CN1871033A - Trefoil factor 3 (TFF3) as a target for anti-cancer therapy - Google Patents

Abstract

The present invention is directed to methds of treating and preventing cancer, such as cancer characterized by differential expression of trefoil factor 3 (TFF3). The methods include administering to a patient an agent that modulates TFF3 activity or expression. The present invention is further directed to reducing the physiological effects of TFF3 expression in cells, including inhibiting cell motility and resistance to apoptosis. Oligonucleotides and antibodies that can modulate expression or activity of TFF3 are also provided.

Description

Trefoil factor 3 (TFF3) as target for anti-cancer therapy

The related application of cross reference

The priority that the application requires to be filed in the U.S. Provisional Patent Application 60/493,173 on August 7th, 2003 and is filed in the U.S. Provisional Patent Application 60/498,438 on August 28th, 2003, they include this paper in as a reference in full with it separately.

Invention field

The present invention partly relates to adjusting trefoil factor 3 (trefoil factor 3, TFF3) reagent of activity or expression.The invention still further relates to the purposes of these reagent in treatment, prevention or cancer diagnosis.

Background of invention

Trefoil factor 3 (TFF3; Or intestine trilobate factor, intestinal trefoil factor (ITF); Or human intestine trilobate factor, human intestinal trefoil factor (HITF)) be a kind of aprotinin, it produces in the small intestinal goblet cell usually, and justacrine is gone into enteric cavity (Thim etc., Biochemistry, nineteen ninety-five, 34,4757).TFF3 brings into play function (Mashimo etc., Science, 1996 in the recovery of damage epithelium posterius, 274,262 and Wong etc., Biochemistry, nineteen ninety-five, 34,4757), and be sure of and can finish this function by number of mechanisms: 1) protective epithelium cell makes it avoid apoptosis (Taupin etc., Proc.Natl.Acad.Sci., the U.S., 2000,97,799); 2) induce border cell (bordering cell) migration and covering wound (Dignass etc., J.Clin.Invest.,, 94,376 in 1994); With 3) stop the deposition (Andoh etc., J Immunol., calendar year 2001,167,3887) that enters epithelial SC system component by wound.The phenotype of knock out mice is normal, and under the impaired situation of their intestinals, they can be died from wound and can't heal this moment.Reorganization TFF3 be proposed to be used in the treatment that relates to irritable bowel syndrome and other intestinal diseases (referring to, as, U.S. Patent number 6,063,755,6,316,218 and U.S. Patent Application Publication No. 20010052483).At present, the receptor of TFF3 is also unknown.

The physiological effect of TFF3 can be considered to help growth, propagation and the transfer of cancerous cell in the enterocyte.For example, anti-apoptosis, induce migration and protection to avoid complement activation to be considered to make cancerous cell to escape adjusting and controlling growth, to invade normal surrounding tissue and to shift or escape the mechanism that immune system monitors.The expression of Clinical Follow-up data show TFF3 can present related (Yamachika etc., Clin.Cancer Res.,, 8,1092 in 2002) with the poor prognosis of gastric cancer.

It is reported, other protein and their nucleic acid of coding also can be used as cancer diagnosis and treatment effective target (referring to, as, U.S. Patent number 6,261,562,6,337,195,6,465,611,6,476,207 and U.S. Patent Application Publication No.: 20020192699).Compositions as herein described and method help to satisfy present new and demand more effective treatment to cancer and relevant disease.

Summary of the invention

The invention provides the method for a kind of treatment or prophylaxis of cancer, this method comprises suffering from or the TFF3 nertralizer of cancer-prone mammal treatment effective dose, and wherein this TFF3 nertralizer is antisense molecule, RNAi molecule, ribozyme or micromolecule.

In some embodiments, described cancer is breast carcinoma, colon cancer, carcinoma of prostate, ovarian cancer or gastric cancer.In some embodiments, TFF3 differential expression in the cell of described cancer.

In some embodiments, nertralizer is an antisense molecule, this antisense molecule comprise or overlapping and SEQ IDNO:5-19 in the corresponding sequence of any sequence.In some embodiments, nertralizer is the RNAi molecule, this RNAi molecule comprise or overlapping and SEQ ID NO:5-19 in the corresponding sequence of any sequence.In some embodiments, nertralizer is the antibody of specificity in conjunction with TFF3.

The present invention also provides the treatment method for cancer, and this method comprises suffering from or the TFF3 nertralizer of cancer-prone mammal treatment effective dose and this mammal carried out conventional treatment of cancer.In some embodiments, Chang Gui treatment of cancer is a chemotherapy.In some embodiments, Chang Gui treatment of cancer is hormone ablation (hormone ablation therapy).In some embodiments, this hormone is an androgen.

The present invention also provides method apoptosis-induced in cell, and this method comprises this cell is contacted with the TFF3 nertralizer.In some embodiments, this cell is mammiferous cell.In some embodiments, this cell is a cancerous cell.In some embodiments, this cell is the cell of mammary gland, prostate, colon, ovary or stomach.

In other embodiments, the present invention also provides and has reduced gross tumor volume, slows down tumor growth or stop the method for tumor growth, and this method comprises this tumor is contacted with the TFF3 nertralizer.In some embodiments, this tumor contains the cell of TFF3 differential expression.

The invention provides the method for regulating at least a physiological effect relevant with TFF3 expression in the cell, this method comprises described cell is contacted with the TFF3 nertralizer.In some embodiments, this physiological effect is to improve cell viability or anti-apoptosis.

The present invention also provides the method that suppresses cell migration, sticks together or breed, and this method comprises described cell contacted with the TFF3 nertralizer.

In some embodiments, the invention provides the method that reduces the cancerous cell infecting potential, this method comprises this cancerous cell is contacted with the TFF3 nertralizer.

The present invention also provides the method that TFF3 expresses in the cell of regulating, and this method comprises this cell contacted with the TFF3 nertralizer.

In other embodiments, the present invention also provides in biological sample the method that detects TFF3, and this method comprises this sample is contacted combining of TFF3 and nertralizer in the test sample also with the TFF3 nertralizer.In some embodiments, the TFF3 nertralizer comprises detectable labelling.

Equally, the invention provides whether there is method for cancer in the detection of biological sample, this method comprise this biological sample contacted with the TFF3 nertralizer and the detection of biological sample in the sign of TFF3 differential expression.There is the sign of TFF3 differential expression to show the existence of cancer.

In some embodiments, should " detections " comprise that the result that will contact compared with the control.In some embodiments, the TFF3 nertralizer comprises detectable labelling.

The present invention also provides and has measured the method for patient to the sensitivity of TFF3 nertralizer, and this method comprises the sign of TFF3 differential expression in the cancer sample that detects the patient.The sign of TFF3 differential expression has shown the sensitivity of patient to the TFF3 nertralizer.In some embodiments, the sign of TFF3 differential expression is the rise of TFF3 in patient's the cancer sample.In some embodiments, patient's cancer sample comes from mammary gland, prostate, colon, ovary or gastric tissue.

In some embodiments, the invention provides the method for cancer progression in the assess patient, when this method comprises when the very first time put the expression of TFF3 and second time point among the patient among the patient expression of TFF3 compare.The expression ratio very first time of TFF3 point the time increases to some extent and to show that cancer makes progress to some extent during second time point.In some embodiments, the TFF3 of the increase " express " is meant has increased at least about 25%, at least about 50%, at least about 75% or at least about 90% expression.

The present invention also provides the method that detects the cancer metastasis risk that increases among the patient, when this method comprises when the very first time put the expression of TFF3 and second time point among the patient among the patient expression of TFF3 compare.Increase the increase that shows the cancer metastasis risk when expression ratio very first time of TFF3 puts during second time point to some extent.

The present invention also provides and has regulated the antisense molecule that TFF3 expresses.In some embodiments, this antisense molecule comprise or overlapping SEQ ID NO:5-19 in a kind of sequence.In some embodiments, this antisense molecule comprises any among the SEQ ID NO:5-19.

The present invention also provides and has regulated the RNAi molecule that TFF3 expresses.In some embodiments, this RNAi molecule comprise or overlapping SEQ ID NO:5-19 in a kind of sequence.In some embodiments, this RNAi molecule comprises any among the SEQ ID NO:5-19.

The present invention also provides the compositions that comprises antisense molecule and/or RNAi molecule and pharmaceutically acceptable carrier.

The present invention also provides isolating anti-TFF3 antibody, and wherein this antibody recognition is corresponding to SEQ ID NO:20, at least one TFF3 sequence area of 21,22,23,24,25,26,27 or 28.In some embodiments, this antibody produces by the following method:

A) synthetic antibody library on phage;

B) by this phage and compositions are contacted the screening of sample being carried out the storehouse, said composition comprises corresponding to SEQ ID NO:20, at least one TFF3 sequence area of 21,22,23,24,25,26,27 or 28;

C) separate and the bonded phage of compositions, being characterized as it and having and the ability that combines corresponding to SEQ IDNO:20,21,22,23,24,25,26, at least one TFF3 sequence area of 27 or 28 of this antibody wherein, its binding affinity is at least 1081/; With

D) analyze this isolating phage to determine to be combined with amino acid coding corresponding to SEQ ID NO:20, at least one TFF3 sequence area of 21,22,23,24,25,26,27 or 28.

In some embodiments, this antibody is antibody (humanized antibody), single-chain antibody or the Fab fragment of monoclonal antibody, polyclonal antibody, chimeric antibody, peopleization.In some embodiments, this antibody is through labelling.In some embodiments, this is labeled as enzyme, radiosiotope or fluorophor.In some embodiments, this antibody is lower than about 1 * 10 to the binding affinity of the polypeptide except that TFF3 ⁵Ka.

The present invention the separation that produces antibody of the present invention also is provided cell and hybridoma.

The present invention also provides isolating polypeptide, and this polypeptide comprises the aminoacid sequence of group under three or following being selected from: SEQ ID NO:13,20,21,22,23,24,25,26,27 or 28.In some embodiments, the length of this polypeptide is about 80 aminoacid of about 8-.In some embodiments, this polypeptid specificity ground and anti-TFF3 antibodies.

The present invention also provides the method for using TFF3 differential expression in the antibody test sample, and this method comprises: allowing antibody: under the condition that TFF3 complex (complex) forms, with antibody of the present invention and described sample mix (combining); Measure the amount of complex; With with the amount of complex with compare.The raising of complex level shows the differential expression of TFF3 in the sample.

The present invention also provides, and epi-position isolating, SEQ ID NO:1-4 polypeptide is carried fragment (epitope-bearingfragment).In some embodiments, this fragment comprises about 20 continuous amino acid of about 6-of SEQ ID NO:1-4.In some embodiments, this fragment comprises about 10 continuous amino acid of SEQ ID NO:1-4.In some embodiments, this polypeptide is SEQ ID NO:2.In some embodiments, this fragment comprises SEQID NO:20,21,22,23,24,25,26,27 or 28.

Simultaneously, the present invention also provides isolating anti-TFF3 antibody, and this antibody comes immune a certain object to obtain by carry fragment with epi-position of the present invention.In some embodiments, the invention provides the isolated antibody in the pharmaceutical composition, this pharmaceutical composition contains one or more pharmaceutically acceptable carriers.In some embodiments, in this antibody and TFF3.

The present invention also provides the method that produces the antibody that is used for the treatment of cancer, and this method comprises in the identification combination also and the antibody of TFF3, and expresses this antibody in recombinant expressed host cell.

In addition, the present invention also provides pharmaceutical composition, and this pharmaceutical composition contains in the combination also and antibody and the pharmaceutically acceptable carrier of TFF3, and wherein this antibody is to use recombinant host cell to produce.In some embodiments, this recombinant host cell is selected from down group: Chinese hamster ovary cell, myeloma cell and bacterial host cell.

The accompanying drawing summary

Figure 1 shows that the differential expression of TFF3mRNA in different complete tissues.

Figure 2 shows that the difference of TFF3 expression in different cell lines.

Figure 3 shows that different TFF3 antisense oligonucleotides effectiveness of the difference aspect the TFF3mRNA expression in lowering colon cancer cell.

Figure 4 shows that the specificity of TFF3 antisense oligonucleotide.

Figure 5 shows that the specificity of another TFF3 antisense oligonucleotide.

Figure 6 shows that antisense oligonucleotide reduces the effectiveness that TFF3mRNA expresses in cancerous cell line.

Figure 7 shows that cytotoxic effect and the anti-proliferative effect of antisense oligonucleotide in colon cancer cell.

Fig. 8 A and 8B are depicted as cytotoxic effect and the anti-proliferative effect of antisense oligonucleotide in prostate gland cancer cell.

Figure 9 shows that the avirulence of TFF3 antisense oligonucleotide in normal cell.

Figure 10 shows that of the inhibition of TFF3 antisense oligonucleotide to prostate gland cancer cell colony growth in the soft agar medium.

Figure 11 shows that the inhibition of using anti-TFF3 antibody on cell proliferation.Will be from anti-TFF3 polyclonal antiserum isolated IgG composition to be diluted to final concentration in containing the growth medium of 1%FBS be 50 mcg/ml, the 0th day and the 4th day at proliferation assay joins in 4 multiple holes then.Mark the 7th day on cell proliferation degree.

Embodiment describes in detail

The present invention provides the TFF3 nertralizer especially and has used these preparation preventions, treatment and detect method for cancer.The TFF3 polypeptide can be at many cancerous tissues and the middle differential expression (for example, on protein or mRNA level) of cancerous cell line (for example, relevant with carcinoma of prostate, breast carcinoma, ovarian cancer, gastric cancer and colon cancer).As illustrated in this paper, the expression of reduction TFF3 (for example, by using the technology based on oligonucleotide) can cause cytotoxicity and suppress the growth (anchorage-independent growth) of non-anchorage dependence.Therefore, the nertralizer that use to be fit to suppresses the expression of active or the reduction TFF3 polypeptide or the polynucleotide of TFF3 polypeptide in cell, can help to prevent or treats for example to express the cancer that TFF3 is a feature.In some embodiments, but this cancerous tissue differential expression (for example, demonstrate raise or downward modulation) TFF3.

As used herein, phrase " differential expression " typically refers in cancerous cell with higher or reduced levels polypeptide expressed or polynucleotide, for example, when finding when comparing with the non-cancerous cell (normal cell) of same type, the existing difference of mRNA in the cancerous cell (for example, higher or lower) is at least about 25%, at least about 50% about 75%, at least about 90%, at least about 95%, at least about 1.5 times, at least about 2 times, at least about 5 times, at least about 10 times or at least about 50 times or more.Relatively can between two kinds of tissues, carry out, for example, if use is that in situ hybridization or another kind can show the test method of difference to a certain degree between the different cell types in tissue.More also can carry out taking from its tissue-derived iuntercellular.TFF3 is a kind of gene that has shown differential expression in colon, mammary gland, prostate and other cancer.In some embodiments, viewed differential expression is higher than normal expression.Be used for determining in the method for the polypeptide of some cell differential expression and polynucleotide in U.S. Patent Application Publication No.: 20020192699 and U.S. Patent number 6,476,207 in further description is arranged.The gene outcome of differential expression is described in United States serial number 10/310,673 to some extent in the canceration prostatic cell.

As used herein, term " about " be meant a certain numerical value+/-10%.

The TFF3 polypeptide

As used herein, " TFF3 " or " TFF3 polypeptide " is meant basically and people's trefoil factor 3 (SEQ ID NO:1-4, GenBank gi:4507453, Q07654, NP003217, AAH17859, BAA95531, BAB13731) homologous protein (polypeptide) or its fragment.The polypeptide that the present invention considers comprises those: by the TFF3 polynucleotide that disclosed (SEQ ID NO:5-8, GenBank gi:4507452, BC017859, AF432265) encoded polypeptides and because the degeneracy of genetic code, and the polypeptide of the nucleic acid coding different with the TFF3 polynucleotide sequence that is disclosed.Therefore, comprise in the scope of the present invention by polynucleotide encoded polypeptide or by its variant encoded polypeptide with any sequence in the polynucleotide sequence that this paper provides.In some embodiments, TFF3 comprises the aminoacid sequence of SEQ ID NO:1-4.Some preferred embodiment in, TFF3 comprises the aminoacid sequence of SEQ IDNO:2.

Term " polypeptide " and " protein " are used interchangeably, and be meant the aminoacid polymerized form of any length, it can comprise aminoacid coding or noncoding, chemistry or biochemical modification or deutero-aminoacid and the polypeptide with modified peptide backbone.This term comprises fusion rotein, includes but not limited to: the fusion rotein that forms with the allogeneic amino acid sequence, with the fusions that allos or homology targeting sequencing form, this fusions contains or does not contain the N-terminal methionine residues; Immune labeled albumen; Deng.This term also comprises " peptide ", and it is that length is about 30 the amino acid whose polypeptide of 2-.Polypeptide can refer to by the full-length polypeptide of described polynucleotide (for example, TFF3 polynucleotide) codings, by polypeptide and the part or the fragment of the gene code of described polynucleotide representative.

" polypeptide " also comprises naturally occurring proteinic variant, wherein these variants and naturally occurring protein homology or similar basically, and can with naturally occurring protein (for example, the people of the described polypeptide of natural expression, Mus or some other species are generally mammalian species) have an identical or different source of species.Usually, the sequence of variant TFF3 polypeptide and the peptide sequence of differential expression as herein described have at least about 80%, usually at least about 90%, be more typically at least about 95%, promptly, 96%, 97%, 98% or 99% sequence homogeneity, this homogeneity can for example be passed through with BLAST, and default parameters (default parameter) is measured.This variant polypeptide can be through natural or non-natural glycosylation (that is, the glycosylation pattern of this polypeptide is different from the glycosylation pattern of being found in naturally occurring respective egg white matter).

The present invention has also comprised the congener (homolog of TFF3 polypeptide, or its fragment), wherein these congeners separate from other species, naturally occurring glycosylation TFF3 can comprise those by the glycosylation TFF3 that normal cell and tumor cell produce in these species, and they may demonstrate different glycosylation patterns, promptly, other animal or plant kind that has this congener, be generally mammalian species, as Rodents, for example mice, rat; Domestic animal is as horse, cattle, Canis familiaris L., cat; And it is human." congener " is used for expression to have at least about 35% with defined concrete protein above, usually at least about 40%, more generally at least about the polypeptide of 60% amino acid sequence identity, wherein sequence homogeneity for example utilizes default parameters to measure with BLAST algorithm (algorithm).

Usually, the TFF3 polypeptide of theme of the present invention is provided in the environment that non-natural exists, and for example, separates from they naturally occurring environment.In some embodiments, this theme protein is present in the compositions that is rich in this protein (compared with the control comparatively).Similarly, the polypeptide of purification is provided, wherein " purification " is meant that this protein is present in the compositions that is substantially free of other polypeptide, wherein " be substantially free of " to contain in the composition that is meant said composition and be less than about 90%, usually be less than approximately 60%, or more generally be less than other polypeptide of 50%.

Variant too within the scope of the present invention; Variant polypeptides comprises mutant, fragment and fusions.Mutant can comprise amino acid replacement, interpolation or disappearance.This amino acid replacement can be conservative amino acid replacement or removes substituting of non-primary amino acid; for example, be used to change glycosylation site, phosphorylation site or acetylation site or by one or more to function and nonessential cysteine residues substitute or disappearance minimizes false folding (misfolding).Conservative amino acid replacement is that those have kept substituting of replaced amino acid whose total electrical charge, hydrophobicity/hydrophilic and/or spatial volume.Can design variable with the biologic activity that keeps or strengthen the protein specific region (for example, functional domain and/or, if this polypeptide is a member of protein families, the zone that it is relevant with concensus sequence).The selection that is used to produce the amino acid change of variant can be based on this amino acid whose accessibility (accessibility, inner for the outside) (referring to, for example, Go etc., Int.J.Peptide Protein Res.1980,15:211), the heat stability of this variant polypeptide (referring to, as, Querol etc., Prot.Eng.1996,9:265, the document is included this paper in as a reference in full with it), required glycosylation site (referring to, as, Olsen and Thomsen, J Gen.Microbiol.1991,137:579), required disulphide bridges (referring to, for example, Clarke etc., Biochemistry, 1993,32:4322; With Wakarchuk etc., Protain Eng.1994,7:1379), required melts combine site (referring to, for example, Toma etc., Biochemistry, 1991,30:97 and Haezerbrouck etc., Protein Eng.1993,6:643) and required substituting in proline ring (proline loop) (referring to, for example, Masul etc., Appl.Env.Microbiol., 1994,60:3579, the document is included this paper in as a reference in full with it).Remove the method that is disclosed in the available U.S. Patent number 4,959,314 of mutain (cysteine-depleted mutein) of all cysteine and produce, the document is included this paper in as a reference in full with it.

Variant also can comprise polypeptide disclosed herein fragment, especially biological active fragment and/or with the corresponding fragment of functional domain.Interested fragment will be generally at least about ten amino acid-at least about the length of fifteen amino acid, at least about 50 amino acid whose length, also can be and reach 300 aminoacid or longer, and in some embodiments, its length is no more than about 1000 aminoacid, wherein this fragment contains the aminoacid of one section extension, and this section aminoacid is with identical by polynucleotide encoded polypeptide or its variant with arbitrary polynucleotide sequence that this paper provides.Protein variants as herein described is coded by the polynucleotide in the scope of the invention.This genetic code can be used for selecting suitable codon to make up corresponding variant.Particularly, fragment can comprise that those contain the fragment of peculiar zone of TFF3 albumen or epi-position.

The TFF3 polynucleotide

In some embodiments of the present invention, the present invention relates to inhibition or detection to the TFF3 polynucleotide of the coding TFF3 of differential expression in some cancer, for example, carcinoma of prostate, breast carcinoma, ovarian cancer or colon cancer.In some embodiments, TFF3 is by the nucleic acid coding with SEQ ID NO:5-8 nucleotide sequence.Some preferred embodiment in, TFF3 is by the nucleic acid coding of the nucleotide sequence with SEQ ID NO:7.

