsequently found certain micro-organisms and also generates maytansinoids, such as maytansinol and C-3 maytansinols ester (United States Patent (USP) 4,151,042).Such as following U.S. Patent Publication synthesis maytansinol and its derivative and analog：4,137,230；4,248,870；4,256,746；4,260,608；4,265,814；4,294,757；4,307,016；4,308,268；4,308,269；4,309,428；4,313,946；4,315,929；4,317,821；4,322,348；4,331,598；4,361,650；4,364,866；4,424,219；4,450,254；4,362,663；And 4,371,533, clearly the disclosure of which is collected herein by reference.

Maytansinoid-antibody coupling matter

In the trial for improving its therapeutic index, by maytansine and maytansinoids and the antibody coupling of specific bond tumor-cell antigen.Such as following patent discloses immune conjugate and its therapeutical uses comprising maytansinoids：United States Patent (USP) 5,208,020；5,416,064；And the B1 of European patent EP 0,425 235, clearly the disclosure of which is collected herein by reference.Liu et al., Proc.Natl.Acad.Sci.USA 93：8618-8623 (1996) describes the immune conjugate for including the maytansinoid for being referred to as DM1 being connected with the monoclonal antibody C242 for human colorectal cancer.It was found that the conjugate has the cytotoxicity of height for the colon cancer cell of culture, and show antitumor activity in tumour growth measurement in vivo.Chari et al., Cancer Research 52：127-131 (1992) describes wherein maytansinoid through disulfide bond joint and the immune conjugate for combining another mouse monoclonal antibody TA.1 couplings of the mouse antibody A 7 of antigen or combination HER-2/neu oncogenes in human colon cancer cell line.The cytotoxicity of TA.1- maytansinoid conjugates, each cell expression 3 × 10 of the cell line are tested on human breast cancer cell system SK-BR-3 in vitro⁵Individual HER-2 surface antigens.Drug conjugates have reached a certain degree of cytotoxicity similar to free maytansinoid drugs, and this can be improved by increasing the maytansinoid molecule amount of each antibody molecule coupling.A7- maytansinoid conjugates show low systemic cellular toxicity in mouse.

Anti- TAT polypeptide antibodies-maytansinoid conjugate (immune conjugate)

Anti- TAT antibody-maytansinoid conjugate can be prepared by being connected anti-TAT antibody with maytansinoid molecular chemistry under conditions of the biological activity of antibody or maytansinoid molecule is not significantly attenuated.It is verified, each antibody molecule, which is averagely coupled 3-4 maytansinoid molecule, can effectively strengthen the cytotoxicity for target cell, and the function or solubility to antibody have no adverse effect, although even if a molecule toxin/antibody than be also expected using exposed antibody strengthen cytotoxicity.Maytansinoids are well known in the art, and can be synthesized or be separated from natural origin by known technology.For example in United States Patent (USP) 5,208,020 and other patents mentioned above and non-Patent Publication thing disclose suitable maytansinoids.It is preferred that maytansinoids be the maytansinol analog of the aromatic rings or other positions of maytansinol and maytansinol molecule by modification, such as various maytansinol esters.

Know that many linking groups can be used for preparing antibody-maytansinoid conjugate, including such as United States Patent (USP) 5,208,020 or B1 and Chari the et al., CancerResearch 52 of European patent 0 425 235 in this area：Disclosed in 127-131 (1992).Linking group includes disulfide bond group, sulfide group, acid-unstable group, photo-labile group, the unstable group of peptase or the unstable group of esterase, as mentioned previously disclosed in patent, preferably disulfide bond and sulfide group.

A variety of bifunctional protein coupling agents can be used to prepare the conjugate of antibody and maytansinoid, such as N- succinimides base -3- (2- pyridine radicals two is thio) propionic ester (SPDP), succinimide base -4- (N- Maleimidomethyls) hexamethylene -1- carboxylates, iminothiolane (IT), imino-ester (such as hydrochloric acid adipyl imido dimethyl phthalate), active esters (such as succinimide base ester of suberic acid two), aldehydes (such as glutaraldehyde), double azido compound (such as double (p- azido benzoyl base) hexamethylene diamines), dual azepine derivatives (such as double (p- diazoniumbenzoyl)-ethylenediamines), diisocyanate (such as toluene 2, 6- diisocyanate), with double activated fluorine compounds (such as 1, 5- bis- fluoro- 2, 4- dinitro benzenes) dual-function derivative.Particularly preferred coupling agent includes N- succinimide bases -3- (2- pyridine radicals two is thio) propionic ester (SPDP) (Carlsson et al., Biochem.J.173：723-737 (1978)) and N- succinimide bases -4- (2- pyridylthios) valerate (SPP), thus disulfide bond is provided.

According to the type of connection, joint can be attached to multiple positions of maytansinoid molecule.For example, conventional coupling techniques can be used to pass through the reaction with hydroxyl to form ester bond.Reaction can occur in the C-3 positions with hydroxyl, C-14 positions, the C-15 positions through hydroxyl modified and the C-20 positions with hydroxyl modified through methylol.In a preferred embodiment, key connection is formed in the C-3 positions of maytansinol or maytansinol analog.

Calicheamicin

Another immune conjugate interested includes the anti-TAT antibody that coupling has one or more calicheamicin molecules.Calicheamicin antibiotic family can generate double-strand DNA cleavage in sub- picomolar concentrations.Preparation on Calicheamicin family conjugate is referring to United States Patent (USP) 5,712,374；5,714,586；5,739,116；5,767,285；5,770,701；5,770,710；5,773,001；5,877,296 (all authorizing Cyanamid companies of the U.S.).Available Calicheamicin analogue includes but is not limited to γ₁ ^I、α₂ ^I、α₃ ^I, N- acetyl group-γ₁ ^I, PSAG and θ_I ¹(Hinman et al., Cancer Research 53：3336-3342(1993)；Lode et al., Cancer Research 58：2925-2928(1998)；And the above-mentioned United States Patent (USP) for authorizing Cyanamid companies of the U.S.).Another can be QFA with the antineoplastic of antibody coupling, and it is a kind of antifolic thing.Calicheamicin and QFA have intracellular action site, and are difficult through plasma membrane.Therefore, these medicaments greatly strengthen their cytotoxic effect via the cellular uptake of antibody-mediated internalization.

Other cytotoxic agents

BCNU, streptozotocin (streptozoicin), vincristine (vincristine), 5 FU 5 fluorouracil, United States Patent (USP) 5 can be included with other antitumor agents of the anti-TAT antibody couplings of the present invention, 053,394th, 5,770, the medicament family for being referred to as LL-E33288 compounds and ai sibo mycin class (esperamicins) (United States Patent (USP) 5 described in 710,877,296).

Available enzyme activity toxin and its fragment include diphtheria toxin A chains, the nonbinding active fragments of diphtheria toxin, exotoxin A chain (comes from pseudomonas aeruginosa Pseudomonas aeruginosa), ricin (ricin) A chains, abrin (abrin) A chains, capsule lotus root toxalbumin (modeccin) A chains, α-broom aspergillin (sarcin), tung oil tree (Aleutites fordii) albumen, carnation (dianthin) albumen, dyers' grapes (Phytolaca americana) albumen (PAPI, PAPII and PAP-S), balsam pear (Momordicacharantia) mortifier, curcin (curcin), crotin (crotin), Saponaria officinalis (sapaonaria officinalis) mortifier, gelonin (gelonin), morphine (mitogellin), restrictocin (restrictocin), phenomycin (phenomycin), enomycin (enomycin) and trichothecin (trichothecenes).The WO 93/21232 announced see, for example, on October 28th, 1993.

Present invention further contemplates antibody and compound (such as ribalgilase or DNA endonucleases, such as deoxyribonuclease with nucleolysis activity；DNA enzymatic) between the immune conjugate that is formed.

For selective destruction tumour, antibody can include high radioactive atom.A variety of radio isotopes can be used for the anti-TAT antibody of generation radiation coupling.Example includes At¹¹、I¹³¹、I¹²⁵、Y⁹⁰、Re¹⁸⁶、Re¹⁸⁸、Sm¹⁵³、Bi²¹²、P³²、Pb²¹²With Lu radio isotope.When the conjugate to be used to diagnose, it can include radioactive atom such as Tc^99mOr I¹²³For scitiphotograph research, or it is used for nuclear magnetic resonance (NMR) imaging (also referred to as magnetic resonance imaging, mri) comprising spin label such as iodo- 123, iodine -131, indium -111, fluoro- 19, carbon -13, nitrogen -15, oxygen -17, gadolinium, manganese or iron.

Radioactivity or other labels can be mixed into conjugate in a known way.For example, can biosynthesis peptide, or by chemical amino acid synthetic method synthetic peptide, wherein using including for example with the fluoro- 19 suitable amino group acid precursors instead of hydrogen.Label, such as Tc can be adhered to through the cysteine residues in peptide^99mOr I¹²³、Re¹⁸⁶、Re¹⁸⁸And In¹¹¹.Yttrium-90 can be adhered to through lysine residue.IODOGEN methods (Fraker etal., Biochem.Biophys.Res.Commun.80：49-57 (1978)) it can be used for mixing iodo- 123.《Monoclonal Antibodies in Immunoscintigraphy》(Chatal, CRC Press, 1989) describes other methods in detail.

A variety of bifunctional protein coupling agents can be used to prepare the conjugate of antibody and cytotoxic agent, such as N- succinimides base -3- (2- pyridine radicals two is thio) propionic ester (SPDP), succinimide base -4- (N- Maleimidomethyls) hexamethylene -1- carboxylates, iminothiolane (IT), imino-ester (such as hydrochloric acid adipyl imido dimethyl phthalate), active esters (such as succinimide base ester of suberic acid two), aldehydes (such as glutaraldehyde), double azido compound (such as double (p- azido benzoyl base) hexamethylene diamines), dual azepine derivatives (such as double (p- diazoniumbenzoyl)-ethylenediamines), diisothio-cyanate (such as toluene 2, 6- diisocyanate), with double activated fluorine compounds (such as 1, 5- bis- fluoro- 2, 4- dinitro benzenes) dual-function derivative.For example, can be such as Vitetta et al., Science 238：Ricin immunotoxin is prepared described in 1098 (1987).The 1- isothiocyanic acid benzyl -3- methyl diethylene-triamine pentaacetic acids (MX-DTPA) of carbon-14 mark are for by the exemplary chelating agent of radioactive nucleotides and antibody coupling.Referring to WO94/11026.Joint can be easy for discharging " the cleavable joint " of cell toxicity medicament in cell.For example, sour unstable joint, peptidase-sensitive linker, photo-labile joint, dimethyl linker or (Chari et al., the Cancer Research 52 of joint containing disulfide bond can be used：127-131(1992)；United States Patent (USP) 5,208,020).

Or, the fusion protein comprising anti-TAT antibody and cytotoxic agent can be prepared for example, by recombinant technique or peptide symthesis.Section of DNA can include the region of each two parts of own coding conjugate, abut one another or separated by the region of encoding linker peptide, the joint peptide does not destroy the desired characteristic of conjugate.

In still another embodiment, antibody can be coupled with " acceptor " (such as streptavidin) to target in advance for tumour, wherein to patient's administration of antibodies-receptor conjugate, then uncombined conjugate is disposed from circulation using scavenger, then using " part " (such as avidin) being coupled with cytotoxic agent (such as radioactive nucleotides).

10. immunoliposome

Anti- TAT antibody disclosed herein can also be configured to immunoliposome." liposome " refers to what is be made up of all kinds lipid, phosphatide and/or surfactant, available for the vesicles that medicine is delivered to mammal.Similar to the lipid arrangement of biomembrane, the composition of liposome is typically arranged to bilayer formation.Liposome containing antibody can be prepared by means known in the art, such as Epstein et al., Proc.Natl.Acad.Sci.USA 82：3688(1985)；Hwang et al., Proc.Natl.Acad.Sci.USA 77：4030(1980)；United States Patent (USP) 4,485,045 and 4,544,545；And WO 97/38731, it is disclosed in described in 23 days October in 1997.The circulation time liposome of extension is disclosed in United States Patent (USP) 5,013,556.

The available lipid composite comprising phosphatidyl choline, cholesterol and PEG derivatization phospholipid acyl monoethanolamines (PEG-PE) of particularly useful liposome is generated by reverse phase evaporation.Liposome is squeezed through into the filter with setting aperture, the liposome with desired diameter is produced.Can be such as Martin et al., J.Biol.Chem.257：Described in 286-288 (1982), the Fab ' fragments of antibody of the present invention are coupled through disulfide bond exchange reaction and liposome.Chemotherapeutics is optionally included in liposome.Referring to Gabizon et al., J.National CancerInst.81 (19)：1484(1989).

B.TAT combination oligopeptides

The TAT combination oligopeptides of the present invention, refers to combine, preferably specifically binds the oligopeptides of TAT polypeptides described herein.TAT combinations oligopeptides can use known oligopeptides synthetic methodology to carry out chemical synthesis, or usable recombinant technique is prepared and purified.The length of TAT combination oligopeptides is typically at least about 5 amino acid, or length is at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 amino acid or longer, wherein such oligopeptides can be combined, it is preferred that specifically binding TAT polypeptides described herein.TAT combinations oligopeptides just known technology can be used to identify without excessively testing.At this point, it is noted that the technology of the oligopeptides for being capable of specific binding polypeptide target to oligopeptides library screening is well known in the art (see, for example, United States Patent (USP) 5,556,762,5,750,373,4,708,871,4,833,092,5,223,409,5,403,484,5,571,689,5,663,143；PCT Publication WO 84/03506 and WO 84/03564；Geysen et al., Proc.Natl.Acad.Sci.U.S.A.81：3998-4002(1984)；Geysen et al., Proc.Natl.Acad.Sci.U.S.A.82：178-182(1985)；Geysen et al., in Synthetic Peptides asAntigens, 130-149 (1986)；Geysen et al., J.Immunol.Meth.102：259-274(1987)；Schoofs et al., J.Immunol.140：611-616(1988)；Cwirla, S.E.et al., (1990) Proc.Natl.Acad.Sci.USA 87：6378；Lowman, H.B.et al., (1991) Biochemistry 30：10832；Clackson, T.et al., (1991) Nature 352：624；Marks, J.D.et al., (1991), J.Mol.Biol. 222：581；Kang, A.S.et al., (1991) Proc.Natl.Acad.Sci.USA 88：8363；Smith, G.P., (1991) Current Opin.Biotechnol.2：668).

At this point, phage display is a kind of can to screen large-scale oligopeptides library to identify the known technology for the member for being capable of specific binding polypeptide target in those libraries.Phage display is a kind of technology (Scott, J.K.andSmith, G.P. (1990) Science 249 that variant polypeptide is illustrated in phage particle surface as the fusion protein with coat protein：386).The effectiveness of phage display is, those can be quickly and efficiently sorted from the large-scale library of selective randomized proteins qualitative change body (or Random clones cDNA) with the sequence of high-affinity binding target molecule.Displayed polypeptide (Cwirla, the S.E.et al., (1990) Proc.Natl.Acad. ' Sci.USA 87 on bacteriophage：Or protein (Lowman, H.B.et al., (1991) Biochemistry 30 6378)：10832；Clackson, T.et al., (1991) Nature 352：624；Marks, J.D.et al., (1991) J.MoI.Biol.222：581；Kang, A.S.et al., (1991) Proc.Natl.Acad.Sci.USA 88：8363) library has been used to those (Smith, G.P. (1991) the Current Opin.Biotechnol.2 of screening with specific binding properties from millions of middle polypeptides or oligopeptides：668).The phage library of sorting random mutant needs to build and expand the strategy of a large amount of variants, the flow of affinity purification is carried out using target acceptor, and assess the means for the result for combining enrichment.Referring to United States Patent (USP) 5,223,409,5,403,484,5,571,689 and 5,663,143.

Although most of phage display methods use filobactivirus, λ classes (lambdoid) phage display system (WO 95/34683；US 5,627,024), T4 phage display systems (Ren et al., Gene, 215：439(1998)；Zhu et al., Cancer Research, 58 (15)：3209-3214(1998)；Jiang etal., Infection ＆ Immunity, 65 (11)：4770-4777(1997)；Ren et al., Gene, 195 (2)：303-311(1997)；Ren, Protein Sci., 5：1833(1996)；Efimovetal., Virus Genes, 10：173 (1995)) and T7 phage display systems (Smith and Scott, Methods in Enzymology 217：228-257(1993)；US 5,766,905) it is also known.

Many other improvement and change to basic phage display concept development now.These improvement enhance display systems and peptide library are screened and selected the combination of target molecule and show the ability of functional protein, and the functional protein has the potentiality that these protein are screened with desired characteristic.The composite reaction device (WO 98/14277) reacted for phage display has been developed, and phage display library has been used to analyze and controls bio-molecular interaction (WO 98/20169；WO 98/20159) and constrained helical peptides characteristic (WO 98/20036).The method that WO 97/35196 describes separation affinity ligand, phage display library is wherein set to contact the part that the first solution and second of solution are combined with Selective Separation, part is by binding target molecule in the first solution, and affinity ligand will not binding target molecule in second of solution.WO 97/46251 describes such a method, i.e., with the antibody biopanning random phage body display storehouse of affinity purification, then separate the bacteriophage of combination, then carries out panning process to separate the bacteriophage that high-affinity is combined using the hole of micro plate.It has been reported that use (Li et al., (1998) Mol.Biotech.9 of staphylococcus aureus (Staphylococcus aureus) albumin A as affinity tag：187).WO 97/47314 describes the purposes that substrate subtracted library is used to distinguish enzyme spcificity, wherein using the combinatorial libraries that can be phage display library.WO 97/09446 describes the method that the enzyme suitable for detergent is selected using phage display.Other methods of the protein of selection specific binding are described in United States Patent (USP) 5,498,538,5,432,018 and WO98/15833.

The method for producing these libraries of peptide library and screening is also disclosed in United States Patent (USP) 5,723,286,5,432,018,5,580,717,5,427,908,5,498,530,5,770,434,5,734,018,5,698,426,5,763,192, and 5,723,323.

C.TAT combination organic molecules

TAT combination organic molecules, refer in addition to oligopeptides or antibody defined herein, with reference to, preferably specifically bind the organic molecules of TAT polypeptides described herein.TAT combinations organic molecule can use the known formula science of law to identify and chemical synthesis (see, for example, PCT Publication WO 00/00823 and WO00/39585).TAT is generally less than about 2000 dalton with reference to organic bulk of molecule, or its size is less than about 1500,750,500,250 or 200 dalton, wherein can so combine, preferably specifically bind the organic molecules of TAT polypeptides described herein and just known technology can be used to identify without excessively test.At this point, it should be noted that the technology for organic molecule libraries to be screened with the molecule for being capable of Binding peptide target is (see, for example, PCT Publication WO 00/00823 and WO 00/39585) well known in the art.TAT combinations organic molecule can be such as aldehyde, ketone, oxime, hydrazone, semicarbazones (semicarbazone), carbonohydrazides (carbazide), primary amine, secondary amine, tertiary amine, the hydrazine of N- substitutions, hydrazides, alcohol, ether, mercaptan, thioether, disulphide, carboxylic acid, ester, acid amides, urea, carbamate (carbamate), carbonic ester (carbonate), ketal, thio ketal ization (thioketal), acetal, mercaptal, aryl halide, aromatic yl sulphonate (aryl sulfonate), alkyl halogen, hydrocarbyl sulfonic ester (alkyl sulfonate), aromatic compound, heterocyclic compound, aniline, alkene, alkynes, glycol, amino alcohol,  oxazolidines,  oxazolines, thiazolidine, thiazoline, enamine, sulfonamide (sulfonamide), epoxides, ethylene imine (aziridine), isocyanates (isocyanate), sulfonic acid chloride, diazonium compound, acid chloride (acid chloride) etc..

D. screening has anti-TAT antibody, TAT combinations oligopeptides and the TAT combination organic molecules of desired characteristic

It is hereinbefore described for producing the antibody for combining TAT polypeptides, oligopeptides and the technology of organic molecule.As needed, the antibody with some biological properties, oligopeptides or other organic molecules can further be selected.

The cell of TAT polypeptides by means commonly known in the art, such as can be expressed using endogenous or after being transfected with TAT genes, to assess anti-TAT antibody of the invention, oligopeptides or the growth inhibitory effect of other organic molecules.For example, suitable tumor cell line and TAT transfectional cells can be handled several days (such as 2-7 days) with anti-TAT monoclonal antibodies of the invention, oligopeptides or the other organic molecules of various concentrations, and dyed with crystal violet or MTT, or analyzed by some other colorimetric methods.Another method of measurement propagation is by comparing the handled cell in the anti-TAT antibody, TAT combinations oligopeptides or TAT combination organic molecules presence or absence of the present invention³H- thymidines are absorbed.After processing, harvesting is simultaneously quantified in scintillation counter to the radioactive amount for mixing DNA.Suitable positive control includes handling the cell line with the known growth inhibiting antibody for suppressing selected cell line growth.The a variety of methods that can be known with this area determine the growth inhibition of interior tumor cell.Preferably, tumour cell is the cell for being overexpressed TAT polypeptides.Preferably, compared with untreated tumour cell, anti- TAT antibody, TAT combinations oligopeptides or TAT combinations organic molecule breed the cell for suppressing to express TAT tumour cell in vitro or in vivo of about 25-100%, more preferably from about 30-100%, very more preferably from about 50-100% or 70-100%, in one embodiment, antibody concentration is about 0.5-30 μ g/ml.Can in cell culture in antibody concentration be about 0.5-30 μ g/ml or about 0.5nM to 200nM under conditions of measure growth inhibition, wherein 1-10 days after tumour cell is exposed to antibody measure growth inhibition.If applied with about 1 μ g/kg to about 100mg/kg body weight, anti-TAT antibody causes in from administration of antibodies first about 5 days to 3 months, tumor mass reduction or tumor cell proliferation are reduced in preferably from about 5 to 30 days, then the antibody is tumor growth inhibition.

In order to select the anti-TAT antibody, TAT combinations oligopeptides or TAT combination organic molecules of inducing cell death, the forfeiture of film integrality can be assessed relative to control, this is indicated by such as propidium iodide (PI), trypan blue or 7AAD intakes.PI intakes determination method can be carried out when lacking complement and immune effector cell.Incubated together with tumour cell and the single culture medium of TAT polypeptides or the culture medium containing suitable anti-TAT antibody (e.g., from about 10 μ g/ml), TAT combinations oligopeptides or TAT combination organic molecules will be expressed.By the 3 day time of cell culture.After per treatment, cleaning cell and decile (aliquot) into (strainer-capped) of the 35mm with filter cover 12 × 75 test tubes (every test tube 1ml, each 3 test tubes for the treatment of group) are used to remove cell mass.Then PI (10 μ g/ml) is added to test tube.Use FACSCAN  flow cytometers and FACSCONVERT  CellQuest softwares (Becton Dickinson) analysis sample.Those may be selected the anti-TAT antibody, TAT combinations oligopeptides or TAT combination organic molecules of inducing cell death are used as by PI anti-TAT antibody, TAT combinations oligopeptides or the TAT combinations organic molecules for absorbing the cell death for being defined as inducing statistical significant level.

In order to screen antibody, oligopeptides or the other organic molecules of the epitope combined with reference to purpose antibody on TAT polypeptides, conventional cross-blocks determination method can be carried out, such as Antibodies, A Laboratory Manual, described in Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988).This determination method can be used for determining test antibody, oligopeptides or other organic molecules whether with known anti-TAT antibody bindings identical site or epitope.Or epitope mapping (mapping) can be carried out by methods known in the art.For example, come matagenized antibody sequence contact residues can be identified by such as Alanine-scanning.Mutant antibodies are tested first with the combination of polyclonal antibody to ensure correctly to fold.In a kind of different method, the peptide for corresponding to TAT polypeptides not same district can be used in competition assay, wherein using several test antibodies or a kind of test antibody and it is a kind of have characterized or known epitope antibody.

F. the prodrug therapy (ADEPT) of the enzyme mediation of antibody is relied on

By the way that antibody and prodrug activation enzyme are coupled, antibody of the invention can be additionally used in ADEPT, and pro-drug (such as peptidyl chemotherapeutic agent, referring to WO 81/01145) is changed into active anticancer medicine by the prodrug activation enzyme.See, for example, WO 88/07378 and United States Patent (USP) 4,975,278.

Enzyme component available for ADEPT immune conjugate includes any enzyme that can act on pro-drug and be transformed into more active cytotoxic form.

Enzyme available for the inventive method includes but is not limited to the pro-drug of phosphate-containing/ester can be changed into the alkaline phosphatase of free drug；The pro-drug of containing sulfate/ester can be changed into the aryl sulfatase of free drug；Nontoxic 5-flurocytosine can be changed into the cytosine deaminase of anticarcinogen 5 FU 5 fluorouracil；Medicine containing propeptide can be changed into the protease of free drug, such as Serratieae protease, thermolysin, subtilopeptidase A, carboxypeptidase and cathepsin (such as cathepsin B and L)；The D- alanylcarboxypeptidases of the pro-drug of the amino acid surrogates containing D- can be converted；Glycosylated prodrugs can be changed into the sugared nickase of free drug, such as beta galactosidase and neuraminidase；It can will be changed into the beta-lactamase of free drug with the medicine of beta-lactam derivatization；And can will be transformed into the penicillin amidase of free drug, such as Penicillin-V-Amidase or Penicillin-G-amidases with the medicine of benzene oxygen acetyl group or phenylacetyl group derivatization respectively at its ammonia nitrogen.Or, the antibody with enzymatic activity can be used, this area is also referred to as " abzyme ", the pro-drug of the present invention is changed into free active medicine (see, for example, Massey, Nature 328：457-458(1987)).Antibody-antibody enzyme conjugates as described herein can be prepared, for abzyme to be delivered into tumor cell group.