Include but not limited to have the polynucleotide (for example, SEQ IDNO:2) of any sequence in the polynucleotide sequence that this paper provides in the scope of the present invention about polynucleotide compositions useful in the method as herein described; By the hybridization of (especially under highly strict condition) under the stringent condition, available from the polynucleotide in biomaterial described herein or other biogenetic derivation (especially people source); Gene corresponding to the polynucleotide that provided; The variant of the polynucleotide that provide and their corresponding gene, especially those variants that kept encoding gene product biologic activity (for example, because gene outcome belongs to a certain or some protein families and/or based on the identification of existing functional domain in the gene outcome, and think the biologic activity that this has corresponding to the gene outcome of providing polynucleotide).After the announcement that this paper is provided, the present invention expects or other nucleic acid compositions within the scope of the present invention will be very conspicuous for those of ordinary skill in the art.Unless stated otherwise, this paper is used for compositions nucleic acid " polynucleotide " and " nucleic acid " does not plan the length or the structure of nucleic acid are limited.

The TFF3 polynucleotide can be expressed in human cancer tissue, for example, and human prostate, mammary gland and colon.Particularly interested nucleic acid compositions as herein described contains arbitrary sequence or its recognition sequence in the polynucleotide sequence that this paper provides." recognition sequence " (identifying sequence) be residue adjoin (contiguous) sequence, it is at least the about 20nt of about 10-on length, normal length is at least the about 100nt of about 50-, it discerns a certain polynucleotide sequence single-mindedly, as, have with any nucleotide sequence that adjoins and to be less than 90%, be generally the sequence homogeneity that is less than about 80%-about 85% greater than about 20nt.Therefore, the theme nucleic acid compositions comprises total length eDNA or the mRNA that comprises a certain contiguous nucleotide recognition sequence, and this contiguous nucleotide comes from the polynucleotide sequence that arbitrary this paper provides.

Useful polynucleotide also comprise the polynucleotide that have sequence similarity or sequence homogeneity with natural TFF3 DNA in method described herein.It comprises relevant 5 ' and the sequence of 3 ' non-translated sequence, promoter and enhancer sequence and justice or antisense orientation.Nucleic acid with sequence similarity can detect by the hybridization under low stringency condition, for example, still keeps combination at 50 ℃ and 10 * SSC (0.9M saline/0.09M sodium citrate) and when washing with 1 * SSC down for 55 ℃.The homogeneity of sequence can detect by the hybridization under stringent condition, for example, and under 50 ℃ or higher temperature, and 0.1 * SSC (9mM saline/0.9mM sodium citrate).The method and the condition of hybridization are well-known in the art, referring to, for example, U.S. Patent number 5,707,829.The nucleic acid substantially the same with the polynucleotide sequence that is provided (as hereditary change form of allelic variant (allelic variant), gene etc.) combines with the polynucleotide sequence that is provided under stringent hybridization condition.By using the probe probe of the DNA sequence of special marking (promptly through), separable homologous or relevant gene.Homogenic source can be any species, and is as primates, especially human; Rodents, for example, rat and mice; Dog class, cat class, cattle, sheep, horse, yeast, nematicide etc.

In one embodiment, used the contiguous nucleotide of 15nt at least at least a in the polynucleotide sequence that this paper provided to hybridize.That is, when at least 15 contiguous nucleotides that use one of the polynucleotide sequence disclosed during as probe, this probe will be preferred and the nucleic acid hybridization that contains complementary series, thereby make the nucleic acid with selected probe specificity hybridization obtain differentiating and reclaiming.If the corresponding same mRNA of source cDNA of the probe of the polynucleotide sequence that provides more than a kind of this paper is provided, so they just can with same nucleic acid hybridization.Can use the probe more than 15nt, as, the probe of size in about 18nt, 25nt, 50nt, 75nt or 100nt scope, but the probe of about 15nt is enough represented the sequence of specific recognition usually.

Polynucleotide in the present invention's design also comprise the naturally occurring variant (for example, degeneracy variant, allelic variant etc.) of nucleotide sequence.Polynucleotide variant in the present invention's design is to identify by variant that this is inferred and nucleotide sequence hybridization disclosed herein, preferably hybridizes under stringent condition.For example, by using suitable wash conditions, can identify the variant of polynucleotide described herein, this equipotential variant demonstrates mismatching of about 25-30% base pair (bp) at most with respect to the polynucleotide probes of selecting.Usually, allelic variant contains mismatching of the about 25%bp of 15-that has an appointment, and also can contain extremely few even about 5-15%, or about 2-5%, or the mismatching of about 1-2%bp, and the mismatching of single bp.

The congener of the TFF3 polynucleotide sequence that provides corresponding to this paper also is provided in the present invention, and wherein this homogenic source can be any mammalian species, as, primates, especially human; Rodents, for example rat; Dog class, cat class, cattle, sheep, horse, yeast, nematicide etc.In mammalian species (for example, the mankind and mice) between, congener usually and TFF3 gene or its part have basic sequence similarity, for example, at least 75% sequence homogeneity, at least 90%, at least 95%, 96%, 97% between nucleotide sequence, 98% or 99% sequence homogeneity.Sequence similarity is based on that reference sequence calculates, and the subclass that this reference sequence can the big sequence in position is preferably the outer coded sequence of born of the same parents, as, as the part of conservative motif, coding region, flanking region (flanking region) etc.The length of reference sequence is generally the nucleotide that adjoins at least about 18, is more typically at least about 30 length of nucleotides, and may extend to the complete sequence that compares.The algorithm of sequence analysis is well known in the art, and for example uses the breach BLAST (gapped BLAST, at Altschul etc., Nucleic Acids Res.1997 describes among the 25:3389-3402 to some extent) of default parameters.

Usually, the sequence homogeneity of the variant institute tool of TFF3 polynucleotide as herein described is greater than at least about 65%, preferably at least about 75%, more preferably at least about 85%, also can be greater than at least about 90%, 95%, 96%, 98%, 99% or higher, this homogeneity is measured by the Smith-Waterman homology searching algorithm of carrying out in MPSRCH program (Oxford Molecular) (homology search algorithm).An example of homogeneity percentage calculation method is the Smith-Waterman algorithm, use as described below: carry out the measured whole DNA sequence homogeneity of Smith-Waterman homology searching algorithm greater than 65% in MPSRCH program (Oxford Molecular), used the accurate breach search of the following search parameter of tool in this algorithm: the open point penalty of breach is 12; It is 1 that breach extends point penalty.

Theme nucleic acid can be cDNA or genomic DNA, also can be its fragment, especially encoding human is learned the active gene product and/or useful fragment (for example, in diagnosis, as the specificity marker of interested difference expression gene etc.) in method disclosed herein.Term used herein " cDNA " will comprise with the sequential element of natural ripe mRNA kind apoplexy due to endogenous wind arranges all identical nucleic acid, and wherein sequential element is exon and 3 ' and 5 ' noncoding region.Usually, the mRNA kind has the exon that adjoins, and has the intron of insertion therebetween, and intron exists, and it is removed by the nRNA montage when expressing, thereby produces the continuous open reading frame of a certain polypeptide of coding.The mRNA kind also can contain exon and intron simultaneously and exist, and wherein intron can be removed through alternative splicing (alternativesplicing).In addition, should also be noted that the different types of mRNA by same genome sequence coding can exist with different levels in cell, and the varying level that detects these mRNA kinds can indicate the differential expression of the gene outcome of encoding in the cell.

Interested genome sequence be included in the nucleic acid that exists between start codon and termination codon (such as in the listed sequence definition), comprise all introns that are present in usually in the natural dyeing body.It also can be included in 3 ' and the 5 ' untranslated region that exists among the ripe mRNA.It also can comprise concrete transcribing and the translational control sequence, as promoter, enhancer etc., in 5 of transcriptional domain ' or 3 ' end comprises the flanking genomic dna of about 1kb (but can be longer).This genomic DNA can be used as 100kbp or littler fragment separated; And be substantially free of the flank chromosome sequence.Side joint coding region, 3 ' or 5 ' or the genomic DNA that includes regulating and controlling sequence that in intron, exists sometimes, the specific expressed required sequence of suitable tissue, phase specificity or morbid state contained.

The theme TFF3 polypeptide that the nucleic acid codified of theme of the present invention is all or part of, maybe can comprise non-coding sequence (for example, from 5 of gene ' or 3 ' noncoding region).Pointed as this paper, these DNA or RNA can be justice or antisense orientation.Carry out chemical synthetic oligonucleotide according to conventional methods, pass through restriction enzyme digestion, pcr amplification etc., can obtain two strands or single-chain fragment from DNA sequence.Isolating polynucleotide in the present invention design and polynucleotide passage comprise be selected from the polynucleotide provided herein at least about 10, about 200, the about 250-about 300 of about 15, about 20, about 35, about 50, about 100, about 150-or about 350 nucleotide that adjoin.With regard to most of, segmental length will be 15nt at least, be generally at least 18nt or 25nt and up to adjoining nt or more at least about 50.In some embodiments, polynucleotide molecule comprises the contiguous nucleotide sequence of the 12nt at least that is selected from the arbitrary sequence in the polynucleotide sequence that this paper provides.

The specific oligonucleotide probe of TFF3 polynucleotide can produce with TFF3 polynucleotide sequence disclosed herein.This probe is preferably at least about the fragment of 12nt, 15nt, 16nt, 18nt, 20nt, 22nt, 24nt or 25nt, this fragment is the fragment of the corresponding contiguous nucleotide sequence of arbitrary polynucleotide sequence provided herein, and length can be less than 2kb, 1kb, 0.5kb, 0.1kb or 0.05kb.This probe can be chemosynthesis or can be and uses Restriction Enzyme to produce from longer polynucleotide.This probe can be with for example, and radioactivity, biotinylated or fluorescent labeling are carried out labelling.Preferably, the design of probe is based on the recognition sequence of the polynucleotide sequence that arbitrary this paper provided.More preferably, the design of probe is based on the contiguous nucleotide sequence of arbitrary theme polynucleotide, at the masking procedure (masking program) of this sequence application being sheltered low-complexity (for example, XBLAST) after, this sequence still keeps not sheltering, promptly can select not blasnket area, the blasnket area is shown by the polynucleotide outside the poly n extension of sheltering sequence that produces through masking procedure for this.

The TFF3 polynucleotide of theme invention can obtain to be different from complete chromosomal form usually through separating and obtaining with the form of substantially pure.Usually, can obtain to be substantially free of the polynucleotide (no matter being DNA or RNA) of other naturally occurring nucleotide sequence, its purity is generally at least about 50%, usually at least about 90%, and be generally " reorganization ", for example, with one or more nucleotide side joints that in naturally occurring chromosome, are not attached thereto usually.

TFF3 polynucleotide as herein described can be used as linear molecule or provide in ring molecule, and can provide in self-replicating molecule (carrier) or in the molecule that does not contain replication sequence.The expression of these polynucleotide can by they itself or regulate and control by other regulating and controlling sequence known in the art.Can these polynucleotide be imported in the proper host cell by multiple available technology in use this area, these technology are for for example, the transfection that transfection, the liposome-mediated DNA that the DNA of siderophillin polycation mediation shifts, carry out with the nucleic acid of naked nucleic acid or parcel shifts, transportation in the born of the same parents of the latex particle (latex bead) of DNA bag quilt, protoplast fusion, viral infection, electroporation, particle gun, calcium phosphate mediate etc.

Nucleic acid compositions described herein for example can be used for, and produces polypeptide (it can be used for obtaining anti-TFF3 antibody); As the probe of mRNA in the detection of biological sample (for example, people's cell extract) with the more multicopy that produces these polynucleotide, be used for producing ribozyme or antisense oligonucleotide; And as ssDNA probe or as the oligonucleotide that forms three chains.Probe described herein can be used for, and for example, whether the existence of arbitrary polynucleotide provided herein or its variant in the working sample.These and other purposes has further description hereinafter.

Term used herein " overlapping " (overlap) is meant the sequence area of sharing by more than polynucleotide or oligonucleotide.For example, a certain oligonucleotide of overlapping another oligonucleotide have with another oligonucleotide in the corresponding basically sequence area of a certain sequence area.In some embodiments, overlappingly be at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 15, at least about 20, at least about 50, at least about 100 or longer length of nucleotides.

Nertralizer

Use in method of the present invention and the goods manufacturing or mixed the TFF3 nertralizer that in cell regulation and control TFF3 is active or express.As used herein, term " regulation and control " is meant in gene, protein or the cell, the matter of any molecule on extracellular or the cell surface or the variation of amount.This variation can be in the expression or the rise or the downward modulation of molecular level.Term " regulation and control " also comprises the change of biological function/active matter or amount, and these biological function/activity are for for example, breeds, secretes, sticks, apoptosis, cell-cell signal transmission etc.In some embodiments, regulation and control active or that express can be greater than about 10%, greater than about 20%, greater than about 30%, greater than about 40% or greater than about 50%.

This paper has comprised multiple nertralizer, as micromolecule, peptide, antibody, antisense molecule, ribozyme, RNAi molecule etc.

Micromolecule

Nertralizer can contain randomly the micromolecule that merges or engage with cytotoxic agent (for example as herein described those).The micromolecule storehouse that can filter out anti-TFF3 or TFF3 express cell is to identify and the bonded micromolecule of this antigen.According to well-known method, also can to this micromolecular antagonism or in and characteristic screen and/or micromolecule engaged with cytotoxic agent.

Peptide

Nertralizer also can be by appropriate design or by phage expression (referring to, for example, the W098/35036) peptide that produces.In one embodiment, the molecule of selection can be " CDR intends like thing " or the antibody analog based on the CDR design of antibody.Though this type of peptide can especially self carry out antagonism, can be randomly this peptide and cytotoxic agent be merged, thereby increase or promote the antagonistic properties of this peptide.Peptide can contain that 2-is about 20,2-about 15 or about 10 aminoacid of 2-.

Antisense oligonucleotide

Under some environment, may need the expression of TFF3 in regulation and control (for example, the downward modulation) cell.Therefore, in another aspect of this invention in, can prepare the TFF3 antisense oligonucleotide, and adopt and to reduce the expression of cell someway TFF3, this method comprises uses one or more TFF3 antisense oligonucleotides.Phrase " TFF3 antisense oligonucleotide " refer to contain by base pair with relate to complementary nucleic acid sequence that TFF3 expresses and carry out the oligonucleotide of interactional nucleotide sequence, make the TFF3 down-regulated expression by this interaction.Preferably, relating to the specific nucleic acid sequence that TFF3 expresses is genomic DNA molecule or the mRNA molecule of coding TFF3.This genomic DNA molecule can comprise the control region and/or the proteic coded sequence of mature T FF3 of TFF3 gene.

The term " complementation " that when describing TFF3 antisense oligonucleotide and method thereof, uses thus be meant and the fully complementary hybridization that allows in the cell with this sequence of this sequence, that is, and under physiological condition.This TFF3 antisense oligonucleotide preferably contains the sequence that has comprised about 100 nucleotide of 8-, and more preferably this antisense oligonucleotide comprises about 30 nucleotide of about 15-or about 26 nucleotide of about 18-.

The TFF3 antisense oligonucleotide also can contain multiple modification, its resistance to nuclear degraded (nucleolyticdegradation) is given in these modifications, for example, link [Uhlmann and Peyman, ChemicalReviews 90:543-548 (1990) between modified nucleoside; Schneider and Banner, Tetrahedron Lett.31:335, (1990)], modified nucleic acid base (as 5,958,773 and the patent wherein mentioned in disclose) and/or sugar etc.Further describing the oligonucleotide modified hereinafter is provided.

Scope of the present invention has comprised modification or the change that is widely used in the antisense molecule of antisense technology known in the art.These modifications comprise that the preparation of phosphorous connection is (as United States Patent (USP) 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; Disclosed in 5,625,050 and 5,958,773).Further describing the oligonucleotide modified hereinafter is provided.

Antisense compounds of the present invention can comprise the base of modification.Antisense oligonucleotide of the present invention also can be connected and modifies by this oligonucleotide and one or more parts or jointer being carried out chemistry, thereby promotes activity, cell distribution or the cellular uptake of this antisense oligonucleotide.This part or jointer comprise lipid, and for example, cholesterol, bile acid, thioether, aliphatic chain, phospholipid, polyamine, Polyethylene Glycol (PEG), soft ester acyl are partly and other is at for example United States Patent (USP) 5,514,758,5,565,552,5,567,810,5,574,142,5,585,481,5,587,371,5,597,696 and 5,958, part described in 773 or jointer.Further describing modified base and antisense oligonucleotide jointer hereinafter is provided.

In the antisense field, the routine test that can carry out a certain degree to select best antisense molecule according to concrete target.In its design, the accessible or exposed portions that antisense molecule can targeting target RNA molecule.Reverse transcription by mRNA is also analyzed the level of cDNA, can be in the cell of treated and control the level of mRNA in the conventional sense cell.Can be with the method conventional sense biological effect of mensuration cell known in the art growth or vigor.

By test with analyze the cDNA level and measure the method that the active specificity of antisense is art-recognized affirmation antisense result.For example, can be from RNA treated and the control cell through reverse transcription, and gained cDNA group analyzed.(Branch，A.D.，T.I.B.S 23：45-50(1998))。

Antisense molecule of the present invention comprises zone or the complementary oligonucleotide of part with the TFF3 polynucleotide, as, have the nucleic acid of the nucleotide sequence of SEQ ID NO:5-8.Can separately or unite some the exemplary antisense oligonucleotide that reduces TFF3 expression in the cell and comprise antisense oligonucleotide or its fragment that those have sequence shown in the following table 1.

Table 1: example TFF3 antisense oligonucleotide

SEQ ID NO	Sequence
		SEQ ID NO：9	5′-TCCTTGGCTGGCACGGCACACT-3
SEQ ID NO：10	5′-CGGGAGCAAAGGGACAGAAAAGC-3′
		SEQ ID NO：11	5′-GAAGAACTGTCCTCGGGTGGAGC-3′

SEQ ID NO：12	5′-TCAGAAAGTCTCAGGCACGAAGAAC-3′
		SEQ ID NO：13	5′-GCAGCAGAAATAAAGCACAACCTCA-3′
SEQ ID NO：14	5′-AACAGTAGCGAGAGTGGTTGTGAAA-3′
		SEQ ID NO：15	5′-CGGCACGGCACACTGGTTTGCA-3′
SEQ ID NO：16	5′-GGTGCATTCTGTCTTCCTAGTCAGG-3′
		SEQ ID NO：17	5′-GGCTCCAGATATGAACTTTCAGCAG-3′
SEQ ID NO：18	5′-GGTGGAGCATGGGACCTTTATTCGT-3′
		SEQ ID NO：19	5′-TGGCACGGCACACTGGTTTGCA-3′

The specificity and the sensitivity of antisense molecule are used for the treatment of in the purposes by those skilled in the art.Antisense oligonucleotide is used in animal and human's the condition of illness treatment as the therapeutic part.Antisense oligonucleotide is applied to the mankind effectively by safety, and the present well afoot of many clinical trials.Thereby can determine that oligonucleotide can be used as effective treatment form and is applied to handle in cell, tissue and the mammal therapy of (comprising the mankind).

Some example of useful antisense compounds comprises the oligonucleotide that contains connection in modified skeleton or the non-natural nucleoside among the present invention.Oligonucleotide with modified skeleton is included in those oligonucleotide that keep those oligonucleotide of phosphorus atoms in the skeleton and do not have phosphorus atoms in skeleton.For the purpose of this description, and sometimes as the reference in this area, the modified oligonucleotide that does not contain phosphorus atoms in its nucleoside skeleton also can be considered to oligonucleotide.

The example of modified oligonucleotide skeleton for example comprises, borine-phosphate with normal 3 '-5 ' connection, thiophosphate (phosphorothioate), the chirality thiophosphate, dithiophosphates, phosphotriester, the aminoalkyl phosphotriester, methyl and other alkylphosphonic, comprise 3 '-alkylene phosphate and chirality phosphonate, phosphate, phosphoramidate comprises 3 '-phosphoramidate and aminoalkyl phosphate ester, the thionic phosphoro-amidate, thionic-alkylphosphonic, the thionic alkyl phosphotriester, and their 2 '-5 ' connection is intended like thing, and those have the skeleton of reversed polarity, wherein the unitary adjacent connection of nucleoside to be 3 '-5 ' to 5 '-3 ' or 2 '-5 ' to 5 '-2 ' connect.Also comprise various salt, blended salt and free acid form.

The representative United States Patent (USP) of lecturing the preparation of above-mentioned phosphorous connection includes but not limited to: U.S. Patent number: 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; With 5,625,050.

Some exemplary modified oligonucleotide skeleton that does not contain phosphorus atoms has the skeleton that is formed by following structure: the interior connection or the interior connection of the heteroatomic or heterocyclic nucleoside of one or more short chains of nucleoside that connects, is mixed with hetero atom and alkyl or cycloalkyl in short-chain alkyl or the cycloalkyl nucleoside.This type of comprises that those have morpholino and connect (part is formed by the glycosyl part of nucleoside); Siloxane backbone; Sulfide, oxysulfide and sulfone skeleton; Formacetyl and thioformacetyl skeleton; Methylene formacetyl and thioformacetyl skeleton; The skeleton that contains alkene; The sulfamate skeleton; Methylene imino group and methylene hydrazine skeleton; Sulfonate and sulfanilamide skeleton; Amide backbone; And other has blended N, O, S and CH ₂The part of forming.

The representative United States Patent (USP) of lecturing the preparation of above-mentioned oligonucleotide includes but not limited to: U.S. Patent number: 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; With 5,677,439.

Intend like in the thing at other oligonucleotide, the glycosyl of nucleotide units is substituted by new group with interior be connected (that is the skeleton) of nucleoside.Can keep the base unit for hybridizing with suitable nucleic acid target chemical compound.So oligomeric compounds promptly demonstrates the oligonucleotide with good hybridization characteristic and intends being called as peptide nucleic acid(PNA) (PNA) like thing.In the PNA chemical compound, the glycosyl skeleton of oligonucleotide is substituted by the skeleton of amide containing (especially aminoethylglycine backbone).This nuclear base is retained and directly or indirectly combines with azepine (aza) nitrogen-atoms of amide moieties in the skeleton.The representative United States Patent (USP) of lecturing the preparation of above-mentioned PNA chemical compound includes but not limited to: U.S. Patent number 5,539,082; 5,714,331; And5,719,262, each patent is hereby incorporated by.Further instruction about the PNA chemical compound can be at Nielsen etc., Science, and 1991,254, find among the 1497-1500.

In other embodiment of the present invention is to have the oligonucleotide of thiophosphate skeleton and have the oligonucleotide of hetero atom skeleton, for example at U.S. Patent number 5,489,677 and U.S. Patent number 5,602,240 in provided.Also provide the oligonucleotide of the morpholino framing structure in the U.S. Patent number 5,034,506 with above reference.

Modified oligonucleotide also can comprise single the replacement or polysubstituted glycosyl part.For example, oligonucleotide can contain one of following radicals: OH in 2 ' position; F; O-, S-or N-alkyl; O-, S-or N-alkenyl; O-, S-or N-alkynyl group; Or O-alkyl-O-alkyl, wherein this alkyl, alkenyl and alkynyl group can be to replace or unsubstituted C ₁-C ₁₀Alkyl or C ₂-C ₁₀Alkenyl and alkynyl group.Also can similarly modify in other site of oligonucleotide, especially in 3 ' terminal nucleotide or 2 '-5 ' 3 ' position of the glycosyl of the oligonucleotide that connects and in 5 of 5 ' terminal nucleotide ' position.Oligonucleotide also can contain glycosyl to be intended for example substituting furan pentose with cyclobutyl moiety like thing.The representative United States Patent (USP) of lecturing the preparation of above-mentioned modified glycosyl structure includes but not limited to: U.S. Patent number 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; With 5,700,920, they include this paper in as a reference in full with it separately.

Oligonucleotide also can comprise the modification of nuclear base (being called " base " in the art usually for short) or substitute.As used herein, " not modified " or " natural " nuclear base comprises purine base adenine (A) and guanine (G) and pyrimidine bases thymus pyrimidine (T), cytosine (C) and uracil (U).Modified nuclear base comprises other synthetic and natural nuclear base, for example 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, the 2-aminoadenine, the 6-methyl of adenine and guanine and other alkyl derivative, the 2-propyl group of adenine and guanine and other alkyl derivative, the 2-thiouracil, 2-sulfur thymus pyrimidine and 2-sulfur cytosine, 5-halo uracil and cytosine, 5-acrylic uracil (5-propynyl uracil) and cytosine, the 6-azauracil, cytosine and thymus pyrimidine, 5-uracil (pseudouracil), the 4-thiouracil, the 8-halo, 8-amino, the 8-sulfo-, the 8-alkylthio, adenine and guanine that 8-hydroxyl and other 8-replace, the 5-halo is the 5-bromo especially, uracil and cytosine that 5-trifluoromethyl and other 5-replace, 7-methyl guanine and 7-methyladenine, 8-azaguanine (8-azaguanine) and 8-nitrogen adenine (8-azaadenine), 7-denitrification guanine (deazaguanine) and 7-denitrification adenine (deazaadenine) and 3-denitrification guanine and 3-denitrification adenine.The representative United States Patent (USP) of lecturing the preparation of above-mentioned some modified nuclear base and other modified nuclear base includes but not limited to: U.S. Patent number 3,687,808 mentioned above, and U.S. Patent number 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; 5,681,941 and 5,750,692, they include this paper in as a reference in full with it separately.

Other modification of oligonucleotide of the present invention relates to this oligonucleotide is connected with one or more parts that can promote its activity, cell distribution or cellular uptake or jointer chemistry.These parts include but not limited to: lipid part, for example cholesterol moiety, bile acid, thioether as: hexyl-S-trityl mercaptan, sulfo-cholesterol, aliphatic chain as: laurane glycol or undecyl residue, phospholipid are as two-cetyl-rac-glycerol or 1,2-two-O-cetyl-rac-glyceryl-3-H-tricresyl phosphate ethyl-ammonium, polyamine or polyglycol chain or adamantane acetic acid, palmitin acyl part or octadecylamine or hexyl amino-carboxyl-oxygen cholesterol moiety.The representative United States Patent (USP) of lecturing the preparation of these oligonucleotide jointers includes but not limited to: U.S. Patent number 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 and 5,688,941, they include this paper in as a reference separately.

Do not need all sites of the chemical compound that provides is modified without exception, and in fact individualized compound or even single nucleoside in oligonucleotide in, can integrate more than a kind of modification mentioned above.

The present invention also comprises it being the antisense compounds of chimeric chemical compound.In background of the present invention, " chimeric " antisense compounds or " chimera " are antisense compounds, especially oligonucleotide, it contains 2 or a plurality of chemical independent zones (chemically distinct regions), each zone is made up of at least one monomeric unit, that is, be nucleotide under the situation of oligonucleotide chemical compound.These oligonucleotide contain at least one zone usually, thereby oligonucleotide modifiedly strengthens the resistance of the anti-ribozyme of this oligonucleotide degraded in this zone, cellular uptake strengthens and/or strengthen with the binding affinity of target nucleic acids.Other zone of this oligonucleotide can be used as the substrate of enzyme, and this enzyme has the ability of dissociating RNA:DNA or RNA:RNA hybridization chain.As an example, RNase H is the Cobra venom endonuclease of cell, its RNA chain in the RNA:DNA two strands that can dissociate.Therefore, the activation of RNaseH has caused the cutting of RNA target, thereby has greatly promoted the effect of oligonucleotide inhibition of gene expression.Therefore, when using chimeric oligonucleotide, can compare favourably with the crossbreeding effect in thiophosphate deoxy-oligonucleotide and same target zone than the crossbreeding effect in short oligonucleotide and target zone.The available gel electrophoresis of the cutting of RNA target carries out conventional sense, and if need, (nucleic acid hybridizationtechnique) carries out conventional sense with associated nucleic acid hybridization technology known in the art.

Chimeric antisense compounds of the present invention can be used as 2 or a plurality of oligonucleotide mentioned above, modified oligonucleotide, oligonucleoside and/or oligonucleotide intended forming like the combinative structure of thing.These chemical compounds also are called as heterocomplex (hybrid) or interval body (gapmer) in the art.The representative United States Patent (USP) of lecturing the preparation of these hybrid structures includes but not limited to: U.S. Patent number 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, they include this paper in as a reference in full with it separately.

Antisense compounds used according to the invention can the convenient and preparation routinely with solid phase synthesis technique well-known in the art.This synthetic required equipment is sold by the how tame seller, for example comprises Applied Biosystems (Foster City, California).Be used for this synthetic any other method known in the art and can add or replace use.Can use similar techniques to prepare oligonucleotide as everyone knows, for example, thiophosphate and alkyl derivative.

Antisense compounds of the present invention can be external synthetic.Chemical compound of the present invention also can with the mixture of other molecule, molecular structure or chemical compound (for example, the molecule of liposome, receptor target, oral, rectally, topical or other preparation) mix mutually, wrap up, in conjunction with or coupling, to assist picked-up, distribute and/or to absorb.The representative United States Patent (USP) of lecturing these picked-ups, distribute and/or absorb the preparation of assisting preparation includes but not limited to: U.S. Patent number 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, they include this paper in as a reference separately.

Antisense compounds of the present invention also comprises the salt of any pharmaceutically acceptable salt, ester or these esters, or any other chemical compound of (directly or indirectly) biologic activity metabolite or its residue can be provided after giving animal (comprising the mankind).Therefore, for example, the pharmaceutically acceptable salt of prodrug that also comprises antisense compounds of the present invention that is disclosed and pharmaceutically acceptable salt, these prodrugs and other bioequivalence thing.

Antisense compounds of the present invention can be used for diagnosis, treatment, prevention and conduct research reagent and test kit.Be used for the treatment of, use antisense compounds of the present invention can for and doubtfully suffer from the animal (especially people) of (available regulation and control TFF3 express treat) disease or disease and treat.Can join by antisense compounds in the suitable pharmaceutically acceptable diluent or carrier and chemical compound of the present invention is used in the pharmaceutical compositions effective dose.Use antisense compounds of the present invention and method also can be effective in the prevention, for example, the formation or the transfer of prevention or delay tumor.

Antisense compounds of the present invention can be effective to research and diagnosis, this be since these chemical compounds can with the nucleic acid hybridization of coding TFF3, analyze thereby can set up sandwich and other easily.The hybridization of antisense oligonucleotide of the present invention and TFF3 code nucleic acid can detect by methods known in the art.These methods can comprise with enzyme combine with this oligonucleotide, with this oligonucleotide of isotopic labeling or other any suitable detection method.Also can prepare and use these detection methods to come the test kit of TFF3 level in the test sample.

The present invention also comprises pharmaceutical compositions and the preparation that comprises antisense compounds of the present invention.In some embodiments, the compositions that contains antisense molecule can be used as Emulsion preparation and prescription.Emulsion is generally a kind of liquid and is scattered in the another kind of liquid and the heterogeneous body system that forms with the form of drop, and the diameter of this drop is usually above 0.1 micron.Emulsion is generally two phase systems, and it contains two kinds and mixes closely and dispersive each other immiscible liquid phase.Usually, Emulsion can be Water-In-Oil (w/o) or oil-in-water (o/w) class.When a kind of water scatter well and be dispensed in a large amount of oil phases as fine droplet, the compositions of gained was called Water-In-Oil (w/o) Emulsion.On the contrary, when a kind of oil phase scatter well and is dispensed into a large amount of aqueous phases as fine droplet, the compositions of gained was called oil-in-water (o/w) Emulsion.Except decentralized photo and active medicine, also can contain other composition in the Emulsion, and active medicine can be used as the solution in water or oil phase or self exists as decentralized photo.Excipient pharmaceutically as, emulsifying agent, stabilizing agent, dyestuff and antioxidant also can exist in Emulsion as required.Pharmaceutical emulsion also can be multiple emulsion, and it contains more than 2 phases, for example, and the situation of Water-In-Oil bag oil (o/w/o) and W/O/W (w/o/w) Emulsion.Some advantage that these compound formulations often can provide single binary Emulsion not had.The multiple emulsion that the single oil droplet of o/w Emulsion wraps up little water droplet formation has constituted w/o/w Emulsion.Equally, stablize the water droplet parcel oil droplet system that is arranged in oily continuous phase and formed o/w/o Emulsion.

In another relevant embodiment, method and composition of the present invention can contain one or more antisense molecules, especially the oligonucleotide of targeting first nucleic acid and one or more targeting second other antisense compounds of nucleic acid target target.Two or more coupling chemical compounds can use or use in order simultaneously.In some embodiments, at least a target nucleic acids coding TFF3.

Ribozyme

Nertralizer for example also can be, and suppresses the ribozyme that TFF3 expresses.Ribozyme is generally have the ribose activity catalytic RNA molecule of (ribonucleic activity), and they can remove the single-chain nucleic acid (as mRNA) that has complementary region with it.Because ribozyme is a kind of enzyme, single ribozyme molecule can cut the molecule of many target RNA.In addition, ribozyme is generally highly narrow spectrum inhibitor, and the specificity of its inhibition had both depended on itself and the bonded base pairing mechanism of target RNA, also depends on the cutting mechanism of target RNA.Design is used for the ribozyme molecule of catalyze cleavage target gene mRNA transcript and also can be used for preventing the translation of target gene mRNA, thereby and prevents the expression of target gene product.(referring to, as, WO90/11364 and Sarver etc., 1990, Science 247,1222-1225).

Ribozyme can be designed for target RNA in different loci anneal.This brachium conjunctivum can with the target site complementation, for example, the TFF3 polynucleotide.But ribozyme chemosynthesis.Exemplary synthetic method can be according to Usman etc.; 1987, J.Am.Chem.Soc., 109; 7845-7854 and Scaringe etc.; 1990, Nucleic Acids Res., 18; the synthetic process of common RNA described in the 5433-5441 is carried out; and can use protection of common nucleic acid and coupling group, for example 5 '-end adds dimethoxytrityl and 3 '-end adds phosphoramidate.The productive rate that on average progressively is coupled can be＞and 98%.Hair fastener shape ribozyme also can divide the synthetic and annealing of two parts with reconstitute active ribozyme (Chowrira and Burke, 1992, Nucleic Acids Res., 20,2835-2840).Ribozyme by ribozyme resistance group (for example, 2 '-amino, 2 '-C-pi-allyl, 2 '-fluorine, 2 '-o-methyl, 2 '-H) modification after, can improve stability (referring to, as, Usman and Gedergren, 1992, TIBS 17,34, and the document is included into this paper as a reference).

Ribozyme can carry out purification with method known in the art, as gel electrophoresis or high pressure lipuid chromatography (HPLC) (HPLC).

Ribozyme activity can be optimized by the ribozyme of chemosynthesis is modified, this modification can prevent they by serum ribonucleic acid enzymatic degradation (referring to, for example, Eckstein etc., WO92/07065; Perrault etc., Nature, 1990,344:565; Pieken etc., Science, 1991,253:314; Usman and Cedergren, Trendsin Biochem.Sci.1992,17:334; Usman etc., WO93/15187; With Rossi etc., WO91/03162 and U.S. Patent number 5,652,094, each document are included into this paper as a reference, and they have described the various chemical modifications that can be used for catalytic RNA molecular saccharides base section.

Therefore, ribozyme (for example, hair fastener shape, hammerhead shape or RNAase P ribozyme, and minimum enzyme (minizyme) (McCall, M. (1992) Proc.Natl.Acad.Sci.USA 89:5710-5714) or other catalytic RNA molecule) thus can be used for catalysis removes the translation that the TFF3 transcript suppresses TFF3mRNA.The design that TFF3 is had specific ribozyme can be based on the nucleotide sequence of TFF3, as, human TFF 3 DNA sequence that this paper provided or relevant polynucleotide.The technology of synthetic ribozyme is at Cech etc., and United States Patent (USP) 4,987 discloses in 071 and 5,116,742 to some extent.In addition, TFF3mRNA can be used for from numerous RNA molecules selecting the having active catalytic RNA of specific ribonucleic acid (referring to, as, Bartel and Stostak, J.W., J.Biol.Chem.1261:1411-1418 (1993)).

Perhaps, can suppress the expression of TFF3 with the following method, i.e. the complementary nucleotide sequence of targeting and TFF3 control region (promoter, enhancer), thus prevent transcribing in the target cell to form triple-helix structure.(referring to, as, Helene etc., Annal.NYAcad Sci.660:27-36 (1992)).

According to some embodiment, nertralizer is a hammerhead ribozyme.Hammerhead ribozyme is believed to be and can by right with said target mrna formation complementary base, thereby cuts mRNA in the position of being instructed by flanking region.Using a characteristic of hammerhead ribozyme is that said target mrna has two following base sequences: 5 '-UG-3 '.The structure of hammerhead ribozyme and generation are well-known in the art, and at Myers, 1995, molecular biology and biotechnology (MolecularBiology and Biotechnology): complete operation is with reference to (A Comprehensive Desk Reference), VCHPublishers, describe to some extent among the New York, (referring to, particularly scheme .4, the 833rd page) and Haseloff and Gerlach, 1988, Nature, 334:585-591.

According to some embodiment, ribozyme through through engineering approaches so that the cutting recognition site near 5 ' end of target gene mRNA, as, accumulation minimizes in the born of the same parents that the mRNA of non-functional transcribes thereby raise the efficiency and make.

Ribozyme of the present invention also comprises RNA Cobra venom endonuclease (hereinafter " Cech-type ribozyme "), naturally occurring ribozyme (being known as IVS or L-19 IVS RNA) in Tetrahymena thermophila for example, it encyclopaedizes (Zaug etc. by Thomas Cech and partner thereof, 1984, Science, 224:574-578; Zaug and Cech, 1986, Science, 231:470-475; Zaug etc., 1986, Nature, 324:429-433; International patent application no WO88/04300 by University Patents Inc. publication; Been and Cech, 1986, Cell, 47:207-216).Cech-type ribozyme has the avtive spot of 8 base pairs usually, and this avtive spot can be hybridized with target RNA, and the cutting to this target RNA takes place then.

Can directly add ribozyme, or the lipid of ribozyme and cationization is compound, liposome or otherwise be delivered to target cell.This RNA or RNA complex can be when its integration or unconformity be in biopolymer, and external topical administration carries out vivo medicine-feeding to respective organization or by injection, aerosol suction, infusion pump or graft.

Ribozyme can be bestowed cell by the method known to the multiple those of ordinary skill in the art, these methods include but not limited to, be wrapped in the liposome, by iontophoresis or by being integrated in other carrier, the microsphere of hydrogel, cyclodextrin, biodegradable Nano capsule and bioadhesive for example.For some indication, ribozyme can use or not use under the situation of above-mentioned carrier, directly the external cell or tissue that is delivered to.In addition, the RNA/ carrier compositions can or use conduit, infusion pump or graft local delivery by direct injection.The mode of sending of other routine includes but not limited to: in the blood vessel, intramuscular, subcutaneous or joint injection, aerosol suck, send in oral (tablet or pill dosage form), part, system, ophthalmic, intraperitoneal and/or the sheath.About ribozyme send with administration be described in more detail in Sullivan etc., provide among the WO94/02595, the document is included this paper in as a reference in full with it.

Can relate in the another kind of method of cell inner accumulation high concentration ribozyme the ribozyme coded sequence is incorporated in the DNA expression vector.Transcribing of this ribozyme sequence can be driven by the promoter of eucaryotic RNA polymerase I (pol I), rna plymerase ii (pol II) or rna plymerase iii (pol III).In all cells, all will be by transcribing of pol II or pol III promoters driven with high level expression; Near the character (enhancer, silencer etc.) of the existing gene regulation sequence level of the given pol II promoter in given cell type depends on.Also can use the prokaryotic rna polymerase promoter, prerequisite is that this prokaryotic rna polymerase is expressed (Elroy-Stein, O. and Moss, B., 1990, Proc in suitable cell.Natl.Acad.Sci.USA, 87,6743-7; Gao, X. and Huang; L, 1993, NucleicAcids Res., 21,2867-72; Lieber, A. etc., 1993, Methods Enzymol., 217,47-66; Zhou, Y. etc., 1990, Mol.Cell.Biol., 10,4529-37).Several researcheres proved the ribozyme of expressing by this class promoters driven can be in mammalian cell functionating (for example (Kashani-Sabet, M. etc., 1992, Antisense Res.Dev., 2,3-15; Ojwang, J.O. etc., 1992, Proc.Natl.Acad.Sci.USA, 89,10802-6; Chen, C.J. etc., 1992, Nucleic Acids Res., 20,4581-9; Yu, M. etc., 1993, Proc.Natl.Acad.Sci.USA, 90,6340-4; L ' Huillier, P.J. etc., 19921, EMBO J., 11,4411-8; Lisziewicz, J. etc., 1993, Proc.Natl.Acad.Sci.U.S.A., 90,8000-4)).Above-mentioned ribozyme transcriptional units can be incorporated in the variety carrier to introduce mammalian cell, include but not limited to: plasmid DNA carrier, viral DNA carrier (for example adenovirus or gland relevant carriers) or viral rna vector (for example, retrovirus retrovirus, Semliki forest virus, Sindbis disease poisonous carrier).

According to some embodiment, express cutting target RNA (for example, the transcriptional units of hair fastener forming core enzyme TFF3mRNA) can be inserted in the plasmid DNA carrier or adenovirus or the relevant dna viral vector of gland in.Two kinds of viral vector all have been used to gene is delivered to pulmonary, and two kinds of carriers all can cause transient gene expression (Zabner etc., 1993 Cell 75,207; Carter, 1992 Curr.Opin.Biotech.3,533).Adenovirus vector is sent as the recombinant adenovirus microgranule.Dna single solely can be sent or with compound the sending of carrier (as above to RNA description).DNA, DNA/ carrier complexes, but or recombinant adenovirus granule topical administration to therapentic part, as, by using injection catheter, graft or infusion pump or directly joining in the cell or tissue external.

The RNAi molecule

It is the reticent mechanism of a kind of revolutionary ground conservative gene that RNA disturbs (RNAi), is (Lee etc., the Cell 75:843 (1993) that finds in the research to nematicide Caenorhabditis elegans the earliest; Reinhart etc., Nature 403:901 (2000)).It is to bring out by dsRNA (double-stranded RNA) being incorporated in the cell of expressing the molecule machine that is fit to that this mechanism is believed to be, and corresponding endogenous mRNA then degrades.This mechanism relates to dsRNA is converted into short rna, and this short rna is directed to (be summarized in Ruvkun, among the Science 2294:797 (2001), the document is included this paper in as a reference) on the homologous mRNA target with ribonuclease.This process relates to the normal defence of virus and the mobilization of transposon.Treating with dsRNA becomes the functional organic more general mode of analyzing gene.RNA disturbs or " RNAi " is the term that is proposed by Fire and colleagues at first, they are viewed after double-stranded RNA (dsRNA) is introduced into nematicide with this term description, its can seal gene expression phenomenon (Fire etc. (1998) Nature 391,806-811).

The RNAi molecule that is used to realize the gene expression regulation of RNAi mediation can comprise the polymerization ribonucleotide of strand or multichain.It can comprise the modification of any number, the modification to antisense molecule for example mentioned above, comprise to phosphate-glycosyl skeleton, to glycosyl or nuclear base modification, with enhanced stability or binding affinity.For example, the phosphodiester bond of natural RNA can be modified to contain at least one nitrogen or sulfur heteroatom.During the common emergency reaction that in avoiding some tissue, produces, can specially adjust modification in the RNA structure so that specific gene is inhibited by dsRNA.Equally, also can modify to seal the activity of ADA Adenosine deaminase base.RNA can produce or produce by part/whole organic synthesiss by enzymatic.By external enzymatic or organic synthesis, any modified ribonucleotide can be incorporated among the RNA.The RNAi molecule can be the dsRNA molecule.

Suppress the size of the theme polynucleotide of its expression as required, the size of available RNAi molecule also can change to some extent, and this RNAi molecule should long enough and can reduce the expression of theme polynucleotide in cell.Usually, it is long that this RNA is at least about about 15 nucleotide of 10-.In some applications, the length of this RNA is less than about 20,21,22,23,24 or 25 nucleotide.In other cases, the length of this RNA is at least about 50,100,150 or 200 nucleotide.During other was used at some, this RNA can be longer, for example, and at least about 300,400,500 or 600 polynucleotide.Typically, this RNA is less than about 3000 nucleotide.Those of ordinary skill in the art need not to use undue experimentation can determine the optimization size of any concrete theme polynucleotide, and its method is to change the size of RNA and determine whether selected size can effectively disturb the expression of theme polynucleotide in systematized mode.