Can be by technology well-known in the art, such as using heterobifunctional crosslinker as discussed above, by the enzyme of the present invention and anti-TAT antibody covalent bond.Or, it recombinant DNA technology well-known in the art can be used to build the fusion protein for the antigen binding domain for comprising at least the antibody of the present invention being connected with least functional activity part of enzyme of the present invention (see, for example, Neuberger et al., Nature 312：604-608(1984)).

F. total length TAT polypeptides

Present invention also offers the nucleotide sequence newly identified and separated, it encodes the polypeptide for being referred to as TAT polypeptides in the application.Specifically, cDNA (part or total length) that is identified and having separated a variety of TAT polypeptides of coding, as Examples below is further disclosed in detail.

As disclosed in Examples below, multiple cDNA clones have been preserved in ATCC.Those clone actual nucleotide sequences can by those skilled in the art using this area conventional method by the way that institute's preservation cloning and sequencing is easily determined.Routine techniques can be used to be determined from nucleotide sequence for the amino acid sequence of prediction.For TAT polypeptides described herein and code nucleic acid, in some cases, applicant, which has identified, to be considered according to the appraisable best reading frame of obtainable sequence information at that time.

G. anti-TAT antibody and TAT polypeptide variants

Except anti-TAT antibody described herein and total length native sequences TAT polypeptides, it is contemplated to anti-TAT antibody and TAT polypeptide variants can be prepared.Anti- TAT antibody and TAT polypeptide variants can be by changing introducing coding DNA by suitable nucleotides and/or being prepared by synthesizing expectation antibody or polypeptide.Skilled artisans will appreciate that, amino acid change can change the post translational processing of anti-TAT antibody or TAT polypeptides, such as change the number or position or change film anchor feature of glycosylation site.

Row variation can be entered in anti-TAT antibody and TAT polypeptides described herein, such as using such as United States Patent (USP) 5, any technology and guilding principle of the conservative and non-conservative mutation described in 364,934.Variation can be the replacement of one or more codons of encoding antibody or polypeptide, delete or insertion that it causes amino acid sequence relative to the change of native sequences antibody or polypeptide.It is optional that, variation is that at least one amino acid is substituted by any other amino acid in one or more domains by anti-TAT antibody or TAT polypeptides.Pass through the sequence and the sequence of homologous known protein molecule of relatively more anti-TAT antibody or TAT polypeptides, and amino acid sequence in high homology area is changed number at least, people can be instructed to determine which amino acid residue be can be inserted into, substituted or be deleted without to it is expected that activity has a negative impact.Amino acid replacement can be the result that is, conserved amino acid is substituted by a kind of another amino acid replacement (such as with serine substitute leucine) of amino acid with similar structure and/or chemical characteristic.Insertion or deletion can be optionally in the range of about 1 to 5 amino acid.Amino acid insertion can be carried out by system in the sequence, substitutes or deletes, and permissible variation is determined by total length or the activity of ripe native sequences displaying to gained mutation testing.

There is provided herein the fragment of anti-TAT antibody and TAT polypeptides.For example, when being compared with total length natural antibody or protein, such fragment can be truncated in N- ends or C- ends, or can lack internal residues.Some fragments lack nonessential amino acid residue for the expectation biological activity of anti-TAT antibody or TAT polypeptides.

Anti- TAT antibody and TAT polypeptide fragments can be prepared by any of a variety of routine techniques.It is expected that fragments of peptides is chemically synthesized.A kind of alternative approach is related to produces antibody or polypeptide fragment by enzymatic digestion, for example by using the enzyme-treated protein of the known scinderin matter at the site limited by particular amino acid residue, or by using suitable limitation enzymic digestion DNA, and separate expectation fragment.Also a kind of suitable technology is related to separation and by PCR (PCR) amplification coding expectation antibody or the DNA fragmentation of polypeptide fragment.Limit DNA fragmentation and it is expected that the oligonucleotides of end is used as 5 ' and 3 ' primers in PCR.Preferably, anti-TAT antibody and TAT polypeptide fragments and natural anti-TAT antibody or TAT polypeptides shared at least one biology and/or immunologic competence disclosed herein.

In a particular embodiment, conservative replacement interested is shown in Table shown under 6 titles " preferably substituting ".If such replacement causes the change of biological activity, then can be introduced into table 6 and be referred to as the more material alterations of " illustrate and substitute ", or following article is described further on amino acid classification, and screens product.

Table 6

Original Residue	Illustrate and substitute	It is preferred that substituting
Original Residue	Illustrate and substitute	It is preferred that substituting	Ala(A) Arg(R) Asn(N) Asp(D) Cys(C) Gln(Q) Glu(E) Gly(G) His(H) Ile(I) Leu(L)	Val；Leu；Ile Lys；Gln；Asn Gln；His；Asp；Lys；Arg Glu；Asn Ser；Ala Asn；Glu Asp；Gln Pro；Ala Asn；Gln；Lys；Arg Leu；Val；Met；Ala；Phe；Nor-leucine Nor-leucine；Ile；Val；Met；Ala；Phe	Val Lys Gln Glu Ser Asn Asp Ala Arg Leu Ile

Lys(K ) Met(M) Phe(F) Pro(P) Ser(S) Thr(T) Trp(W) Tyr(Y) Val(V)

Arg；Gln；Asn Leu；Phe；Ile Trp；Leu；Val；Ile；Ala；Tyr Ala Thr Val；Ser Tyr；Phe Trp；Phe；Thr；Ser Ile；Leu；Met；Phe；Ala；Nor-leucine

Arg Leu Leu Ala Thr Ser Tyr Phe Leu

Confrontation TAT antibody or the function of TAT polypeptides or the substantive sex modification of immunology identity are by selecting dramatically different replacement in the effect for keeping following aspect to complete：(a) structure of the polypeptide backbone of replacement area, such as pleated sheet or helical conformation, (b) target site punishment son electric charge or hydrophobicity, or (c) side chain volume.According to common side chain properties, naturally occurring residue can be grouped as follows：

(1) it is hydrophobic：Nor-leucine, Met, Ala, Val, Leu, Ile；

(2) it is neutral, hydrophilic：Cys、Ser、Thr、Asn、Gln；

(3) it is acid：Asp、Glu；

(4) it is alkaline：His、Lys、Arg；

(5) residue of influence side chain orientation：Gly、Pro；With

(6) it is aromatic：Trp、Tyr、Phe.

Non-conservative replacement will need to exchange another classification with a member in one of these classifications.Such replacement residue can also be introduced to conservative substitution sites, or it is further preferred that introduce remaining (non-conservative) site.

Variation can be used the method that this area is known to carry out, such as oligonucleotide mediated (fixed point) mutagenesis, Alanine-scanning and PCR mutagenesis.Direct mutagenesis (Carter et al., Nucl.Acids Res.13 can be carried out to the DNA of clone：4331(1986)；Zoller et al., Nucl.Acids Res.10：6487 (1987)), cassette mutagenesis (Wells et al., Gene 34：315 (1985)), limitation Sexual behavior mode mutagenesis (restriction selection mutagenesis) (Wells et al., Philos.Trans.R.Soc.LondonSerA 317：415 (1986)) or other known technology to produce anti-TAT antibody or TAT polypeptide variants DNA.

One or more amino acid along neighbouring sequence can be also identified using scanning amino acid analysis.There is relatively small neutral amino acid in preferred scanning amino acid.This amino acid includes alanine, glycine, serine and cysteine.Alanine is typically the preferred scanning amino acid in this group, because it eliminates the side chain on β-carbon, and unlikely Conformation of the main chain (the Cunninghamand Wells, Science 244 for changing variant：1081-1085(1989)).Alanine is generally also preferably as it is most common amino acid.In addition, usually its (Creighton, The Proteins, W.H.Freeman ＆ Co., N.Y. can be found in concealed location and exposure position；Chothia, J.Mol.Biol.150：1(1976)).If alanine substitutes the change scale of construction produced and is insufficient to, then can be used and wait row's (isoteric) amino acid.

It is any be not related to keep the cysteine residues of anti-TAT antibody or the correct conformation of TAT polypeptides also alternative, generally substituted with serine, to improve the oxidation stability of molecule and prevent abnormal crosslinking.On the contrary, cysteine key can be added into anti-TAT antibody or TAT polypeptides to improve its stability (particularly when antibody is antibody fragment such as Fv fragments).

Particularly preferred class alternative variations are related to be substituted to one or more some hypervariable region residues (such as humanization or human antibody) of parental antibody.Be typically chosen for the gained variant further developed relative to produce their parental antibody will have improved biological characteristics.A kind of facilitated method for producing such alternative variations is directed to use with the affinity maturation of phage display progress.In brief, several hypervariable region sites (such as 6-7 site) are mutated, all possible amino acid replacement is produced in each site.The antibody variants so produced are illustrated on filamentous phage particle as the fusions with the M13 gene III products of each particle inner packing with monovalent fashion.Then the biological activity (such as binding affinity) of the variant of phage display is screened as disclosed herein.In order to identify the candidate hypervariable region site for modification, alanine scanning mutagenesis can be carried out to identify some hypervariable region residues of significant contribution are combined with to antigen.Or analyze the crystal structure of antigen-antibody complex to identify that the contact point between antibody and TAT polypeptides is probably beneficial.Such contact residues and neighbouring residue are according to the candidate locus that detailed description technology is substituted herein.Once such variant is produced, it is as described herein that this group of variant is screened, it may be selected there is the antibody of good characteristic to be used to further develop in one or more relevant assays.

Encoding the nucleic acid molecules of the amino acid sequence variation of anti-TAT antibody can be prepared by a variety of methods known in the art.These methods include but is not limited to from natural origin separation (in the case of naturally occurring amino acid sequence variation), or carry out oligonucleotide mediated (or fixed point) mutagenesis, PCR mutagenesis and cassette mutagenesis to prepare by the variant or the anti-TAT antibody of non-variant form that prepare early stage.

H. the modification of confrontation TAT antibody and TAT polypeptides

The covalent modification of confrontation TAT antibody and TAT polypeptides is included within the scope of the invention.A type of covalent modification includes making the targeted amino acid residues of anti-TAT antibody or TAT polypeptides react with organic derivatization reagent, and organic derivatization reagent can react with the selected side chain or N- or C- terminal residues of anti-TAT antibody or TAT polypeptides.The derivatization carried out with bifunctional reagent can be used for for example making anti-TAT antibody or TAT polypeptides and water insoluble supported matrix or surface-crosslinked, and for the purification process of anti-TAT antibody, vice versa.Conventional crosslinking agent includes such as 1; ester for example with the formation of 4- azidosalicylic acids of 1- double (diazonium-acetyl group) -2- vinylbenzenes, glutaraldehyde, N-hydroxy-succinamide esters, with difunctional imino-ester include two succinimide esters such as 3; the reagent such as such as double-N- maleimides -1, the 8- octanes of 3 '-two thiobis (Succinimidyl Propionate), difunctional maleimide and methyl -3- [(p- azidophenyl) two is thio] third imide ester.

It is glutamy and aspartyl residue that other modifications, which are included glutaminyl and asparaginyl difference deamidation; the hydroxylating of proline and lysine; the phosphorylation of the hydroxyl of seryl or threonyl residues; alpha-amino (T.E.Creighton, the Proteins of methylating of lysine, arginine and histidine side chains：Structure and Molecular Properties, W.H.Freeman ＆ Co., SanFrancisco, pp.79-86 (1983)), the acetylation of N- terminal amines, and any C- terminal carboxyl groups amidatioon.

The another kind of covalent modification of included confrontation TAT antibody or TAT polypeptides includes the Natively glycosylated pattern for changing antibody or polypeptide in the scope of the invention." change Natively glycosylated pattern " refers to delete herein one or more sugar moieties (moiety) being present in the anti-TAT antibody of native sequences or TAT polypeptides (or by eliminating potential glycosylation site, or glycosylated by being deleted with chemistry and/or enzymatic means), and/or add one or more non-existent glycosylation sites in the anti-TAT antibody of native sequences or TAT polypeptides.In addition, this phrase includes the glycosylated qualitative change of native protein, it is related to the change of the property and ratio of existing a variety of sugar moieties.

It is the glycosylation of antibody and other polypeptides generally N- connections or O- connections.N- connections refer to the side chain that sugar moieties are attached to asparagine residue.Tripeptide sequence asparagine-X-serine and asparagine-X-threonine (wherein X is any amino acid in addition to proline) are the recognition sequences that sugar moieties enzymatic is attached to asparagine side chain.Thus, the presence of these tripeptide sequence any of which produces potential glycosylation site in polypeptide.The glycosylation of O- connections refers to is attached to hydroxy-amino-acid, most commonly serine or threonine by one of sugars N-aceylgalactosamine, galactolipin or xylose, but 5-OxoPro or 5- hydroxylysines can also be used.

Glycosylation site is added into anti-TAT antibody or TAT polypeptides makes it easily be completed comprising one or more above-mentioned tripeptide sequences (glycosylation site for being used for N- connections) by changing amino acid sequence.This change can also be carried out (glycosylation site for being used for O- connections) by adding or substituting one or more serines or threonine residues in the sequence to original anti-TAT antibody or TAT polypeptides.The amino acid sequence of anti-TAT antibody or TAT polypeptides can optionally be changed by the change of DNA level, especially by the DNA of the anti-TAT antibody of mutation coding or TAT polypeptides at the base being pre-selected, so that the codon for expecting amino acid will be translated into by producing.

Another method for increasing the number of sugar moieties on anti-TAT antibody or TAT polypeptides is by making glucosides and chemiluminescent polypeptide or enzymatic of glucosides.This area is described to such method, such as WO 87/05330 disclosed in 1987 on Septembers 11, and Aplin and Wriston, CRC Crit.Rev.Biochem., pp.259-306 (1981).

Removing sugar moieties present on anti-TAT antibody or TAT polypeptides can be realized by chemistry or enzymatic method, or be substituted by the mutation for the codon for encoding the amino acid residue for serving as glycosylation target.Chemical deglycosylation technology is known in the art, and is described in such as Hakimuddin et al., Arch.Biochem.Biophys.259：52 (1987) and Edge et al., Anal.Biochem.118：131(1981).Sugar moieties on enzymatic cutting polypeptide can be realized by using a variety of inscribes and exoglycosidase, such as Thotakura et al., Meth.Enzymol.138：350 (1987) are described.

The another kind of covalent modification of confrontation TAT antibody or TAT polypeptides includes, with United States Patent (USP) 4,640,835；4,496,689；4,301,144；4,670,417；4,791,192 or 4, one of antibody or polypeptide and polymer of a variety of non-proteinaceous are connected, such as polyethylene glycol (PEG), polypropylene glycol or polyoxyalkylene (polyoxyalkylene) by mode described in 179,337.Antibody or polypeptide can also contain in the microcapsules prepared for example by condensation technique or by interfacial polymerization (being for example hydroxymethyl cellulose or gelatin-microcapsule and poly- (methyl methacrylate) microcapsules respectively), in colloidal drug delivery system (such as liposome, albumin microspheres, microemulsion, nano particle and Nano capsule) or in macro emulsion.Such technology is disclosed in such as Remington ' s Pharmaceutical Sciences, 16th edition, Osol, A.Ed., 1980.

The mode that chimeric molecule can also be formed modifies the anti-TAT antibody or TAT polypeptides of the present invention, and the chimeric molecule includes the anti-TAT antibody or TAT polypeptides merged with another heterologous polypeptide or amino acid sequence.

In one embodiment, such chimeric molecule includes the fusions of anti-TAT antibody or TAT polypeptides with tag polypeptide, and the tag polypeptide provides the epitope that the antibody alternative of anti-label is combined.Epitope tag is usually located at the amino or carboxyl terminal of anti-TAT antibody or TAT polypeptides.The antibody for the tag polypeptide can be used to detect for the presence of the anti-TAT antibody or TAT polypeptides of such epitope tag form.Moreover, the presence of epitope tag causes anti-TAT antibody or the affinity substrate of TAT polypeptides anti-tag antibody easy to use or the another kind of epitope tag with reference to described in be purified by affinity purification.A variety of tag polypeptides and its respective antibody are well known in the art.Example includes polyhistidine (poly-his) or many-HIS-GLY (poly-his-gly) label；Influenza HA tag polypeptide and its antibody 12CA5 (Field et al., Mol.Cell.Biol.8：2159-2165(1988))；C-myc labels and its antibody 8F9,3C7,6E10, G4, B7 and 9E10 antibody (Evan et al., Molecular and Cellular Biology 5：3610-3616(1985))；And herpes simplex virus glycoprotein D (gD) labels and its antibody (Paborsky et al., ProteinEngineering 3 (6)：547-553(1990)).Other tag polypeptides include Flag peptides (Hopp et al., BioTechnology 6：1204-1210(1988))；KT3 epitope peptides (Martin et al., Science 255：192-194(1992))；Alpha-tubulin epitope peptide (Skinner et al., J.Biol.Chem.266：15163-15166(1991))；And the protein peptide tag of T7 genes 10 (Lutz-Freyermuth et al., Proc.Natl.Acad.Sci.USA 87：6393-6397(1990)).

In an alternative embodiment, chimeric molecule may include anti-TAT antibody or TAT polypeptides and immunoglobulin or the fusions of immunoglobulin specific region.For the chimeric molecule (also referred to as " immunoadhesin ") of bivalent form, such fusions can be merged with IgG molecule Fc areas.Ig fusions preferably comprise the replacement of at least one variable region of anti-TAT antibody or TAT polypeptides displacement Ig intramoleculars with soluble form (membrane spaning domain is deleted or inactivated).In an especially preferred embodiment, immunoglobulin fusions include hinge area, the CH of IgG1 molecules₂Area and CH₃Area, or hinge area, CH₁Area, CH₂Area and CH₃Area.The United States Patent (USP) 5,428,130 that preparation on immunoglobulin fusions was authorized referring also to 27 days June nineteen ninety-five.

I. the preparation of anti-TAT antibody and TAT polypeptides

Following description relates generally to prepare anti-TAT antibody and TAT polypeptides by cultivating with the cell of the carrier conversion comprising the nucleic acid for encoding anti-TAT antibody and TAT polypeptides or transfection.It has been certainly contemplated as that anti-TAT antibody and TAT polypeptides can be prepared using alternative approach well known in the art.For example, solid phase technique can be used to pass through direct peptide symthesis to generate suitable amino acid sequence or part thereof (see, for example, Stewart et al., Solid-Phase Peptide Synthesis, W.H.Freeman Co., San Francisco, CA, 1969；Merrifield, J.Am.Chem.Soc.85：2149-2154(1963)).Protein synthesis in vitro can be used manual skill or be carried out by automating.Fully automated synthesis can be completed for example using Applied BiosystemsPeptide Synthesizer (Foster City, CA) according to the specification of manufacturer.The some of anti-TAT antibody or TAT polypeptides can separate chemical synthesis, and be combined using chemistry or enzymatic method to generate desired anti-TAT antibody and TAT polypeptides.

1. the DNA of the anti-TAT antibody of coding or TAT polypeptides separation

Encoding the DNA of anti-TAT antibody or TAT polypeptides can obtain from cDNA library, and the cDNA library is from thinking to express its tissue preparation with anti-the TAT antibody or TAT polypeptides mRNA and with detectable level.Therefore, the anti-TAT antibody or TAT polypeptid DNAs of people can be obtained easily from the cDNA library of people's tissue preparation.Anti- TAT antibody or TAT polypeptide coding genes can also be obtained from genomic library or by known synthesis flow (such as automatic nucleic acid synthesis).

It can use designed for identification target gene or library is screened by the probe (oligonucleotides of such as at least about 20-80 base) of its protein encoded.CDNA is screened with selected probe or normal process can be used to carry out for genomic library, such as Sambrook et al., Molecular Cloning：A LaboratoryManual, New York, Cold Spring Harbor Laboratory Press, described in 1989.A kind of alternative approach of the gene of the anti-TAT antibody of separation coding or TAT polypeptides is that (Sambrooket al., see above using PCR method；Dieffenbach et al., PCR Primer：A Laboratory Manual, ColdSpring Harbor Laboratory Press, 1995).

Technology for screening cDNA library is well known in the art.Be elected to be probe oligonucleotide sequence should long enough and enough clearly (unambiguous) so that false positive is preferably minimized.Oligonucleotides is preferably mark so that it can detect that when with DNA hybridization in screened library.Labeling method be it is known in the art that including the use of radio-labeled thing, as³²ATP, biotinylation or the enzyme mark of P- marks.Hybridization conditions, including medium stringency and High stringency, are shown in Sambrook et al, see above.

The sequence identified in such library screening methods can with preservation and available other known sequence is compared and contrasted in the privately owned sequence libraries of public database such as GenBank or other.That in molecule limited area or across full length sequence sequence identity (on amino acid levels or on nucleotide level) can be used that this area knows and method described herein is determined.

Nucleic acid with protein coding sequence can screen selected cDNA or genomic library by using deduction amino acid sequence first public herein to obtain, and if necessary, using Sambrook et al., see above the custom primer extension flow detect may not reverse transcription for cDNA mRNA precursor and processing intermediate product.

2. the selection and conversion of host cell

Host cell is transfected or converted with the expression described herein generated for anti-TAT antibody or TAT polypeptides or cloning vector, and is cultivated in for evoked promoter, the conventional nutrient culture for selecting transformant or the gene of amplification coding expectation sequence and appropriately adjusting.Condition of culture, such as culture medium, temperature, pH etc., can just be selected by those skilled in the art without excessively experiment.It is commonly used for making the maximized principle of cell culture biological productivity, scheme and practical technique reference can be made to Mammalian CellBiotechnology：A Practical Approach, M.Butler, ed., IRL Press, 1991 and Sambrook et al., see above.

The method that eukaryotic cell transfection and prokaryotic are converted is such as CaCl known to those of ordinary skill₂、CaPO₄, liposome-mediated and electroporation.According to host cell used, conversion is carried out using the standard technique suitable to such cell.Using the Calcium treatment of calcium chloride, such as Sambrook et al. see above described, or electroporation is generally used for prokaryotic.It is used for certain plants transformation, such as Shaw et al., Gene 23 with the infection of Agrobacterium tumdfaciens (Agrobacteriumtumefaciens)：Described in WO 89/05859 disclosed in 315 (1983) and 29 days June in 1989.For the mammalian cell of not such cell membrane, Graham and van der Eb, Virology 52 can be used：456-457 (1978) calcium phosphate precipitation.The ordinary circumstance of mammalian cell host system transfection is referring to United States Patent (USP) 4,399,216.Conversion into yeast is generally according to Van Solingen et al., J.Bact.130：946 (1977) and Hsiao et al., Proc.Natl.Acad.Sci. (USA) 76：The method of 3829 (1979) is carried out.It is also possible, however, to use other methods for DNA to be imported to cell, such as core microinjection, electroporation, bacterial protoplast are merged or polycation such as polybrene, poly ornithine with intact cell.See Keown et al., Methods inEnzymology 185 on the multiple technologies for transformed mammalian cell：527-537 (1990) and Mansour et al., Nature 336：348-352(1988).

Host cell suitable for cloning or expressing the DNA in this paper carriers includes prokaryotes, yeast or higher eucaryotic cells.Suitable prokaryotes include but is not limited to eubacteria, such as Gram-negative or gram-positive organism, such as such as enterobacteriaceae, Escherichia coli.A variety of coli strains are publicly available, such as e. coli k12 strain MM294 (ATCC 31,446)；Escherichia coli X1776 (ATCC 31,537)；Coli strain W3110 (ATCC 27,325) and K5 772 (ATCC53,635).Other suitable prokaryotes host cells include enterobacteriaceae, such as Escherichia (Escherichia) such as ETEC (E.coli), Enterobacter (Enterobacter), Erwinia (Erwinia), Klebsiella (Klebsiella), proteus (Proteus), Salmonella (Salmonella) such as salmonella typhimurium (Salmonella typhimurium), Serratia (Serratia) such as serratia marcescens (Serratia marcescans), with Shigella (Shigella), and bacillus (Bacilli) such as bacillus subtilis (B.subtilis) and bacillus licheniformis (the B.licheniformis) (DD 266 that on April 12nd, 1 publishes, bacillus licheniformis 41P disclosed in 710), pseudomonas (Pseudomonas) such as pseudomonas aeruginosa (P.aeruginosa), with streptomyces (Streptomyces).These examples are illustrative rather than restricted.Bacterial strain W3110 is particularly preferred a host or parent host, because it is the conventional host strain for recombinant DNA product fermentations.Preferably, host cell secretes the proteolytic enzyme of minimum.For example, bacterial strain W3110 can be modified, genetic mutation is produced in the gene for encoding protein endogenous for host, the example of such host includes Escherichia coli W3110 bacterial strain 1A2, and it has complete genotype tonA；Escherichia coli W3110 bacterial strain 9E4, it has complete genotype tonA ptr3；Escherichia coli W3110 bacterial strains 27C7 (ATCC 55,244), it has the degP ompT kanr of complete genotype tonA ptr3 phoA E15 (argF-lac) 169；Escherichia coli W3110 bacterial strain 37D6, it has the degP ompT rbs7 UvG kan of complete genotype tonA ptr3 phoA E15 (argF-lac) 169^r；Escherichia coli W3110 bacterial strain 40B4, it is the bacterial strain 37D6 that mutation is deleted with non-kalamycin resistance degP；And the coli strain of the mutant periplasmic protease disclosed in the United States Patent (USP) 4,946,783 authorized for 7th with nineteen ninety August.Or, body outer clone method, such as PCR or other nucleic acid polymerase reactions are also suitable.