The amount that this RNAi molecule can make each cell delivery deliver to the RNAi of at least one copy is introduced.The double-stranded material of higher dosage (for example, at least 5,10,100,500 or 1000 copy/cells) can obtain more effectively to suppress; Also can be used in the concrete application than low dosage.Inhibition is sequence-specific, and promptly the nucleotide sequence corresponding to this RNA double stranded region is the target of gene inhibition.In tissue, introduce enough RNA, but the change detected (the supposition candidate gene is actual expression the in the cell of introducing this RNA) to cause expression of target gene, the detection method that use can get.Therefore, in some cases, compare, introduce enough RNA to obtain the reduction that candidate gene is expressed at least about 5-about 10% with the cell of not introducing this RNA.In other cases, suppress to be at least about 20,30,40 or 50%.Under other situation that also has, this inhibition is at least about 60,70,80,90 or 95%.But expression in some cases is suppressed to fully basically and is lower than detection level.

The amount of introducing RNA depends on various factors, for example, and the size of the employed pattern of administration, RNA, the cell number that RNA fed and if dsRNA is introduced a certain animal, age of this animal and size.For example, those of ordinary skill in the art can determine suitable dosage by the RNA that gives a plurality of variable concentrations at first.In some cases, when being incorporated into RNA in the cultured cell, the amount of introducing the RNA of cell can change at the about microgram of about 0.5-/106 iuntercellulars.

There are multiple choices available for the interference that detects the candidate gene expression (that is, detecting the silence of candidate gene).Usually, can change the inhibition that detects in the expression by level and/or the detection phenotype relevant of the mRNA of this genetic transcription by reduction, the mensuration that detects by the level of candidate gene encoded protein matter with the candidate gene expression.

The RNA that contains identical with the part of target gene (for example, TFF3 polynucleotide) basically nucleotide sequence can be used for RNA and suppresses.The RNA sequence that has insertion, disappearance and single site mutation with respect to target sequence also can be effective to suppress.Therefore, as known in the art, can be optimized sequence homogeneity, the method of optimizing is by the percentage difference between sequence alignment and array algorithm and calculating nucleotide sequence, for example in the BESTFIT software program, use the Smith-Waterman algorithm (for example, University ofWisconsin Genetic Computing Group) of default parameters execution.In some embodiments, the sequence homogeneity that suppresses between the part of RNA and target gene can be greater than 90%, or even reaches about 100%.In addition, the double stranded region of RNA can by functional be defined as the nucleotide sequence that can partly hybridize with the target gene transcript (for example, 400mM NaCl, 40mMPIPES pH 6.4,1mM EDTA is in 50C or 70 ℃ hybridization 12-16 hour down; Washing then).The length of this RNA nucleotide sequence can be at least about 15,20,25,50,100,200,300 or 400 bases.

As disclosed herein, 100% sequence homogeneity is unwanted putting into practice when of the present invention between RNA and target gene.Therefore, the present invention has the advantage that can tolerate sequence difference, and this sequence difference can be gene mutation, chain polymorphism or evolutionary divergence to be caused.Yet,, between the part that suppresses RNA and target gene, have 100% sequence homogeneity according to some embodiment.In addition, the RNA that has greater than about 70%, 80%, 90% or 95% sequence homogeneity can be used for the present invention, can tolerate the sequence difference that expectation is caused by gene mutation, chain polymorphism or evolutionary divergence thus.

RNA can be in vivo or is external synthetic.The endogenous RNA polymerase of cell can mediate intravital transcribing, or clone's RNA polymerase can be used in the body or external transcribing.In order to transcribe from transgenic in vivo or in the expression construct, can use control region (for example, promoter, enhancer, silencer, joint donor and receptor, polyadenylic acid) to transcribe one or more RNA chain.Can come targeting to suppress by the specific transcriptional in organ, tissue or the cell type; The stimulation of environmental condition (for example, infection, pressure, temperature, chemical inducer); And/or carry out through engineering approaches at developmental stage or age and transcribe.The RNA chain can be randomly by poly-adenosine; The RNA chain can randomly can be translated into polypeptide by the translation unit of cell.RNA also can be synthetic by the reactive chemistry or the enzymatic of artificial or automatization.This RNA can be by cell RNA polymerase or bacteriophage RNA polymerase synthetic (for example, T3, T7, SP6).Expression construct use and production be well known in the art (referring to, as, WO97/32016; U.S. Patent number: 5,593,874; 5,698,425; 5,712,135; 5,789,214 and 5,804,693).If RNA be chemosynthesis or external synthetic by enzymatic, then this RNA can be purified before introducing cell.For example, can from mixture, come purifying RNA with solvent or resin extraction, precipitation, electrophoresis, chromatography or its combined method.In addition, RNA can be not purified or be used through minimum purification, with the loss of avoiding causing owing to sample treatment.But the RNA drying is for storing or being dissolved in the aqueous solution.This solution can contain buffer agent or salt is annealed with promotion, and/or make double-stranded stable.

According to the present invention, have with the RNA of the similar or substantially the same sequence in a certain zone of TFF3 polynucleotide sequence and be used in regulation and control TFF3 polypeptide expression in the cell, for example by the RNA-interference mechanism.The selection in target sequence zone can be carried out according to any standard that above-mentioned antisense regulation and control TFF3 expresses.In some embodiments, RNA can comprise and the substantially the same or complementary basically sequence of arbitrary sequence among the SEQ ID NO:5-19.RNA also can be overlapping corresponding to certain a part of sequence or its complementary series in arbitrary sequence among the SEQ ID NO:5-19.

RNA can be given on the organ or patient that give antisense molecule as described herein.According to some embodiment, RNA can directly introduce cell (that is, in the born of the same parents); Or introduce the circulation of cavity, space, introducing organism, oral introducing outward by born of the same parents and maybe can introduce RNA by organism is immersed in the solution that contains RNA.The method of oral introducing comprises: directly RNA is mixed with the food of organism, and use the through engineering approaches approach, even as the species through engineering approaches ground expressed rna of food, give the organism that will introduce RNA with its feeding then.Also can use physical method to introduce nucleic acid, for example, be injected directly into cell or born of the same parents and be injected into organism outward.In the blood vessel or blood vessel outer circulation, blood or lymphsystem, phloem, root and cerebrospinal fluid all can be the site that RNA introduces.Can derive from zygote, embryonic stem cell or other pluripotent stem cell that is fit to organism by construct is introduced, and produce transgenic organism by the recombinant precursor expressed rna.

The physical method of introducing nucleic acid comprises: injection contain RNA solution, with the microparticle bombardment of RNA bag quilt, with cell organism immerses in the RNA solution or under the situation that RNA exists cell membrane carry out electroporation.The virus formulation body that wraps up virion can reach effectively introduces cell and the purpose of transcribing by the RNA of expression construct coding with expression construct.Can use other known in the art nucleic acid to be incorporated into method in the cell, for example liposome-mediated carrier transportation, the transportation of chemicals mediation, for example calcium phosphate etc.Therefore, RNA can with have one or more following active compositions and introduce jointly: strengthen cell to the picked-up of RNA, promote the double-stranded of double-stranded annealing, stabilizing annealing or strengthen inhibition in addition target gene.

The check that expression of target gene suppresses can be carried out by the following method: observe or detect shortage or observable decline (it can detect by for example specific antibody or other method known to those of skill in the art) by target gene encoded protein matter level; And/or the mRNA product of observation or detection target gene (it can be hybridized research and detect by for example); And/or observation or the detection phenotype relevant with gene expression.Under the Drug therapy background, the variation that the check that expression of target gene is suppressed can be by object of observation morbid state (for example the variation of the alleviating of symptom, disappearance, morbid state etc.) is carried out.According to some embodiment, the variation of morbid state can be reducing etc. of the reducing of the reducing of the slowing down of tumor growth, gross tumor volume, cellular activity, cellular invasion power.Preferably, this inhibition is specific, that is, other gene that the inhibition of target gene can pair cell causes remarkable influence.

Giving the amount that mammal is used for effectively carrying out the RNA of gene inhibition can change according to many factors, comprises approach, this mammiferous age, size and situation, the gene that will suppress, the disease that will treat or the imbalance etc. of administration.According to some embodiment, can give dsRNA in each cell, to provide 0.1-400 pik, preferably 1-40 pik or the more preferably dsRNA of 1-20 pik.

Be well known in the art (respectively referring to Schubiger and Edgar by transcribing the method for using RNAi in external source adding or the body, Methods in Cell Biology (1994) 44:697-713, with PCT application WO99/32619, these documents are included this paper in as a reference separately), for example, in C.elegans, RNAi has demonstrated (Lu and the Horvitz of knocking out to tumor suppressor gene in the pudendum precursor, Cell (1998) 95:981-991, the document is incorporated herein by reference in full with it).

U.S. Patent number 6,506,559 and U.S. Patent Application Publication No. 20030027783 (relate in the mammal gene expression suppress) further provide and use RNAi to reduce or suppress the method for some gene product expression, each document is incorporated herein by reference in full with it.

Antibody

Nertralizer also can be immunoglobulin, for example, can with the protein bound antibody of TFF3 or its fragment.Immunoglobulin can comprise that antibody and other lack the antibody sample molecule of antigenic specificity.One type the polypeptide in back is, for example, and the polypeptide that produces by the myeloma high level with low-level generation by lymphsystem.

" natural antibody and immunoglobulin " is generally about 150,000 daltonian different four dimerization glycoproteins, and it contains two identical light (L) chains and two identical weights (H) chain.Every light chain is connected with a heavy chain by a covalent disulfide bonds, and the quantity of disulfide bond is different in different homotypic immunity globulin between heavy chain.Each heavy chain and light chain also have the interior disulphide bridges of chain of regular spaces.Each heavy chain at one end has variable region (VH), is thereafter many constant regions.

From the antibody (immunoglobulin) of arbitrary vertebrates kind, its " light chain " can be designated as a kind of (being called κ and λ) in two kinds of visibly different types, and this is based on the aminoacid sequence of their constant regions and is fixed.Based on the aminoacid sequence of their CH, complete antibody can be designated as different " class ".

Each light chain has variable region (VL) at the one end and has constant region at its other end; First constant region of the constant region of light chain and heavy chain is in line, and the variable region of light chain then is in line with the variable region of heavy chain.Special amino acid residue be believed to be can between the variable region of light chain and heavy chain, form the interface (Chothia etc., J.Mol.Biol.186:651 (1985; Novotny and Haber, Proc.Natl.Acad.Sci.U.S.A.30 82:4592 (1985); Chothia etc., Nature 342:877-883 (1989)).

What term " antibody " used is its implication the most widely, and has contained the polyclonal antibody and the monoclonal antibody of assembling fully, and antibody fragment that can conjugated antigen (for example, Fab ', F ' (ab) 2, Fv, single-chain antibody, binary (diabody)).

Term used herein " monoclonal antibody " is meant that promptly, the antibody individuality of forming this colony is all identical available from the antibody of homologous antibody population basically, except there being a small amount of possible spontaneous mutation.

Hereinafter described example technique is used for producing antibody nertralizer used according to the invention.

Antibody is called as " specificity combination ", if: 1) they demonstrate in conjunction with active threshold level, and/or 2) they with known related polypeptide molecule significant cross reaction do not take place.Those of ordinary skill in the art can measure the binding affinity of antibody easily, for example, analyzes (Scatchard, Ann.NYAcad.Sci.51:660-672,1949) by Scatchard.In some embodiments, antibody of the present invention is higher than at least 103 times of other TFF3 associated protein with combining of TFF3, and more preferably at least 104 times, more preferably at least 105 times, and even more preferably at least 106 times.

In some embodiments, antibody of the present invention not with known relevant peptide molecule specificity in conjunction with (or identification), for example, if the Western engram analysis (Ausubel etc.) of the standard of use, they combine with the TFF3 polypeptide but do not combine with known related polypeptide.In some embodiments, can from known related polypeptide, filter out antibody, to isolate the antibody population of specificity in conjunction with the TFF3 polypeptide.For example, under suitable buffer condition, specificity can flow through in conjunction with the antibody of TFF3 polypeptide and contain attached to the post of (not containing TFF3) of the TFF3 family polypeptides on the insoluble matrix.This screening makes and is not separated (antibody (Antibodies): laboratory manual, Harlow and Lane (volume), Cold Spring Harbor LaboratoryPress, 1988 with the polyclone of closely-related polypeptide generation cross reaction with monoclonal antibody; Current Protocols in Immunology, Cooligan etc. (volume), Naional Institutesof Health, John Wiley and Sons, Inc., 1995).The screening of specific antibody with separate be in the art well-known (referring to, basic immunology (Fundamental Immunology), Paul (volume), RavenPress, 1993; Getzoff etc., Adv.in Immunol.43:1-98,1988; Monoclonal antibody (Monoclonal Antibodies): principle and put into practice (Principles and Practice), Goding, J.W. (volume), Academic Press Ltd., 1996; Benjamin etc., Ann.Rev.Immunol.2:67-101,1984).The exemplary of these analyses comprises: synchronous immunoelectrophoresis (concurrent immunoelectrophoresis), radioimmunoassay (RIA), radioimmunoprecipitation, Enzyme Linked Immunoadsorbent Assay (ELISA), dot blot or the analysis of the Western marking, inhibition or competition analysis and sandwich assay.

In some embodiments, antibody of the present invention is higher at least about 1000 times than known relevant family member for the affinity of TFF3, and is high at least about 10,000 times.In some embodiments, antibody of the present invention,, less than about 1 * 104Ka and is preferably less than 1 * 103Ka less than 1 * 105Ka the binding affinity of the related polypeptide except TFF3.

Polyclonal antibody

By repeatedly subcutaneous (sc), intraperitoneal (ip) or intramuscular (im) injection related antigen and auxiliary agent, can in animal, produce polyclonal antibody.It is favourable that related antigen is combined with protein (having immunogenic protein in the species of wanting immunity); as; with keyhole type hemocyanin (keyhole limpet hemocyanin); serum albumin; the bovine thyroglobulin combination; or combine with soybean trypsin inhibitor; used difunctional dose or derivating agent are for example; maleimide benzoyl sulfosuccinimide ester (maleimidobenzoyl sulfo succinimideester (by the cysteine residues combination)); N-hydroxy-succinamide (by the lysine residue combination); glutaraldehyde; succinic anhydrides; SOC12 or R1N=C=NR, wherein R is different alkyl groups with R1.Animal is carried out immunity with antigen, immunogenic conjugates or derivant, and method is with for example, and 100 pik protein or conjugate (respectively to rabbit or mice) mix with the complete auxiliary agent of Fu Shi of 3 volumes, and at this solution of multidigit point intradermal injection.After one month, in the complete auxiliary agent of Fu Shi, this animal is carried out booster immunization by multidigit point subcutaneous injection with peptide or the conjugate of primary quantity 1/5-1/10.After 7-14 days,, and serum carried out the antibody titer analysis with this animal blood-letting.Animal is carried out enhance immunity reach plateau until titre.Preferably, this animal carries out enhance immunity with same antigen with the conjugate of different proteins and/or by different cross reaction reagent.Conjugate also can produce as the protein blend compound in the reorganization cultured cell.Equally, polymerizer (aggregating agent), for example Alumen also is suitable for promoting immunne response.

Monoclonal antibody

Monoclonal antibody is available from homologous antibody population basically, that is, the antibody individuality of forming this colony is all identical, except there being a small amount of possible spontaneous mutation.Therefore, modifier " monoclonal " is meant that the character of this antibody is not the mixture of discrete antibody.For example, available hybridoma legal system is equipped with monoclonal antibody, and this method is the earliest by description (Nature, 256:495 (1975)) or available recombinant DNA method (U.S. Patent number 4,816,567) preparations such as Kohler.

In the hybridoma method, the host animal (for example rabbit or hamster) that mice or other are fit to carries out immunity according to mentioned above, with stimulate lymphocyte produce maybe can generation meeting specificity in conjunction with the proteinic antibody that be used for immunity.In addition, can carry out immunity to lymphocyte external.(for example use suitable fusion reagent then, Polyethylene Glycol), lymphocyte and myeloma cell are merged, to form hybridoma [Goding, monoclonal antibody (MonoclonalAntibodies): principle and put into practice (Principles and Practice), 59-103 page or leaf (Academic Press, 1986)].

The hybridoma that makes is thus inoculated and is grown in the suitable culture medium, and this culture medium preferably contains one or more and suppresses the not parental generation myeloma cell's of fusion the growth and the material of survival.For example; if the parental generation myeloma cell lacks hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT); usually can contain hypoxanthine, aminopterin-induced syndrome and thymidine (HAT substrate) in this hybridoma culture medium, these materials have been organized the growth of HGPRT deficient cell.

Representational myeloma cell is effective fusion, support the stable high level of antibody to produce by the antibody produced cell of selecting, and those myeloma cells responsive to culture medium (HAT medium matrix), comprise myeloma cell line, the myeloma cell line of muroid for example, comprise that the myeloma cell line derived from MOPC-21 and MPC-11 mouse tumor (can be available from Salk Institute Cell Distribution Center, Santiago, California, the U.S.) and SP-2, NZ0 or X63-Ag8-653 cell (can be available from American Type Culture Collection, Luo Keweier, Maryland, the U.S.).Human myeloma and mice-people's heterozygosis myeloma cell line also has been described and has been used to produce human monoclonal antibodies [Kozbor, J.Immunol., 133:3001 (1984); Brodeur etc., the production technology of monoclonal antibody and application (Monoclonal Antibodies Production Techniques andApplications), 51-63 page or leaf (Marcel Dekker, Inc., New York, 1987)].

The culture medium that hybridoma grows in is wherein analyzed the generation that has required specific monoclonal antibody with detection, as, by external binding analysis for example, Enzyme Linked Immunoadsorbent Assay (ELISA) or radioimmunoassay, RIA (RIA).The position of the cell of expressing antibodies can be detected with FACS.Then, the hybridoma clone can be formed sub-clone (subcloned) by the limiting dilution step, and by standard method growth (Goding, monoclonal antibody (Monoclonal Antibodies): principle and put into practice (Principles and Practice), AcademicPress (1986) 59-103 page or leaf).The culture medium of using in order to reach this purpose that is fit to comprises, for example, and DMEM or RPMI-1640 culture medium.In addition, hybridoma can be grown as ascites tumor in animal body.

Suitably obtain separating by the excretory monoclonal antibody of sub-clone immunoglobulin purification technology by routine from culture medium, ascites or serum, these purifying process are for for example, protein A-agarose method (proteinA-Sepharose), hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatograph.

The DNA of coding monoclonal antibody can use conventional technology to obtain at an easy rate separating and order-checking (for example, by using oligonucleotide probe, this probe can combine with the heavy chain of rodent antibody and the encoding gene of light chain specifically).This hybridoma is as the source of these DNA.This DNA just can be placed in the expression vector once separating, then with this expression vector transfection in host cell (for example, escherichia coli (E.coli) cell, ape COS cell, Chinese hamster ovary (CHO) cell or do not produce the myeloma cell of other immunoglobulin), in recombinant host cell, to obtain the synthetic of monoclonal antibody.The summary document of expressing about the DNA of encoding antibody in the antibacterial comprises: Skerra etc., Curr.Opinion in Immunol., 5:256-262 (1993) and Phickthun, Immunol.Revs., 130:151-188 (1992).

In other embodiments, separation antibody or antibody fragment from the antibody phage storehouse, this antibody phage storehouse is to produce in order to the technology of being put down in writing in the following document: McCafferty etc., Nature, 348:552-554 (1990).Clackson etc., Nature, 352:624-628 (1991) and Marks etc., J.Mol.Biol., 222:581-597 (1991) have described respectively and have used phage library separating mice and people's antibody.Thereafter publication has been described by chain and has been replaced the method (Marks etc. that (chain shuffling) produces people's antibody of high-affinity (the nM order of magnitude), BiolTechnology, 10:779-783 (1992), the document is included this paper in as a reference in full) and to recombinate as the strategy (Waterhouse etc. that make up very big phage library in combination infection and the body, Nuc.Acids.Res., 21:2265-2266 (1993), the document is included this paper in as a reference in full).Therefore, these technology can substitute traditional monoclonal antibody hybridoma technology feasiblely and be used to separate monoclonal antibody.

DNA also can be modified, and for example, replaces homologous mice sequence (U.S. Patent number 4,816,567 by the coded sequence of personnel selection heavy chain and constant region of light chain; Morrison etc., Proc.Natl Acad.Sci.USA, 81:6851 (1984) includes this paper in as a reference in full with it), or all or part of coded sequence by covalently bound NIg polypeptide on immunoglobulin coding sequence.Usually these NIg polypeptide are used to replace the constant region of antibody, or they are used to replace the variable region of an antigen binding site of antibody, thereby produce chimeric bivalent antibody, this bivalent antibody contain to a certain antigen have a specific antigen binding site and to another not synantigen have specific another antigen binding site.

In addition, the recombinant antibodies of anti-TFF3 can produce in transgenic animal, as, described in a plurality of patents.For example, recombinant antibodies can be expressed in transgenic animal (as, Rodents), as disclosing in following arbitrary United States Patent (USP): U.S. Patent number: 5,877,397; 5,874,299; 5,814,318; 5,789,650; 5,770,429; 5,661,016; 5,633,425; 5,625,126; 5,569,825; 5,545,806; 6,162,963; 6,150,584; 6,130,364; 6,114,598; 6,091,001; 5,939,598.In addition, recombinant antibodies can be expressed in the milk of transgenic animal, as U.S. Patent number 5,849, is discussed in 992 or 5,827,690.

The antibody of peopleization

Be used for the existing in the art description of the method for non-human antibody's peopleization.Preferably, the antibody of peopleization has one or more inhuman source amino acid residue of introducing.These inhuman source amino acid residues often are called as " input " (import) residue, and it is taken from " input " variable region usually.The peopleization reaction can be passed through hypervariable region implementation and operation sequence (Jones etc., Nature, the 321:522-525 (1986) of the corresponding human antibody sequence of displacement basically according to the method for Winter and colleague's announcement thereof; Riechmann etc., Nature, 332:323-327 (1988); Verhoeyen etc., Science, 239:1534-1536 (1988)).Therefore, these " peopleization " antibody are chimeric antibody (U.S. Patent number: 4,816,567), wherein be less than a complete people variable region basically and replaced by the corresponding sequence of inhuman source of species.In practice, the humanized antibodies is people's antibody usually, and wherein some hypervariable region residue and some possible FR residue are derived from that the residue in similar site replaces in the rodent animal antibody.

The selection that is used to prepare humanized antibodies's people variable region (light chain and heavy chain) has influence on antigenicity.According to so-called " the suitableeest " method, can be to the variable region sequences of whole known people's variable region sequences library screening Rodents antibody.Just be accepted as humanized antibodies's people's framework region (frameworkregion, FR) (Sims etc., J.Immunol, 151:2296 (1993) with the immediate human sequence of Rodents sequence; Chothia etc., J.Mol.Biol, 196:901 (1987)).The special frames district that another kind method is used is derived from the consensus sequence of everyone antibody of light chain with certain specific hypotype or heavy chain.Identical framework region can be used for a plurality of different humanized antibodieses (Carter etc., Proc.Nad.Acad.Sci.USA, 89:4285 (1992); Presta etc., J:Immunol, 151:2623 (1993)).

Can be under situation about keeping to antigenic high-affinity and other favourable biological characteristics, antagonist advances the pedestrianization.For reaching this purpose, according to some embodiment, the peopleization product of analyzing auxiliary sequence and various designs by the threedimensional model that uses auxiliary sequence and peopleization sequence prepares the humanized antibodies.Three-dimensional immunoglobulin model is that commercialization is obtainable, and is known by those skilled in the art.Can buy computer program in order to illustrate and to show the possible three-dimensional conformation structure of selected candidate's immunoglobulin sequences.Can analyze the effect that residue may play to these demonstration inspections in the function performance of candidate's immunoglobulin sequences, that is, the residue that can have influence on candidate's immunoglobulin and its antigen binding capacity be analyzed.By this method, can from receptor (recipient) and list entries, select and make up the FR residue, thereby obtain the required antibody character raising of target antigen affinity (for example, to).Usually, the residue of hypervariable region directly and relate to the bonded influence of antigen the most at all.

People's antibody

As substituting of peopleization reaction, can produce people's antibody.As discussed above, it is known producing antibody (especially people's antibody) in transgenic animal.For example, can produce transgenic animal (for example, mice), after to these animal immunes, it can produce complete people's antibody library, and does not produce endogenic immunoglobulin.For example, described in chimera and system genitale (germ-line) mutant mice, the homozygosity of heavy chain of antibody land (JH) gene disappearance can cause the inhibition fully of endogenous antibody producing.People's system genitale immunoglobulin gene array is transferred to this system genitale mutant mice, can when antigenic stimulus (challenge), produce people's antibody like this.Referring to, for example, Jakobovits etc., Proc.Mad.Acad.Sci.USA, 90:2551 (1993); Jakobovits etc., Nature, 362:255-258 (1993); Bruggermann etc., Year in Immuno., 7:33 (1993); And U.S. Patent number: 5,591,669,5,589,369 and 5,545,807.In addition, display technique of bacteriophage (phage displaytechnology) (McCafferty etc., Nature 348:552-553 (1990), the document is included this paper in as a reference in full with it) be used in and externally from immunoglobulin variable (V) the district gene bank that obtains from donor, produce people's antibody and antibody fragment without immunity.According to this technology, antibody V district gene (in-frame) synchronously is cloned in the main or less important capsid protein gene of filobactivirus (filamentous bacteriophage), for example M13 or fd, and on the phage particle surface, show as the functional antibodies fragment.Because filamentous particle contains the single stranded DNA copy of phage genome, also can choose the gene that coding demonstrates the antibody of those characteristics based on the selection of antibody function characteristic.Therefore, this phage has been simulated some characteristic of B cell.Phage display can carry out in a variety of forms; About they summary referring to, as Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology 3:564-571 (1993).The V genetic fragment in multiple source can be used in the phage display.Clackson etc., Nature, 352:624-628 (1991) makes up the anti-azolactone antibody of isolating different series the little library at random from the V gene that derives from the immune mouse spleen.Can make up V gene bank from not immune people's donor, and the antibody corresponding to different series antigen (comprising hapten) can substantially be separated according to the described technology of following document: Marks etc., J Mol.Biol.222:581-597 (1991), or Griffith etc., EMBO is (1993) J.12:725-734.Also can be referring to U.S. Patent number 5,565,332 and 5,573,905.People's antibody also can produce (referring to U.S. Patent number 5,567,610 and 5,229,275) by external activatory B cell.

Antibody fragment

Develop multiple technologies and produced antibody fragment.Conventional, these fragments be by the proteolytic digestion of complete antibody derive come (referring to, for example, Morimoto etc., Journal of Biochemical andBiophysical Methods 24:107-117 (1992) and Brennan etc., Science, 229:81 (1985)).Yet these fragments also can directly be produced by recombinant host cell.For example, this antibody fragment can separate from the antibody phage storehouse of above being discussed and obtains.In addition, Fab '-SH fragment can directly reclaim from escherichia coli (E.coli) and form F (ab ') 2 fragments [document is included this paper in as a reference in full with it for Carter etc., Bio/Technology 10:163-167 (1992)] through chemical coupling.According to another approach, F (ab ') 2 fragments can directly be separated acquisition from the recombinant host cell culture of cultivating.Other technology that is used to produce antibody fragment is conspicuous for skilled practitioner.In other embodiments, selected antibody is strand Fv fragment (scFv).Referring to, WO93/16185; U.S. Patent number 5,571,894; With U.S. Patent number 5,587,458.This antibody fragment also can be " a linear antibody ", and for example, as United States Patent (USP) 5,641,870 is described.These linear antibody fragments can be monospecific or bispecific (bispecific).In addition, from the phage display storehouse, discern antibody fragment, can clone, check order and be engineered to encoding and can expressing the part of the nucleic acid of any isotypes full length antibody its code nucleic acid.

Bi-specific antibody

The method that is used to prepare bi-specific antibody is well known in the art.The conventional method that produces the total length bi-specific antibody is based on the right coexpression of two heavy chain immunoglobulin-light chains, wherein these two chains have different specificity (Millstein etc., Nature, 305:537-539 (1983) document is included this paper in as a reference in full with it).Because the sorting at random of heavy chain immunoglobulin and light chain, (quadroma quadromas) can produce by 10 kinds of molecular possible mixture of different antibodies these hybridomas, wherein has only a kind of correct bispecific structure that has.This correct molecule of purification (can be undertaken by the affinity chromatograph step) is trouble very, and the productive rate of product is low.Similarly technology is at WO93/08829 and Traunecker etc., and EMBO J. describes among the 10:3655-3659 (1991) to some extent.

According to different approach, has the constant region sequence fusion of antibody variable region (antibody-antigen binding site) with the immunoglobulin of required binding specificity.Merge between preferred and the immunoglobulin heavy chain constant region and carry out, this constant region contains the hinge region to small part, CH2 and CH3 district.In some embodiments, contain light chain and in fusions, have one at least in conjunction with first CH (CH1) in required site.If necessary, the DNA of the heavy chain immunoglobulin fusions of can encoding inserts in the different expression vectors with the DNA of coding light chain immunoglobulin, and cotransfection is gone in the suitable hosts organism.Like this, when geometric ratio ground did not use three peptide species fragments to make up, this method provided very big motility to the mutual ratio of regulating three polypeptide fragments in embodiment, provide the optimization productive rate with this.Yet, when at least two polypeptide chains of equal proportion can obtain high yield, maybe when this ratio is not particular importance, can in same expression vector, insert the coded sequence of two or all three kinds polypeptide chain.

In some embodiments, bi-specific antibody in a certain arm, contain heterozygosis heavy chain immunoglobulin with first binding specificity and the heavy chain immunoglobulin light chain that in another arm, contains heterozygosis to (second binding specificity is provided).Discover, owing to only have light chain immunoglobulin in half of this bispecific molecule, just for separating the method for providing convenience, this dissymmetrical structure makes easier the carrying out of the separation of required bispecific chemical compound from unwanted immunoglobulin chain compositions for this.This method discloses in WO94/04690 to some extent.About further describing of production bi-specific antibody, referring to, for example, Suresh etc., Methods in Enzymology, 121:210 (1986).

According to another at U.S. Patent number 5,731, described in 168, antibody molecule between the interface can be through through engineering approaches to obtain maximized heterodimer percentage ratio, this heterodimer reclaims from the reconstitution cell culture.In some embodiments, at least a portion in the CH3 district of antibody constant region can be contained in this interface.In the method, the one or more p1 amino acid side chains that come from first antibody molecule interface can be substituted (for example tyrosine or tryptophan) by bigger side chain.Compensatory " cavity " (Compensatory " cavities ") to the identical or similar size of bulky side chain can form by substitute big amino acid side chain with less amino acid side chain (for example alanine or threonine) on the interface of second antibody molecule.This just provides a kind of mechanism for the productive rate (with respect to other non-required end-product (for example homodimer)) that improves heterodimer.

Bi-specific antibody comprises crosslinked or " different joint " (heteroconjugate) antibody.For example, a kind of antibody in different joint can with the avidin coupling, another antibody then with the biotin coupling.This antibody by, for example, propose to be used for (U.S. Patent number 4,676,980) and be used for the treatment of HIV and infect (WO91/00360 and WO92/200373) with immune system cell targeting unwanted cells.The preparation of different binding antibody can be adopted any cross-linking method easily.The cross-linking agent that is fit to is well-known in the art, and at U.S. Patent number 4,676, is disclosed together with many crosslinking technologicals in 980.

The technology that produces bi-specific antibody from antibody fragment is also described in the literature to some extent.For example, can use chemical crosslinking to prepare bi-specific antibody.Brennan etc., Science, 229:81 (1985) has described that complete antibody is produced F (ab ') 2 segmental methods through the protease hydrolysis cutting.These fragments are reduced under the situation that dimercapto complexing agent sodium arsenite exists, stable contiguous dithiol and the formation that prevents intermolecular disulphide.Then Fab ' the fragment that is produced is converted into sulfo-nitrobenzoyl acid esters (thionitrobenzoate, TNB) derivant.By reducing one of Fab '-TNB derivant is converted into Fab '-mercaptan then, and mixes to form bi-specific antibody with another Fab '-TNB derivant of equivalent with mercaptoethylmaine.The bi-specific antibody that is produced can be used as the selectivity fixating reagent of enzyme.

Present progress makes that directly reclaiming Fab '-SH fragment from escherichia coli (E.coli) becomes convenient, and this Fab '-SH fragment can form bi-specific antibody through chemical coupling.Shalaby etc. have described the generation of total length peopleization bi-specific antibody F (ab ') 2 molecules at J.Exp.Med. among the 175:217-225 (1992).Each Fab ' fragment secretes independently from escherichia coli (E.coli), and it is directly carried out chemical coupling to form bi-specific antibody external.Therefore, formed bi-specific antibody can combine with crossing the cell and the normal human T-cell that express the ErbB2 receptor, and causes the lytic activity of people's cellulotoxic lymphocyte to HBT's target.

Also be described with the method for separating bispecific antibody fragment multiple directly the preparation from the reconstitution cell culture.For example, use leucine zipper to produce bi-specific antibody.Kostelny etc., J.Immunol., 148 (5): 1547-1553 (1992).To come from Fos by gene fusion is connected with the Fab ' part of two kinds of different antibodies with the proteic leucine zipper peptide of Jun.This antibody morphism dimer at hinge region through reduction to form monomer, then again oxidation to form the antibody heterodimer.This method also can be used for producing the antibody morphism dimer.Hollinger etc., Proc.Natl.Acad.Sci.USA, 90:6444-6448 (1993) have described this " bispecific antibody " (diabody) technology, and this technology provides the another kind of mechanism of preparation bispecific antibody fragment.This fragment comprises the variable region of heavy chain (VH) that is connected with variable region of light chain (VL) by catenation sequence, and this catenation sequence is short to and can not forms pairing between two zones of same chain.

Therefore, segmental VH and VL district are forced to and another segmental complementary VL and VH pairing, thereby form two antigen binding sites.Another prepares the also existing report of strategy of bispecific antibody fragment by using strand Fv (sFv) dimer.Referring to Gruber etc., J.Immunol., 152:5368 (1994).What conceived is the antibody that is higher than bivalence.For example, can prepare three-specific antibody.Tutt etc., J.Immunol.147:60 (1991).

The combination of nertralizer and other modification

The nertralizer that contains in use or the goods in this paper method can randomly combine with cytotoxin or therapeutic agent.Example comprises chemotherapeutics.These chemotherapeutics have definite effect in the specific cancer of treatment.

Nertralizer and one or more micromolecule toxin (also interim in advance at this paper as calicheamycin, maytansine (U.S. Patent number 5,208,020), trichothecin (trichothene) and conjugate CCl065).According to some embodiment, nertralizer combines (combining with about 10 the maytansine molecules of about 1-as each nertralizer molecule) with one or more maytansine molecules.Maytansine can, for example, change May-SS-Me into, it is reducible to be May-SH3 and to generate maytansine sample-nertralizer (maytansinoid-neutralizing agent) conjugate with the nertralizer (Chari etc., Cancer Research 52:127-131 (1992)) of modified.

In addition, nertralizer can combine with one or more thorn spore lps molecules.Antibiotic thorn spore toxin family can produce the double-stranded DNA fracture under Asia-picomole concentration.The structural similarity thing of thorn spore toxin also is known.(Hinman etc., Cancer Research 53:3336-3342 (1993) and Lode etc., Cancer Research 58:2925-2928 (1998)).

Available toxin and fragment thereof with enzymatic activity comprises: diphtheria toxin, diphtherotoxin A chain, the disconnected active fragment of diphtheria toxin, diphtherotoxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, α-sarcina, caryophyllin albumen, Virgina poke albumen (Phytolaca americana protein, PAPI, PAPII and PAP-S), Fructus Momordicae charantiae inhibitive factor (momordica charantia inhibitor), curcin, crotin, Salvia japonica Thunb. inhibitive factor (sapaonaria officinalis inhibitor), gelonin, silk splits element (mitogellin), restrictocin, phenomycin, enomycin and trichothecene (tricothecenes).Referring to as WO 93/21232.

The present invention has also comprised and the bonded nertralizer of the chemical compound with nucleolytic activity (for example ribonuclease or DNA restriction endonuclease such as deoxyribonuclease; DNase).Multiple radiosiotope can be used for preparing the bonded nertralizer of radioactivity.The radiosiotope that comprises Y90, At211, Re186, Re188, Sm153, Bi212, P32 and Lu for example.Nertralizer and cytotoxic conjugate can use multiple bifunctional protein coupling agent to be prepared; for example: N-succinimido-3-(2-dithio pyridine-) propionic ester (SPDP); succinimido-4-(N-maleimide ylmethyl) cyclohexane extraction-1-carboxylate; imino group thiol alkane (mercapto alcohol imines; iminothiolane; IT); the dual-function derivative of polyurethane (for example; dimethyl oxalyl imines HCL); active ester (for example; the hot diester of two butanimides); aidehydes (for example; glutareldehyde); two-fold nitrilo compound (for example; two (right-the azido benzoyl base) hexamethylene diamine), two-the diazol derivant (for example, two-(right-the diazobenzene formoxyl)-ethylenediamines); diisocyanate or ester are (for example; toluene 2,6-vulcabond) and two active fluorine compounds (as 1; 5-two fluoro-2, the 4-dinitro benzene).For example, the ricin immunotoxin can be by Vitetta etc., the method preparation that Science 238:1098 (1987) describes.The 1-isothiocyanato benzyl of carbon-14 labelling-3-methyl diethylene triamine pentacetic acid (DTPA) (MX-DTPA) is a kind of being used for radioactive nucleus thuja acid and the bonded typical chelating agen of nertralizer.Referring to, as W094/11026.Catenation sequence can be " catenation sequence that can cut ", and it is easy to discharge cell toxicity medicament in cell.For example, can use catenation sequence, the dimethyl catenation sequence of sour unsettled catenation sequence, peptidase sensitivity or contain the catenation sequence (Chari etc., Cancer Research 52:127-131 (1992)) of disulphide.The fusion rotein that perhaps, can comprise nertralizer and cytotoxic agent by for example recombinant technique or the preparation of peptide synthetic technology.

In some embodiments, nertralizer can combine with " receptor " (as streptavidin) to be used for the pre-targeting (pretargeting) of tumor, wherein give the patient with this nertralizer-receptors bind thing, then remove unconjugated conjugate in the circulation, give then and cytotoxic agent (for example radioactive nucleus thuja acid) bonded " part " (for example avidin) with clarifier.Nertralizer of the present invention also can combine with the prodrug activating enzymes, and this enzyme can be converted into active cancer therapy drug with prodrug (for example, the peptidyl chemotherapeutics is referring to W081/01145).Referring to, for example, WO88/07378 and U.S. Patent number 4,975,278.

Enzyme component in this class conjugate comprises any enzyme that can act on prodrug, and its action pathway is prodrug to be converted into than prodrug have more active and toxic form.The useful enzyme that can be used for the inventive method includes but not limited to: alkali phosphatase is used for phosphatic prodrug is converted into free drug; Arylsulfatase is used for the prodrug of sulfur-bearing acid esters is converted into free drug; The cytosine deaminase is used for avirulent 5-flurocytosine is converted into the cancer therapy drug 5-fluorouracil; Protease, for example Bacterium prodigiosum protease, thermolysin, subtilisin, carboxypeptidase and cathepsin (for example, cathepsin B and L), the prodrug that is used for containing peptide is converted into free drug; D-alanyl carboxypeptidase is used to transform the prodrug that contains D-aminoacid replacement base; The sugar lyases, for example b-tilactase and neuraminidase are used for glycosylated prodrug is converted into free drug; (the 3-lactamase is used for medicine with (the 3-lactam derivative becomes free drug; And penicillin (taking off) amidase, for example, penicillin V (taking off) amidase or benzylpenicillin (taking off) amidase are used for and will be separately converted to free drug with benzene oxygen acetyl or the deutero-medicine of phenylacetyl group at the amine nitrogen place.Perhaps, have the antibody (being also referred to as " abzyme " in the art) of enzymatic activity, can be used for prodrug of the present invention be converted into active medicine (referring to, for example, Massey, Nature 328:457-458 (1987)).Can method as described herein preparing nertralizer-abzyme jointer is used for abzyme is delivered in the tumor cell group.

Enzyme also can with the TFF3 nertralizer by technology known in the art (for example utilize above discussed isodigeranyl functional cross-link agent) covalent bond.Perhaps, can use recombinant DNA technology known in the art to make up the fusion rotein that partly is connected with the functional activity of at least one enzyme of the present invention, this fusion rotein contain at least one nertralizer of the present invention antigen binding domain [referring to, for example, Neuberger etc., Nature, 312:604-608 (1984)].

This paper has also comprised other modification of nertralizer.For example, nertralizer can with a kind of connection the in the multiple non-albuminous polymer, for example copolymer of Polyethylene Glycol, polypropylene glycol, polyoxy alkene or Polyethylene Glycol and polypropylene glycol.Nertralizer disclosed herein also can be formulated as liposome.Can contain the liposome of nertralizer by the methods known in the art preparation, for example in following document, describe: Epstein etc., Proc.Mad.Acad Sci.USA, 82:3688 (1985); Hwang etc., Proc.Natl Acad.Sci.USA, 77:4030 (1980); U.S. Patent number 4,485,045 and 4,544,545; And W097/38731.The liposome of circulation time with prolongation is at U.S. Patent number 5,013, discloses to some extent in 556.

Can produce particularly useful liposome with the lipid composition that contains GranulestinLecithin, cholesterol and PEGization PHOSPHATIDYL ETHANOLAMINE (PEG-PE) by anti-phase method of evaporating.Liposome is filtered by the filter that limits the aperture, to obtain to have the liposome of required diameter.Fab ' the fragment of antibody of the present invention can combine (as Martin etc., J.Biol.Chem.257:286-288 (1982) described in) by the disulphide mutual exchange reaction with liposome.Chemotherapeutics can randomly be included in the liposome.Referring to J.National Cancer Inst.81 (19) 1484 (1989) such as Gabizon.Expected and modified for the aminoacid sequence of protein described herein or peptide nertralizer.For example, may wish to improve binding affinity and/or other biological nature of nertralizer.

The preparation of the aminoacid sequence variant of nertralizer is by introducing suitable nucleotide variation or undertaken by peptide is synthetic in nertralizer nucleic acid.These modifications include but not limited to the residue in the nertralizer aminoacid sequence is lacked and/or inserts and/or substitutes.Can implement to lack, insertion and alternate any combination to be to obtain formal construct, prerequisite is that final construct has required character.Amino acid whose variation also can change process after the translation of nertralizer, for example changes the quantity or the position of glycosylation site.

Differentiate in the nertralizer and be called as " alanine scanning mutagenesis " (alanine scanning mutagenesis) as the residue in mutation site or the effective ways in zone, as Cunningham and Wells at Science, described in the 244:1081-1085 (1989).Differentiate single residue or target residue group (for example, charged residue such as arg herein,, asp, his, lys, and glu), and substituted, thereby influenced aminoacid and antigenic interaction with neutrality or electronegativity aminoacid (most preferably being alanine or Poly(Ala) Alanine homopolymer).Pass through then in alternate site or more or other variation to the alternate site introducing, those reveal the amino acid sites of functional sensitivity to substitution tables with refine.Therefore, be when presetting when introducing site that aminoacid sequence changes, the character of this sudden change itself then need not to preset.For example, in order to analyze, to carry out alanine scanning or random mutagenesis at target codon or zone, and the nertralizer variant of expressing is carried out required active screening to the effect that suddenlys change on the anchor point.

Aminoacid sequence inserts and to comprise that length range is 1 residue-contain 100 or the aminoterminal of more residue polypeptide and/or the fusion of c-terminus, and inserts in the sequence of single or multiple amino acid residues.The terminal example that inserts comprises nertralizer that has N-end methionine residues or the nertralizer that merges with the cell toxicant polypeptide.Other of nertralizer inserts variant and comprises that enzyme maybe can improve this nertralizer serum polypeptide and the N-of nertralizer or the fusant of C-end of half life.

Another kind of variant is amino acid whose alternative variations.Amino acid residue in these variants at least one nertralizer molecule is substituted by different residues.The most interested site of the alternative mutation of antibody nertralizer comprises hypervariable region, yet has also imagined the change of FR.

By selecting that following effect is had substituting of notable difference, finish alternative modification to the nertralizer biological characteristics, these effects are: the structure that (i) keeps polypeptide backbone in the replacement area, for example, as folding or helical conformation, (ii) keep the charging property or the hydrophobicity of this molecule at target site, or the space occupy-place that (iii) keeps side chain.According to common side chain characteristic, naturally occurring residue is divided into following each group:

Hydrophobic: norleucine, met, ala, val, leu, ile;

Neutral hydrophilic: cys, ser, thr;

Tart: asp, glu;

Alkalescence: asn, gln, his, lys, arg;

Influence the residue of chain space orientation: gly, pro; With

Aromatic: trp, tyr, phe.