Full length antibody, antibody fragment and antibody fusions protein can be prepared in bacterium, particularly when that need not glycosylate with Fc effector functions, such as when treatment is coupled with antibody and cytotoxic agent (such as toxin) and immune conjugate itself shows the effect of tumor cell destruction.Full length antibody has relatively long half-life in the circulating cycle.Generation in Escherichia coli is faster and more economically.For the expression of antibody fragment and polypeptide in bacterium, see, for example, US 5,648,237 (Carter et al.), US 5,789,199 (JoIy et al.), and US 5,840,523 (Simmons et al.), they describe the Translation initiator (TIR) and signal sequence for Optimal Expression and secretion, these patents are taken in herein as reference.After expression, from Bacillus coli cells paste separation antibody in soluble fraction, and for example it can be purified according to isotype by albumin A or G posts.Final purifying can be similar with the method for purifying the antibody for example expressed in Chinese hamster ovary celI progress.

In addition to prokaryotes, eukaryotic microorganisms, such as filamentous fungi or yeast are also the suitable clones or expressive host of the carrier of the anti-TAT antibody of coding or TAT polypeptides.Saccharomyces cerevisiae (Saccharomycescerevisiae) is conventional low eucaryon host microorganism.Others include grain wine pombe (Schizosaccharomyces pombe) (Beach and Nurse,Nature 290：140(1981)；EP139, on May 2nd, 383,1985 is open)；Kluyveromyces (Kluyveromyces) host's (United States Patent (USP) 4,943,529；Fleer et al.,Bio/Technology 9：968-975 (1991)) such as Kluyveromyces lactis (K.lactis) (MW98-8C, CBS683, CBS4574；Louvencourt et al.,J. Bacteriol.154(2)：737-742 (1983)), Kluyveromyces fragilis (K.fragilis) (ATCC12,424), Bulgaria kluyveromyces (K.bulgaricus) (ATCC 16,045), Brunswick kluyveromyces (K.wickeramii) (ATCC 24,178), K.waltii (ATCC 56,500), drosophila kluyveromyces (K.drosophilarum) (ATCC 36,906；Van den Berg et al.,Bio/Technology 8：135 (1990)), Kluyveromyces thermotolerans (K.thermotolerans) and Kluyveromyces marxianus (K.marxianus)；Yarrow saccharomyces (Yarrowia) (EP 402,226)；Pichia pastoris phaff (Pichiapastoris) (EP 183,070；Sreekrishna et al.,J.Basic Microbiol.28：265-278(1988))；Candida (Candida)；Filamentous fungi (Trichoderma reesia) (EP 244,234)；Neuraspora crassa (Neurospora crassa) (Case et al.,Proc.Natl.Acad.Sci.USA 76：5259-5263(1979))；Perhaps all so prosperous yeast (Schwanniomyces occidentalis) of prosperous saccharomyces (Schwanniomyces) (EP 394,538, October 31 nineteen ninety is open)；With filamentous fungi such as Neurospora (Neurospora), Penicillium (Penicillium), Tolypocladium (Tolypocladium) (WO 91/00357, on January 10th, 1991 is open) and aspergillus (Aspergillus) host such as aspergillus nidulans (A.nidulans) (Balance et al.Biochem.Biophys.Res. Commun.112：284-289(1983)；Tilburn et al.,Gene 26：205-221(1983)；Yeltonet al.,Proc.Natl.Acad.Sci.USA 81：1470-1474 (1984)) and aspergillus niger (A.niger) (Kelly and Hynes,EMBO J.4：475-479(1985)).Methylotrophic yeast (Methylotropicyeast) be suitable to the present invention, include but is not limited to can be grown on methanol, selected from the yeast of subordinate：Hansenula anomala category (Hansenula), candida (Candida), gram Le kirschner saccharomyces (Kloeckera), pichia category (Pichia), saccharomyces (Saccharomyces), Torulopsis (Torulopsis) and Rhodotorula (Rhodotorula).C.Anthony, The Biochemistry of Methylotrophs, 269 (1982) are can be found in as the specific species list of the example of this kind of yeast.

Multicellular organisms are derived from suitable for the host cell of the anti-TAT antibody of expression glycosylation or TAT polypeptides.The example of invertebral zooblast include insect cell such as drosophila S2 and noctuid Sf9, and plant cell such as cotton, corn, potato, soybean, petunia, tomato, tobacco cell culture..Many baculoviral strains and variant are identified and have allowed insect host cell accordingly, they are from hosts such as fall army worm Spodoptera frugiperda (caterpillar), Aedes aegypti Aedes aegypti (mosquito), aedes albopictus Aedes albopictus (mosquito), Drosophila melanogaster Drosophila melanogaster (drosophila) and silkworm Bombyx mori.The public, which can obtain a variety of Strain, to be used to transfect, the Bm-5 strains of such as autographa california Autographa californica NPV L-1 variants and BmSNPV, and this viroid can be used as this paper virus according to the present invention, particularly for transfecting Spodopterafrugiperda cells.

However, what people were most interested in is vertebrate cells, and the breeding of vertebrate cells has become old process in culture (tissue cultures).The example of useful mammalian host cell line is the monkey kidney CV1 systems (COS-7, ATCC CRL 1651) converted with SV40；Human embryonic kidney cell line (293 or in order to suspend culture in growth and be subcloned 293 cells, Graham et al., 1977, J.Gen Virol.36：59)；Baby hamster kidney cell (BHK, ATCC CCL 10)；Chinese hamster ovary cell/- DHFR (CHO, Urlaub et al., 1980, Proc.Natl.Acad.Sci.USA 77：4216)；Mouse Sai Tuoli (sertoli) cell (TM4, Mather, 1980, Biol.Reprod.23：243-251)；MK cells (CV1, ATCCCCL 70)；African green monkey kidney cell (VERO-76, ATCC CRL-1587)；Human cervical carcinoma cell (HELA, ATCC CCL 2)；MDCK (MDCK, ATCC CCL 34)；Ox mouse (buffalorat) liver cell (BRL 3A, ATCC CRL 1442)；Human pneumonocyte (W138, ATCC CCL 75)；Human liver cell (Hep G2, HB 8065)；Mouse mammary tumor (MMT 060562, ATCC CCL 51)；TRI cells (Mather et al., 1982, Annals N.Y.Acad.Sci.383：44-68)；MRC5 cells；FS4 cells；With people's hepatoma system (Hep G2).

Host cell is converted with the above-mentioned expression generated for anti-TAT antibody or TAT polypeptides or cloning vector, and is cultivated in for evoked promoter, the conventional nutrient culture for selecting transformant or the gene of amplification coding expectation sequence and appropriately adjusting.

3. the selection of replicating vector and use

Nucleic acid (such as cDNA or genomic DNA) the insertion replicating vector for encoding anti-TAT antibody or TAT polypeptides can be used to clone (DNA cloning) or expression.Variety carrier is publicly available.Carrier can be such as plasmid, clay, virion or the form of bacteriophage.Can be by a variety of methods by suitable nucleotide sequence insertion vector.Generally, DNA is inserted to suitable restriction endonuclease sites using techniques known in the art.Support element typically includes, but not limited to following one or more：Signal sequence, replication orgin, one or more marker gene, enhancer element, promoter and transcription terminator.The structure of suitable carrier comprising these one or more components uses standard ligation techniques known to technical staff.

TAT not only can directly recombinant production, and can be as the fused polypeptide with heterologous polypeptide, the heterologous polypeptide can be the signal sequence or other polypeptides for having specific cleavage site in the N- ends of mature protein or polypeptide.Generally, signal sequence can be the component of carrier, or it can be the DNA for encoding anti-TAT antibody or TAT polypeptides of an insertion vector part.Signal sequence can be prokaryotic signal sequence, selected from such as alkaline phosphatase, penicillase, lpp or heat-staple enterotoxin II leaders.For yeast secretary, signal sequence can be that for example yeast invertase leader, α factor leaders (include α-factor leaders of sugar yeast and kluyveromyces, the latter sees United States Patent (USP) 5,010, or the signal described in acid phosphatase leader, Candida albicans glucoamylase targeting sequencing (EP 362,179 disclosed in April 4 nineteen ninety) or WO 90/13646 disclosed in 15 days November nineteen ninety 182).In mammalian cell expression, mammalian signal sequences can be used to instruct the secretion of protein, such as signal sequence from identical or relative species secreted polypeptides, and viral secretory leaders.

Expression and cloning vector are all comprising the nucleotide sequence that carrier can be made to be replicated in the host cell of one or more selection.It is known that such sequence of various bacteria, yeast and virus.Replication orgin from pBR322 plasmid is suitable for most of gramnegative bacteriums, 2 μ plasmid origins are suitable for yeast, and various viral origins (SV40, polyomavirus, adenovirus, VSV or BPV) are available for the cloning vector in mammalian cell.

Expression and cloning vector will generally include Select gene, also referred to as selection marker.Typical Select gene encodes following protein：(a) antibiotic or other toxin resistances, such as ampicillin, neomycin, methotrexate (MTX) or tetracycline are assigned；(b) auxotrophy is supplied；Or (c) provides the critical nutrients that can not be obtained by complex medium, such as encodes the gene of bacillus D-alanine racemase.

An example suitable for the selection marker of mammalian cell is those selection markers of the cell for the nucleic acid that can identify the have the ability anti-TAT antibody of intake coding or TAT polypeptides, such as DHFR or thymidine kinase.When using wild type DHFR, suitable host cell is the Chinese hamster ovary celI system of DHFR active defects, and it is prepared and breeding such as Urlaub et al.,Proc.Natl.Acad.Sci.USA 77：4216 (1980) are described.It is the trpl genes (the Stinchcomb et al., 1979, Nature 282 that are present in yeast plasmid YRp7 suitable for the Select gene of yeast：39；Kingsman et al., 1979,Gene 7：141；Tschemper et al., 1980,Gene 10：157).Trpl bases are in default of the yeast mutant of the growth ability in tryptophan, and such as ATCC No.44076 or PEP4-1 are there is provided selection marker (Jones, 1977, Genetics 85：12).

Expression and cloning vector generally comprise the promoter that is operatively connected with the nucleotide sequence of the anti-TAT antibody of coding or TAT polypeptides to instruct mRNA to synthesize.The promoter recognized by a variety of potential host cells is well-known.Suitable for prokaryotic hosts promoter include beta-lactamase and lactose promoter system (Chang et al.,Nature 275：615(1978)；Goeddel et al.,Nature 281：544 (1979)), alkaline phosphatase, tryptophan (trp) promoter systems (Goeddel,Nucleic acids Res.8：4057(1980)；EP 36,776) and hybrid promoter such as tac promoters (deBoer et al.,Proc.Natl. Acad.Sci.USA 80：21-25(1983)).Promoter for bacterial system is also by comprising with encoding Shine-Dalgarno (S.D.) sequence that the DNA of anti-TAT antibody or TAT polypeptides is operatively connected.

Suitable for yeast host promoter sequence example include glycerol 3-phosphate acid kinase (Hitzemanet al.,J.Biol.Chem.255：2073 (1980)) or other glycolytic ferments (Hess et al.,J.Adv. Enzyme Reg.7：149(1968)；Holland,Biochemistry 17：4900 (1978)) promoter, such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, GPI, 3-phoshoglyceric acid mutase, pyruvate kinase, phosphotriose isomerase, glucose phosphate isomerase and glucokinase.

It is the promoter region of the enzyme of alcohol dehydrogenase 2, different cell pigment C, acid phosphatase, the digestive enzyme relevant with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase and responsible maltose and galactose utilization as other Yeast promoters of the inducible promoter with the additional advantage that transcription is controlled by growth conditions.EP 73,657 is further described in suitable for the carrier and promoter of Yeast expression.

Anti- TAT antibody or TAT polypeptides are transcribed by for example by viral such as polyomavirus by carrier in mammalian host cell, fowlpox virus (UK 2 disclosed in 5 days July in 1989, 211, 504), adenovirus (such as adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B and simian virus 40 (SV40) genome, by heterologous mammal promoter such as actin promoter or immunoglobulin promoter, and the control of the promoter obtained by heat-shock promoters, if if such promoter is compatible with host cell systems.

Transcription of the higher eucaryotic cells to the anti-TAT antibody of coding or the DNA of TAT polypeptides can be improved by inserting enhancer sequence in the carrier.Enhancer is DNA cis-acting elements, and generally about 10 to 300bp, acts on promoter to increase transcription.Known many enhancer sequences from mammalian genes (globulin, elastoser, albumin, α-fetoprotein and insulin).However, usually using the enhancer from eukaryotic cell virus.Example includes enhancer (bp100-270), the sub- enhancer of cytomegalovirus early promoter, the enhancer and adenovirus cancers of polyomavirus replication orgin late period side of SV40 replication orgins late period side.Enhancer can be with montage into carrier, positioned at 5 ' or 3 ' positions of anti-TAT antibody or TAT polypeptid coding sequences, it is preferred that positioned at 5 ' sites of promoter.

Expression vector for eukaryotic host cell (yeast, fungi, insect, plant, animal, people or karyocyte from other multicellular organisms) will also be comprising terminating transcription and sequence necessary to stable mRNA.Such sequence can generally be obtained by 5 ' ends of eucaryon or viral DNA or cDNA non-translational regions with 3 ' ends once in a while.These regions are included in the nucleotide segment that polyadenylated fragments are transcribed into the untranslated part for the mRNA for encoding anti-TAT antibody or TAT polypeptides.

It is adapted to synthesize anti-TAT antibody in recombinant vertebrate cell culture after change or other methods, carrier and the host cell of TAT polypeptides is shown in Gething et al., Nature 293：620-625(1981)；Mantei et al., Nature 281：40-46(1979)；EP 117,060；With EP 117,058.

4. cultivate host cell

The host cell for generating anti-TAT antibody of the invention or TAT polypeptides can be cultivated in a variety of culture mediums.Commercially available culture medium such as HamShi F10 (Sigma), minimum essential medium (MEM, Sigma), the EagleShi culture mediums (DMEM, Sigma) of RPMI-1640 (Sigma) and DulbeccoShi improvement are suitable to culture host cell.Further, it is possible to use the culture medium of any culture medium described in following documents as host cell：Ham et al., 1979, Meth.Enz.58：44；Barnes et al., 1980, Anal.Biochem.102：255；United States Patent (USP) 4,767,704；4,657,866；4,927,762；4,560,655；Or 5,122,469；WO 90/03430；WO 87/00195；Or United States Patent (USP) review 30,985.These any culture mediums can hormone supplemented and/or other growth factors (such as insulin, transferrin or EGF), salt (such as sodium chloride, calcium, magnesium and phosphate), buffer (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotic (such as GENTAMYCIN as needed^TMMedicine), trace element (being defined as the inorganic compound generally existed with the final concentration of micro-molar range) and glucose or the equivalent energy.Can also with debita spissitudo include those skilled in the art will know that any other required supplement.Condition of culture temperature, pH etc. had previously been selected for host cell for expression, and this is obvious for those of ordinary skill.

5.Detect gene magnification/expression

The amplification and/or expression of gene can direct measurements in the sample, for example according to provided herein is sequence, using suitable label probe, by conventional Southern traces, quantitative Northern traces (Thomas, Proc.Natl.Acad.Sci.USA 77 is transcribed to mRNA：5201-5205 (1980)), point trace (DNA analysis) or in situ hybridization.Or, can be using the antibody of specific duplex can be recognized, the duplex includes DNA duplex, RNA duplexs and DNA-RNA hybrid duplexes or DNA- protein duplexes.Then can labelled antibody, and can be measured method, wherein duplex is attached on surface so that when duplex is formed on the surface, the presence of the detectable antibody combined with duplex.

Or, in order to which the expression directly to gene outcome is quantified, the determination method of gene expression, such as immunohistochemical staining of cell or tissue section and cell culture or body fluid can be measured by immunological method.It can be monoclonal or polyclonal available for immunohistochemical staining and/or the antibody of sample liquids determination method, and can be prepared in any mammal.It is expedient to, can for native sequences TAT polypeptides or for based on provided herein is DNA sequence dna synthetic peptide or for merging with TAT DNA and the exogenous array of encoding particular antibodies epitope prepares antibody.

6.Purify anti-TAT antibody and TAT polypeptides

Various forms of anti-TAT antibody and TAT polypeptides can be reclaimed from nutrient solution or from host cell lysats.If film combination, then suitable detergent solution (such as Triton-X 100) can be used or it is discharged from film by enzymatic lysis.Cell employed in anti-TAT antibody and TAT expression of polypeptides can be ruptured by a variety of physically or chemically means, such as Frozen-thawed cycled, ultrasonically treated, mechanical disruption or cell lytic agent.

It may be desirable to from recombinant cell protein matter or the anti-TAT antibody of peptide purification and TAT polypeptides.Following flow is the illustration of appropriate purification flow：In ion exchange column fractionation；Ethanol precipitation；Reversed-phase HPLC；Chromatography on tripoli or cationic ion-exchange resin such as DEAE；Chromatofocusing；SDS-PAGE；Ammonium sulfate precipitation；Use such as Sephadex G-75 gel filtration；Albumin A Sepharose posts are to remove pollutant such as IgG；And combine the metal chelating column of the epitope tag of anti-TAT antibody and TAT polypeptides.Multiple proteins purification process can be used, such method is known in the art, and is described in such as Deutscher,Methods in Enzymology, 182 (1990)；Scopes,Protein Purification：Principes and Practice, Springer-Verlag, New York (1982).The selection of purification step is by depending on the property of generating process for example used and produced specific anti-TAT antibody or TAT polypeptides.

When using recombinant technique, can in the cell, antibody is generated in periplasmic space, or be directly secreted into culture medium.If generating antibody in the cell, then as the first step, the particle debris of host cell or crack fragment is removed for example, by centrifugation or ultrafiltration.Carter et al., Bio/Technology 10：163-167,1992 describe the flow of the antibody for being secreted into colibacillus periplasm space.Briefly, cell paste is made to melt when there is sodium acetate (pH 3.5), EDTA and phenylmethylsulfonyl fluoride (PMSF) about 30 minutes.Cell fragment can be removed by centrifugation.If by antibody-secreting into culture medium, then generally first by commercialization protein concentration filter, such as supernatant of the Amicon or Millipore Pellicon ultra filtration units concentration from such expression system.In any above-mentioned steps, protease inhibitors such as PMSF can be included to suppress proteolysis, and antibiotic can be included to prevent the growth of external contaminant.

Such as hydroxyapatite, gel electrophoresis, dialysis and affinity chromatography can be used to purify the antibody compositions prepared by cell, purification technique preferably is affinity chromatography.Albumin A depends on the species and isotype of any immunoglobulin fc region present in antibody as the suitability of affinity ligand.Albumin A can be used for antibody (Lindmark et al., 1983, J.Immunol.Meth.62 of the purifying based on people γ 1, γ 2 or the heavy chains of γ 4：1-13).Protein G recommends to be used for all mouse isotypes and people γ 3 that (Guss et al., 1986, EMBO J.5：1567-1575).Matrix accompanying by affinity ligand is most commonly used that agarose, but can use other matrix.The matrix of physically stable such as controlled pore glass or poly- (styrene divinyl) benzene result in flow velocity more faster than agarose and shorter process time.For including C_HFor the antibody of 3 domains, Bakerbond ABX can be used^TMResin (J.T.Baker, Phillipsburg, NJ) is purified.According to antibody to be recycled, it is possible to use classification, ethanol precipitation, reversed-phase HPLC on other oroteins purification technique, such as ion exchange column, the chromatography on tripoli, heparin SEPHAROSE^TMOn chromatography, anion or cationic ion-exchange resin (such as poly-aspartate post) on chromatography, chromatofocusing, SDS-PAGE and ammonium sulfate precipitation.

After any preliminary purification step, the mixture comprising purpose antibody and pollutant can use pH about 2.5-4.5 elution buffer, preferably carry out low pH hydrophobic interaction chromatographies in low salt concn (e.g., from about 0-0.25M salt).

J.Medicinal proportional preparation

It can be prepared as follows the treatment preparaton of the anti-TAT antibody, TAT combinations oligopeptides, TAT combinations organic molecule and/or the TAT polypeptides that are used according to the present invention, i.e. in the form of freeze-dried formulation or the aqueous solution, (Remington ' s Pharmaceutical Sciences will be mixed with optional pharmaceutically acceptable carrier, excipient or stabilizer with antibody, polypeptide, oligopeptides or the organic molecule of expecting purity, 16th edition, Osol, A.Ed., 1980).Acceptable carrier, excipient or stabilizer are nontoxic, including buffer, such as acetate, Tris, phosphate, citrate and other organic acids to recipient in the dosage and concentration used；Antioxidant, including ascorbic acid and methionine；Preservative (such as octadecyl dimethyl benzyl ammonium chloride；Hexamethonium chloride；Benzalkonium chloride, benzethonium chloride；Phenol, butanol or phenmethylol；Alkyl paraben, such as methyl p-hydroxybenzoate or propyl ester；Catechol；Resorcinol；Cyclohexanol；3- amylalcohols；And metacresol)；Low molecule amount (less than about 10 residues) polypeptide；Protein, such as serum albumin, gelatin or immunoglobulin；Hydrophilic polymer, such as polyvinylpyrrolidone；Amino acid, such as glycine, glutamine, asparagine, histidine, arginine or lysine；Monose, disaccharides and other sugar, including glucose, mannose or dextrin；Chelating agent, such as EDTA；Tension regulator (tonicifier), such as trehalose and sodium chloride；Carbohydrate, such as sucrose, mannitol, trehalose or sorbierite；Surfactant, such as polysorbate；Into salt counter ion, such as sodium；Metal composite (such as Zn- protein complexes)；And/or nonionic surfactant, such as TWEEN^TM、PLURONICS^TMOr polyethylene glycol (PEG).Antibody preferably comprises concentration for 5-200mg/ml, preferably 10-100mg/ml antibody.

Preparaton herein can also contain have more than it is a kind of treat reactive compound necessary to specific indication, preferably complementary activities and not adversely affect each other.For example, in addition to anti-TAT antibody, TAT combinations oligopeptides or TAT combination organic molecules, may be it is also expected to containing another antibody in a kind of preparaton, for example with reference to the second anti-TAT antibody of different epitopes on TAT polypeptides, or for some other targets such as influence particular cancer grow growth factor antibody.Or composition can also include chemotherapeutics, cytotoxic agent, cell factor, growth inhibitor, antihormone agent and/or heart protective agent.Suitably, this quasi-molecule exists to be combined for the effective amount of predetermined purpose.

Active component can also contain in the microcapsules prepared for example by condensation technique or by interfacial polymerization (being for example hydroxymethyl cellulose or gelatin-microcapsule and poly- (methyl methacrylate) microcapsules respectively), in colloidal drug delivery system (such as liposome, albumin microspheres, microemulsion, nano particle and Nano capsule) or in macro emulsion.Such technology is disclosed in such as Remington ' sPharmaceutical Sciences, 16th edition, Osol, A.Ed., 1980.

Extended release preparation can be prepared.The suitable example of extended release preparation includes the solid hydrophobic polymers semipermeable matrices containing antibody, and the matrix is the form of approved product, such as film or microcapsules.The example of sustained-release matrix includes polyester, hydrogel (such as poly- (2- ethoxys-methacrylate) or poly- (vinyl alcohol)), polyactide (United States Patent (USP) 3,773,919), the copolymer of Pidolidone and Pidolidone γ-ethyl ester, nondegradable ethane-acetic acid ethyenyl, degradable lactic acid-ethanol copolymer such as LUPRONDEPOT^TM(the Injectable microspheres body being made up of lactic acid-ethanol copolymer and leuprorelin acetate) and poly- D- (-) -3-hydroxybutyrate.

Preparaton for applying in vivo must be sterile.This readily can be realized by using sterilised membrane filter filtering.

K.Use the diagnosis and treatment of anti-TAT antibody, TAT combinations oligopeptides and TAT combination organic molecules

In order to determine the expression of the TAT in cancer, using a variety of diagnostic assay methods.In one embodiment, TAT polypeptides overexpression can be analyzed by SABC (IHC).IHC determination methods can be carried out to the paraffin-embedded tissue section from tumor biopsy, and give following TAT protein matter staining intensity criteria：Score 0：Not it was observed that dyeing or film dyeing being observed in the tumour cell less than 10%.

Score 1+：Faint/just perceptible film dyeing is detected in the tumour cell more than 10%.The cell only has dyeing on its part film.

Score 2+：Faint to medium complete film dyeing is observed in the tumour cell more than 10%.

Score 3+：Medium to strong complete film dyeing is observed in the tumour cell more than 10%.

Those TAT expression of polypeptides scores 0 or 1+ tumour can be accredited as and not be overexpressed TAT, and those scores 2+ or 3+ tumour can be accredited as overexpression TAT.

Or/in addition, formalin can be fixed, the tumor tissues of FFPE carry out FISH determination methods such as INFORM  (by Ventana, Arizona is sold) or PATHVISION  (Vysis, Illinois) be overexpressed degree (if any) to determine TAT in tumour.

In-vivo diagnostic determination method can be used and is overexpressed or expands to assess TAT, for example by the way that molecules detected can be combined using one kind, and the molecule (such as antibody, oligopeptides or organic molecule) of detectable (such as radio isotope or fluorescent marker) is marked with, then patient is carried out external scan to position the label.