Non-conservative substituting is with the seed amino acid in wherein a kind of another classification of amino acid replacement of a certain class in these classifications.Conservative substituting relates to amino acid whose exchange in the same classification.

Any do not relate to the cysteine residues of keeping the correct conformation of nertralizer also can be replaced (normally substituting) with serine, with the oxidation stability that improves molecule with prevent crosslinked unusually.Otherwise, can in nertralizer, add the cysteine key with the stability that improves it (especially when this nertralizer is antibody fragment, for example Fv fragment).

In some embodiments, a class alternative variations relates to the one or more hypervariable region residues that substitute parental antibody.Common, the gained variant that is selected for further exploitation has better biological nature with respect to the parental antibody that produces this variant.A kind of method easily that produces this alternative variations is to use phage display to carry out affine maturation.In brief, several hypervariable region sites (for example, 6-7 site) suddenlyd change substitute to produce all possible amino in each site.Consequent antibody variants is displayed from the filobactivirus granule with the unit price form, and itself and M13 gene III product form fusions and are wrapped in each granule.Pressing the announcement of this paper then screens the biologic activity (for example, binding affinity) of the variant of phagocytosis displaying.In order to identify the candidate's hypervariable region site that is used to modify, can carry out alanine scanning mutagenesis to identify antigen in conjunction with hypervariable region residue with outstanding contributions.Perhaps, or in addition, analyze the crystal structure of antigen-antibody complex to identify that the contact point between antibody and antigen also may be favourable.According to the technology that this paper describes in detail, this contact residues and adjacent residue are alternate candidate's residues.In case this variant produces, just these variants are screened according to described herein, and select in one or more related checks, have better characteristic antibody for further exploitation.

The another kind of amino acid variant of nertralizer described herein has changed the original glycosylation pattern of nertralizer." change " be meant and remove one or more glycosyl parts that can find in nertralizer, and/or add one or more scripts and be not present in glycosylation site in the nertralizer.

The glycosylation of polypeptide is generally the N-connection or O-connects.The N-connection is meant that glycosyl part is connected with the side of asparagine residue is interchain.Tripeptide sequence, promptly agedoite-X-serine and agedoite-X-threonine (wherein X is any aminoacid except proline) is that glycosyl part is carried out the recognition sequence that enzymatic is connected with the agedoite side chain.Therefore, in the polypeptide in these tripeptide sequences the existence of arbitrary sequence all form potential glycosylation site.O-connects glycosylation and is meant being connected of a kind of and hydroxy-amino-acid in N-acetylamino galactosamine, galactose or the xylose, and the most frequently used is serine or threonine, although also can use 5-hydroxyproline or 5-hydroxylysine.Thereby make it contain one or more above-mentioned tripeptide sequences (connecting glycosylation site) by changing aminoacid sequence, can reach the purpose that increases glycosylation site in the nertralizer for N-.Also can reach change purpose (for the glycosylation site of O-connection) by in primary nertralizer sequence, adding one or more serines or threonine residues or substituting other residue with one or more serines or threonine residues.

Can be by the nucleic acid molecules of several different methods preparation coding nertralizer aminoacid sequence variant known in the art.These methods include but not limited to, separate from natural origin (under the naturally occurring situation of aminoacid sequence variant) or are prepared by mutation, PCR mutation and cassette mutagenesis that the non-variant form to the nertralizer variant of early stage preparation or nertralizer carries out oligonucleotide mediated (or site instruct).

In some embodiments, consider the function of effector, may need nertralizer of the present invention is modified, thereby as the cytotoxicity (ADCC) and/or the complement-dependent cytotoxicity (CDC) of the antigen dependent cell mediation that promotes nertralizer.These can be realized by introduce one or more amino acid replacements in the Fc district of antibody nertralizer.Perhaps or in addition, can in the Fc district, introduce cysteine residues, thereby form interchain disulfide bond in this zone.Consequent homotype dimerization antibody can have the cell killing effect and the antibody-dependent cytotoxicity effect (ADCC) of taking the photograph the complement-mediated of ability and/or raising in the cell of improvement.Referring to Caron etc., J.Exp Med.176:1191-1195 (1992) and Shopes, B.J.Immunol.148:2918-2922 (1992).The homotype dimerization antibody of the anti-tumor activity that also can use isodigeranyl functional cross-link agent (heterobifunetional cross-linkers) preparation of Wolff described in Cancer Research 53:2560-2565 (1993) to have to strengthen.Perhaps, can carry out through engineering approaches to antibody with two Fc districts, and the complement lysis and the ADCC ability that can be enhanced thus.Anti-Cancer Drug Design 3:219-230 (1989) referring to Stevenson etc.

Improve the half-life of nertralizer in serum, can be by as United States Patent (USP) 5,739,277 methods of describing will be remedied receptors bind epi-position (salvage receptor binding epitope) and be incorporated into nertralizer (particularly antibody fragment).As used herein, the epi-position in the Fc zone of IgG molecule (as IgG1, IgG2, IgG3 or IgG4) " remedied the receptors bind epi-position " and refer in term, and this epi-position plays and improves the effect of half-life in the serum in vivo of IgG molecule.

The present invention also provides the screening technique that is used to identify the anti-TFF3 antibody with expection treatment or diagnostic feature.Screening technique comprise differentiate influence analysis, ADCC or the CDC activity analysis of tumor cell proliferation and/or adherent anti-TFF3 antibody, anti-apoptosis analysis, the cell cycle test point is analyzed and to the body inner analysis of genetically modified non-human animal (as mice and other rodent).The present invention has also comprised in order to differentiate the screening technique of the antibody that combines with proteinic specific part as mentioned above, and these antibody have required characteristic, as blocking-up calcium in conjunction with, blocking-up dimer form, the arrangement in blocking-up chain formation and/or interference TFF3 district.Can identify aforesaid antibody to anti-TFF3 albumen or its segmental antibody population that is provided.

The nertralizer of other form

Nertralizer also can adopt the form of prodrug.Term " prodrug " refers to a kind of therapeutic agent with inactive form preparation, and it is subjected to the effect of endogenous enzyme or other chemical drugs and/or condition, and changes activity form (being medicine) in vivo or in the intravital cell.Particularly; the prodrug forms that contains the nertralizer (as antisense oligonucleotide, ribozyme and RNAi molecule etc.) of nucleic acid can be by WO93/24510 or WO94/26764 and U.S. Patent number 5; 770,713 methods that disclose are prepared into SATE[(S-acetyl group-2-thio-ethyl) phosphate ester] derivant.

Nertralizer also can adopt the form of pharmaceutically acceptable salt.Term " pharmaceutically acceptable salt " is meant the physiology and the pharmaceutically acceptable salt of The compounds of this invention: promptly keep the required biologic activity of parent compound and can not bring the salt of other unwanted toxic effect to it.For the nertralizer (as antisense oligonucleotide, ribozyme, RNAi molecule etc.) that contains nucleic acid, the example of pharmaceutically acceptable salt includes but not limited to, (a) salt that forms with cation is as sodium, potassium, ammonium, magnesium ion, calcium, polyamine such as spermine and spermidine etc.; (b) acid-addition salts that forms with mineral acid, for example hydrochloric acid, hydrobromic acid, sulphuric acid, phosphoric acid, nitric acid etc.; (c) salt that forms with organic acid, for example acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannin, Palmic acid, alginic acid, polyglutamic acid, LOMAR PWA EINECS 246-676-2, methanesulfonic acid, right-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid etc.; (d) salt that forms with the anion of element is as chlorine, bromine and iodine.

Polynucleotide constructs

The polynucleotide molecule of coding TFF3, TFF3 fragment or TFF3 nertralizer (as antibody) can be inserted in the polynucleotide constructs, as DNA or RNA construct.Polynucleotide molecule of the present invention can be used in, for example, and in the expression construct and at the part or all of protein of host cell inner expression, variant, fusion rotein or single-chain antibody.Expression construct comprises a promoter that has function in selected host cell.Those skilled in the art can select suitable promoter at an easy rate from a large amount of cell type specificity promoteres of known in the art and use.Expression construct also can contain a transcription terminator that has function in host cell.Expression construct has comprised coded portion or the proteic polynucleotide passage of target complete.This polynucleotide passage is positioned at the downstream of promoter.The transcription initiation of polynucleotide passage is in promoter.Expression construct can be linearity or cyclic, can also contain the required sequence of self-replicating if needed.

Host cell

The expression construct of coding TFF3 or TFF3 nertralizer can be imported in the host cell.The host cell that contains expression construct can be any suitable protokaryon or eukaryotic cell.Expression system in the antibacterial comprises Chang etc., Nature 275:615 (1978); Goeddel etc., Nature 281:544 (1979); Goeddeletal., Nucleic Acids Res.8:4057 (1980); EP 36,776; United States Patent (USP) 4,551,433; DeBoer etc., Proc.Natl.Acad Sci.USA 80:21-25 (1983); With Siebenlist etc., the system of describing among the Cell20:269 (1980).

Expression system in the yeast comprises Hinnnen etc., Proc.Natl.Acad.Sci.USA 75:1929 (1978); Ito etc., JBacteriol 153:163 (1983); Kurtz etc., Mol.Cell.Biol.6:142 (1986); Kunze etc., J BasicMicrobiol.25:141 (1985); Gleeson etc., J:Gen.Microbiol.132:3459 (1986), Roggenkamp etc., Mol.Gen.Genet.202:302 (1986)); Das etc., JBacteriol.158:1165 (1984); De Louvencourt etc., J Bacteriol.154:737 (1983), Vanden Berg etc., Bio/Technology 8:135 (1990); Kunze etc., J.Basic Microbiol.25:141 (1985); Cregg etc., Mol.Cell.Biol.5:3376 (1985); United States Patent (USP) 4,837,148; United States Patent (USP) 4,929,555; Beach and Nurse, Nature 300:706 (1981); Davidow etc., Curr.Genet.10:380 (1985); Gaillardin etc., Curr.Genet.10:49 (1985); Ballance etc., Biochem.Biophys.Res.Commun.112:284-289 (1983); Tilburn etc., Gene 26:205-22 (1983); Yelton etc., Proc.Natl.Acad, Sci.USA 81:1470-1474 (1984); Kelly and Hynes, EMBO be (1985) J.4:475479; EP 244,234; And the system described in the WO 91/00357.

Can use United States Patent (USP) 4,745,051; " molecular biology of baculovirus " (THE MOLECULARBIOLOGY OF BACULOVIRUSES, W.Doerfler, compile) in, Friesen etc. (1986) " regulation and control of baculovirus gene expression " (The Regulation of Baculovirus Gene Expression); EP127,839; EP 155,476; Vlak etc., J.Gen.Virol.69:765-776 (1988); Miller etc., Ann.Rev.Microbiol.42:177 (1988); Carbonell etc., Gene 73:409 (1988); Maeda etc., Nature 315:592-594 (1985); Lebacq-Verheyden etc., Mol.Cell Biol.8:3129 (1988); Smith etc., Proc.Natl.Acad.Sci.USA 82:8404 (1985); Miyajima etc., Gene58:273 (1987); With Martin etc., the described method of DNA 7:99 (1988) realizes heterogeneic expression in the insecticide.Multiple baculovirus strain and variant and the corresponding insect host cell of being received from the host thereof are described in Luckow etc., Bio/Technology (1988) 6:47-55, among the Miller etc., see " genetic engineering " (GENETIC ENGINEERING, Setlow, volumes such as J.K.), the 8th volume, 277-279 page or leaf (Plenum publishes, 1986); And Nature such as Maeda, 315:592-594 (1985).

Can be as Dijkema etc., EMBO is (1985) J.4:761; Gorman etc., Proc.Natl.Acad.Sci.USA 79:6777 (1982b); Boshart etc., Cell 41:521 (1985); With United States Patent (USP) 4,399,216 describedly realize the mammal expression.Can be as Ham and Wallace, Meth Enz.58:44 (1979); Barnes and Sato, Anal.Biochem.102:255 (1980); United States Patent (USP) 4,767,704; United States Patent (USP) 4,657,866; United States Patent (USP) 4,927,762; United States Patent (USP) 4,560655; WO 90/103430, WO87/00195 and United States Patent (USP) RE 30, the 985 described further features that promote that mammal is expressed.

Can use any technology known in the art that expression construct is imported in the host cell.These technology comprise that the DNA of transferrins-polycation mediation shifts, carries out with nucleic acid exposed or parcel the transfection of intracellular transport, protoplast fusion, viral infection, electroporation, " particle gun " and calcium phosphate mediation of the latex bead (latex bead) of transfection, liposome-mediated cell fusion, DNA bag quilt.

Pharmaceutical preparation

According to TFF3 nertralizer treatment preparation of the present invention, nertralizer that can be by will having required purity and optional pharmaceutically acceptable carrier, excipient or stabilizing agent (Remington ' s PharmaceuticalSciences the 16th edition, Osol, A.Ed. (1980)) to mix mutually and prepare and be used for to store, its form is lyophilized formulations or aqueous solution.Acceptable carrier, excipient or stabilizing agent are nontoxic to the experimenter under dosage that is adopted and concentration, and contain buffer such as phosphoric acid, citric acid and other organic acid; Antioxidant comprises ascorbic acid and methionine; Antiseptic is (as chlorination octadecyl dimethylamino benzylidene ammonia; Bistrium chloride; Benasept, benzethonium chloride; Phenol, butanols or benzyl alcohol; Alkyl is to benzene, as methyl or propyl group to benzene; Catechol; Resorcinol; Hexalin; The 3-amylalcohol; With m-cresol); Low-molecular-weight (being less than about 10 residues) polypeptide; Protein is as serum albumin, gelatin or immunoglobulin; Hydrophilic polymer such as polyvinylpyrrolidone; Aminoacid such as glycine, glutamic acid, aspartic acid, histidine, arginine or lysine; Monosaccharide, disaccharide and other carbohydrate comprise glucose, mannose or dextrin; Chelating agen such as EDTA; Sugar is as sucrose, mannitol, trehalose or Sorbitol; Salifiable counter ion of shape such as sodium; Metal complex (for example zinc-protein complex); And/or nonionic surfactant such as TWEENTM, PLURONICSTM or Polyethylene Glycol (PEG).In addition, acceptable carrier, excipient or stabilizing agent do not disturb the activity of nertralizer.

Preparation can also contain and surpass a kind of reactive compound.In some embodiments, the additional activity that reactive compound had can not play negative effect mutually.For example, may wish further to provide a kind of cytotoxic agent, chemotherapeutics or cytokine.The effective dose of these other preparations depends on the amount of the nertralizer that exists in the preparation, type and other foregoing factor of treatment of cancer.Usually, with the route of administration that identical dosage uses these chemical compounds and uses this paper to use previously, perhaps be about 1% to 99% of former using dosage.

Active component also can be embedded in the microcapsule of preparation, for example, adopt condensation technique or adopt interfacial polymerization such as hydroxy methocel or gelatin microcapsule and poly-(methyl acrylic acid) microcapsule, be respectively applied for colloid drug delivery system (for example, liposome, albumin microsphere spheroid, microemulsion, nanoparticle and Nano capsule) or be used for huge Emulsion (macroemulsion).These technology are disclosed in Remington ' s Pharmaceutical Sciences the 16th edition, Osol, and A. compiles (1980).

Also can prepare sustained release formulation.The appropriate example of extended release preparation comprises the semi permeability substrate of the solid-state hydrophobic polymer that contains nertralizer, and such substrate has the form of molding, as film or microcapsule.The example that continues release matrix (for example comprises polyester, hydrogel, poly-(2-ethoxy-methacrylate) or poly-(vinyl alcohol)), polyactide (U.S. Patent number 3,773,919), the copolymer of L-glutamic acid and ethyl-L-glutamate, Glu, the ethane-acetic acid ethyenyl ester that can not degrade, degradable poly lactic coglycolic acid such as LUPRON DEPOTTM (the injectable microsphere of forming by lactic-co-glycolic acid (lactic acid glycolic acidcopolymer) and leuprolide acetate or ester) and poly--D-(-)-3-hydroxybutyric acid.

In some embodiments, can pass through the mode administration of the pharmaceutical composition of use sterile injectable.Phrase used herein " injectable pharmaceutical composition ", or its variant are meant and satisfy the pharmaceutical composition that American Pharmacopeia " injectable agent " requires, promptly aseptic, apyrogeneity and microgranule and have specific pH and osmotic pressure value.Can obtain the sterile solution preparation with the method for aseptic membrane filtration.

Treat with nertralizer

Can to suffer from cancer or cancer-prone mammal (for example, people) impose the treatment effective dose the TFF3 nertralizer carry out treatment for cancer and prevention.Similarly, can realize reducing gross tumor volume with similar method, slow down tumor growth and/or prophylaxis of tumours growth.

Term used herein " treatment (treatment, treating, treat) " etc. typically refers to pharmacology and/or the physiologic effect that obtains expectation.This effect can be the prophylactic effects that reaches in the mode of prevent disease or its symptom wholly or in part, and/or can be to disease and/or reach the therapeutic effect of partially or completely stable or therapeutic effect owing to the ill effect of this disease." treatment " used herein contained mammal, especially any treatment that the people carried out, and it comprises: (a) disease or symptom appear in object of prevention, and this object may easily be suffered from this disease or symptom, is not suffered from this disease or symptom but also be diagnosed; (b) suppress disease symptoms, promptly block its development; Or the alleviation disease symptoms, promptly cause disappear (regression) of disease or symptom, as colon or other alimentary tract cancer, for example gastric cancer or hepatocarcinoma or breast carcinoma, ovarian cancer or carcinoma of prostate.

Term " individuality ", " object ", " host " and " patient " can exchange use, refer to the mammalian object that any needs are diagnosed, handled or treat, especially the people.Other object can comprise cattle, Canis familiaris L., cat, Cavia porcellus, rabbit, rat, mice, horse or the like.

Can with the corresponding to mode of good medical practice to containing the compositions of TFF3 nertralizer, for example, antibody, peptide, antisense molecule, RNAi molecule, ribozyme or micromolecule carry out preparation, formulate dosage, and administration.For example, the TFF3 nertralizer is scFv, antibody fragment, peptide or the micromolecule that can suppress anti--TFF3 antibody of the active people of TFF3, chimeric or peopleization; Or can suppress antisense oligonucleotide, RNAi molecule or the ribozyme that TFF3 expresses.The factor of Kao Lving comprises the factor known to release position, medication, administration process and other medical worker of cause, preparation of clinical setting, disease or the disease of the specific cancer of receiving treatment, the specific mammal of receiving treatment, individual patients herein.Consider that these factors determine the treatment effective dose of the nertralizer of administration.

The treatment effective dose of nertralizer is to be enough to improve in patient's body of accepting the nertralizer treatment or palliate a disease or the related indication dosage of disease.In some embodiments, the treatment effective dose is the nertralizer consumption that can alleviate cancer symptoms, as the migrating, adhere to and/or breed of aggressive, anticancer by stoping tumor growth, the tumor growth that slows down, reducing gross tumor volume, reduce cancerous cell, or the like.Determine that the treatment effective dose of nertralizer and the method for its therapeutic effect of assessment are known in the art, and be described further in this article.

With the nertralizer of the treatment effective dose of the outer mode administration of gastrointestinal tract, its each dosage can be, for example, in the scope of about 0.1 to 30mg/kg weight in patients every day, the initial scope of the typical nertralizer that uses about 2 to 10mg/kg.Yet as mentioned above, the recommendation consumption of these nertralizers changes with a lot of treatment Considerations.As mentioned above, selecting a factor of suitable dose and process is the result that will obtain.For example, that worsen in treatment or during acute illness, beginning to need higher dosage.In order to obtain the most effective result, according to disease or disease, nertralizer use should be as much as possible near first sign, diagnosis, the appearance of disease or disease or when taking place, or in the recurrence process of disease or disease.

Available any suitable method is used nertralizer, comprises that gastrointestinal tract is outer, subcutaneous, in the intraperitoneal, lung and intranasal administration, and if partial immunosuppressant therapy need, then carry out intralesional (intralesional) administration.Gastrointestinal tract is inculcated outward and is comprised intramuscular, intravenous, intra-arterial, intraperitoneal or subcutaneous administration.

In addition, available pulse is inculcated nertralizer is carried out administration suitably, for example, reduces the dosage of nertralizer gradually.Preferably, the injection system administration, intravenous or subcutaneous injection optimum depend in part on administration and are temporary transient or secular.

The nertralizer of this paper also can be used with other chemical compound, as cytotoxic agent, chemotherapeutics, immunosuppressant and/or cytokine.Administering drug combinations comprises co-administered, uses independent preparation or continues release with single pharmaceutical preparation and in arbitrary mode, wherein preferably, brings into play its biologic activity simultaneously at a period of time two kinds of (or all) active ingredients.

Have in a lot of bodies in this area and stripped method with nucleic acid (as antisense molecule, RNAi molecule or ribozyme (randomly being included in the carrier)) insertion patient cell.For release in the body, nucleic acid can be injected directly into the patient, normally is injected at the position that needs nertralizer.For the treatment of exsomatizing, isolate patient's cell, nucleic acid is imported in these isolated cells, and with the cell of modified directly to being administered to the patient or as being wrapped in perforated membrane and being implanted into (referring to U.S. Patent number 4,892,538 and 5,283,187) among the patient.The technology that much nucleic acid is imported living cells has been arranged.According to nucleic acid is to transfer in the cultured cell external, or transfers in the body in the host cell, and these technology are different.Be suitable for the external technology of transferring in the mammalian cell of nucleic acid is comprised liposome, electroporation, microinjection, cell fusion, DEAE-glucosan, the calcium phosphate precipitation method used, or the like.The common carrier that is used for the ex vivo delivered gene is a retrovirus.Nucleic acid also can come administration with hydraulic pressure release (intravenous injection of increase pressure).

An example of nucleic acid in vivo transfer techniques comprise with viral vector carry out transfection (as adenovirus, herpes simplex virus I or adeno-associated virus) and fat based system (be used for the lipid mediation gene transfer lipid as, DOTMA, DOPE and DC-Chol).In some cases, need provide the preparation that can be targeted to target cell, have specific antibody, part of receptor or the like on the targeted cells as pair cell surface membrane protein or targeted cells to nucleic acid source.When adopting liposome, the albumen that can use the cell surface memebrane protein relevant with endocytosis to combine comes targeting and/or is beneficial to cellular uptake, for example, the capsid protein or its fragment that certain particular cell types are had tropism, the proteic antibody of experience internalization in cyclic process, and interior position of targeted cells and the albumen of interior half-life of enhancing cell.The technology of receptor-mediated endocytosis by, for example, Wu etc., J.Biol.Chem.262:4429-4432 (1987); With Wagner etc., Proc.Natl.Acad.Sci.USA 87:3410-3414 (1990) describes.

According to the present invention, treatment can also comprise conjoint therapy." conjoint therapy " used herein is meant the patient of a kind of medicine of needs and nertralizer united and treats or use another kind of Drug therapy disease.Conjoint therapy can be a sequential therapy, promptly earlier with one or more Drug therapys patient, and then uses another kind, perhaps uses two or more medicines simultaneously.Some can be used in exemplary drugs in the conjoint therapy comprise chemotherapeutics (cisplatin, amycin, danurubicin, tamoxifen (tamoxiphen), paclitaxel, methotrexate etc.), aromatase inhibitor, use angiogenesis inhibitor, specifically with non-immunomodulator specifically, biological response modifier (BRMs), colony stimulating factor (CSFs), interferon, interleukin, from body homology tumour-cell vaccine, hormone, or the like.Preferably, co-administered medicine or other preparation therapeutic activity of not disturbing nertralizer.

In some embodiments, the administration of nertralizer can combine with traditional treatment of cancer.Preferably, the effectiveness of nertralizer is not disturbed or reduces in traditional treatment of cancer.The example of the treatment of cancer that some are traditional comprises that operation (comprises, for example cryosurgery, part resection operation, radical prostatectomy, lumpectomy, mammectomy, or the like), chemotherapy, radiotherapy (for example, inner radiotherapy, external beam radiotherapy), brachytherapy (for example, with radiation directly to the primary tumor site effect and use single conduit to reduce the radiotherapy time and carry out breast cancer treatment), hormone resectional therapy (reduction hormonal readiness), or the like.

The present invention further provides by tumor is contacted with the TFF3 nertralizer method that reduces gross tumor volume, slows down tumor growth and/or prevention tumor growth.The contact enforcement can, for example, carry out in vivo, use any suitable method that tumor is exposed under the TFF3 nertralizer.For example, can directly contain the compositions of TFF3 nertralizer, perhaps use the compositions bag that contains the TFF3 nertralizer, perhaps can use the TFF3 nertralizer object or the patient who suffers from tumor by tumor to tumor injection.Can measure gross tumor volume and rate of growth thereof at an easy rate with methods known in the art.

In addition, can be by the patient being used the TFF3 nertralizer or cell and TFF3 nertralizer being contacted TFF3 at tissue or intracellular physiological effect (as overexpression TFF3).Above discussed owing to TFF3 expresses several physiological effects bring, comprised, for example, the enhancing of cell mobility (for example, transfer ability) and to the resistance of apoptosis.

Can assess vigor, migration and the potential aggressivity of cell with a test, this test comprises a ware cell of wound confluent growth, and the measurement cell is to the migration (for example, by the time-lapse photography microscopy or by inserting the time detecting of wound) of wound.Other two kinds of tests have been used and have been striden hole (transwell) filter membrane, in the test cell are laid on the top that porous is striden the hole insert, and the cell of moving to end well or move on the filter membrane of Fn Fiberonectin bag quilt is counted.Two tests of this of back are improvement of " Boyden chamber " test.Can further improve crosshole test and measure chemotaxis (by chemoattractant is placed bottom outlet), chemistry short live effect (being placed on the top and the bottom in hole by chemical substance) and aggressivity (by wrapping by the hole) with regenerated extracellular matrix (as matrigel) with induced movement.

Can detect apoptosis and to the resistance of apoptosis with any cell toxicity test commonly used.A variety of method known in the art is as the change of cellular morphology, distinctive 180bp dna ladder shape banding pattern appearance situation, TUNEL (TdT-mediated dUTP nick end labeling otch end labelling) test, flow cytometry, dna fragmentation ELISA and the cell membrane biophysical properties on agarose gel.Caspase family in the cysteine proteinase also has been accredited as the general amboceptor of cell suicide approach, and the activity test of caspase has also joined the method base that is used for detecting apoptosis.Lactic acid dehydrogenase (LDH) leaks outside, and test also is used for detecting or the measurement apoptosis.Can buy the test kit of multiple test in these methods of enforcement.

Cell, as the cell of differential expression TFF3, can with the TFF3 nertralizer in external, body or contact with each other under the isolated condition.Can implement contact with any preparation/medication as herein described, perhaps adopt, for example, with the method for cellular exposure in the culture medium that contains the TFF3 nertralizer.For example, washed cell, cultivation be suspended in solution or agar culture medium in certain hour, make the TFF3 nertralizer be enough to partial penetration cell membrane at least, and/or contact with intracellular TFF3 polypeptide or TFF3 polynucleotide.

Preparation, dosage, pharmaceutical composition

Can be according to expectation be the position of part or systematic treating and treatment as required and use pharmaceutical composition of the present invention with several different methods.Administration can be local (comprising eye or mucosa such as vagina and rectum release), through lung, for example by sucking or being blown into powder or aerosol, comprises and passes through aerosol apparatus; In the trachea, intranasal, epidermis and transdermal), outside the oral or gastrointestinal tract.The gastrointestinal tract external administration comprises intravenous, intra-arterial, subcutaneous, intraperitoneal or intramuscular injection or inculcates; Perhaps intracranial is for example in the sheath or the Intraventricular administration.

Be used for the pharmaceutical composition of topical and patch, ointment, emulsion, emulsifiable paste, gel, drop, suppository, spraying, liquid and the powder that preparation can comprise transdermal.May need or expect to use conventional pharmaceutical carrier, water, powder or butyrous substrate, thickening agent or the like.Also can use rubber case, glove of bag quilt or the like.

Be used for liquid preparations for oral administration and preparation comprise powder or granule, at suspension or solution, capsule, wafer (sachets) or the tablet of water or non-aqueous media.May need to use thickening agent, flavoring agent, diluent, emulsifying agent, dispersing aid or binding agent.

Be used for that gastrointestinal tract is outer, the compositions and the preparation of intracranial or Intraventricular administration can comprise sterile water solution, it also can contain buffer, diluent and other suitable additive, as but be not limited to penetration enhancer, carrier compound and other pharmaceutically acceptable carrier or excipient.

Pharmaceutical composition among the present invention includes, but not limited to solution, Emulsion and contains the preparation of liposome.These compositionss can be produced by a series of components, include, but not limited to the solid of previously prepared liquid, self emulsifying and the semisolid of self emulsifying.

Pharmacy thing preparation of the present invention can be represented with the form of unit dose easily, can be prepared according to the conventional art that pharmaceuticals industry is known.These technology comprise active component and pharmaceutical carriers or the contacted step of excipient.Normally make active component with liquid-carrier or fine grain solid phase carrier or both, mixing and contact mutually closely prepare preparation with this, and be if desired, moulding to goods again.

Composite preparation of the present invention can be become in the multiple available dosage form any, as but be not limited to tablet, capsule, capsule sheet, liquid sugar sirup, soft capsule, suppository and enema.Compositions of the present invention also can preparation be formulated as the suspension in water, non-water or the blending agent.Suspension liquid of aqueous phase can be with further comprising the material that increases suspension viscosity, comprise as, sodium carboxymethyl cellulose, Sorbitol and/or glucosan.Suspension also can contain stabilizing agent.

In some embodiments, pharmaceutical composition can be mixed with foam is used.The preparation that pharmaceutical foam comprises as, but be not limited to Emulsion, microemulsion, emulsifiable paste, gel and liposome.Although these preparations are similar substantially in essence, (consistency) is different for the concentration of their component and end product.The preparation of these compositionss and preparation normally known to the technical staff of pharmacy and formulation art, can be applied in preparation of the present invention and the compositions.

" pharmaceutical carriers " or " excipient " is pharmacy acceptable solvent, suspending agent or any other inert carrier of pharmacology, and they are used for one or more delivery of nucleic acids to animal.Excipient can be liquid or solid, then considers the predetermined way of administration during selection so that its consumption that reaches expectation with nucleic acid and other given pharmaceutical composition when co-administered, concentration (bulk, consistency) etc.Typical pharmaceutical carriers includes, but not limited to binding agent (for example pregelatinized corn starch, polyvinylpyrrolidone or hypromellose); Filler (for example lactose and other sugar, microcrystalline Cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylate or ester or calcium hydrogen phosphate or the like); Lubricant (for example magnesium stearate, Talcum, silicon, silica sol, stearic acid, metallic stearate, hydrogenated vegetable oil, corn starch, Polyethylene Glycol, sodium benzoate, sodium acetate, or the like); Disintegrating agent (for example starch, primojel (sodium starchglycolate) or the like); And wetting agent (for example, sodium lauryl sulphate or the like).

The preparation that also can not be used for the present composition with the acceptable organic or inorganic excipient of the pharmacy that is applicable to the gastrointestinal tract external administration of nucleic acid generation adverse reaction.Suitable pharmaceutical acceptable carrier includes, but not limited to water, saline solution, ethanol, Polyethylene Glycol, gelatin, lactose, amylose, magnesium stearate, Talcum, viscous paraffin, hydroxy methocel, polyvinylpyrrolidone or the like.

The preparation that is used for local application nucleic acid can be with comprising sterilization and unsterilised aqueous solution or be dissolved in the non-aqueous solution of common solvent (as ethanol), or the nucleic acid solution in the liquid or solid oleaginous base.Solution can also contain buffer, diluent and other suitable additive.Can use not and the acceptable organic and inorganic excipients of the pharmacy that is applicable to the gastrointestinal tract external administration of nucleic acid generation adverse reaction.

Suitable pharmaceutical acceptable excipient includes, but not limited to water, saline solution, ethanol, Polyethylene Glycol, gelatin, lactose, amylose, magnesium stearate, Talcum, viscous paraffin, hydroxy methocel, polyvinylpyrrolidone or the like.

Compositions of the present invention can also contain other the annexing ingredient that is common in pharmaceutical composition, the level that its dosage level is formulated for this field.Therefore, for example, compositions can with contain other, compatible, pharmaceutical active material, as antipruritic, astringent, local anesthetic or antiinflammatory, maybe can contain other material useful when compositions of the present invention being carried out the physical preparation of various dosage forms, as dyestuff, flavoring agent, antiseptic, antioxidant, opacifier, thickening agent and stabilizing agent.Yet when adding these materials, they should not can exceedingly disturb the biologic activity of present composition component.Can sterilize to preparation, also can mix mutually if desired with adjuvant, for example lubricant, antiseptic, stabilizing agent, wetting agent, emulsifying agent, the salt that influences osmotic pressure, buffer, coloring agent, flavoring agent and/or aromatic substance or the like, deleterious interaction can not take place with the nucleic acid of preparation in them.

The aqueous suspension agent can contain the material that strengthens suspending agent viscosity, for example comprises sodium carboxymethyl cellulose, Sorbitol and/or glucosan.Suspending agent also can contain stabilizing agent.

The pharmaceutical composition that some embodiments of the present invention provide contains: (a) one or more antisense compounds and one or more other chemotherapeutics that (b) play a role by non-antisense mechanism.The example of such chemotherapeutics comprises, but be not limited to, cancer therapy drug is as daunorubicin, dactinomycin, amycin, bleomycin, mitomycin, chlormethine, chlorambucil, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytosine arabinoside (CA), 5-fluorouracil (5-FU), floxuridine (5-FUdR), methotrexate (MTX), Colchicine, vincristine, vincaleucoblastine, etoposide, teniposide, cisplatin and diethylstilbestrol (DES).Usually referring to The Merck Manual of Diagnosis and Therapy, the 15th edition, Berkow etc. compile, and 1987, Rahway, N.J., 1206-1228 page or leaf).Anti-inflammatory agent includes but not limited to NSAID (non-steroidal anti-inflammatory drug) and corticosteroid, and antiviral drugs, includes but not limited to ribavirin, vidarabine, acyclovir and Cymevan, and compositions also available and of the present invention is united use.Usually referring to The Merck Manual ofDiagnosis and Therapy, the 15th edition, Berkow etc. compile, and 1987, Rahway, N.J. is respectively 2499-2506 page or leaf and 46-49 page or leaf).Other non-antisense chemotherapeutics is also included within the scope of the present invention.The two or more combination of compounds of can using together or use in order.

Assessment of the diagnosis of cancer, prognosis, therapy (therapy) and control

TFF3 polynucleotide as herein described and gene outcome thereof can be further used as heredity or biochemical marker (for example in blood or the tissue), are used for detecting the variation of the earliest period on the carcinogenic approach and/or monitor various therapies and the curative effect of prevention intervention.For example, the expression of TFF3 can be the sign of poorer prognosis, therefore just need use to have more aggressive chemotherapy or radiotherapy to the patient, otherwise perhaps.The indication index that novel alternative tumour-specific feature and the relatedness between the therapeutic outcome that the reaction for the treatment of and patient are obtained can be established prognosis so just can be designed suitable therapy according to the molecular linkage map of tumor.These therapies comprise antibody target, nertralizer (for example micromolecule) and gene therapy.Determine the expression of TFF3 and patient's collection of illustrative plates compared with the expression in known normal structure and the disease variant just to determine possible therapeutic regimen that the specificity aspect that this not only is meant treatment also refers to patient's comfort level aspect for this patient.Substitute tumor marker (surrogate tumor marker), express, also can be used for better the multi-form and morbid state of cancer being classified as polynucleotide.Widely used two kinds of sorting techniques can benefit from the evaluation of the gene expression dose to the polynucleotide correspondence as herein described among the oncology, and they can be to the cancer disease in addition by stages whereby, and the character of cancerous tissue is carried out classification.

Measure TFF3 be expressed in to suffer from or cancer-prone patient's monitoring aspect valuable, can detect under the gross morphology level less than situation under detect potential malignant event from molecular level.In addition, TFF3 polynucleotide and can be used as therapy and work corresponding to the gene of these polynucleotide, for example use the gene outcome of polynucleotide or their coding to assess curative effect, as before the assessment treatment, in, back patient's tumor load (tumor burden).

In addition, be accredited as the pairing polynucleotide of gene of differential expression in one type cancer, it also may indicate the development of other types of cancer or the danger of development also to have association, for example, represent a kind of gene when polynucleotide and in various various cancers types, had differential expression.Therefore, for example corresponding to certain polynucleotide of a gene, it is expressed has clinical relatedly with the transitivity colon cancer, and it also may have clinical association to gastric cancer or carcinoma of endometrium equally.

By stages

Be the method that the doctor is used for describing patient's cancerous state process by stages, help the doctor to determine prognosis, planned treatment and the effect of assessing this treatment by stages.By stages system with the difference of cancer types difference to some extent, but generally include following " TNM " system: tumor type, represent by T; Whether cancer has been transferred to is closed on lymph node, is represented by N; And whether cancer transferred to the more distal part of health, represented by M.Usually, if only in the primary lesion location detection to cancer, and it is not diffused into any lymph node, this just is called the I phase.If it only is diffused into nearest lymph node, then be called the II phase.Interim at III, cancer has been diffused into the lymph node that the primary lesion position is closed on usually.Being diffused into health is the IV phase than the cancer of distal part (as liver, bone, brain or other position), just late period.

Polynucleotide as herein described can the identical well-regulated process by stages of cancer occur and play with the health different parts by the labelling such as the potential transitivity of identification tumor aggressiveness (aggressiveness).Therefore,, the II phase tumor on border can be transformed into III phase tumor, then need to have more aggressive therapy if II phase cancer is attended by a kind of polynucleotide that indicating highly potential metastatic cancer.On the contrary, if there are a kind of lower potential metastatic polynucleotide that indicating, so just can make comparatively and guarding by stages tumor.

The cancer classification

Rank (grade) is one and how has the term of similarity degree in order to the normal structure of describing tumor and its same type.According to cellular morphology, cell tissue and other differentiation sign, discern the tumor rank with the appearance of tumors of microscopically.As common rule, the rank of tumor is corresponding to its rate of growth or aggressiveness, not differentiation or high level tumor usually than fully break up or low-level tumor more aggressive.Usually following guilding principle is used for tumor grade: 1) GX can not level of evaluation; 2) G1 fully breaks up; The differentiation of G2 moderate; 3) the insufficient differentiation of G3; 4) G4 does not break up.The polynucleotide that the present invention considers are particularly useful when definite tumor rank, because they not only can assist the differentiation state of determining tumor cell, also can discern the useful other factors aspect the aggressiveness of definite tumor beyond the differentiation, as potential transitivity.

The detection of cancer

Can come detected object intravital cancer, especially colon cancer, breast carcinoma and carcinoma of prostate with the TFF3 expression pattern.Colorectal cancer is one of human most common tumor, also may be the form that hereditary tumor forms the most normal generation.Prevention and earlier detection are control and the key element of curing colorectal cancer.Colorectal cancer originates in polyp, and polyp is the very little optimum growth of the cell that forms on the colon inwall.In the time in several years, some in these polyps have accumulated more sudden change and canceration have taken place.Identified multiple family colorectal cancer, be summarized as follows: 1) family's adenomatous polyp (FAP); 2) Gardner syndrome (Gardner ' ssyndrome); 3) hereditary nonpolyposis colon cancer (HNPCC); With 4) familial colorectal cancer in the Ashkenazi Jew.The expression of suitable polynucleotide can be used in diagnosis, prognosis and the cancer control.The definite of cancer detection can use the expression of TFF3 sequence separately or unite use with other gene.Can pass through the level of one or more gene outcomes of polynucleotide correspondence more described herein, and relatively another known in cancerous tissue the aggregate level of discrepant sequence determine the aggressivity and/or the potential transitivity of colon cancer, for example, the expression of p53, DCC, ras, FAP (referring to, Fearon ER for example, etc., Cell (1990) 61 (5): 759; Hamilton SR etc., Cancer (1993) 72:957; Bodmer W, etc., Nat Genet. (1994) 4 (3): 217; Fearon ER, Ann N YAcad Sci. (1995) 768:101).For example, can with corresponding to the expression of the TFF3 of polynucleotide described herein and oncogene (for example, ras) or the level of tumor suppressor gene (for example, FAP or p53) compare and detect the development of cancer.Therefore, the expression of the polynucleotide of specific marker can be used to distinguish the colon of normal and canceration, is used to distinguish the different cancer in cell source, is used to distinguish cancer with different potential rates of transform or the like.For the summary of cancer markers, referring to, for example (2000) Cell 100:57-70 such as Hanahan is incorporated herein by reference in full.

Treatment for cancer

The invention provides method with the anticancer growth that is expressed as feature and/or the adhesion of regulation and control cancer, migration and/or the transfer of TFF3.The example comprises alimentary tract cancer, as colon cancer, gastric cancer and hepatocarcinoma, and other cancer, as pulmonary carcinoma, breast carcinoma, ovarian cancer and carcinoma of prostate.In some embodiments, cancer shows different TFF3 expressions.In other embodiments, TFF3 is up-regulated in cancer.In also having some embodiments, cancer is not a colon cancer, and in other embodiments, cancer is not a carcinoma of prostate.

Present invention includes any treatment for cancer, wherein the following at least a situation of regulation and control (for example, suppressing) of using of TFF3 nertralizer: the propagation of cancerous cell, cancerous cell migration, cancerous cell adhesion and/or transfer.Shown in embodiment, the TFF3 nertralizer has demonstrated the inhibition for cancerous cell adhesion and cancer cell multiplication.Can be to adherent inhibition by anticancer to the adhesion at position beyond the primary tumo(u)r and develop into tumor and transfer is had regulating effect.Usually, this method comprise with cancerous cell with regulation and control following aspect material contact: (1) is corresponding to the expression of the polynucleotide of TFF3; Or the level and/or the activity of (2) TFF3 polypeptide.These methods reduced TFF3 in cancerous cell expression or reduced level and/or the activity of TFF3.This inhibition can reduce propagation, migration and/or the adhesion of cancerous cell, reduces tumor growth, reduces gross tumor volume, reduces aggressive of tumor or the like.

" reduce the growth of tumor or cancerous cell " and include, but not limited to reduce the propagation of cancer (or tumor) cell, and reduce the incidence rate that non-cancerous cell is transformed into cancerous cell.Can determine at an easy rate whether the growth of cancerous cell decreases, and such test includes, but not limited to [3H]-thymidine and integrates with any known test; Pair cell is counted in a period of time; Detect and/or measure the relevant labelling (for example, CEA, CA19-9, and LASA) of colon cancer, or the like.

The present invention provides the method for treatment TFF3 associated cancer especially, and as colon cancer, breast carcinoma and/or carcinoma of prostate, this method comprises uses the material that reduces growth of cancer cells to the individuality that needs are arranged, and application dosage is enough to reduce the growth and the treatment cancer of cancerous cell.Any certain material of assessing in can testing with known a series of cancer diagnosis, or certain concrete consumption of this material the cancer that whether can treat the patient effectively, these diagnostic tests comprise, but be not limited to, sigmoidoscopy, proctoscopy, examination per rectum, living tissue colonoscopy, the inspection of the tumor marker relevant in the research of contrast pneumoradiography, cat scan, angiography and the individual blood with colon cancer.Can be systematically or this material of local application.Local application can be effective when treating as solid tumor.

The diagnosis and other detection method that relate to TFF3

The invention provides the method and some other method that TFF3 nertralizer as herein described, TFF3 polypeptide and TFF3 polynucleotide are used for diagnostic purpose.Concrete but in the nonrestrictive embodiment, these methods detect aspect the relevant cancerous cell of TFF3 useful, as colon, mammary gland, ovary, stomach and prostate gland cancer cell, these methods also are beneficial to the diagnosis of cancer in the subject and seriousness thereof (for example, tumor rank, tumor load, or the like), and the prognosis and evaluation object (for example the replying that are beneficial to definite object to therapy, by the measurement to curative effect is provided, for example, by assessing in the chemotherapy and the tumor load after the chemotherapy).Detection can be based upon on the level of TFF3 in the cell, for example colon cancer cell and/or to the detection of the TFF3 polypeptide in the cancerous cell.Detection method of the present invention can be carried out in external or body, can be the detection to isolated cell, or to the detection of complete tissue or body fluid (for example blood, blood plasma, serum, urine or the like).