As described above, anti-TAT antibody, oligopeptides and organic molecule of the invention have a variety of non-therapeutic applications.Anti- TAT antibody, oligopeptides and the organic molecule of the present invention can be used for the diagnosis of the cancer of expression TAT polypeptides and by stages (such as in radiophotography).Antibody, oligopeptides and organic molecule can be additionally used in purifying or immunoprecipitation TAT polypeptides from cell, for the vitro detection of TAT polypeptides and quantitatively for example in ELISA or western blot, and the cell for expressing TAT is killed and eliminated from mixed cellularity group as a step for purifying other cells.

Now, according to the stage of cancer, the treatment of cancer is related to one kind or combination in following therapy：Operation removes cancerous tissue, radiation and chemotherapy.Anti- TAT antibody, oligopeptides or organic molecule therapy for can not bear very well the Side effect of chemotherapy gerontal patient and the little metastatic disease of radiotherapy use be probably especially valuable.Anti- TAT antibody, oligopeptides or the organic molecule of the target tumor of the present invention can be used for the cancer for mitigating expression TAT in the initial diagnosis of disease or during recurring.For therapeutical uses, can be used alone anti-TAT antibody, oligopeptides or organic molecule, either treated by it with the compound of such as hormone, antiangiogenic agent (antiangiogen) or radio-labeled or with operation, cold therapy and/or chemotherapy combined radiotherapy.Anti- TAT antibody, the treatment of oligopeptides or organic molecule can be administered in combination with the routine treatment of other forms, and routine treatment continuous administration, or be applied before or after routine treatment.Chemotherapeutics such as TAXOTERE  Taxoteres (doxetaxel), TAXOL  taxols (paclitaxel), Estramustine (estramustine) and mitoxantrone (mitoxantrone) are used for the patient for the treatment of cancer, particularly devoid of risk (goodrisk).In the method for present invention treatment or mitigation cancer, the treatment that can combine one or more foregoing chemotherapeutic agents applies anti-TAT antibody, oligopeptides or organic molecule to cancer patient.Specifically, it is contemplated to taxol and the conjoint therapy of improvement derivative (see, for example, EP 0 600 517).Anti- TAT antibody, oligopeptides or organic molecule are applied together with the chemotherapeutics for the treatment of effective dose.In another embodiment, combined chemotherapy applies anti-TAT antibody, oligopeptides or organic molecule to improve the activity and effect of chemotherapeutics such as taxol.Physicians ' Desk Reference (PDR) disclose the dosage of these medicaments used in the treatment of kinds cancer.Effective dosage regimen and dosage will can be determined depending on other factorses known to the internist of the specific cancer, the degree of disease and capable field technique treated and by internist these foregoing chemotherapeutic medicines in the treatment.

In a specific embodiment, the conjugate comprising anti-TAT antibody, oligopeptides or organic molecule with cytotoxic agent couplings is applied to patient.Preferably, the immune conjugate combined with TAT protein matter causes the therapeutic efficiency that immune conjugate kills its cancer cell combined to improve by cell internalization.In a preferred embodiment, cytotoxic agent targets or disturbed the nucleic acid in cancer cell.The example of such cytotoxic agent has been described above, including maytansinoids, Calicheamicin, ribalgilase and DNA endonucleases.

Anti- TAT antibody, oligopeptides, organic molecule or its toxin conjugated thing are administered to human patientses according to known method, it is such as intravenous to apply, for example as inject or a period of time continuous infusion, by intramuscular, intraperitoneal, myelencephalon, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, part or suction path.It is preferred that intravenously or subcutaneously administration of antibodies, oligopeptides or organic molecule.

Anti- TAT antibody, oligopeptides or organic molecule can be administered in combination with other therapeutic schemes.Be administered in combination co-application including the use of separated multiple preparatons or single medicinal proportional preparation, and random order continuous administration, wherein it is preferred that all two kinds of (or a variety of) activating agents play its biological activity simultaneously for some time.Preferably, such conjoint therapy causes synergistic therapeutic effect.

May be it is also expected to the administration of one or more anti-TAT antibody, oligopeptides or organic molecule be joined together with the administration of the antibody for another tumour antigen relevant with particular cancers.

In another embodiment, the therapeutic treatment method of the present invention is related to, and (one or more) anti-TAT antibody, oligopeptides or organic molecule are administered in combination with one or more chemotherapeutics or growth inhibitor, including are co-administered from the mixture (cocktail) of different chemotherapeutics.Chemotherapeutics includes EMP (estramustine phosphate), prednimustine (prednimustine), cis-platinum, 5 FU 5 fluorouracil, melphalan (melphalan), endoxan, hydroxycarbamide and hydroxycarbamide taxane (hydroxyureataxane) (such as taxol and Taxotere (doxetaxel)) and/or anthracycline (anthracycline) antibiotic.The preparation and dosage regimen of such chemotherapeutics can be used or empirically determined by skilled practitioner according to the specification of manufacturer.The preparation of this based chemotherapy and dosage regimen also see ChemotherapyService Ed., M.C.Perry, Williams Wilkins, Baltimore, MD (1992).

Antibody, oligopeptides or organic molecule can be combined with anti-hormonal compound with the known dose of this quasi-molecule, for example antiestrogenic compound, such as TAM (tamoxifen)；Antiprogestin compound, such as Onapristone (onapristone) (see EP 616,812)；Or Anti-androgenic compounds, such as Drogenil (flutamide).When cancer to be treated is androgen independence cancer, patient may previously receive anti androgenic therapy, and when cancer becomes androgen independence, anti-TAT antibody, oligopeptides or organic molecule (and optional other medicaments described herein) can be applied to patient.

Sometimes, return patient and co-administer heart protective agent (to prevent or reduce the myocardial dysfunction relevant with treatment), or one or more cell factors, may be also beneficial.Except above-mentioned therapeutic scheme, before the treatment of antibody, oligopeptides or organic molecule, simultaneously or afterwards, operation can be carried out to patient and remove cancer cell and/or radiotherapy.Any of above suitable dose for co-administering medicament is exactly those used dosage at present, and can be reduced due to the synergy (synergy) of the medicament and anti-TAT antibody, oligopeptides or organic molecule.

In order to prevent or treat disease, applied dose and pattern can be selected by internist according to known standard.The optimal dose of antibody, oligopeptides or organic molecule by depending on the as defined above type of disease to be treated, the order of severity of disease and process, be to prevent or therapeutic purposes administration of antibodies, oligopeptides or organic molecule, previous therapy, the clinical history of patient and response and the judgement of attending doctor to antibody, oligopeptides or organic molecule.Appropriate disposably or in a series of treatments is administered to patient by antibody, oligopeptides or organic molecule.Preferably, applied by antibody, oligopeptides or organic molecule by intravenous infusion or by being subcutaneously injected.According to the type and the order of severity of disease, about 1 μ g/kg can be administered to patient to the antibody of about 50mg/kg body weight (e.g., from about 0.1-15mg/kg/ agent) as initial candidate dosage, either for example by one or many separated administrations, or pass through continuous infusion.Dosage regimen may include the initial loading dose using about 4mg/kg, the maintenance dose weekly of the rear anti-TAT antibody of renewed treaty 2mg/kg.It is also possible, however, to use other dosages.According to above-mentioned factor, typical daily dosage can be in the range of about 1 μ g/kg to 100mg/kg or more.For take several days or the longer time repetitive administration, according to situation, maintaining treatment until occur the expectation to disease symptomses containment.The progress of this therapy can readily by conventional method and determination method, and based on internist or it is other those skilled in the art will know that standard monitor.

Except antibody protein is administered into patient, the application is contemplated by gene therapy come administration of antibodies.Such apply of the nucleic acid of encoding antibody is covered in statement " antibody for applying therapeutically effective amount ".On producing the purposes of intracellular antibody using gene therapy see, for example, WO96/07321 disclosed in 14 days March in 1996.

There are the cell that two kinds of main methods make nucleic acid (being optionally included in carrier) enter patient, i.e. internal and ex vivo (ex vivo).For delivery in vivo, generally the position of antibody is being needed to be injected directly into nucleic acid in patient's body.For ex vivo therapy, the cell of patient is gathered, nucleic acid is imported to the cell of these separation, and by the cell by modification or patient is directly applied to, or for example load the interior simultaneously implantation within a patient of perforated membrane (see, for example, United States Patent (USP) 4,892,538 and 5,283,187).There are multiple technologies to can be used for nucleic acid importing living cells.These technologies are according to being that nucleic acid is transferred into the cultured cell in vitro or internal cell of expected host and is varied from.Suitable for nucleic acid is transferred into technology in mammalian cell including the use of liposome, electroporation, microinjection, cell fusion, DEAE- dextrans, calcium phosphate precipitation etc. in vitro.The carrier for being usually used in ex vivo delivery gene is retrovirus.

Currently preferred nucleic acid in vivo transfer techniques include the transfection carried out with viral vector (such as adenovirus, I herpes simplex virus types or adeno-associated virus) and the system based on lipid (lipid for the gene transfer that can be used for lipid to mediate has such as DOTMA, DOPE and DC-Chol).On the genetic marker and the summary of gene therapy approach that are currently known referring to Anderson et al., Science 256：808-813(1992).Referring also to WO 93/25673 and its bibliography quoted.

The anti-TAT antibody of the present invention can be that " antibody " herein defines covered multi-form.Therefore, antibody includes total length or complete antibody, antibody fragment, native sequences antibody or amino acid variant, humanization, chimeric or fusion antibody, immune conjugate, and its functional fragment.In fusion antibody, antibody sequence is merged with allogeneic polypeptide sequence.Can modified antibodies Fc areas to provide desired effector function.Part such as this paper is discussed more fully, by suitable Fc areas, cytotoxicity is can induce with reference to the exposed antibody on cell surface, for example via antibody dependent cellular toxic action (ADCC), or by raising complement, or some other mechanism in being acted in complement dependent cytotoxicity.Or, when expecting to eliminate or reduction effector function so that reduce side effect or complication as far as possible, some other Fc areas can be used.

In one embodiment, antibody and antibody competition of the present invention to the combination of same epitope or substantially with antibody binding identical epitope of the present invention.The antibody of the biological property with anti-TAT antibody of the invention is also contemplated, in-vivo tumour targeting is specifically included and any cell proliferation inhibiting or cytotoxicity feature.

The method for generating above-mentioned antibody is described in detail herein.

The anti-TAT antibody of the present invention, oligopeptides and organic molecule can be used for one or more symptoms for the treatment of expression TAT cancer or the mitigation cancer in mammal.Such cancer includes prostate cancer, carcinoma of urethra, lung cancer, breast cancer, colon cancer and oophoroma, more specifically there is adenocarcinoma of the prostate, clear-cell carcinoma, colorectal adenocarcinoma, the gland cancer of lung, the squamous cell carcinoma and mesothelioma of pleura of lung.Cancer covers the metastatic carcinoma of any aforementioned cancer.Antibody, oligopeptides or organic molecule can combine at least a portion of the cancer cell of expression TAT polypeptides in mammal.In a preferred embodiment, antibody, oligopeptides or organic molecule in vitro or in vivo TAT polypeptides on cell is combined when effectively destruction or kill expression TAT tumour cell or suppress the growth of such tumour cell.This antibody-like includes exposed anti-TAT antibody (not being coupled with any medicament).Exposed antibody with cytotoxicity or cell growth inhibiting property can be combined further (hamess) with cytotoxic agent so that they more effectively destroy tumour cell.Can be for example, by antibody and cytotoxic agent couplings be formed into immune conjugate as described herein, so as to assign anti-TAT antibody with cytotoxic properties.Cytotoxic agent or growth inhibitor are preferably small molecule.It is preferred that toxin such as Calicheamicin or maytansinoids and the like or derivative.

The invention provides a kind of composition, it includes anti-TAT antibody of the invention, oligopeptides or organic molecule, and carrier.For the purpose for the treatment of cancer, can be applied to composition needs the patient of such treatment, and wherein composition can be rendered as immune conjugate or the anti-TAT antibody of exposed antibody comprising one or more.In another embodiment, composition can include these antibody, oligopeptides or organic molecule and combine other therapeutic agents such as cytotoxic agent or growth inhibitor, including chemotherapeutics.Present invention also offers the preparaton for including anti-TAT antibody of the invention, oligopeptides or organic molecule and carrier.In one embodiment, preparaton is the treatment preparaton for including pharmaceutical acceptable carrier.

Another aspect of the present invention is the seperated nuclear acid for encoding anti-TAT antibody.Contemplate the nucleic acid of both encoding heavy chain and light chain, especially some hypervariable region residues, the chain of the variant of corresponding native sequence antibody and the antibody, modification and humanization pattern.

Present invention also offers the cancer available for expression TAT polypeptides in treatment mammal or the method for the one or more symptoms for mitigating the cancer, including give anti-TAT antibody, oligopeptides or organic molecule of the mammal using therapeutically effective amount.Antibody, oligopeptides or organic molecule therapeutic composition can be instructed by internist, and short-term or long-term or interruption is applied.Additionally provide the cell growth for suppressing expression TAT polypeptides and kill its method.

Present invention also offers the kit and product for including at least one anti-TAT antibody, oligopeptides or organic molecule.Kit comprising anti-TAT antibody, oligopeptides or organic molecule can be used for the killing determination method of such as expression TAT cell, purifying or immunoprecipitation TAT polypeptides from cell.For example, in order to separate and purify TAT, kit can include anti-TAT antibody, oligopeptides or the organic molecule being coupled with pearl (such as sepharose pearls).The kit for including antibody, oligopeptides or organic molecule can be provided, for TAT vitro detection and quantitative, such as in ELISA or western blot.Such antibody, oligopeptides or organic molecule that can be used for detection can have label, such as fluorescence or radio-labeled thing.

L.Product and kit

Another aspect of the present invention is the product of the material comprising the cancer that can be used for treatment expression TAT.Product includes the label or package insert accompanied on container and the container or with it.Suitable container is included such as bottle, tubule, syringe.Container can be made from a variety of materials, such as glass or plastics.The composition of effective treating cancer illness is housed in container, and there can be sterile access port (such as container can be the intravenous solution bag or tubule for the plug that can pierce with hypodermic needle).At least one of composition activating agent is anti-TAT antibody, oligopeptides or the organic molecule of the present invention.Label or package insert indicate that said composition is used for treating cancer.Label or package insert are further comprising the specification to cancer patient's administration of antibodies, oligopeptides or organic molecule composition.In addition, product may also include second container, wherein equipped with pharmaceutically acceptable buffer, such as injection bacteriostatic water (BWFI), phosphate buffered saline (PBS), woods grignard (Ringer) solution and dextrose solution.It may also include the other materials needed in business and user's position, including other buffers, diluent, filter, syringe needle and syringe.

The kit available for a variety of purposes is additionally provided, such as the killing determination method for the cell for expressing TAT, for purifying or immunoprecipitation TAT polypeptides from cell.In order to separate and purify TAT, kit can include anti-TAT antibody, oligopeptides or the organic molecule being coupled with pearl (such as sepharose pearls).The kit for including antibody, oligopeptides or organic molecule can be provided, for the vitro detection of TAT polypeptides and quantitative, such as in ELISA or western blot.Identical with product, kit includes on container and the container or relative label or package insert.Equipped with the composition for including at least one anti-TAT antibody of the invention, oligopeptides or organic molecule in container.It may include other container, wherein equipped with such as diluent and buffer, control antibodies.Label or package insert can provide the description to composition and the specification for expected external or diagnostic uses.

M.The purposes of the nucleic acid of TAT polypeptides and coding TAT polypeptides

The nucleotide sequence (or its complementary series) of coding TAT polypeptides has a variety of applications in biology field, including as hybridization probe, for chromosome and gene mapping, and for generating antisense RNA and DNA probe.Coding TAT nucleic acid can also be used to prepare TAT polypeptides by recombinant technique described herein, and those in which TAT polypeptides can be used for for example preparing anti-TAT antibody described herein.

Total length native sequences TAT genes or part thereof can be used for cDNA library as hybridization probe, have other cDNA (cDNA of such as those codings TAT naturally occurring variant or TAT from other species) of expectation sequence homogeneity to separate total length TAT cDNA or separation and natural TAT sequences disclosed herein.It is optional that, the length of probe is about 20 to about 50 bases.Hybridization probe can the new region of at least part derived from total length native nucleotide sequence, those in which region need not excessively test and be assured that, or carry out the genome sequence of self-contained native sequences TAT promoter, enhancer element and introne.For example, screening technique will separate the code area of TAT genes including the use of known dna sequence to synthesize the selected probe of about 40 bases.Hybridization probe can use a variety of mark substance markers, including radioactive nucleotides, such as³²P or³⁵S, or enzyme marker, such as pass through avidin/biotin coupling system and the alkaline phosphatase of probe conjugate.Label probe with the complementary sequence of the sequence with TAT genes of the present invention can be used for screening people cDNA, genomic DNA or mRNA libraries to determine that probe and which member in such library hybridize.The following examples have described in more detail hybridization technique.Using method disclosed herein, what any est sequence disclosed herein can be similar is used as probe.

Encoding other useful fragments of TAT nucleic acid includes such antisense or has MODN, and they, which are included, can combine target TAT mRNA (having justice) or the single strand nucleotide sequence (RNA or DNA) of TAT DNA (antisense) sequence.According to the present invention, antisense or the fragment for thering is MODN to include TAT DNA encodings area.Such fragment generally comprises at least about 14 nucleotides, preferably from about 14 to 30 nucleotides.Derive antisense according to the cDNA sequence of the given protein of coding or have the ability description of MODN in such as Stein and Cohen, Cancer Res.48：2659,1988 and van der Krol et al., BioTechniques 6：958,1988.

Antisense or the combination for having MODN and target nucleic acid sequence cause the formation of duplex, and it blocks the transcription or translation of target sequence by one of multiple means, and the means include the premature end or other means of degraded enhancing, transcription or the translation of duplex.Such method covers in the present invention.ASON is therefore available for the expression for blocking TAT protein matter, and those in which TAT protein matter may play induced cancer in mammal.Antisense has MODN also to include the oligonucleotides with sugar-phosphodiester backbone (or other sugared keys (linkage), such as described in WO 91/06629) by modification, and wherein such sugared key is resistant to endogenous nuclease.This class oligonucleotide of resistant sugared key is stable (can resist enzymatic degradation) in vivo, but remain can with reference to target nucleotide sequences sequence-specific.

Include the region of the translation initiation codon (5 '-AUG/5 '-ATG) containing the gene open reading frame (ORF) or terminator codon (5 '-UAA, 5 '-UAG and 5-UGA/5 '-TAA, 5 '-TAG and 5 '-TGA) for the preferred sites that antisense is combined in gene.These regions, which refer to, to be covered from translation initiation or terminator codon (i.e. 5 ' or 3 ') in either direction and counts about 25 parts to about 50 contiguous nucleotides in mRNA or gene.The other favored areas combined for antisense include：Introne；Extron；Intron-exon junction；ORFs (ORF) or " code area ", i.e. translation initiation codon and translation stop codon give between region；MRNA 5 ' caps, it is included is methylated G residue, including 5 ' caps itself and preceding 50 nucleotides adjacent with cap by N7- of 5 ' -5 ' triphosphoric acid key and mRNA most 5 ' end residues connections；In 5 ' non-translational regions (5 ' UTR), i.e. mRNA from translation initiation codon 5 ' directions part, therefore including the corresponding nucleotide on the nucleotides or gene in mRNA between 5 ' capsites and translation initiation codon；With 3 ' non-translational regions (3 ' UTR), i.e. in mRNA from translation termination codon 3 ' side, therefore including translation termination codon in mRNA and 3 ' ends between nucleotides or the corresponding nucleotide on gene part.

Specific example available for the preferred antisense compounds for suppressing the expression of TAT protein matter is included containing skeleton or the oligonucleotides of non-natural nucleoside bond through modification.With modification skeleton oligonucleotides include those retain in skeleton phosphorus atoms and those there is no the oligonucleotides of phosphorus atoms in skeleton.For the purpose of this specification, and sometimes also mention in the art, the modified oligonucleotide for not having phosphorus atoms in its intemucleoside backbone is also believed to oligonucleotides (oligonucleosides).It is preferred that modified oligonucleotide skeleton include thiophosphate for example with normal 3 ' -5 ' key, chiral phosphorothioates, phosphorodithioate, phosphotriester, the ester of aminoalkyl three (aminoalkylphosphotriester), methyl and other alkyl phosphonates include 3 '-alkylene phosphonate ester (3 '-alkylene phosphonate), 5 '-alkylene phosphonate ester (5 '-alkylene phosphonate) and chiral phosphonate, phosphite ester, phosphoramidate includes 3 '-amino phosphoramidate (3 '-amino phosphoramidate) and ammonia hydrocarbylamino phosphate (aminoalkylphosphoramidate), thion phosphoramidate (thionophosphoramidate), thion alkyl phosphonate (thionoalkylphosphonate), the ester of thion hydrocarbyl phosphate three (thionoalkylphosphotriester), phosphoroselenoate (selenophosphate) and brominated phosphate (borano-phosphate), their 2 ' -5 ' connection analog, and those have the analog of opposite polarity, between wherein one or more nucleotides key be 3 ' -3 ', 5 ' -5 ', or 2 ' -2 ' key.Preferred oligonucleotides with opposite polarity key between most 3 ' terminal nucleotides includes single 3 ' -3 ' key, you can be the single reverse nucleotide residues of no base (core base lack or with hydroxyl replaced).In the form of various salt, salt-mixture and free acid is also included within.The representative United States Patent (USP) of the preparation of phosphorous key is instructed to include but is not limited to United States Patent (USP) 3,687,808；4,469,863；4,476,301；5,023,243；5,177,196；5,188,897；5,264,423；5,276,019；5,278,302；5,286,717；5,321,131；5,399,676；5,405,939；5,453,496；5,455,233；5,466,677；5,476,925；5,519,126；5,536,821；5,541,306；5,550,111；5,563,253；5,571,799；5,587,361；5,194,599；5,565,555；5,527,899；5,721,218；5,672,697；With 5,625,050, each single item is taken in herein and is used as reference.

Wherein the preferred modified oligonucleotides skeleton without phosphorus atoms has the skeleton that key is formed between short-chain hydrocarbon group or cyclic hydrocarbon radical nucleoside bond, mixing hetero atom and alkyl or cyclic hydrocarbon radical nucleoside bond or one or more short chain heteroatomics or heterocycle nucleosides.These, which include those, has morpholino key (partly being formed by the sugar moieties of nucleosides)；Siloxane backbone；Sulfide, sulfoxide and sulfone skeleton；Formoxyl (formacetyl) and thioformyl (thioformacetyl) skeleton；Methylene formacetyl (formacetyl) and thioformyl (thioformacetyl) skeleton；Riboacetyl skeleton；Skeleton containing alkene；Sulfamate (sulfamate) skeleton；Methylene imino group and methylene diazanyl (methylenehydrazino) skeleton；Sulphonic acid ester and sulfonamide (sulfonamide) skeleton；Amide backbone；And it is other with mixing N, O, S and CH₂The skeleton of part.The representative United States Patent (USP) of the preparation of such oligonucleotides is instructed to include but is not limited to United States Patent (USP) 5,034,506；5,166,315；5,185,444；5,214,134；5,216,141；5,235,033；5,264,562；5,264,564；5,405,938；5,434,257；5,466,677；5,470,967；5,489,677；5,541,307；5,561,225；5,596,086；5,602,240；5,610,289；5,602,240；5,608,046；5,610,289；5,618,704；5,623,070；5,663,312；5,633,360；5,677,437；5,792,608；5,646,269；With 5,677,439, each single item is taken in herein and is used as reference.

In other preferred ASONs, the sugar and nucleoside bond of nucleotide units, i.e. skeleton are substituted by new group.Retain base units to be used for hybridizing with suitable nucleic acid target compound.One of such oligomeric compounds are a kind of oligonucleotide mimetic for having shown that and having remarkable hybrid trait, referred to as peptide nucleic acid (PNA).In PNA compounds, the sugared skeleton of oligonucleotides is replaced by amide containing skeleton, particularly aminoethylglycine backbone.Core base is retained, and directly or indirectly combines the aza nitrogen atom of framework amide part.The representative United States Patent (USP) of the preparation of PNA compounds is instructed to include but is not limited to United States Patent (USP) 5,539,082；5,714,331；With 5,719,262, each single item is taken in herein and is used as reference.More teachings of PNA compounds can be found in Nielsen et al., 1991, Science 254：1497-1500.

It is preferred that ASON include phosphorothioate backbone and/or heteroatom backbones, particularly-CH₂-NH-O-CH₂-、-CH₂-N(CH₃)-O-CH₂- (being referred to as methylene (methyl-imino) or MMI skeletons) ,-CH₂-O-N(CH₃)-CH₂- ,-CH described in above-mentioned United States Patent (USP) 5,489,677₂-N(CH₃)-N(CH₃)-CH₂- and-O-N (CH₃)-CH₂-CH₂- (wherein natural phosphodiester skeleton representation is-O-P-O-CH₂-) and above-mentioned United States Patent (USP) 5,602,240 amide backbone.The ASON with morpholino backbone structures of above-mentioned United States Patent (USP) 5,034,506 is also preferred.

Oligonucleotides through modification can also include one or more substitution sugar moieties.It is preferred that oligonucleotides in 2 ' positions comprising one of following：OH；F；O- alkyl, S- alkyl, or N- alkyl；O- alkenyls, S- alkenyls, or N- alkenyls；O- alkynyls, S- alkynyls, or N- alkynyls；Or O- alkyl-O- alkyl, wherein alkyl, alkenyl and alkynyl can be substituted or unsubstituted C₁To C₁₀Alkyl or C₂To C₁₀Alkenyl and alkynyl.Particularly preferably O [(CH₂)_nO]_mCH₃、O(CH₂)_nOCH₃、O(CH₂)_nNH₂、O(CH₂)_nCH₃、O(CH₂)_nONH₂And O (CH₂)_nON[(CH₂)_nCH₃)]₂, wherein n and m are 1 to about 10.Other preferred ASONs are in 2 ' positions comprising one of following：C₁To C₁₀Low alkyl group, substituted low alkyl group, alkenyl, alkynyl, alkaryl, aralkyl, O- alkaryls or O- aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂Heterocyclylalkyl, heteroalkylaryl, aminoalkyl amino (aminoalkylamino), poly- alkyl amino (polyalkylamino), substituted silicyl, RNA cutting groups (cleaving group), reporter group (reporter group), intercalator, for the group for the pharmacokinetics for improving oligonucleotides, or for the group for the pharmacodynamic properties for improving oligonucleotides, and other substituents with similar quality.It is preferred that modification include 2 '-methoxy ethoxy (2 '-O-CH₂CH₂OCH₃, also referred to as 2 '-O- (2- methoxy ethyls) or 2 '-MOE) and (Martin et al., 1995, HeIv.Chim.Acta 78：486-504) it is alkyloxy-alkoxy (alkoxyalkoxy).Further preferred modification includes 2 '-dimethylaminooxyethoxy, i.e. O (CH₂)₂ON(CH₃)₂Group, also referred to as 2 '-DMAOE, described in following article embodiment, and 2 '-Dimethylaminoethoxy ethyoxyl (this area is also referred to as 2 '-O- Dimethylaminoethoxies ethyls or 2 '-DMAEOE), i.e., '-O- (CH₂)₂-O-(CH₂)₂-N(CH₃)₂。

Further preferred modification includes locked nucleic acid (Locked Nucleic Acid, LNA), wherein 2 '-hydroxyl is connected with 3 ' or 4 ' carbon atoms of sugared ring, is consequently formed bicyclic sugar moieties.Key is preferably bridging methylene (methelyne) (- CH of 2 ' oxygen atoms and 4 ' carbon atoms₂-)_nGroup, wherein n are 1 or 2.LNA and its preparation are referring to WO 98/39352 and WO 99/14226.

Other preferred modifications include 2 '-methoxyl group (2 '-O-CH₃), 2 '-ammonia propoxyl group (2 '-OCH₂CH₂CH₂NH₂), 2 '-pi-allyl (2 '-CH₂- CH=CH₂), 2 '-O- pi-allyls (2 '-O-CH₂- CH=CH₂) and 2 '-fluorine (2 '-F).2 '-modification can be in arabinose (arabino) (on) position or ribose (ribo) (under) position.It is preferred that 2 '-arabinose modification be 2 '-F.Also other positions that can be on oligonucleotides carry out similar modification, the 3 ' sugared positions of particularly 3 ' terminal nucleotides or the 5 ' positions in the oligonucleotides of 2 ' -5 ' connection with 5 ' terminal nucleotides.Oligonucleotides also can use sugared analogies, such as with cyclobutyl moiety substituted furan pentose base (pentofuranosyl) sugar.The representative United States Patent (USP) of the preparation of the such sugared structure by modification of teaching includes but is not limited to United States Patent (USP) 4,981,957；5,118,800；5,319,080；5,359,044；5,393,878；5,446,137；5,466,786；5,514,785；5,519,134；5,567,811；5,576,427；5,591,722；5,597,909；5,610,300；5,627,053；5,639,873；5,646,265；5,658,873；5,670,633；5,792,747；With 5,700,920, complete income each single item is used as reference herein.

Oligonucleotides may also include core base (in the art often referred to simply as " base ") modification or substitute.As used herein, " unmodified " or " naturally " core base include purine base adenine (A) and guanine (G), and pyrimidine base thymine (T), cytimidine (C) and uracil (U).Core base by modification includes other synthesis and natural core base, such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2- aminoadenines, the 6- methyl and other alkyl derivatives of adenine and guanine, the 2- propyl group and other alkyl derivatives of adenine and guanine, 2- thiouracils, 2- sulphur thymidine and 2- sulphur cytimidines, 5- halo uracils and cytimidine, other alkynyl derivatives of 5- propinyls (- C ≡ C-CH3 or-CH2-C ≡ CH) uracil and cytimidine and pyrimidine bases, 6- azos (azo) uracil, cytimidine and thymidine, 5- uracils (pseudouracil), 4- thiouracils, 8- halos, 8- amino, 8- mercaptos (thiol), 8- sulfanyls (thioalkyl), 8- hydroxyls and the adenine and guanine of other 8- substitutions, 5- halos particularly 5- bromines, 5- trifluoromethyls and the uracil and cytimidine of other 5- substitutions, 7- methyl guanines and 7- methyl adenines, 2-F- adenines, 2- amino-adenines, guanozola and 8- azaadenines, 7- deazaguanines and 7- denitrogenations adenine and 3- deazaguanines and 3- denitrogenation adenines.The core base further modified includes tricyclic pyrimidine, such as fen  piperazines cytidine (1H- pyrimidines [5, 4-b] [1, 4] benzoxazine -2 (3H) -one), phenthazine cytidine (1H- pyrimidines [5, 4-b] [1, 4] benzothiazine -2 (3H) -one), fen  piperazines cytidine (such as 9- (2- ammonia ethyoxyl)-H- pyrimidines [5 that G- clamp rings such as replace, 4-b] [1, 4] benzoxazine -2 (3H) -one), carbazole cytidine (2H- pyrimidines [4, 5-b] indol-2-one), pyridine diindyl cytidine (H- pyridines [3 ', 2 '：4,5] pyrroles [2,3-d] pyrimid-2-one).Core base through modification may also include those core bases that wherein purine or pyrimidine bases are replaced by other heterocycles, such as 7- denitrogenations-adenine, 7- deazaguanines, PA and 2- pyridones.More core bases include those disclosed in United States Patent (USP) 3,687,808；The Concise Encyclopedia Of Polymer Science And Engineering, the 858-859 pages, Kroschwitz, J.L, ed.John Wiley ＆ Sons, those disclosed in 1990；With Englisch et al., Angewandte Chemie, International Edition, those disclosed in 1991,30,613.Some of these core bases are particularly useful in enhancing the binding affinity of oligomeric compounds of the present invention.These include the purine of the pyrimidine that 5- replaces, 6- aza-pyrimidines and N-2, N-6 and O-6 substitution, including 2- aminopropyl adenines, 5- propynyluracils and 5- propynylcytosines.5-methylcytosine substitution has been demonstrated the stability of nucleic acid duplex can be improved into 0.6-1.2 DEG C of (Sanghvi et al., AntisenseResearch and Applications, CRC Press, Boca Raton, 1993, pp.276-278), it is preferred base substitution, especially more preferred when with the sugar-modified combination of 2 '-O- methoxy ethyls.The representative United States Patent (USP) of the preparation through modifying core base is instructed to include but is not limited to United States Patent (USP) 3,687,808, and United States Patent (USP) 4,845,205；5,130,302；5,134,066；5,175,273；5,367,066；5,432,272；5,457,187；5,459,255；5,484,908；5,502,177；5,525,711；5,552,540；5,587,469；5,594,121,5,596,091；5,614,617；5,645,985；5,830,653；5,763,588；6,005,096；5,681,941；With 5,750,692, each single item is taken in herein and is used as reference.

Another modification for the ASON being connected with oligonucleotides chemistry is one or more parts or the conjugate (conjugate) of the activity, cell distribution or cellular uptake that improve oligonucleotides.The compound of the present invention can include the coupling group (conjugate group) being covalently attached with functional group (such as primary hydroxyl or secondary hydroxyl).The coupling group of the present invention includes intercalator, reporter molecule, polyamines, polyamide, polyethylene glycol, polyethers, the group for improving oligomer pharmacodynamic properties and the group for improving oligomer pharmacokinetics.Typical coupling group includes cholesterol, lipid, cation lipid, phosphatide, cationic phospholipid, biotin, azophenlyene, folic acid (folate), phenanthridines, anthraquinone, acridine, fluorescein, rhodamine, cumarin and dyestuff.In the context of the invention, improving the group of pharmacodynamic properties includes improving oligomer intake, improves oligomer to the resistance of degraded, and/or enhancing and the group of RNA sequence specific hybridization.In the context of the invention, improving the group of pharmacokinetics includes improving the group of oligomer intake, distribution, metabolism or excretion.Conjugate part includes but is not limited to lipid part, such as cholesterol moiety (Letsinger et al., 1989, Proc.Natl.Acad.Sci.USA 86：6553-6556), cholic acid (Manoharan et al., 1994, Bioorg.Med.Chem.Let.4：1053-1060), thioether such as hexyl-S- trityls mercaptan (tritylthiol) (Manoharan et al., 1992, Ann.N.Y.Acad.Sci.660：306-309；Manoharan et al., 1993, Bioorg.Med.Chem.Let.3：2765-2770), thio cholesterol (Oberhauser et al., 1992, Nucl.Acids Res.20：533-538), (Saison-Behmoaras et al., 1991, EMBO J.10 for aliphatic chain such as dodecanediol or undecyl residues：1111-1118；Kabanov et al., 1990, FEBS Lett.259：327-330；Svinarchuk et al., 1993, Biochimie 75：49-54), phosphatide such as two-hexadecyl-rac-glycerol or triethyl group-ammonium 1,2- bis--O- hexadecyl-rac-glycerol -3-H- phosphonate esters (Manoharan et al., 1995, Tetrahedron Lett.36：3651-3654；Shea et al., 1990, Nucl.Acids Res.18：3777-3783), polyamines or polyglycol chain (Manoharan et al., 1995, Nucleosides ＆Nucleotides 14：969-973), or acetic acid adamantane (Manoharan et al., 1995, TetrahedronLett.36：3651-3654), palm base section (Mishra et al., 1995, Biochim.Biophys.Acta1264：229-237), or octadecylamine or hexylamino-carbonyl-oxycholesterol part.The oligonucleotides of the present invention can be also coupled with active drug substance, such as aspirin (aspirin), warfarin (warfarin), bute (phenylbutazone), brufen (ibuprofen), suprofen (suprofen), fenbufen (fenbufen), Ketoprofen (ketoprofen), (S)-(+)-pranoprofen (pranoprofen), Carprofen (carprofen), red sulphonyl methyl amimoacetic acid (dansylsarcosine), 2,3,5- Triiodobenzoic acids, Flufenamic acid (flufenamic acid), folinic acid (folinic acid), benzothiadiazine (benzothiadiazide), chlorothiazide (chlorothiazide), the miscellaneous  (diazepine) of phenodiazine grass, Indomethacin (indomethacin), barbiturate or ester (barbiturate), cynnematin (cephalosporin), sulfa drug (sulfa drug), antidiabetic (antidiabetic), antimicrobial (antibacterial) or antibiotic.Oligonucleotides-drug conjugates and its preparation are referring to U.S. Application Serial 09/334,130 (submission on June 15th, 1999) and United States Patent (USP) 4,828,979；4,948,882；5,218,105；5,525,465；5,541,313；5,545,730；5,552,538；5,578,717,5,580,731；5,580,731；5,591,584；5,109,124；5,118,802；5,138,045；5,414,077；5,486,603；5,512,439；5,578,718；5,608,046；4,587,044；4,605,735；4,667,025；4,762,779；4,789,737；4,824,941；4,835,263；4,876,335；4,904,582；4,958,013；5,082,830；5,112,963；5,214,136；5,082,830；5,112,963；5,214,136；5,245,022；5,254,469；5,258,506；5,262,536；5,272,250；5,292,873；5,317,098；5,371,241,5,391,723；5,416,203,5,451,463；5,510,475；5,512,667；5,514,785；5,565,552；5,567,810；5,574,142；5,585,481；5,587,371；5,595,726；5,597,696；5,599,923；5,599,928；With 5,688,941, each single item is taken in herein and is used as reference.

Homogeneous modification need not be made to giving all positions in compound, incorporation exceedes a kind of above-mentioned modification at single nucleosides that in fact can be in single compound or even in oligonucleotides.Present invention additionally comprises the antisense compounds as Chimeric compounds.In the context of the invention, " chimeric " antisense compounds or " block polymer " refer to such antisense compounds, particularly oligonucleotides：It includes two or more different regions in chemistry, and each region is made up of at least one monomeric unit (being nucleotides in oligonucleotide compound).These oligonucleotides generally comprise at least one such region, and wherein oligonucleotides is modified so that resistance enhancing, cellular uptake raising, and/or the binding affinity to target nucleic acid that the oligonucleotides is degraded to nuclease are improved.Another region of oligonucleotides may act as that RNA can be cut:DNA or RNA:The substrate of the enzyme of RNA heterocomplexs.For example, RNase H is cutting RNA:The cellular endonuclease of the RNA chains of DNA duplex.Therefore, RNase H activation causes the cutting to RNA target thing, thus greatly improves the efficiency of oligonucleotides inhibition of gene expression.Therefore, when using chimeric oligonucleotide, compared with hybridizing in the thiophosphate deoxy-oligonucleotide of identical target area, generally available shorter oligonucleotides obtains similar result.The Chimeric antisense compounds of the present invention are formed as the composite construction of two or more oligonucleotides as described above, the oligonucleotides through modification, few nucleosides and/or oligonucleotide mimetic.It is preferred that Chimeric antisense oligonucleotides mixed in 3 '-end at least one 2 ' modification sugar (preferably '-O- (CH₂)₂-O-CH₃), to assign its nuclease resistant；And with the region for the 2 '-H sugar being connected containing at least four, to assign its RNase H activity.Such compound is also referred to as heterocomplex (hybrid) or binding element (gapmer) in this area.It is preferred that binding element (gapmer) there are sugar (preferably '-O- (CH of 2 ' modifications in 3 '-end and 5 ' ends₂)₂-O-CH₃) region, therebetween by with least four be connected 2 '-H sugar at least one distinguish every, and preferably comprise phosphorothioate backbone connection.The representative United States Patent (USP) of the preparation of such heterocomplex structure is instructed to include but is not limited to United States Patent (USP) 5,013,830；5,149,797；5,220,007；5,256,775；5,366,878；5,403,711；5,491,133；5,565,350；5,623,065；5,652,355；5,652,356；With 5,700,922, complete income each single item is used as reference herein.

Conventional it can be prepared conveniently and by well-known solid phase synthesis technique according to the antisense compounds that use of the present invention.There are many retailers to sell the equipment for such synthesis, including such as AppliedBiosystems (Foster City, Calif.).Additionally or alternatively, any other means known in the art for such synthesis can be used.It is known that preparing oligonucleotides, such as thiophosphate and hydrocarbylation derivative using similar techniques.The compound of the present invention can also mix with the mixture of other molecules, molecular structure or compound, it is encapsulated, be coupled or be otherwise jointly formed such as liposome, receptor target molecule, oral, rectum, local or other formulations to help to absorb, be distributed and/or absorb.The representative United States Patent (USP) of the such intake of teaching, distribution and/or the preparation for absorbing formulation auxiliary includes but is not limited to United States Patent (USP) 5,108,921；5,354,844；5,416,016；5,459,127；5,521,291；5,543,158；5,547,932；5,583,020；5,591,721；4,426,330；4,534,899；5,013,556；5,108,921；5,213,804；5,227,170；5,264,221；5,356,633；5,395,619；5,416,016；5,417,978；5,462,854；5,469,854；5,512,295；5,527,528；5,534,259；5,543,152；5,556,948；5,580,575；With 5,595,756, each single item is taken in herein and is used as reference.

Other examples of sense or antisense oligonucleotides include those and (described in such as WO90/10048 those) and improve the oligonucleotides that oligonucleotides is covalently attached to the other parts (such as poly- (1B)) of the affinity of target nucleic acid sequence with organic moiety.Moreover, intercalator such as ellipticine (ellipticine) and alkylating agent or metal complex can be attached on sense or antisense oligonucleotides, to adjust antisense or have binding specificity of the MODN to target nucleotide sequences.

Can be by the way that any gene transfer method is by antisense or has MODN to import the cell for including target nucleic acid sequence, including such as CaPO₄The DNA transfections of mediation, electroporation or viral (Epstein-Barr virus) by using gene transfer vector such as angstrom bar Er Shi.In a kind of preferred flow, by antisense or there is MODN to insert suitable retroviral vector.Make the cell comprising target nucleic acid sequence in vivo or ex vivo contact recombinant retroviral vector.Suitable retroviral vector include but is not limited to those be derived from mouse retrovirus M-MuLV those, N2 (retrovirus for being derived from M-MuLV), or it is named as DCT5A, DCT5B and DCT5C double copy carriers (see WO 90/13641).

Also the cell for including target nucleotide sequences can be imported by with ligand binding molecules formation conjugate by sense or antisense oligonucleotides, as described in WO 91/04753.Suitable ligand binding molecules include but is not limited to cell surface receptor, growth factor, other cell factors or the other parts for combining cell surface receptor.Preferably, do not disturb the ligand binding molecules to combine the ability of its corresponding molecule or acceptor substantially to the coupling of ligand binding molecules or block sense or antisense oligonucleotides or its conjugate pattern to enter cell.

Or, can be by forming the cell that the importing of sense or antisense oligonucleotides is included target nucleic acid sequence by oligonucleotides-lipid complex, as described in WO 90/10448.Sense or antisense oligonucleotides-lipid complex is preferably dissociated by endogenous lipase in the cell.

Antisense or the length for having adopted RNA or DNA molecular are typically at least about 5 nucleotides,Or length is at least about 6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,35,40,45,50,55,60,65,70,75,80,85,90,95,100,105,110,115,120,125,130,135,140,145,150,155,160,165,170,175,180,185,190,195,200,210,220,230,240,250,260,270,280,290,300,310,320,330,340,350,360,370,380,390,400,410,420,430,440,450,460,470,480,490,500,510,520,530,540,550,560,570,580,590,600,610,620,630,640,650,660,670,680,690,700,710,720,730,740,750,760,770,780,790,800,810,820,830,840,850,860,870,880,890,900,910,920,930,940,950,960,970,980,990,Or 1000 nucleotides,Term " about " refers to the nucleotide sequence length and adds deduct the 10% of the length wherein in this content.

Also probe can be used to round pcr produce the arrangement set for identifying closely related TAT coded sequences.

Coding TAT nucleotide sequence can also be used to build hybridization probe, for the assignment of genes gene mapping for encoding the TAT and for making genetic analysis to the individual for suffering from hereditary conditions.Can be used known technology by provided herein is nucleotide sequence be positioned at chromosome and specific chromosome regions, such as in situ hybridization, the linkage analysis for known chromosome marker and the hybridization for screening library.

When TAT coded sequence coding combines the protein of another protein (such as when TAT is acceptor), the other oroteins or molecule for participating in binding interactions can be identified using TAT in the assay.Pass through such method, it is possible to identify the mortifier of receptor/ligand binding interactions.The protein for participating in such binding interactions can be additionally used in the peptide or little molecules in inhibiting thing or activator for screening binding interactions.Equally, acceptor TAT can also be used to separate associated ligands.The screening test method can be designed with the lead compound for the biological activity for finding to simulate natural TAT or TAT acceptors.Determination method including adapting to high flux screening chemistry library is make them particularly suitable for use in identification small molecule drug candidates by such the screening test method.Contemplated small molecule includes the organic or inorganic compound of synthesis.The protein-protein binding assay being fully described in method, including this area, biochemistry the screening test method, immunoassay and the determination method based on cell can be measured in a variety of forms.

The nucleic acid or its modified forms for encoding TAT can be additionally used in generation transgenic animals or " knockout " animal, and they then can be used for exploitation and the upper useful reagent of screening treatment.Transgenic animals (such as mouse or rat) refer to the animal with the cell comprising transgenosis, and wherein transgenosis is that, before birth, such as embryo stage imports the animal or the ancestors (ancestor) of the animal.Transgenosis refers to the DNA being incorporated into the genome for the cell for developing into transgenic animals.In one embodiment, mouse can be compiled according to the technology set up) refer to the animal with the cell comprising transgenosis, wherein transgenosis is that, before birth, such as embryo stage imports the animal or the ancestors (ancestor) of the animal.Transgenosis refers to the DNA being incorporated into the genome for the cell for developing into transgenic animals.In one embodiment, TAT genomic DNA can be encoded with coding TAT cDNA clone according to the technology set up, and the genome sequence is encoded into TAT DNA cell comprising expression for generating transgenic animals, the transgenic animals.For generating transgenic animals, particularly the method for the animal such as mouse or rat has been conventional in this area, see, for example, United States Patent (USP) 4,736,866 and 4,870,009.Generally, using tissue-specific enhancer, using specific cells as TAT integrated transgenes target.The transgenic animals of the transgenosis (transgenosis imports animal germline in embryo stage) of the coding TAT comprising a copy can be used to carry out the test code TAT enhanced effect of DNA expression.Such animal can be used as testing animal, for testing the reagent of (such as the protective effect for being overexpressed relevant pathological condition with it) of being considered as shielding.According to this aspect of the present invention, with the agent therapy animal, if the incidence of disease of the pathological condition has declined compared to the untreated animal for carrying the transgenosis, show to there may be the therapeutic intervention effect for the pathological condition.

Or, TAT non-human homologue can be used to build TAT " knockout " animal, it has coding TAT that is defective or changing gene, this be coding TAT endogenous gene and between being imported into the animal embryonic stem cell, coding TAT by change genomic DNA generation homologous recombination result.For example, TAT genomic DNA can be encoded using the cDNA clone for encoding TAT according to the technology set up.Another gene is deleted or replaced with to the part that can will encode TAT genomic DNA, such as encodes the gene of selection marker (mark can be used for monitoring to integrate).Generally, in the carrier comprising many kilobases (description is see, for example, Thomas and Capecchi, 1987, Cell 51 as described in homologous recombination vector for unchanged flanking DNA (5 ' and 3 ' ends have)：503).By the vector introduction embryonic stem cell line (such as by electroporation), and wherein imported DNA is selected to occur the cell of homologous recombination (see, for example, Li et al., 1992, Cell 69 with interior source DNA：915).Then by the blastocyst of selected cell infusion to animal (such as mouse or rat) to form aggregation chimera (see, for example, Bradley, inTeratocarcinomas and Embryonic Stem Cells：A Practical Approach, E.J.Robertson, ed.IRL, Oxford, 1987, pp.113-152).Then chimeric embryo can be implanted into suitable pseudopregnant female foster animal, and make embryo is mature to produce " knockout " animal.The offspring comprising homologous recombination DNA can be identified by standard technique in its reproduction cell, and be used for the animal that all cells of breeding wherein animal all include homologous recombination DNA.Ability and occur some pathological conditions to be identified that knock-out animal can resist some pathological conditions according to having due to TAT polypeptides.

Lasting effect is obtained, the latter then once or repeatedly applies treatment upper effective DNA or mRNA.Antisense RNA and DNA can be used for the expression for blocking some genes in vivo as therapeutic agent.It is verified, short ASON can be transfused to the cell that they play inhibitor wherein, although because cell membrane is limited to their intake, their relatively low (the Zamecnik et al. of intracellular concentration, 1986, Proc.Natl.Acad.Sci.USA 83：4143-4146).Their intake can be improved with modified oligonucleotide, such as by using their negatively charged phosphodiester groups of uncharged substituent group.

There are multiple technologies to can be used for nucleic acid importing living cells.These technologies are according to being transferred to nucleic acid in external culture cell, or in the cell of internal expection host and are varied from.Suitable for nucleic acid is transferred into technology in mammalian cell including the use of liposome, electroporation, microinjection, cell fusion, DEAE- dextrans, calcium phosphate precipitation etc. in vitro.Transfection (Dzau the et al., 1993, Trends in Biotechnology 11 of transfection and virus capsid protein of the currently preferred vivo gene transfer technology including the use of virus (be typically retrovirus) carrier-liposome-mediated：205-210).In some cases, expect to provide nucleic acid source together with the reagent for targetting target cell, antibody, the part of receptor on target cells special to cell surface membrane protein or target cell etc..When using liposome, with reference to the cell surface membrane protein relevant with endocytosis protein can be used for targeting and/or promote intake, such as capsid protein to particular cell types aeoplotropism or its fragment, in the circulating cycle experience internalization protein antibody, target inner cellular localization and strengthen the protein of intracellular half-life period.The technology of receptor-mediated endocytosis is see, for example, Wu et al., 1987, J.Biol.Chem.262：4429-4432；With Wagner et al., 1990, Proc.Natl.Acad.Sci.USA 87：3410-3414.Summary on genetic marker and gene therapy protocol is shown in Anderson et al., 1992, Science 256：808-813.

The nucleic acid molecules or its fragment of coding TAT polypeptides described herein can be used for Chromosome Identification.On this point, just need to find new chromosome marker at present, because being currently available that the chromosome marking reagent based on actual sequence data is seldom.Every kind of TAT nucleic acid molecules of the present invention are used as chromosome marker.

The TAT polypeptides and nucleic acid molecules of the present invention can also be used for tissue typing in diagnosis, wherein the present invention TAT polypeptides may in one kind tissue compared with another organize, preferably in illing tissue compared with the normal structure of identical organization type differential expression.TAT nucleic acid molecules can be used for production probe, for PCR, Northern analysis, Southern analyses and Western analyses.

The present invention covers screening compounds to identify those simulation TAT polypeptides (activator) or prevent TAT polypeptides from telling on (the method for the compound of (antagonist).The screening of design antagonist drug candidates then determines method to identify such compound：It is combined or compound with the TAT polypeptides for the coded by said gene being mentioned herein, or otherwise disturbs the interaction of the polypeptide that these are encoded and other cell proteins, including for example suppresses cell expression TAT polypeptides.Such the screening test method includes the determination method suitable for high flux screening chemistry library so that they are particularly suitable for use in identifying small molecule drug candidates.

Measure can be carried out by various modes, including protein-protein combination mensuration, biochemistry screening test, immunoassays and the measure based on cell that this area has been well known.

All determination methods to antagonist have in common that they need the TAT polypeptide time enough for making drug candidates contact the encoded by nucleic acid being mentioned herein under the conditions of sufficiently, both components is interacted.

In binding assay, " interaction " refers to combine, and the compound formed can be separated or detected in the reactive mixture.In a specific embodiment, by the TAT polypeptides or drug candidates of the coded by said gene identified herein by being covalently or non-covalently attached to fixed in solid phase, such as on microtiter plate.Non-covalent attachment is generally realized by using TAT polypeptide solutions coating solid phase surface and drying.Or, the immobilized antibody such as monoclonal antibody to TAT polypeptides to be fixed can be used for anchoring to it on solid phase surface.Determination method is carried out as follows, and can will be added to the on-fixed component of detectable label substance markers on the immobilization component such as coating surface comprising grappling component.When reacting completion, for example, unreacted component is removed by cleaning, and detect the compound anchored on solid phase surface.If initial on-fixed component carries detectable, detect that the label being fixed on surface shows to there occurs compound action.If initial on-fixed component does not carry label, compound action for example can be detected by using the labelled antibody of fixed complex is specifically bound.

If candidate compound and many peptide interactions of specific T AT of coded by said gene identified herein but do not combined, then the interaction of it and the polypeptide can be by determining for detecting the known method of protein-protein interaction.Such determination method includes conventional method, such as crosslinking, co-immunoprecipitation and pass through gradient or the copurification of chromatographic column.Furthermore it is possible to such as Chevray and Nathans, 1991, Proc.Natl.Acad.Sci.USA 89：Disclosed in 5789-5793, Fields and its colleague (Fields andSong, 1989, Nature (London) 340 are used：245-246；Chien et al., 1991, Proc.Natl.Acad.Sci.USA 88：9578-9582) genetic system based on yeast of description monitors protein-protein interaction.Many transcriptional activators, such as yeast GAL4, discrete modular structural domains are constituted in two spaces, and one is played DNA binding domain, and another plays the function of transcription activating domain.Yeast expression system (being commonly referred to as " two-hybrid system ") described in above-mentioned publication make use of this characteristic, using two kinds of hybrid proteins, target protein is merged with GAL4 DNA binding domain in one, and merged candidate's activator protein matter with activation domain in another.The reconstruction that expression of the GAL1-lacZ reporters under the promoter control that GAL4 is activated passes through protein-protein interaction dependent on GAL4 activity.The bacterium colony of interaction polypeptide is included with the chromogenic substrate detection of beta galactosidase.Complete kit (the MATCHMAKER of the protein-protein interaction between two kinds of specified proteins is identified using two-hybrid techniques^TM) can be bought from Clontech.This system may also extend into the mapping of the protein domain to participating in specified protein interaction and point out to these vital amino acid residues of interaction.

The gene and the compound of the interaction of other intracellulars or extracellular fraction of the coding TAT polypeptides being mentioned herein is disturbed to test as follows：Generally it is being enough to make two kinds of product interactions and the condition combined and the reactant mixture comprising gene outcome and intracellular or extracellular fraction is prepared under the time.The ability combined to test candidate compound to suppress, is reacted when lacking and there is test compound.Furthermore it is possible to add placebo into the 3rd part of reactant mixture, positive control is used as.Combination present in monitoring mixture between test compound and intracellular or extracellular fraction as described above (compound is formed).Formed in control reaction thing and do not form compound in compound but reactant mixture containing test compound and show, test compound disturbed test compound reacts the interaction of gametophyte.

In order to determine antagonist, TAT polypeptides can be added in cell together with the compound of given activity to be screened, if the compound can suppress the activity of concern when there is TAT polypeptides, it is the antagonist of TAT polypeptides to show the compound.Or, antagonist can be detected as follows：TAT polypeptides and potential antagonist are mixed with the TAT polypeptide receptors or recombinant receptor of film combination under conditions of being determined suitable for Reverse transcriptase.TAT polypeptides can be marked, and such as pass through radioactivity so that the number of the TAT peptide molecules combined with acceptor can be used for the effect for determining potential antagonist.Encode acceptor gene can by those skilled in the art will know that a variety of methods identify, such as part elutriation and FACS sortings.Coligan et al., 1991, Current Protocols in Immun.1 (2)：Chapter 5.Preferably, using expression cloning, wherein preparing poly+RNA to the cell that TAT polypeptides have response, multiple set will be divided into from this RNA cDNA libraries built, the other cells not responded to for rotaring redyeing COS cell or to TAT polypeptides.The transfectional cell cultivated on slide is set to be exposed to the TAT polypeptides by mark.TAT polypeptides, including iodate or the recognition site for including site-specific protein kinases can be marked by multiple means.After fixed and incubation, slide is subjected to radioautographic analysis.The positive set of identification, prepares subclass, and carries out transfection and again screening process again with the subclass of interaction, finally produces the single clone of coding presumption acceptor.

As the alternative approach of receptor identification, the TAT polypeptides of mark can be made to couple with the cell membrane of expressed receptor molecule or extraction prepared product photoaffinity.Cross-linking agent is parsed by PAGE and x-ray film is exposed.The labeled complex comprising acceptor can be cut, fragments of peptides is dissociated into, and carry out Protein microassay sequencing.The amino acid sequence obtained from microsequencing can be used for one group of degenerate oligonucleotide probe of design, estimate the gene of acceptor with identification code for screening cDNA library.

In the method for another measure antagonist, the mammalian cell of expressed receptor or film preparation thing are incubated together with the TAT polypeptides by mark under conditions of candidate compound presence.Then the measurable compound strengthens or blocked the ability that this interacts.

More specific examples of potential antagonist include the oligonucleotides of binding domain-immunoglobulin and the fusions of TAT polypeptides, particularly antibody, including but not limited to polyclonal and monoclonal antibody and antibody fragment, single-chain antibody, anti-idiotype, with this chimeric or humanization pattern antibody-like or fragment, and human antibody and antibody fragment.Or, potential antagonist can be closely related protein, the TAT polypeptides of such as certain mutant form, its identification receptor but not work, so as to competitively suppress the effect of TAT polypeptides.

Another potential TAT polypeptide antagonists are the antisense RNAs or DNA constructions prepared using antisense technology, wherein such as antisense RNA or DNA molecular directly block mRNA to translate by hybridizing with said target mrna and preventing protein translation.It antisense technology can be used to pass through triple helix formation or antisense DNA or RNA to control gene expression, both approaches are all based on polynucleotides and DNA or RNA combination.For example, using 5 ' coded portions of the polynucleotide sequence for encoding this paper mature T AT polypeptides come antisense rna oligonucleotide that design length is about 10-40 base-pair.DNA oligonucleotides is designed to the regional complementarity (triple helix-referring to Lee et al., 1979, Nucl.Acids Res.6 with participating in transcription in gene：3073；Cooney et al., 1988, Science 241：456；Dervan et al., 1991, Science 251：1360), thus prevent from transcribing and producing TAT polypeptides.Antisense rna oligonucleotide and mRNA hybridize and block the translation of mRNA molecules to turn into TAT polypeptides (antisense-Okano, 1991, Neurochem.56 in vivo：560；Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press：Boca Raton, FL, 1988).Also above-mentioned oligonucleotides can be delivered to cell so that antisense RNA or DNA can express to suppress the generation of TAT polypeptides in vivo.When using antisense DNA, the oligodeoxyribonucleotide of the translation initiation site (e.g., from about between -10 to+10 positions) derived from target gene nucleotide sequence is preferred.

Potential antagonist includes combining avtive spot, receptor binding site or the growth factor or other relevant binding sites of TAT polypeptides, so as to block the small molecule of the normal bioactivity of TAT polypeptides.The example of small molecule includes but is not limited to small peptide or peptide sample molecule, preferably soluble peptide, and the non-peptidyl linker organic or inorganic compound synthesized.

Ribozyme is can be catalyzed the enzymatic RNA molecules of the specificity cutting to RNA.Ribozyme then carries out endonuclease hydrolysis (endonucleolytic) cutting to work by occurring sequence specific hybridization with complementary target rna.The specific Ribozyme cleavage site in potential rna target can be identified by known technology.More details are see, for example, Rossi, 1994, Current Biology 4：469-471 and PCT Publication WO97/33551 (disclosure on the 18th of September in 1997).

For nucleic acid molecules suppressing transcription, existing with triple helix structure, it may be that single-stranded and be made up of deoxynucleotide.Design the base composition of these oligonucleotides so that it promotes triple helix to be formed by Hoogsteen basepairing rules, and this usually requires have big section purine or pyrimidine on a chain of duplex.More details are seen above see, for example, PCT Publication WO 97/33551.

These small molecules can be by one or more the screening test methods discussed above and/or by identifying well known to a person skilled in the art any other triage techniques.

The nucleic acid of the coding TAT polypeptides of separation may be used in technology well known in the art herein while as described herein generate TAT polypeptides to recombinate.Then, the TAT polypeptides generated may be used in technology well known in the art and as described herein generate anti-TAT antibody.

The antibody for the specific binding TAT polypeptides being mentioned herein, and the other molecules identified by above-disclosed the screening test method, can be applied in the form of Pharmaceutical composition, for treating various disease conditions, including cancer.

If TAT polypeptides are in intracellular, and use complete antibody as inhibitor, then the antibody (internalizing antibodies) of preferred internalization.However, it is also possible to which antibody or antibody fragment are delivered in cell with fat transfection or liposome.When using antibody fragment, the minimum inhibition fragment of the binding domain of target protein is preferably specifically bound.For example, according to the variable region sequences of antibody, the peptide molecule for retaining the ability combined with target protein sequence can be designed.Such peptide can be generated with chemical synthesis and/or by recombinant DNA technology.See, for example, Marasco et al., 1993, Proc.Natl.Acad.Sci.USA 90：7889-7893.

Preparaton herein can also contain has treated more than one reactive compound necessary to specific indication, preferably those complementary activities and not adversely affects each other.Or composition can include the medicament of its function of enhancing, such as cytotoxic agent, cell factor, chemotherapeutics or growth inhibitor.Suitably, this quasi-molecule exists to be combined for the effective amount of predetermined purpose.

The following example is provided just to illustrate purpose, and is not intended to limit the scope of the present invention in any way.

All patents and document quoted in this complete income this specification are as reference.

Embodiment

Unless otherwise indicated, the commercial reagents referred in embodiment are used according to the specification of manufacturer.The source for numbering those cells differentiated in Examples below and entire description with ATCC is American type culture collection (American Type Culture Collection, Manassas, VA).

Embodiment 1：Expression profile analysis is carried out using GeneExpress 

Proprietary database (GeneExpress  of the analysis bag containing gene expression information, Gene Logic Inc., Gaithersburg, MD), attempt to identify the polypeptide (and its code nucleic acid) that its expression has notable and detectable up-regulation in specific purpose human tumour tissue compared with other human tumours and/or health adult tissue.Specifically, use the analysis of proprietary software progress GeneExpress  databases being obtained by Gene Logic companies (Gaithersburg, MD), with software that GeneExpress  databases are used together or Genentech companies writing and develop, being used together with GeneExpress  databases.The evaluation of positives hit is analyzed based on the expression in some standards, including such as tissue specificity, tumour-specific and normal elementary organization and/or normal proliferating tissues.Such tissue expression overview is presented in following molecule：It is in specific one or more human tumours, compared with other human tumours and/or health adult tissue, there are high tissue expression and significant, reproducible and detectable up-regulated expression, and optionally, have relatively low expression in normal basic people's tissue and/or normal proliferative people tissue.

Using expression analysis described above it was determined that coding this paper SEQ ID NO：The mRNA of TAT113 polypeptides shown in 2 has significant, reproducible and detectable overexpression compared with corresponding normal human colonic and rectal tissue respectively in the carcinous colon of certain form of people and rectal neoplasm.

A. colon

In first experiment, the TAT113 expression in one group 237 parts independent normal human colonic's tissue samples is analyzed.The result of these analyses is proved, TAT113 mRNA expressions in all normal human colonic's tissue samples analyzed are shockingly consistent, and fall into very narrow distribution, the TAT113 expression without normal human colonic's tissue sample has more than 2 times of rise compared with the overall average TAT113 expressions of this group of sample.

For the purpose of quantitative comparison, also independent to many parts and different types of carcinous people's colon tissue samples analyze TAT113 expression.The result that these analyses are obtained is proved, TAT113 expression variabilities in carcinous sample are quite big, and the carcinous sample for having significant number shows that TAT113 expression has the rise of at least 2 times (until being up to about 16 times) compared with the average TAT113 expressions for the normal colonic tissue's sample sets analyzed.In particular, in following colon cancer type, observe that detectable and reproducible TAT113 is overexpressed (numeral in the bracket of wherein every kind of cancer types represents that TAT113 expresses the sum of the number of at least 2 times elevated independent samples of display with the average TAT113 expressions for the normal colonic tissue's sample sets analyzed compared with/analyze independent tumors sample) compared with Normal Colon：The gland cancer (21/34) of the gland cancer (25/35) of the unspecified adenocarcinoma of colon in position (4/9), caecum and right liter section colon, the gland cancer (5/6) of transverse colon and left drop section colon and sigmoid colon.The experiment being additionally carried out confirms these results.

B. rectum

In another experiment, the TAT113 expression in one group 46 parts independent normal person's rectal tissue samples is analyzed.The result of these analyses is proved, surprising consistent of TAT113mRNA expressions in all normal person's rectal tissue samples analyzed, and fall into very narrow distribution, show that TAT113 expression has more than 2 times of rise compared with the overall average TAT113 expressions of this group of sample without normal person's rectal tissue sample.

For the purpose of quantitative comparison, the TAT113 expression also to 25 parts of independent human rectal adenocarcinoma tissue sample analysis.The result that these analyses are obtained is proved, TAT113 expression variabilities in carcinous sample are quite big, have 15 parts of display TAT113 expression to have the rise of at least 2 times (until being up to about 13 times) compared with the average TAT113 expressions for the normal rectal tissue sample group analyzed in the sample that 25 parts are tested.

In summary, this paper SEQ ID NO：TAT113 polypeptides shown in 2 and encode the nucleic acid of the polypeptide and may be used as excellent target and come qualitative and quantitative determine this paper SEQ IDNO in various mammalian tissue samples：TAT113 polypeptides shown in 2 and the expression for encoding its mRNA, so as to carry out qualitative and quantitative comparison between these samples.Therefore, this paper SEQ ID NO：TAT113 polypeptides shown in 2 and encode the polypeptide nucleic acid be its uniqueness expression overview can be used for as described above diagnosis mammal in some type cancerous tumours molecule.In addition, due to this analytical proof TAT113 polypeptides in some human tumours its corresponding health adult tissue compared to have it is notable, reproducible and it is detectable be overexpressed, so TAT113 polypeptides can be used as carrying out such tumour in mammal the excellent target of therapeutic treatment.

Embodiment 2：The up-regulation of TAT polypeptides in microarray analysis detection cancerous tumour

Nucleic acid microarray usually includes thousands of gene orders, and they can be used for the gene than normal homologue differential expression in identification illing tissue.In order that with nucleic acid microarray, self-test in future and the test of control tissue sample and control mRNA samples reverse transcription simultaneously mark to produce cDNA probes.Then by cDNA probes and the nucleic acid array hybridisations being fixed on solid support.The setting of array make it that the sequence of each member of array and position are known.For example, a collection of known sequence in the gene expressed under some morbid states may be selected to solid support.If label probe hybridizes with specific array member, show that the sample table for deriving the probe reaches the gene.If the hybridization signal for carrying out the probe of self-test (diseased tissue) sample is more than the hybridization signal of the probe from control (normal structure) sample, the one or more genes being overexpressed in diseased tissue are identified.The meaning of this result is that the protein being overexpressed in illing tissue can be used not only as the diagnosis marker of disease condition presence, and can be used as the therapeutic target of disease condition treatment.

Nucleic acid hybridizes and the methodology of microarray technology is well-known in the art.In the present embodiment, the PCT Patent Application serial number PCT/US01/10482 submitted on March 30th, 2001 is all described in detail in for the nucleic acid of hybridization and specific prepared product, slide and the hybridization conditions of probe, income is used as reference herein.

In the present embodiment, the gene expression of up-regulation is organized relative to the cancerous tumour from histological types and/or non-cancerous people to the cancerous tumour research organized derived from a variety of people, to attempt to identify the polypeptide that those are overexpressed in specific cancerous tumour.In some experiments, the carcinous human tumour tissue and non-cancerous human tumour tissue of same organization type (often from same patient) are obtained and TAT expression of polypeptides is analyzed.In addition, obtain carcinous human tumour tissue any in a variety of different human tumours and be compared with " general " epithelium control sample, wherein prepared by " general " epithelium control sample is by organizing to gather the non-cancerous people of epithelium genesis (including liver, kidney and lung).The mRNA separated from epithelial tissue set represents the mixture of the expressed gene product from a variety of different epithelial tissues, thus provides a kind of excellent negative control for being used for carrying out quantitative comparison with the gene expression dose in epithelium genesis tumour.Tested using the microarray hybridization of set control sample and generate linearity curve in double-colored analysis.Then ratio (the test during each time is tested using the slope of the line generated in double-colored analysis：Control test) standardization.Then the ratio by standardization by each experiment is compared, and for identifying the cluster of gene expression.Therefore, " universal control " sample of set is not only allowed in the effective Relative gene expression judgement of the middle progress of simple two sample, and the Multi-example that it also allows for carrying out between some experiments compares.

In this experiment, create microarray using the nucleic acid probe from TAT polypeptide encoding nucleic acids sequence described herein and be hybrid with it using the RNA from kinds of tumors tissue.Normalized Ratio will be based on：One numerical value of experiment ratio is referred to as " retention ratio " (cutoffratio).Value only higher than the retention ratio is just determined as significantly.The conspicuousness of ratio is assessed according to each related noise of experiment or scattered amount, but identified usually using 1.8 times to 2 times or more large retention ratio has the candidate gene of relative overexpression compared with corresponding normal structure and/or the normal epithelial universal control of set in tumor sample.The ratio being accredited as by this way with respect to the gene being overexpressed in tumor sample is 2 times to 40 times, or even more big.By contrast, in the control experiment for being hybridized same RNA with each color mark and with itself, nearly all signal is substantially less than 1.8 times higher than the ratio observed by the gene of background.This shows, 1.8 times or bigger of the multiple change that experimental noise is extremely low and observed when ratio is more than 1.8 times is significant, and is expected to represent true, detectable between the sample analyzed and compared and reproducible differential expression.

The result of these experiments is proved, encodes the application SEQ ID NO：The mRNA of TAT113 polypeptides shown in 2 has significant overexpression (i.e. at least 2 times) in tested 75 parts of independent human colon tumor's samples more than 50% compared with both epithelium control samples that normal human colonic organizes and gathers.These data also confirm, observed overexpression compared with both corresponding normal human colonic's sample and people's epithelium control sample of set is significant, detectable and reproducible in many parts of human colon tumor's sample rooms.As described above, these numbers are it was demonstrated that the TAT113 polypeptides and code nucleic acid of the present invention can be used not only as the diagnostic criteria thing that there is human colon tumor, and also act as the potential therapeutic targets for treating these human tumours.

Embodiment 3：The quantitative analysis of TAT mRNA expression

In this determination method, use 5 ' nuclease assays (such as TaqMan ) and real-time quantitative PCR (such as Sequence Detection System  (the Perkin Elmer of ABI Prizm 7700, AppliedBiosystems Division, Foster City, CA)) find have the gene being significantly overexpressed compared with other cancerous tumours or normal non-cancerous tissue in one or more cancerous tumours.The reaction of 5 ' nuclease assays is the technology based on fluorescent PCR, and it utilizes the real-time gene expression of 5 ' exonuclease activities of Taq archaeal dna polymerases.The amplicon of PCR response features is produced using two kinds of Oligonucleolide primers (its sequence is based on target gene or est sequence).The third oligonucleotides or probe is designed to detect the nucleotide sequence between described two PCR primers.The probe can not be extended by Taq archaeal dna polymerases, and be marked with reporter (reporter) fluorescent dye and quencher (quencher) fluorescent dye.When both dyestuffs are located proximate to together as them on probe, the transmitting of any induced with laser from reporter dyestuff is that quencher dyestuff is quenched.During pcr amplification reaction, Taq archaeal dna polymerases cut probe with template dependent manner.Gained probe fragment is dissociated in the solution, and the signal of the reporter dyestuff from release is not by the quenching effect of the second fluorogen.The reporter dyestuff that a recruit then discharges a molecule is often synthesized, the qualitative and quantitative deciphering for being detected as data that reporter dyestuff is not quenched is provided the foundation.This determination method is well known in the art and conventionally used for the gene expression difference between two parts of different human tissue samples of Quantitative measurement, see, for example, Higuchi et al., Biotechnology10：413-417(1992)；Livak et al., PCR Methods Appl.4：357-362(1995)；Heid etal., Genome Res.6：986-994(1996)；Pennica et al., Proc.Natl.Acad.Sci.USA95 (25)：14717-14722(1998)；Pitti et al., Nature 396 (6712)：699-703(1998)；Bieche et al., Int.J.Cancer78：661-666(1998).

5 ' nuclease flows are carried out on real-time quantitative PCR device, such as ABI Prism 7700^TMSequence Detection.The system is made up of thermal cycler, laser, charge coupling device (CCD) camera and computer.The system expands sample on thermal cycler with 96 well formats.In amplification procedure, detected by the fluorescence signal of the fibre optics cable real-time collecting induced with laser for all 96 holes, and at CCD.The system includes being used for operational outfit and the software for analyze data.

Parent material for screening is the mRNA from a variety of different cancerous tissue separation.Accurate quantification is carried out to the mRNA, for example, passes through fluorescence.As negative control, RNA is separated from a variety of normal structures that there is identical organization type with surveyed cancerous tissue.Usually tumor sample is directly compared with the normal specimens of " matching " same organization type, means that tumour and normal specimens are derived from same individual.

5 ' nuclease assay data are initially represented as Ct, in other words threshold cycle number (threshold cycle).It is defined as the period when accumulation of reporter signal exceeds background fluorescence level.During using ACt values as comparing cancer mRNA results and normal person's mRNA results in nucleic acid samples the relative starting copy number of specific target sequence quantitative target.Relative increase due to 1 Ct unit equivalent to 1 wheel PCR cycle or relative to normally about 2 times, therefore two units are equivalent to 4 times of relative increase, 3 units are equivalent to 8 times of relative increase, the rest may be inferred, and researcher can relative fold's increase that is qualitative and quantitatively detecting mRNA expression between two or more different tissues.At this point, art-recognized this determination method is technically sensitive enough, it is sufficient to reproducibly detect at least 2 times of rise of the mRNA expression relative to normal control in human tumour sample.

Using this technology, determine in all 9/9 parts independent human colon tumor's samples, encode SEQ ID NO of the present invention：The mRNA of TAT113 polypeptides shown in 2 has notable and reproducible overexpression (i.e. at least 2 times) compared with two kinds of samples as described below：Normal human colonic's sample from different people tissue donor, and with various " match " normal human colonic tumor samples of the tumor sample from same people tissue donor.Therefore, as described above, these numbers are it was demonstrated that the TAT113 polypeptides and code nucleic acid of the present invention can be used not only as the diagnosis marker that there is human colon tumor, and also act as the potential therapeutic targets for treating those human tumours.

Embodiment 4：In situ hybridization

In situ hybridization is the strong and general technology for detecting and positioning for cell or tissue prepared product nucleic acid sequence.It can be used for the position for for example identifying gene expression, analyzes the Tissue distribution of transcription, identification and positioning virus infection, tracks the change of specific mRNA synthesis, and helps chromosome mapping.

In situ hybridization follows Lu and Gillett, Cell Vision 1：The optimization version of code is carried out in 169-176 (1994), is generated using PCR³³P mark riboprobes (riboprobe).In brief, formalin is fixed, people's histotomy of FFPE, take off paraffin, in 37 DEG C of isolating proteins 15 minutes in Proteinase K (20g/ml), and such as Lu and Gillett, further processing same as above is to carry out in situ hybridization.From PCR primer generation [³³- P] UTP marks antisense RNA probe, and in 55 DEG C of hybridized overnights.Slide is immersed into Kodak NTB2 core mark emulsions and exposed 4 weeks.

³³P- riboprobes are synthesized

By 6.0 μ l (125mCi)³³P-UTP (Amersham BF 1002, SA ＜ 2000Ci/mmol) traditional vacuum evaporation drying.Contain drying to each³³Following component is added in P-UTP pipe：

2.0 μ l 5x transcription buffers

1.0μl DTT(100mM)

2.0 μ l NTP mixtures (2.5mM：10 μ l every kind of 10mM GTP, CTP ＆ ATP+10 μ l H2O)

1.0μl UTP(50μM)

1.0μl Rnasin

1.0 μ l DNA profilings (1 μ g)

1.0μl H₂O

1.0 μ l RNA polymerases (for PCR primer, typically T3=AS, T7=S)

Pipe is incubated 1 hour in 37 DEG C.1.0 μ l RQ1 DNA enzymatics are added, are then incubated 15 minutes in 37 DEG C.90 μ l TE (10mM Tris pH 7.6/1mM EDTA pH 8.0) are added, by mixed liquor with being pipetted on DE81 paper.Surplus solution is added in Microcon-50 ultra filtration units, rotated (6 minutes) with program 10.Filter element is upside down on second pipe, and rotated (3 minutes) with program 2.After last rotation is reclaimed, 100 μ l TE are added.1 μ l end-products are pipetted on DE81 paper, and counted in 6ml Biofluor II.

By probe on TBE/ urea gels electrophoresis.1-3 μ l probes or 5 μ l RNA Mrk III are added in 3 μ l sample-loading buffers.After being heated 3 minutes on 95 DEG C of heat blocks, probe is immediately placed on ice.Gel pore is rinsed, sample is added, and in 180-250 volts of electrophoresis 45 minutes.Gel is wrapped in saran wrapping papers, and in -70 DEG C of refrigerators with intensifying screen to XAR exposures 1 hour to overnight.

³³P- hybridizes

A.The pretreatment of freezing microtome section

Slide is taken out from refrigerator, is placed in aluminium dish, and is melted 5 minutes in room temperature.Disk is placed 5 minutes in 55 DEG C of incubators and condenses (condensation) to reduce.Slide is fixed 10 minutes on ice in fume hood in 4% paraformaldehyde, and in 0.5x SSC (25ml 20x SSC+975ml SQ H₂O cleaned 5 minutes in room temperature in).In 0.5 μ g/ml Proteinase Ks (12.5 μ l 10mg/ml liquid storages dilute in the RNase buffer solution without RNase that 250ml is preheated) in after 37 DEG C of isolating proteins 10 minutes, section is cleaned 10 minutes in 0.5x SSC in room temperature.Section is dehydrated in 70%, 95%, 100% ethanol, 2 minutes every time.

B.The pretreatment of specimens paraffin embedding slices

Slide is taken off into paraffin, SQ H are placed on₂In O, and rinsed twice in room temperature in 2x SSC, 5 minutes every time.Will section in 20 μ g/ml Proteinase Ks (500 μ l 10mg/ml dilute in RNase buffer solutions of the 250ml without RNase, 37 DEG C 15 minutes)-Human embryo；Or isolating protein in 8x Proteinase Ks (100 μ l dilute in 250mlRNA enzyme buffer liquids, 37 DEG C 30 minutes)-formalin tissue.Then rinse, and be dehydrated as described above in 0.5xSSC.

C.Prehybridization

Slide is placed on and is lined with the plastic casing with the filter paper of Box buffer solutions (4x SSC, 50% formamide) saturation.

D.Hybridization

By each slide 1.0 × 10⁶Cpm probes and 1.0 μ l tRNA (50mg/ml liquid storages) are heated 3 minutes in 95 DEG C.By slide in cooled on ice, each slide adds 48 μ l hybridization buffers.After vortex oscillation, 50 μ l are added into 50 μ l prehybridization solutions on slide³³P mixed liquors.Slide is incubated overnight in 55 DEG C.

E.Cleaning

Use 2x SSC, EDTA (400ml 20x SSC+16ml 0.25M EDTA, V_f=4L) in room temperature clean within 2 times 10 minutes, handled 30 minutes with RNaseA (500 μ l 10mg/ml dilute=20 μ g/ml in 250ml RNase buffer solutions) with after 37 DEG C.Slide 2x SSC, EDTA are cleaned 2 times 10 minutes in room temperature.Stringent wash condition is as follows：55 DEG C, 0.1x SSC, EDTA (20ml 20x SSC+16mlEDTA, V_f=4L), 2 hours.

F.Oligonucleotides

In-situ study is carried out to a variety of DNA sequence dnas disclosed herein.Obtain makes it complementary with nucleic acid shown in accompanying drawing (or its complementary series) for the oligonucleotides of these analyses.

G.As a result

To encoding SEQ ID NO herein：The nucleic acid of TAT113 polypeptides shown in 2 has carried out situ Analysis.The result of these analyses shows that in the independent primary human colon carcinoma's sample of 10/22 part analyzed and 6/9 part of independent metastatic colon cancer sample strongly expressed occurs for TAT113 genes.On the contrary, TAT113 detectable expression (or significantly lower expression) is not observed in all normal colonic tissue's samples of analysis.

Embodiment 5：The differential expression of TAT polypeptides is verified and analyzed by GEPIS

The TAT polypeptides that may be as above accredited as tumour antigen described in one or more embodiments are carried out to verify and analyze as follows.EST (EST) DNA databases (LIFESEQ , Incyte Pharmaceuticals, Palo Alto, CA) are searched for using GEPIS and identify purpose est sequence.Insilico gene expression profiles analysis (GEPIS) is the bioinformatics method of Genentech companies research and development, and for identifying, gene interested is to obtain new treatment of cancer target.GEPIS determines gene expression profile using substantial amounts of est sequence and library information.GEPIS can determine the expression overview of gene according to it with the mutual proportionate relationship of the occurrence number in est database, and it is operated by integrating LIFESEQ  EST relational databases and Genentech Proprietary Informations in strict and statistically significant mode.In this embodiment, the tumour antigen new with cross validation is identified with GEPIS, although GEPIS can perform very special analysis or widely screen task by setting.For initial screening, the est sequence related to the expression in a certain particular organization interested or multiple tissues (often tumor tissues interested) is identified from LIFESEQ  databases using GEPIS.Then the est sequence (or consensus sequence obtained from being compared by a variety of related and overlapping est sequence obtained to initial screening) identified in this initial screening is screened, at least one membrane-spanning domain is whether there is to identify in coded protein.Finally, overview is expressed using the GEPIS complete tissues for generating various sequences interested.Using this kind of screening bioinformatics, identify a variety of TAT polypeptides (and its coding nucleic acid molecule) has significant overexpression in certain specific cancer or some cancers compared with other cancers and/or normal non-cancerous tissue.The assessment of GEPIS hits is based on the expression in some standards, including such as tissue specificity, tumour-specific and normal necessary tissue and/or normal proliferating tissues.

Utilize GEPIS expression analysis described above, it is determined that coding SEQ ID NO：The mRNA of TAT113 polypeptides shown in 2 has notable, reproducible and detectable overexpression in human colon tumor's sample compared with corresponding normal colonic tissue.Therefore, this paper SEQ ID NO：TAT113 polypeptides shown in 2 and encode the nucleic acid of the polypeptide and can be used to qualitatively and quantitatively determine this paper SEQ ID NO as excellent target：TAT113 polypeptides shown in 2 and expressions of its mRNA in various mammalian tissue samples is encoded, so as to carry out qualitative and quantitative comparison between these tissues.Therefore, this paper SEQ ID NO：TAT113 polypeptides shown in 2 and encode the polypeptide nucleic acid can be used for diagnose mammal in tumour excellent target.In addition, due to this analytical proof TAT113 polypeptides in some human tumours its corresponding health adult tissue compared to have it is notable, reproducible and it is detectable be overexpressed, so TAT113 polypeptides turn into the excellent target that can be used for the therapeutic treatment of such tumour in mammal.

Embodiment 6：With reference to the preparation of TAT113 antibody

Technology for generating monoclonal antibody is known in the art, and is described in such as Goding, ibid.Adoptable immunogene includes the TAT polypeptides, the fusion protein of the polypeptide containing TAT and the cell that restructuring TAT polypeptides are expressed on cell surface of purifying.Those skilled in the art can complete the selection of immunogene without excessively experiment.

It is subcutaneous or mouse, such as Balb/c is immunized in intraperitoneal injection with 1-100 Micrograms using the TAT immunogenes emulsified in complete Freund's adjuvant.Or, immunogene is emulsified in MPL-TDM adjuvants (RibiImmunochemical Research, Hamilton, MT) and is expelled in the rear palmula of animal.After 10 to 12 days, the supplementary immunization emulsified in selected adjuvant is former to immune mouse progress booster immunization.Hereafter, in time several weeks, also booster immunization can be carried out to mouse by supplementary immunization injection.Blood serum sample periodically can be obtained from mouse by bloodletting after eye socket, for being tested in being determined in ELISA to detect anti-TAT antibody.

After suitable antibody titer is detected, last TAT can be carried out in the animal of " positive " to antibody and be injected intravenously.After 3 to 4 days, put to death mouse and harvest splenocyte.Then by splenocyte and selected rat bone marrow tumour cell system (such as available from ATCC, No.CRL 1597 P3X63AgU.1) fusion (using 35% polyethylene glycol).Fusion produces hybridoma, then these hybridomas can be assigned in 96 hole tissue culturing plates, wherein equipped with HAT (hypoxanthine, aminopterin-induced syndrome and thymidine) culture mediums to suppress the propagation of non-fused cell, myeloma heterozygote and splenocyte heterozygote.

Examination hybridoma is directed to TAT reactivity in ELISA.Secretion is directed to the determination of " positive " hybridoma of TAT required monoclonal antibody within the skill of the art.

Positive hybridoma cell intraperitoneal injection can be generated the ascites containing anti-TAT monoclonal antibodies into homogenic Balb/c mouse.Or, hybridoma can be cultivated in tissue culture flasks or roller bottle.The follow-up gel exclusion chromatography of ammonium sulfate precipitation can be used to complete for the purifying of the monoclonal antibody generated in ascites.Or, can be using the affinity chromatography combined based on antibody with albumin A or Protein G.

Using techniques described above, 12 kinds of respectively different hybridoma cell lines are generated, each is all produced with reference to SEQ ID NO：The monoclonal antibody of TAT113 polypeptides shown in 2.This 12 kinds of hybridoma cell lines are herein referred to as 12E5.1.1 (producing monoclonal antibody 12E5), 6G10.1.1 (produces monoclonal antibody 6G10), (17C12.7.8 producing monoclonal antibody 17C12), (1B5.1.3 producing monoclonal antibody 1B5), 6H10.3.11 (produces monoclonal antibody 6H10), (17H5.25.1 producing monoclonal antibody 17H5), 15B6.4.1 (produces monoclonal antibody 15B6), (11A12.3.2 producing monoclonal antibody 11A12), 13A7.1.8 (produces monoclonal antibody 13A7), (4D1.1.2.1 producing monoclonal antibody 4D1), 14A3.1.1 (producing monoclonal antibody 14A3) and 10F11.11.11.11 (producing monoclonal antibody 10F11).Use the well-known and conventional technology used, such as western blot, elisa assay, the FACS sortings for the cell for expressing TAT113 polypeptides are analyzed and/or immunohistochemical analysis, it has proved that the monoclonal antibody combination SEQ ID NO that this 12 kinds of hybridoma cell lines are produced：TAT113 polypeptides shown in 2.In this 12 kinds hybridoma cell lines for producing the anti-TAT113 monoclonal antibodies of feature, there is the clause of 2 kinds (hybridoma clone 12E5.1.1 and 6G10.1.1) according to budapest treaty to be preserved in American type culture collection (American Tissue Type Collection, Manassas, VA), as detailed further below.

Embodiment 7：Competitive binding analysis and epitope mapping

TAT113 epitopes (Fendly et al., the Cancer Research 50 that the monoclonal antibody is combined is determined by standard competition binding analysis：1550-1558(1990)).Use PANDEX^TMScreenMachine carries out fluorescent quantitation, and cross-blocks research has been carried out by the direct fluorescent challenge body on expression TAT113 entire engineered PC3 cells.Using set up flow (Wofsy et al., SelectedMethods in Cellular Immunology, p.287, Mishel and Schiigi (eds.) San Francisco：WJ.Freeman Co. (1980)) various monoclonal antibodies and fluorescein isothiocynate (FITC) are coupled.The confluent monolayer Trypsin Induced of TAT113 PC3 cells will be expressed, will be cleaned once, and with 1.75 × 10⁶Individual cell/ml, which is resuspended in, cold contains 0.5% bovine serum albumin (BSA) and 0.1%NaN₃PBS in.The latex particle (IDC, Portland, OR) of final concentration 1% is added to reduce PANDEX^TMThe obstruction (clogging) of plate film.20 μ l cell suspending liquids and 20 μ the l monoclonal antibody (100 μ g/ml to 0.1 μ g/ml) purified are added to PANDEX^TMIn the hole of plate, and in incubated on ice 30 minutes.The FITC labeled monoclonal antibodies of the 20 predetermined dilution factors of μ l are added in each hole, incubated 30 minutes, cleaning, and pass through PANDEX^TMScreen Machine quantitative fluorescences.If the control of nothing to do with monoclonal antibody is compared, in identical antibody concentration, the combination of another monoclonal antibody is blocked 40% or more by certain monoclonal antibody, then think that they have common epitope.In this experiment, TAT113 epitopes B, A, D, E, F, A, B, A, A, A and C are assigned to monoclonal antibody 12E5,17C12,1B5,6H10,17H5,15B6,11A12,13A7,4D1,14A3 and 10F11 respectively.Using this determination method, those of ordinary skill in the art can identify other monoclonal antibodies with monoclonal antibody combination same epitope described above.

Embodiment 8：It is coupled the preparation of the combination TAT113 of toxin antibody

(the Baldwin et al. when the medicament that systemic administration is not coupled may produce unacceptable cytotoxicity levels to normal cell as the tumour cell to attempting to eliminate, (1986) Lancet (Mar.15,1986) pp.603-05；Thorpe, (1985) " Antibody Carriers Of Cytotoxic Agents InCancer Therapy：A Review, " in Monoclonal Antibodies ' 84：Biological AndClinical Applications, Pinchera et al. (eds.), pp.475-506), topical delivery cytotoxic agent or cytostatics (medicine that tumour cell is killed or suppressed i.e. in treatment of cancer) (Payne (2003) Cancer Cell 3 are come using antibody-drug conjugates (ADC) (i.e. immune conjugate)：207-212；Syrigosand Epenetos(1999)Anticancer Research 19：605-614；Niculescu-Duvaz andSpringer(1997)Adv.Drug Del.Rev.26：151-172；US 4,975,278) tumour is delivered to while drug moiety targeting can be made, and in its intracellular accumulation.Thus attempt to realize the maximum effect under the conditions of minimum toxicity.Design and improvement ADC effort are concentrated in the selectivity and medicine connection and drug release characteristics of monoclonal antibody (mAb).Both polyclonal antibody and monoclonal antibody have report (the Rowland et al., (1986) Cancer Immunol.Immunother., 21 tactful for these：183-87).Medicine used in these methods includes daunomycin, Doxorubicin, methopterin and eldisine (Rowland et al., (1986) ibid).Toxin used in Antibody-toxin conjugate includes bacteriotoxin, such as diphtheria toxin, phytotoxin such as ricin, small molecule toxins such as geldanamycin (Mandler et al. (2000) J.of the Nat.Cancer Inst.92 (19)：1573-1581；Mandler et al.(2000)Bioorganic & Med.Chem.Letters 10：1025-1028；Mandler et al.(2002)Bioconiugate Chem.13：786-791), maytansinoids (EP 1391213；Liu et al., (1996) Proc.Natl.Acad.Sci.USA 93：8618-8623) with Calicheamicin (Lode et al. (1998) Cancer Res.58：2928；Hinman et al.(1993)Cancer Res.53：3336-3342).

It is well-known in the art and conventional use by the technology that toxin and the antibody purified are connected to produce antibody-drug conjugates.The realization for example, monoclonal antibody of purifying and toxin DM1 coupling can such as get off.By the antibody of purifying with N- succinimide bases -4- (2- pyridylthios) valerate derivatizations to introduce dithiopyridines base.Antibody (376.0mg, 8mg/mL) in 50mM kaliumphosphate buffers (pH 6.5) of the 44.7ml containing NaCl (50mM) and EDTA (1mM) is handled with SPP (5.3 molar equivalents in 2.3ml ethanol).Under argon gas after environment temperature is incubated 90 minutes, by reaction mixture by using 35mM sodium citrates, the Sephadex G25 post gel filtrations of 154mM NaCl and 2mM EDTA balances.It is then combined with containing antibody fractions and determines.Antibody-SPP-Py (337.0mg, with releasable 2- thiopyridines group) is diluted to final concentration 2.5mg/ml with above-mentioned 35mM sodium citrate buffer solutions pH6.5.Then the DM1 (1.7 equivalents, 16.1 moles) added into antibody-solutions in 3.0mM- dimethyl acetamides (DMA, the 3%v/v in final reaction mixture).Homologation reaction is carried out 20 hours in environment temperature under argon gas.Reaction solution is loaded into and uses 35mM sodium citrates, on the Sephacryl S300 solvent resistant columns (5.0cm × 90.0cm, 1.77L) that 154mM NaCl, pH 6.5 is balanced.Flow velocity is 5.0ml/min, have collected 65 parts of fractions (every part of 20.0ml).Merge fraction and determine, wherein determining the number (p ') of the DM1 drug molecules of each antibody molecule connection by measuring 252nm and 280nm absorbance.

For exemplary purposes, the monoclonal antibody of purifying and toxin DM1 coupling can also such as get off realization.By the antibody of purifying with succinimide base 4- (N- maleimidomehyls) hexamethylene -1- carboxylates (SMCC, Pierce Biotechnology, Inc) derivatization to introduce SMCC joints.Antibody is handled with 20mg/ml in 50mM potassium phosphates/50mM sodium chloride/2mM EDTA, pH 6.5 with 7.5 molar equivalent SMCC (20mM in DMSO, 6.7mg/ml).Under argon gas after environment temperature is stirred 2 hours, reaction mixture is filtered by using 50mM potassium phosphates/50mM sodium chloride/2mM EDTA, pH 6.5 Sephadex G25 posts balanced.Merge containing antibody fractions and determine.Then antibody-SMCC is diluted to final concentration 10mg/ml with 50mM potassium phosphates/50mM sodium chloride/2mM EDTA, pH 6.5, and reacted with the 10mM DM1 solution (1.7 equivalents, it is assumed that 5 SMCC/ antibody, 7.37mg/ml) in dimethyl acetamide.Reaction solution is stirred 16.5 hours in environment temperature under argon gas.Then coupling reaction mixed liquor is filtered with 1x PBS pH 6.5 by Sephadex G25 solvent resistant columns (1.5 × 4.9cm).Then DM1/ antibody ratio (p ') is measured by 252nm and 280nm absorbance.

Typically, cytotoxic drug is coupled on antibody by the lysine residue usually largely existed on antibody.People also by be present in or the engineered antibody to purpose in mercapto realize coupling.For example, cysteine residues are imported protein to form site (Better et al. (1994) J.Biol.Chem.13 for part covalent attachment by technique for gene engineering：9644-9650；Bernhard et al.(1994)Bioconjugate Chem.5：126-132；Greenwood et al.(1994)TherapeuticImmunology 1：247-255；Tu et al.(1999)Proc.Natl.Acad.Sci.USA96：4862-4867；Kanno et al.(2000)J.of Biotechnology 76：207-214；Chmura et al.(2001)Proc.Nat.Acad.Sci.USA 98(15)：8480-8484；U.S. Patent No. 6,248,564).Once there are free cysteine residues in purpose antibody, so that it may which toxin is connected to the site.For example; it will be dissolved in after DMSO medicine linker reagents maleimide capryl-monomethyl auristatin E (MMAE) i.e. MC-MMAE, maleimide capryl-monomethyl auristatin F (MMAF) i.e. MC-MMAF, MC-val-cit-PAB-MMAE or MC-val-cit-PAB-MMAF dilute with concentration known in acetonitrile and water, add in the cysteine derivatives antibody in the phosphate buffered saline (PBS) of cooling.After about 1 hour, add that excessive maleimide is with terminating reaction and closes any unreacted antibody mercapto.Reaction mixture is concentrated by centrifugal ultrafiltration, the antibody for being coupled toxin is purified and desalination in PBS by the elution of G25 resins, aseptically filtered by 0.2 μm of filter, and refrigerated storage.

Furthermore, it is possible to which the free cysteine modified with double-maleimide reagent BM (PEO) 4 (Pierce Chemical) on selected antibody, leaves unreacted Maleimido on the surface of antibody.This realization that can such as get off, it is 10mM that BM (PEO) 4 is dissolved in into 50% ethanol/water mixed liquor to concentration, added to 10 times of molar excess in phosphate buffered saline (PBS) in the solution of the antibody containing about 1.6mg/ml (10 micromole) concentration, and homologation reaction 1 hour.Pass through the excessive BM (PEO) 4 of the gel filtration removing in 30mM citrates, 150mM NaCl, pH6 buffer solution.The DM1 of about 10 times of molar excess is dissolved in dimethyl acetamide (DMA), and added in the antibody-BMPEO intermediate products.Also drug moiety reagent can be dissolved using dimethylformamide (DMF).The reaction of homologation reaction mixed liquor is stayed overnight, and then gel filtration or is dialysed to PBS to remove unreacted medicine.High molecular weight aggregates are removed by the gel filtration in PBS on S200 posts, the antibody-BMPEO-DM1 conjugates of purifying are produced.

Using this paper and elsewhere description, the well-known and conventional technology used, anti-TAT113 monoclonal antibodies 12E5 (prepared as described in example 6 above and be preserved in ATCC as described below) and MC-val-cit-PAB-MMAE are coupled.By the toxin conjugated antibody purification, and it is referred to herein as " 12E5-MMAE ".

Embodiment 9：Cell in vitro kills determination method

Standard expression vectors and clone technology can be used to obtain for the mammalian cell for expressing purpose TAT polypeptides.Or, many tumor cell lines for having expression purpose TAT polypeptides are publicly available, such as by ATCC, and standard ELISA or facs analysis can be used to carry out general survey.Then the anti-TAT polypeptides monoclonal antibody derivative of toxin (and its coupling) can be used to be measured, to determine ability that the antibody kills the cell of expression TAT polypeptides in vitro.

With regard to the present invention specifically, the PC3 derived cells system (herein referred to as PC3-gD-MDP) of the stable expression TAT113 polypeptides on its cell surface is obtained using standard technique engineering, and uses the TAT113 expression of polypeptides of standard FACS cell sortings, ELISA and immunohistochemical analysis confirmation PC3-gD-MDP cells.The monoclonal antibody 12E5-MMAE that killing determination method measure coupling using cell in vitro has MMAE causes the ability of PC3-gD-MDP cell deaths, and the determination method uses following scheme (Promega Corp.Technical Bulletin TB288；Mendoza et al.(2002)Cancer Res.62：5485-5488)：

1. the cell culture in the growth medium of 50 μ l equal portions is assigned in each hole of 96 transparent orifice plates of wall, the cell culture contains about 10⁴Individual cell (PC3-gD-MDP cells or the untransfected PC3 cells for not expressing TAT113).Set in addition and contain 50 μ l growth mediums but not celliferous control wells.

2. the anti-interleukin-8 monoclonal antibody for not combining TAT113 that antibody 12E5-MMAE or MMAE are coupled is added in each hole respectively, the μ l of volume 50, concentration range 0.0001-100 μ g/ml, and by plate in 37 DEG C and 5%CO₂Incubate 3-5 days.

3. plate is balanced to room temperature, it is necessary to about 30 minutes.

4. CellTiter-GloLuminescent Cell Viability Reagent (the luminescent cell viability reagents with cell culture growth medium volume equal volume in hole are added into each hole, Promega Corp.), and plate is shaken on orbital shaker to 2 minutes to induce lysis.

5. by plate in incubation at room temperature 10 minutes with stabilized illumination signal.

6. recorded on the photometer equipped with Tropix Winglow programs and light and report into RLU=relative light units.

The result obtained from said determination proves that the cell that 12E5-MMAE antibody is capable of induced expression TAT113 polypeptides is dead in the way of antibody dependent.In particular, 12E5-MMAE and IL-8-MMAE does not induce the notable death of untransfected PC3 cells in 1 μ g/ml and following antibody concentration.In the antibody concentration higher than 1 μ g/ml, the amount of untransfected PC3 cell deaths is linearly increasing with antibody concentration, and is in the way of independent of antibody.Therefore, it appears that untransfected PC3 cells are the elevated non-specific results of MMAE endotoxin levels present in reaction mixture in the death of the antibody concentration higher than 1 μ g/ml, rather than the binding specificity of used antibody function.But, for the PC3-gD-MDP cells of stable expression TAT113 polypeptides, although IL-8-MMAE is to kill the ability identical pattern inducing cell death of untransfected PC3 cells with antibody, 12E5-MMAE induces significant cell killing in the low antibody concentration up to 0.001 μ g/ml.In fact, in 1 μ g/ml antibody concentration (now the specific IL-8-MMAE antibody of non-TAT113 does not show significant cell killing), 12E5-MMAE kills almost all of PC3-gD-MDP cells.Therefore, these numbers are it was demonstrated that monoclonal antibody 12E5 combines those cell deaths expressed the TAT113 polypeptides on cell surface and it can be induced to be combined.

In Section 2 experiment, the ability that monoclonal antibody 12E5-MMAE kills COLO205 cells derived from colon cancer (applicant proves that the cell expresses TAT113 polypeptides by FACS cell sorting assays) in vitro is determined using said determination method.It is consistent with the result presented above for PC3 derived cells system, IL-8-MMAE just starts to show significant COLO205 cell killings until cell concentration higher than about 1 μ g/ml, wherein cell killing level is linearly raised with antibody concentration, and is in the way of independent of antibody.However, monoclonal antibody 12E5-MMAE is to start to induce significant COLO205 cell deaths in the low antibody concentration up to about 0.1 μ g/ml.In fact, in 1 μ g/ml antibody concentration (now the specific IL-8-MMAE antibody of non-TAT113 does not show significant cell killing), it was observed that the COLO205 cell killing levels of the 12E5-MMAE inductions of highly significant.Therefore, these data are demonstrated again that, monoclonal antibody 12E5 combines those cell deaths expressed the TAT113 polypeptides on cell surface and it can be induced to be combined.

Embodiment 10：Interior tumor cell kills determination method

In order to test coupling or be not coupled toxin anti-TAT polypeptides monoclonal antibody effect inducing death of neoplastic cells in vivo ability, following scheme can be used.

To one group of nude mouse in flank portion subcutaneous vaccination 5 × 10⁶The tumor promotion cell of individual expression TAT polypeptides.When tumour reaches 100-200mm³Between mean tumour volume when, mouse is divided equally into 5 groups and is handled as follows：

1st group-PBS control solvent, weekly using once, continuing 4 weeks；

2nd group-non-specific control antibody, 1mg/kg, weekly using once, continuing 4 weeks；

3rd group-non-specific control antibody, 3mg/kg, weekly using once, continuing 4 weeks；

4th group-specific anti-TAT polypeptide antibodies, 1mg/kg, weekly using once, continuing 4 weeks；

5th group-specific anti-TAT polypeptide antibodies, 3mg/kg, weekly using once, continuing 4 weeks.

Then mean tumour volume can be determined in the mouse of each treatment group during certain and determines effect of antibody.

Embodiment 11：TAT as hybridization probe purposes

The nucleotide sequence that following method describes coding TAT is used for the purposes of tumour presence in such as diagnosis mammal as hybridization probe.

The homologous dna (such as DNA of those codings TAT naturally occurring variant) that the DNA of coded sequence comprising total length disclosed herein or mature T AT can also be used to screen in people's tissue cDNA library or people's tissue gene group library as probe.

The hybridization and cleaning of filter membrane containing any library DNA are carried out under following high stringency conditions.Radiolabeled TAT derives the hybridization of probe and filter membrane in 50% formamide, 5x SSC, 0.1%SDS, 0.1% sodium pyrophosphates, 50mM sodium phosphates pH 6.8, is carried out 20 hours in 42 DEG C in the solution of 2x DenhardtShi solution and 10% dextran glucosides.The cleaning of filter membrane is in the 0.1x SSC and 0.1%SDS aqueous solution in 42 DEG C of progress.

Then the DNA that standard technique known in the art can be used to identify with encoding full leng native sequences TAT has the DNA of expectation sequence homogeneity.

Embodiment 12：Expression of the TAT in Escherichia coli

This embodiment passes through preparation of the recombination expression in Escherichia coli exemplified with the TAT of nonglycosylated form.

First by selected PCR primer amplification coding TAT DNA sequence dna.Primer should include restriction enzyme sites corresponding with the restriction enzyme sites on selected expression vector.A variety of expression vectors can be used.One example of suitable carrier is that pBR322 (is derived from Escherichia coli；Refering to Bolivar et al., Gene, 2：95 (1977)), its gene comprising ampicillin and tetracyclin resistance.By carrier limitation enzymic digestion and dephosphorylation.Then the sequence that PCR is expanded is connected in carrier.Carrier preferably comprises the sequence of coding antibiotics resistance gene, trp promoters, polyhistidine targeting sequencing (including preceding 6 STII codons, polyhistidine sequence and enterokinase cleavage site point), TAT code areas, λ transcription terminators and argU genes.

Then by Sambrook et al., ibid described in the method selected coli strain of connection mixed liquor conversion.Transformant is identified by the ability grown on LB flat boards, antibiotic resistant colonies are then selected.Separable DNA, and confirmed by restriction analysis and DNA sequencing.

Selected clone can liquid medium within, be such as supplemented with overnight incubation in the LB culture mediums of antibiotic.Then available overnight culture is inoculated with large-scale culture thing.Then make cell growth to desired optical density, promoter is expressed during this period and is opened.

Cell was further cultured for after some hours, can be by the way that cell be harvested by centrifugation.It various reagents known in the art can be used to dissolve by the cell pellet being harvested by centrifugation, then can use metal chelating column to purify the TAT protein of dissolving under conditions of allowing protein to combine closely.

Following flow can be used, in expression in escherichia coli TAT in the form of with polyhistidine label.First with selected PCR primer amplification coding TAT DNA.Primer includes restriction enzyme sites corresponding with the restriction enzyme sites on selected expression vector, and provides other useful sequences effectively with the proteolytic cleavage of reliable translation initiation, the fast purifying on metal chelating column and enterokinase.Then the sequence of polyhistidine mark PCR expanded is connected in expression vector, and the escherichia coli host based on bacterial strain 52 (W3110 fuhA (tonA) lon galE rpoHts (htpRts) clpP (lacIq)) is converted with the carrier.Transformant is reached into 3-5 in the LB culture mediums of the carbenicillin containing 50mg/ml in 30 DEG C of shaken cultivations to O.D.600 first.Then culture (is led to and prepared mixing following component in CRAP culture mediums：3.57g (NH in 500mL water₄)₂SO₄, 0.71g sodium citrates 2H₂O, 1.07g KCl, 5.36g Difco yeast extracts, 5.36g Sheffield hycase SF, and 110mM MPOS pH 7.3,0.55% (w/v) glucose and 7mM MgSO₄) in 50-100 times of dilution, and in 30 DEG C of shaken cultivations about 20-30 hours.Sample is taken out to reach come proof list by SDS-PAGE analyses, and by the centrifugation of culture main body with sedimentation cell.Cell pellet is frozen up to purifying and refolding.

E. coli cell masses (6-10g sediments) from 0.5 to 1 liter of zymotic fluid are resuspended in the 7M guanidines of 10 times of volumes (w/v), the buffer solutions of 20mM Tris pH 8.It is respectively 0.1M and 0.02M to add solid sodium sulfite and sodium tetrathionate to final concentration, and solution is stayed overnight in 4 DEG C of agitations.This step produces the denatured protein that all cysteine residues are closed by sulfurous acylating acid (sulfitolization).Solution is centrifuged 30 minutes in Beckman ultracentrifuges with 40,000rpm.Supernatant is diluted with the metal chelate column buffer (6M guanidines, 20mM Tris pH 7.4) of 3-5 times of volume, and passes through 0.22 zut filter to clarification.The extract of clarification is added on the 5mlQiagen Ni-NTA metal chelating columns balanced in metal chelate column buffer.With imidazoles containing 50mM (Calbiochem, Utrol grade) pH7.4 other buffer solution for cleaning pillar.With the buffer solution eluted protein matter of the imidazoles containing 250mM.Merge containing the fraction for expecting protein and be stored in 4 DEG C.Using the extinction coefficient calculated according to the amino acid sequence of protein, its concentration is estimated in 280nm absorbance according to it.

Make its refolding by the way that protein is diluted in the refolding buffers of Fresh, refolding buffers Tris containing the 20mM pH 8.6,0.3M NaCl, 2.5M urea, 5mM cysteines, 20mM glycine and 1mM EDTA.Selection refolding volume causes final protein concentration between 50 to 100 μ g/ml.Refolding solution is gently stirred 12-36 hours in 4 DEG C.By adding TFA to final concentration 0.4% (pH about 3) termination refolding reaction.Before protein is further purified, by solution by 0.22 zut filter, and acetonitrile is added to final concentration 2-10%.By refolding protein in the anti-phase column chromatographies of PorosR1/H, using 0.1%TFA as mobile phase buffer solution, and with 10 to 80% acetonihile gradient elution.The aliquot of fraction with A280 absorbances is analyzed on sds page, and merges the fraction containing homogeneous refolding protein.Generally, the correct refolding form of most protein is eluted in minimum acetonitrile concentration, because those forms are most compact, makes its hydrophobic interior from the interaction with reversed-phase resin.Aggregated forms are usually eluted in higher acetonitrile concentration.Except by the false folding form of protein, with expecting form dissociation, phase inverting step also removes the endotoxin in sample.

Merge the fraction containing the TAT polypeptides for expecting to fold, and acetonitrile is removed with the gentle nitrogen stream of alignment solution.Protein is formulated into the 20mM HepespH 6.8 of sodium chloride containing 0.14M and 4% mannose, and be sterile filtered by dialysing or carrying out gel filtration using G25 Superfine (Pharmacia) resin balanced in buffer solution is prepared.

Embodiment 13：Expression of the TAT in mammalian cell

This embodiment passes through preparation of the recombination expression in mammalian cell exemplified with the TAT of potential glycoforms.

Expression vector is used as using carrier pRK5 (referring to EP 307,247 disclosed in 15 days March in 1989).Be optional that, using such as Sambrook et al., ibid described in connection method, TAT DNA are connected into selected limitation enzymic digestion to allow to insert in TAT DNA pRK5.Resulting vehicle is referred to as pRK5-TAT.

In one embodiment, selected host cell can be 293 cells.By the cell of people 293 (ATCC CCL 1573) in tissue culture dishes be supplemented with hyclone and the culture medium such as the DMEM of optional nutritional ingredient and/or antibiotic in cultivate to converging.About 10 μ g pRK5-TAT DNA and about 1 μ g are encoded to DNA (Thimmappaya the et al., Cell, 31 of VA rna genes：543 (1982)) mixing, and it is dissolved in 500 μ l 1mM Tris-HCl, 0.1mM EDTA, 0.227M CaCl₂In.500 μ l 50mM HEPES (pH 7.35), 280mM NaCl, 1.5mM NaPO are added dropwise into this mixed liquor₄, and form precipitation within 10 minutes in 25 DEG C of processes.Sediment is suspended and is added in 293 cells, it is settled 4 hours in 37 DEG C.Suck culture medium and add the PBS that 2ml contains 20% glycerine with 30 second time.Then clean 293 cells with serum free medium, add fresh culture, and by cell culture about 5 days.

About 24 hours after transfection, culture medium is removed, and with culture medium (independent) or containing 200 μ Ci/ml³⁵S- cysteines and 200 μ Ci/ml³⁵The culture medium displacement of S- methionines.After incubating 12 hours, collection condition culture medium is concentrated, and be added on 15%SDS gels on rotary filter.Can be by the gel drying after processing, and a period of time selected to exposure is to show the presence of TAT polypeptides.Culture containing transfectional cell can be further incubated for (in serum free medium), and the test media in selected bioassary method.

In a kind of alternative technique, Somparyrac et al., Proc.Natl.Acad.Sci., 12 can be used：The dextran glucosides method of 7575 (1981) description instantaneously imports TAT in 293 cells.293 cells are cultivated in Spinner flask to maximal density, 700 μ g pRK5-TAT DNA are added.First by centrifuging from Spinner flask concentrating cells and using PBS.DNA- dextran sediments are incubated 4 hours in cell pellet.Cell is handled 90 seconds with 20% glycerine, is cleaned with tissue culture medium (TCM), is placed again into the Spinner flask equipped with tissue culture medium (TCM), 5 μ g/ml bovine insulins and 0.1 μ g/ml ox transferrins.After about 4 days, conditioned medium is centrifuged and filtered to remove cell and fragment.Then it can such as be dialysed by any selected method and/or column chromatography contains expressed TAT sample to concentrate and purify.

In another embodiment, TAT can be expressed in Chinese hamster ovary celI.Usable known agent such as CaPO₄Or pRK5-TAT is transfected into Chinese hamster ovary celI by DEAE- dextrans.As described above, cell culture can be incubated, and with culture medium (single) or containing radioactively labelled substance such as³⁵The culture medium replacement medium of S- methionines.After the presence for determining TAT polypeptides, serum free medium replacement medium can be used.Preferably, culture is incubated about 6 days, then harvests conditioned medium.Then it can concentrate and purify the nutrient solution containing expressed TAT by any selected method.

The TAT of epitope tag can be also expressed in host CHO cell.TAT can be subcloned out from pRK5 carriers.Epitope tag (such as polyhistidine label) of the Insert Fragment with selecting in rhabdovirus expression vector can will be subcloned by PCR to merge to reading frame altogether.In the carrier that then the TAT Insert Fragments of additional polyhistidine label can be subcloned into SV40 drivings, the carrier is used to select stable clone comprising selected marker such as DHFR.Finally, the carrier transfection CHO cell (as described above) that can be driven with SV40.It can as described above be marked and be reached with proof list.Then any selected method such as Ni can be passed through²⁺- chelate affinity chromatography to concentrate and purify the nutrient solution of the TAT containing expressed polyhistidine label.

TAT can also be expressed by instant expression method in CHO and/or COS cells, or be expressed by other stable expressions in Chinese hamster ovary celI.

Stable expression in Chinese hamster ovary celI makes to carry out with the following method.Expressed protein as IgG constructions (immunoadhesin), wherein the coded sequence of the soluble form (such as extracellular domain) of corresponding protein is merged with the IgG1 constant-region sequences containing hinge, CH2 and CH3 domains, and/or is the form of additional polyhistidine label.

After PCR amplifications, use such as Ausubel et al., each DNA is subcloned into CHO expression vectors by the standard technique described in Current Protocols of Molecular Biology, Unit 3.16, John Wiley and Sons (1997).CHO expression vector establishments are respectively provided with Compatible restriction sites into the 5 ' of target DNA and 3 ' sides, to allow that cDNA is easily passed in and out.For the carrier such as Lucas et al., Nucl.Acids Res.24 expressed in Chinese hamster ovary celI：Described in 9 (1774-1779) (1996), the expression of purpose cDNA and dihyrofolate reductase (DHFR) is driven using SV40 early promoters/enhancer.DHFR is expressed as selecting the stable of plasmid to maintain there is provided possible after transfection.

Commodity in use transfection reagent SUPERFECTt^(Quiagen)、DOSPER^Or FUGENE^12 microgram target plasmid DNA are imported about 1 × 10 by (Boehringer Mannheim)⁷Individual Chinese hamster ovary celI.Such as Lucas et al., ibid described in cultivate cell.To about 3 × 10⁷Individual cell cryopreservation is in ampoule for further culture as described below and production.

Melt the ampoule containing DNA and whirlpool concussion mixing by being placed in water-bath.Content is pipetted into the centrifuge tube equipped with 10mL culture mediums, and centrifuged 5 minutes with 1000rpm.Supernatant is suctioned out, and cell is resuspended in 10mL Selective agar mediums (0.2 μm of filtering PS20 containing 5%0.2 μm of diafiltration hyclones).Then cell is distributed in the 100mL rolling bottles equipped with 90mL Selective agar mediums.After 1-2 days, cell is transferred in the 250mL revolving bottles equipped with 150mL selective growth culture mediums, and in 37 DEG C of incubations.After after 2-3 days, with 3 × 10⁵Individual cell/mL inoculation 250mL, 500mL and 2000mL rolling bottles.Fresh culture is replaced into by being resuspended in the method for production medium after centrifugation by cell culture medium.Although any suitable CHO culture mediums can be used, the United States Patent (USP) 5 that actually on June 16th, 1992 can be used to authorize, the production medium described in 122,469.With 1.2 × 10⁶Individual cell/mL inoculation 3L production rolling bottles.0th day, determine cell number and pH.1st day, since rolling bottle sample and sprayed into air filtering.2nd day, from rolling bottle sampling, by temperature transition into 33 DEG C, and add 30mL 500g/L glucose and the defoamers of 0.6mL 10% (such as 35% aqueous emulsion of dimethyl polysiloxane fluid, the medical emulsions of Dow Corning 365).In whole production process, regulation pH keeps it in 7.2 or so as needed.After 10 days, or when viability is down to less than 70%, by the way that cell culture is harvested by centrifugation and is filtered by 0.22 μm of filter.Filter liquor is stored in 4 DEG C or is added on post to be purified immediately.

The construction marked for polyhistidine, uses Ni-NTA posts (Qiagen) protein purification.Before purification, imidazoles is added into conditioned medium to concentration 5mM.Conditioned medium was added to the flow velocity of 4-5ml/ minutes on the 6ml Ni-NTA posts balanced in the 20mM Hepes pH 7.4 of NaCl containing 0.3M and 5mM imidazoles with pump in 4 DEG C.After sample-adding, pillar is cleaned with extra level pad, and protein is eluted with the level pad of the imidazoles containing 0.25M.Then by 25ml G25 Superfine (Pharmacia) the posts desalination to Hepes containing 10mM, 0.14M NaCl and 4% mannitol of highly purified protein, in pH 6.8 storage buffer, and -80 DEG C are stored in.

Immunoadhesin (containing Fc) construction is purified from conditioned medium as described below.CMC model thing is added on the 5ml albumin As post (Pharmacia) balanced in 20mM sodium phosphate buffers, pH 6.8 with pump.After sample-adding, with level pad thoroughly cleaning pillar, 100mM citric acids are then used, pH 3.5 is eluted.1ml fraction collectors are neutralized into the protein of elution to being equipped with 275 μ L 1M Tris buffer solutions, pH 9 pipe immediately.Then as described in the protein above for additional polyhistidine label by highly purified protein desalination into storage buffer.Homogeneity is assessed in the -terminal amino acid sequencing degraded by sds page and through Edman.

Embodiment 14：Expression of the TAT in yeast

Following method describes recombination expressions of the TAT in yeast.

First, building Yeast expression carrier is used for by the intracellular generation of ADH2/GAPDH promoters or secretion TAT.By suitable restriction enzyme sites in the selected plasmid of DNA insertions for encoding TAT and promoter to instruct TAT cell inner expression.For secretion, it can will encode TAT DNA and be cloned into together with encoding the ADH2/GAPDH promoters expressed for TAT, natural TAT signal peptides or the DNA of other mammalian signal peptides such as yeast alpha factor or invertase secretory signal/targeting sequencing and joint sequence (if desired) in selected plasmid.

Then expression plasmid transformed yeast cell described above such as yeast strain AB110 can be used, and is cultivated in selected fermentation medium.The supernatant of inverted yeast can be analyzed by following methods：Separated with 10% trichloroacetic acid precipitation, with SDS-PAG, then carried out using Coomassie blue it is gel-colored.

Then restructuring TAT can be separated after yeast cells using selected Cassette filter Concentrated culture fluids by centrifuging to remove from fermentation culture.Selected column chromatography resin can be used to be further purified for concentrate containing TAT.

Embodiment 15：Expression of the TAT in the insect cell of baculovirus infection

Following method describes recombination expressions of the TAT in the insect cell of baculovirus infection.

The sequence for encoding TAT is fused to the upstream of the epitope tag included in rhabdovirus expression vector.Such epitope tag includes polyhistidine label and immunoglobin tags (as IgG Fc areas).A variety of plasmids, including the plasmid derived from commercialization plasmid such as pVL1393 (Novagen) can be used.In brief, sequence or the expectation part of TAT coded sequences with the primer PCR amplification coding TAT complementary with 5 ' and 3 ' areas, the sequence of transmembrane protein extracellular domain is such as encoded, or if the protein is extracellular protein, the sequence of encoding mature protein.5 ' primers can include flank (selected) restriction enzyme sites.Then with those selected restriction enzyme digestion products, and it is subcloned into expression vector.

Using lipofectin reagent (lipofectin can be bought from GIBCO-BRL) by above-mentioned plasmid and BaculoGold^TMViral DNA (Pharmingen) cotransfection produces recombinant baculovirus into fall army worm (Spodopterafrugiperda) (" Sf9 ") cell (ATCC CRL 1711).After 28 DEG C incubate 4-5 days, the virus of harvest release simultaneously is used to further expand.Virus infection and protein expression such as O ' Reilley et al., Baculovirus expression vectors：A Laboratory Manual, Oxford：Oxford University Press (1994) are described to be carried out.

Then the TAT of expressed additional polyhistidine tag can be purified, for example, passes through following Ni²⁺- chelating affinity chromatography.Such as Rupert et al., Nature, 362：175-179 (1993) is described to prepare extract from the Sf9 cells of recombinant virus infection.In brief, Sf9 cells are cleaned, sonication buffer (25mL Hepes pH 7.9,12.5mM MgCl is resuspended in₂, 0.1mM EDTA, 10% glycerine, 0.1%NP-40,0.4M KCl), it is each 20 seconds at ultrasonically treated 2 times on ice.Sonicates are clarified by centrifugation, by supernatant is in middle 50 times of the dilution of sample loading buffer (50mM phosphate, 300mM NaCl, 10% glycerine, pH 7.8) and passes through 0.45 μm of filter filtering.Prepare bed volume 5mL Ni²⁺- NTA agarose columns (can be bought) from Qiagen, cleaned with 25mL water and used 25mL sample loading buffers to balance.Cell extract after filtering was added on post with 0.5mL/ minutes.Pillar is cleaned with sample loading buffer to baseline A₂₈₀, now start classification and collect.Then, pillar is cleaned with the second cleaning buffer solution (50mM phosphate, 300mM NaCl, 10% glycerine, pH 6.0), the buffer solution elutes the protein of non-specific binding.A is reached again₂₈₀After baseline, pillar is rinsed with 0 in the second cleaning buffer solution to 500mM imidazole gradients.Collect 1mL fractions, and the Ni for having alkaline phosphatase by SDS-PAGE and silver staining or by using being coupled²⁺- NTA (Qiagen) western blot is analyzed.Merge the band His containing elution₁₀The TAT of label fraction, and sample loading buffer is dialysed.

Or, known chromatographic technique can be used to carry out for the TAT of additional IgG labels (or Fc labels) purifying, including such as albumin A or Protein G column chromatography.

Embodiment 16：TAT polypeptides are purified using specific antibody

Can be by the multiple standards technology in protein purification field come purifying natural or restructuring TAT polypeptides.For example, purifying original TAT polypeptides, mature T AT polypeptides or preceding TAT polypeptides by immunoaffinity chromatography using the antibody to purpose TAT polypeptides.Generally, immune affinity column is by the way that anti-TAT polypeptide antibodies and the chromatographic resin covalent coupling of activation are constructed.

Polyclonal immunoglobulin is prepared from immune serum by ammonium sulfate precipitation or immobilization albumin A (Pharmacia LKB Biotechnology, Piscataway, N.J.) purifying.Similarly, monoclonal antibody is prepared from mouse ascites by ammonium sulfate precipitation or immobilization Protein A Chromatography.Partially purified immunoglobulin is covalently attached to the SEPHAROSE of chromatographic resin such as CnBr activation^TM(PharmaciaLKB Biotechnology).Antibody and resin are coupled by the specification according to manufacturer, close resin, and clean derivative resin.

Prepare fraction to purify TAT polypeptides from the cell of the polypeptides of TAT containing soluble form using such immune affinity chromatographic column.This prepared product is dissolved by adding detergent in the subcellular fraction that is obtained to full cell or through differential centrifugation, or by derived from other methods well-known in the art.Or, the soluble T AT polypeptides containing signal sequence can be secreted into the culture medium of culture cell in useful quantities.

The prepared product of the polypeptides of AT containing soluble T is set to flow through immune affinity column, and (the high ionic strength bufferses liquid for for example having detergent) cleans pillar under conditions of TAT polypeptide Preferential adsorptions are allowed.Then, (such as low pH buffer solutions, such as about pH 2-3, or high concentration chaotropic agent, such as urea or thiocyanate ion) elution pillar under conditions of destruction antibody/TAT polypeptides are combined, and collect TAT polypeptides.

Material preservation

Following material has been preserved in American type culture collection (American Type CultureCollection, 10801 University Blvd., Manassas, VA 20110-2209, USA) (ATCC)：

Table 7

Material	ATCC preserving numbers	Preservation day
Material	ATCC preserving numbers	Preservation day	Hybridoma cell line 12E5.1.1 Hybridoma cell line 6G10.1.1	PTA-6625 PTA-6623	2005.3.3 2005.3.3

These preservations are the regulation progress of microbial preservation budapest treaty (Budapest Treaty) and its (budapest treaty) detailed rules for the implementation for being used for proprietary program according to international recognition.It ensure that preserving the survival culture 30 years of preservation from the preservation.Preserved material can be obtained according to the clause of budapest treaty by ATCC, and obey the agreement between Genentech companies and ATCC, it ensure that after relevant United States Patent (USP) mandate or in any U.S. or foreign patent application to after public, it is defined prior in both, the public can it is permanent and it is unrestricted obtain preservation culture offspring, and ensure that according to 35 USC § 122 and according to its management article (including 37 CFR § 1.14, especially quoting 886 OG638) individual that is ratified by United States Patent and Trademark Office head will be eligible to obtain the offspring of preservation culture.

Present assignee has agreed to, if dead when the culture of preserved material is cultivated under suitable conditions, lose or destroyed, he will be after having notice rapidly with another material replacing of same culture.The availability of institute's preserved material is not construed as according to the right that its Patent Law is authorized implementing to violating any government organs the license of the present invention.

Think that foregoing written explanation is enough to enable those skilled in the art to implement the present invention.The present invention is not limited to the scope of institute's preservation construction, because institute's preservation embodiment is intended to the single illustration as some aspects of the invention, and functionally suitable any construction is within.Material preservation herein can not be construed to being limited to the scope of claim into the particular instantiation described by it is not an admission that written explanation contained herein is not enough to that any aspect of the present invention, including its optimal mode can be implemented.In fact, as described above, except a variety of modifications shown and described herein, of the invention will be readily apparent to one having ordinary skill, and within the scope of the appended claims.

Claims

1. the nucleic acid of separation, it is included：

(c) nucleotide sequence, it encodes SEQ ID NO：The extracellular domain of polypeptide shown in 2；

(d)SEQ ID NO：Nucleotide sequence shown in 1；

(e)SEQ ID NO：The full length coding region of nucleotide sequence shown in 1；Or

(f) complementary series of (a), (b), (c), (d) or (e).

2. the expression vector of the nucleic acid comprising claim 1.

3. the expression vector of claim 2, wherein the nucleic acid is operatively connected with control sequence, the control sequence is recognized by the host cell converted with the carrier.

4. host cell, it includes the expression vector of claim 2.

5. the host cell of claim 4, it is Chinese hamster ovary celI, Bacillus coli cells or yeast cells.

6. the method for producing polypeptide, is included in the host cell suitable for cultivating claim 4 under conditions of the expression polypeptide, and reclaim the polypeptide from cell culture.