Therefore, the invention provides the method for TFF3 in the detection of biological sample,, come the situation that exists of cancer in the detection of biological sample by the sign that TFF3 differentiation in contact sample and the test sample is expressed.Can to be the TFF3 nertralizer combine with TFF3 in the sample form of expression of sign.Multiple detection known in the art and measure method in conjunction with level comprises as based on test of ELISA or the like.The expression that can will be compared in conjunction with level and standard sample and show TFF3 is lower than or is higher than level in the normal specimens.In some embodiments, detected TFF3 expression is higher than the normal level indication and has cancer.

The present invention further provides by the differential expression sign that detects TFF3 in patient's cancer sample and determined the method patient of patient TFF3 nertralizer sensitivity.Term used herein " sensitivity " is used to describe uses the TFF3 nertralizer to its patient for the method for can receiving treatment, and just, the patient of this term description may make energetically the treatment of TFF3 nertralizer and replying.With compare treating insensitive patient, may show the differential expression of TFF3 to the cancer patient of Therapeutic Method sensitivity of the present invention, for example in diseased tissue.

Detection method of the present invention can be used as the part of test kit.Therefore, the present invention further provides detect TFF3 in the cancerous cell the test kit that has situation and/or its expression (for example, by detecting mRNA by interested difference expression gene coding), and/or the test kit that has situation and/or its expression of TFF3 encoded polypeptides in the detection of biological sample is provided.The service routine of these test kits can be operated separately by clinical laboratory, laboratory, medical personnel or individual.The test kit that is used for detecting polynucleotide (it is at the colon cancer cell differential expression) encoded polypeptides of the present invention contain one specifically with the bonded part of polypeptide, it can be special antibody, antisense molecule, RNAi molecule, ribozyme or micromolecule.Of the present inventionly contain a part with this polynucleotide specific hybridization in order to detect in the cancerous cell test kit of the polynucleotide of differential expression.This test kit can be chosen wantonly provides other useful components in operation, includes but not limited to buffer, developing agent, labelling, reaction surface, detection means, control sample, standard, guidance and descriptive information.

Detect the TFF3 polypeptide in the cancerous cell

In some embodiments, provide by detecting the method that the TFF3 overexpressing cell detects the TFF3 associated cancer.In the known serial of methods any may be used to detect, and includes but not limited to, immunoassay uses the specific antibody of coded polypeptide tool, as enzyme-linked immunosorbent assay (ELISA), and radioimmunoassay (RIA) or the like; And, for example live in conjunction with activity or enzyme for the function test of coded polypeptide.

For example, can under the situation of not separating coded polypeptide in advance, implement immunofluorescence test by pair cell.Earlier with cell fixation to solid support, as microscope slide or microtiter well.This fixing step can make cell membrane have permeability.The permeability of cell membrane allows the antibodies of polypeptid specificity to get on.Then, with fixed cellular exposure in the specific antibody of encoded polypeptide.In order to increase the sensitivity of test, can be further with cellular exposure in second antibody, two anti-carried out labelling and be combined in one anti-on, one anti-ly then has specificity to encoded polypeptide.Usually, can detect, for example, carry out labelling with fluorescent labeling to two anti-labellings.The cell of expressing coded polypeptide will be by on the fluorescent labeling, and is easy to observe at microscopically.Referring to, (1992) Biochem.Bioplzys.Res.Comm.187:1241-1248 such as Hashido for example.

After reading this description, those skilled in the art are readily understood that detection method as herein described and other method can make change easily.Such change belongs in the purpose scope of the present invention.For example, in above-mentioned detection scheme, the probe that is used to detect can be fixed on solid support, and testing sample is contacted with immobilized probe.Then, can detect combining of testing sample and probe, for example,, be beneficial to detect the complex of testing sample-immobilization probe by detecting the detectable label that combines with testing sample with serial of methods.

The present invention further provides the method that there is situation in TFF3 polypeptide in the detection of biological sample and/or measures TFF3 polypeptide level, use be the specific antibody of TFF3.Such method generally includes: a) sample is contacted with the specific antibody of TFF3; And b) detect between antibody and the sample molecule in conjunction with situation.

Detect the specific bond of TFF3 specific antibody if compare, then represent to exist in the sample TFF3 with suitable contrast.Suitable contrast comprises the known sample that does not contain TFF3; With with for the contacted sample of the nonspecific antibody of encoded polypeptide, for example, anti-id AB.The method of a series of detection specificity antibody-AIs known in the art may be used in this method, includes, but not limited to SABC method, immunoprecipitation, EIA enzyme immunoassay and the radioimmunoassay, RIA of standard.Common specific antibody is connected with detectable labelling directly or indirectly.Direct-connected labelling comprises radiosiotope; Can detect the enzyme (for example luciferase, tilactase or the like) of its product; Fluorescent labeling (for example fluorescein isothiocyanate, rhodamine, phycoerythrin or the like); The fluorescent emission metal, for example 152Eu, or other lanthanide series can be connected on the antibody by metal-chelating group such as EDTA; Chemiluminescence compound, for example, luminol, different luminol (isoluminol), acridine salt, or the like; Bioluminescent compound, for example, fluorescein, aequorin (green fluorescent protein), or the like.Antibody can be connected (coupling) to insoluble holder, as polystyrene board or pearl.Indirect labelling comprises two of the specific antibody of encoded polypeptides tool (" first specific antibody ") is resisted, and wherein resists two and carries out labelling with above-mentioned method; And the right member of specific bond, for example, biotin-avidin, or the like.Can and be fixed on solid support or the carrier the biological sample contact, as NC Nitroncellulose, its energy immobilized cell, cell granulations or soluble protein.Can contact with first specific antibody that is connected with detectable label again with suitable buffer washing holder then.Detection method is known in the art, and selects the suitable detection method of signal to detectable label sent.Common and suitable contrast and suitable standard are compared and are finished detection.

In some embodiments, change these methods, for example, have the position of TFF3 in conjunction with cancerous cell with location or identification to adapt to the needs that use in the body.In these embodiments, the part of detectable label will be connected with, for example, the specific antibody of TFF3 is applied to individual (for example, with the method for injection), and positions with the imaging technique of standard cell to labelling, such imaging technique comprises, but be not limited to, nuclear magnetic resonance, X ray CT scan photography, or the like.Like this, the cell of expression TFF3 then has different labellings.

Detect the TFF3 polynucleotide in the cancerous cell

The method that detects the TFF3 cancerous cell by TFF3 transcript in the detection cancerous cell at intracellular expression is provided.In a series of known methods any may be used to detect, and includes, but not limited to use hybridize with the polynucleotide of TFF3 multi-nucleotide hybrid and detect transcription product; Use special oligonucleotide primers to detect transcript with the polymerase chain reaction; The polynucleotide of the gene recombination of differential expression carry out in situ hybridization as probe in use and the colon cancer cell.Can use these methods to detect and/or measure the mRNA level of TFF3 gene expression in the cancerous cell.In some embodiments, these methods comprise: a) under the condition that allows hybridization sample is contacted with the TFF3 polynucleotide; And b) if present, detect hybridisation events.

Detect differential hybridization if compare, then be illustrated in the polynucleotide that have differential expression in the cancerous cell in the sample with suitable contrast.Suitable contrast comprises, for example, and the known sample that contains the TFF3 polynucleotide.It is known in the art allowing the condition of hybridization.Can any known method use the polynucleotide that are connected with appropriate flags to finish detection, comprise, but be not limited in situ hybridization, PCR (polymerase chain reaction), RT-PCR (reverse transcription-PCR) and the combination of " Northern " or RNA hybridization or these technology.The labeling method of a series of labelling known in the art and polynucleotide can be used in the test method of the present invention.Can by with appropriate control relatively come to determine specific hybrid.

The invention provides and contain at least 12 polynucleotide that adjoin nucleotide in the TFF3 polynucleotide usually, can be used for a series of purpose, as the probe of the transcriptional level of the polynucleotide of differential expression in colon cancer cell as detection and/or measurement.The probe of the polynucleotide specific hybrid that discloses with this paper should be than the detection signal of background hybridization high at least 5-, 10-or 20-doubly, background hybridization is provided by other incoherent sequence.It is worthy of note that " probe " used herein is meant the polynucleotide sequence that is used for detecting testing sample TFF3 gene outcome.What those skilled in the art were readily appreciated that is, can add detectable labelling to probe, and with, for example contain that (for example, the microarray of immobilization polypeptide mRNA) contacts from testing sample.Perhaps can be on microarray with probe-immobilized, and testing sample is added detectable labelling.These of the inventive method and other version are all in the technical scope of this area, also within the scope of the invention.

Can detect and the corresponding expression of gene of polynucleotide that is provided with nucleotide probe.In Northern hybridization, electrophoretic separation mRNA, and contact with probe.Detect the probe of hybridizing with a certain size mRNA kind.The amount that can measure hybridization is determined the relative quantity expressed, for example under given conditions, with the in situ hybridization of cell in use probe to detect expression.Probe also can be used in the body and hybridization sequences is done diagnostic assays.Usually use the labelled with radioisotope probe.Also can use the detectable label of other type, as chromophoric group, fluorophor and enzyme.Other embodiment of nucleotide cross experiment is described in WO92/02526 and U.S. Patent number 5,124,246.

PCR be the another kind of method that detects a small amount of target nucleic acid (referring to, for example, Mullis etc., Meth.Enzymol. (1987) 155:335; U.S. Patent number 4,683,195 and 4,683,202).Use two kinds of primer polypeptide nucleotides to start reaction with target nucleic acid hybridization.Primer can contain in the polynucleotide that this paper discloses 3 ' or one section sequence in 5 ' zone.Perhaps, if primer is exactly 3 of these polynucleotide ' and 5 ' part, they do not need hybridization or complementary.After with heat-staple polymeric enzymatic amplification target, can detect the target nucleic acid of amplification with methods known in the art, for example, Southern hybridization.Also can be (for example with traditional hybridization technique, Southern hybridization, Northern hybridization, or the like) detect mRNA or cDNA, be described in Sambrook etc., " Molecular Cloning:A Laboratory Manual " New York, Cold Spring HarborLaboratory, 1989) (for example, not carrying out pcr amplification).Usually, mRNA or use polymerase can carry out purification by the cDNA that mRNA produces, and separate with gel electrophoresis, transfer on the solid support, as NC Nitroncellulose again.Solid support is exposed in the probe that labelling crosses, and washing contains the underlined two strands of crossing probe to remove any not probe of hybridization, to detect.

Can use the method for pcr amplification to DNA, although more convenient use is at least about 105 cells from individual cells.The use of polymerase chain reaction is described in (1985) Science 239:487 such as Saiki, can be at Sambrook, Deng Molecular Cloning.A Laboratory Manual, CSH Press 1989, pp.14.2-14.33, in find the summary of present technology, both all are incorporated herein by reference.Can in amplified reaction, add detectable labelling.Suitable detectable label comprises that fluorophor (for example, fluorescein isothiocyanate (FITC), rhodamine, texas Red, phycoerythrin, allophycocyanin, 6-CF 5(6)-Carboxyfluorescein (6-FAM), 2 ', 7 '-dimethoxy-4 ' ', 5 '-two chloro-6-CF 5(6)-Carboxyfluorescein, 6-carboxyl-X-rhodamine (ROX), 6-carboxyl-2 ', 4 ', 7 ', 4,7-chlordene fluorescein (HEX), 5-CF 5(6)-Carboxyfluorescein (5-FAM) or N, N, N ', N '-tetramethyl-6-carboxyl rhodamine (TAMRA)), radioactive label (for example, 32P, 35S, 3H, or the like), or the like.Labelling can be the system in two stages, wherein polynucleotide are conjugated on biotin, hapten or the like, have a highly affine bonded part, for example, avidin, specific antibody or the like, bound fraction wherein is conjugated on the detectable labelling.Labelling can be conjugated on one or both primers.Perhaps, can labelling used nucleotide storehouse in the amplification, go and labelling is incorporated in the amplified production.

Microarray

The polynucleotide microarraies provide can test sample in the high-throughput techniques of a large amount of polynucleotide or polypeptide.This technology can be used as the instrument that detects differential expression.The method of a series of production microarray known in the art and the version of these methods can be considered to use in the present invention.For example, polynucleotide probes can be put on the egative film of two-dimentional substrate (for example, glass, NC Nitroncellulose, or the like) produce microarray, perhaps produce with the microarray that is combined with probe.Combining of probe and egative film can also can be with non-special interaction, as hydrophobic interaction with covalent bond.Can connect detectable labelling (for example, using radioactivity or fluorescent labeling) to polynucleotide, and then and probe hybridization.Sample can detect double-stranded polynucleotide after bound fraction is not washed, it comprises the labelling sample polynucleotide that are attached to the probe polynucleotide.Perhaps, the polynucleotide of testing sample can be fixed on the microarray, and probe is carried out detectable labelling.Make up the technology of microarray and use the method for these microarraies to be described in, for example, Schena etc. (1996) ProcNatl Acad SciU S is (20): 10614-9 A.93; Schena etc. (1995) Science 270 (5235): 467-70; Shalon etc. (1996) Genome Res.6 (7): 639-45, U.S. Patent number 5,807,522, EP799 897; WO 97/29212; WO 97/27317; EP785280; WO 97/02357; U.S. Patent number 5,593,839; U.S. Patent number 5,578,832; EP 728 520; U.S. Patent number 5,599,695; EP 721016; U.S. Patent number 5,556,752; WO95/22058; With U.S. Patent number 5,631,734.

Microarray can be used for, and for example, detects the differential expression of gene, also can be used for detecting gene function.For example, can use microarray to detect the differential expression of TFF3 gene, wherein the expression to cell to be measured and control cells (for example, cancerous cell and normal cell) compares.For example, the high expressed of specific mRNA in the cancerous cell, and in the normal cell of correspondence, do not observe this phenomenon, then be designated as the cancer specific gene outcome.The typical use of microarray is further described in, for example, and Pappalarado etc., Sem.RadiationOncol. (1998) 8:217; With Ramsay Nature Biotechnol. (1998) 16:40.In addition, a lot of versions of detection method that use microarray are also all in the technical scope of this area, and within the scope of the invention.For example, testing sample can be fixed on the solid support, and then with probe contact, rather than with probe stationary on solid support.

Goods

In other embodiment of the present invention, the goods that contain (useful) TFF3 nertralizer are provided to treating disease as herein described or disease.These goods contain a container and insertion wherein or labelling that is attached thereto or packing.Suitable containers comprises, for example, and bottle, vial, syringe or the like.Can form container with multiple material, as glass or plastics.Container be equipped with or the compositions that contains effective to treating selected disease or disease, also can contain the passage of getting it filled (for example, container can be an intravenous fluids bag or a vial, and it contains can be by the saturating stopper of subcutaneous injection acupuncture) of sterilization.At least a active ingredient is the TFF3 nertralizer in the compositions, as TFF3 antisense molecule, RNAi molecule, ribozyme, micromolecule or antibody.Labelling or package insert show that said composition is used for the treatment of and suffers from or cancer-prone patient, for example, and colon, mammary gland or carcinoma of prostate.Goods can further contain second container, and it contains the acceptable dilution buffer liquid of pharmacy, the bacteriostatic water (BWFI) as being used to inject, phosphate buffered saline(PBS), Ringer ' s solution and dextrose solution.It can also further contain other from commercial and the useful material of user angle, comprises other buffer, diluent, pin and syringe.

Illustrate details more specifically of the present invention with following non-restrictive example.All disclosed quoted passages all are incorporated herein by reference full text clearly in the description.

Embodiment

Listing following examples to provide about how to prepare and use detailed announcement of the present invention and description for those of ordinary skill in the art, but this does not also mean that their scope of invention that the inventor is thought makes restriction, and also not representing following experiment is whole or only experiments of being carried out.We endeavour to ensure the accuracy (for example, quantity, temperature or the like) of data, but need explanation to have some experimental erroies and deviation.Unless otherwise indicated, mark is meant mass fraction, and molecular weight is an average molecular mass, and the unit of temperature is ℃, and pressure then is atmospheric pressure or near atmospheric pressure.

Embodiment 1: the source of biomaterial

Be used for test to realize that biomaterial of the present invention is described below.

The source of patient tissue samples

Use laser capture microdissection (LCM) technology to gather the tissue of normal and canceration in patient's body, such technology be well known in the art (referring to, for example, Ohyama etc. (2000) Biotechniques 29:530-6; Curran etc. (2000) Mol.Pathol.53:64-8; Suarez-Quian etc. (1999) Biotechniques 26:328-35; Simone etc. (1998) Trends Genet 14:272-6; Conia etc. (1997) J.Clin.Lab.Anal.11:28-38; Emmert-Buck etc. (1996) Science 274:998-1001). following table 2 provides each patient's information, isolates the prostata tissue sample on one's body from them, comprising: 1) " patient ID " is to give the specified number of patient for distinguishing purpose; 2) " types of organization; With 3) " the Gleason rank " of tumor.The histopathology of all primary tumo(u)rs shows that tumor is an adenocarcinoma.

Table 2. prostate patient data

Patient ID	Types of organization	The Gleason rank	Patient ID	Types of organization	The Gleason rank
						93	Carcinoma of prostate	3+4	391	Carcinoma of prostate	3+3
94	Carcinoma of prostate	3+3	420	Carcinoma of prostate	3+3
						95	Carcinoma of prostate	3+3	425	Carcinoma of prostate	3+3

96	Carcinoma of prostate	3+3	428	Carcinoma of prostate	4+3
						97	Carcinoma of prostate	3+2	431	Carcinoma of prostate	3+4
100	Carcinoma of prostate	3+3	492	Carcinoma of prostate	3+3
						101	Carcinoma of prostate	3+3	493	Carcinoma of prostate	3+4
104	Carcinoma of prostate	3+3	496	Carcinoma of prostate	3+3
						105	Carcinoma of prostate	3+4	510	Carcinoma of prostate	3+3
106	Carcinoma of prostate	3+3	511	Carcinoma of prostate	4+3
						138	Carcinoma of prostate	3+3	514	Carcinoma of prostate	3+3
151	Carcinoma of prostate	3+3	549	Carcinoma of prostate	3+3
						153	Carcinoma of prostate	3+3	552	Carcinoma of prostate	3+3
155	Carcinoma of prostate	4+3	858	Carcinoma of prostate	3+4
						171	Carcinoma of prostate	3+4	859	Carcinoma of prostate	3+4
173	Carcinoma of prostate	3+4	864	Carcinoma of prostate	3+4
						231	Carcinoma of prostate	3+4	883	Carcinoma of prostate	4+4
232	Carcinoma of prostate	3+3	895	Carcinoma of prostate	3+3
						251	Carcinoma of prostate	3+4	901	Carcinoma of prostate	3+3
282	Carcinoma of prostate	4+3	909	Carcinoma of prostate	3+3
						286	Carcinoma of prostate	3+3	921	Carcinoma of prostate	3+3
294	Carcinoma of prostate	3+4	923	Carcinoma of prostate	4+3
						351	Carcinoma of prostate	5+4	934	Carcinoma of prostate	3+3
361	Carcinoma of prostate	3+3	1134	Carcinoma of prostate	3+4
						362	Carcinoma of prostate	3+3	1135	Carcinoma of prostate	3+3
365	Carcinoma of prostate	3+2	1136	Carcinoma of prostate	3+4
						368	Carcinoma of prostate	3+3	1137	Carcinoma of prostate	3+3
379	Carcinoma of prostate	3+4	1138	Carcinoma of prostate	4+3
						388	Carcinoma of prostate	5+3

Embodiment 2: use the microarray assay differential expression

In embodiment 1 described patient's cell, separate total RNA, and prepare the cDNA probe thus.Because LCM can isolate special cell type,, so just obtain approximate pure RNA sample so the cell sample of homogenizing basically just can be provided.

With the primer that contains T7 rna polymerase promoter total RNA reverse transcription is become cDNA earlier, then carry out the synthetic of second DNA chain.The expression of using T7 promoter mediation is then carried out in vitro transcription to cDNA and is produced antisense RNA (referring to, for example, Luo etc. (1999) Nature Med 5:117-122), again antisense RNA is transformed into cDNA.Re-use the T7 promoter second group cDNA in vitro transcription is produced antisense RNA.Randomly, again RNA is transformed into cDNA, the amplification that allows the nearly T7 mediation of three-wheel is to produce more antisense RNA.Like this, this step provides 2 or 3 in vitro transcriptions of taking turns, and produces to be used for fluorescently-labeled whole RNA.

Earlier contrast RNA is added in the antisense RNA mixture, produce fluorescently-labeled cDNA from the RNA parent material again, produce probe with this.Compare with fluorescent labeling cDNA originating from the fluorescent labeling cDNA of tumor RNA sample from normal cell RNA sample preparation.For example, from Normocellular cDNA probe, then use Cy5 fluorescent dye (redness) labelling from the cDNA probe of tumor cell preparation with Cy3 fluorescent dye (green) labelling, vice versa.

The microarray of each use all has identical spatial arrangements and control point group.Always counting for each is about 9,216 microarray, and each microarray is divided into two parts, and the microarray that each part has partly contains 12 group 32 * 12 points at each.Two part point samples are identical, and each microarray all has two repetitions to each clone at least like this.

Be used in polynucleotide on the microarray all from public information sources and the cDNA storehouse that produces from aforesaid selected cell line and patient tissue.According to manufacturer's recommendation, use Molecular Dynamics GenIII point sample instrument that the length in these sources is put on the microarray at the pcr amplification product of about 0.5kb to 2.0kb.First row of each on the microarray in 24 parts has 32 control points approximately, comprises 4 negative controls and 8 polynucleotide to be measured.Before labeled reactant, polynucleotide point to be measured to be advanced in each sample, concentration range is every of 2-600pg/, and ratio is 1: 1.For the design of each microarray, with two all with labeled reactant in the testing sample of reverse labelling hybridize.Each clone just has 4 duplicate detection approximately like this, is a kind of color for two in each sample, and two is another color.

The implementation method of differential expression test is as follows, with mixing mutually with Normocellular probe from same patient tumors cell of equivalent.Under 60 ℃ of conditions, place 5X SSC/0.2% SDS/1mM EDTA to hatch microarray and carried out prehybridization in 2 hours, in water, wash three times washed twice in isopropyl alcohol then.After the microarray prehybridization, reuse probe mixture and microarray hybridization adopt highly strict hybridization conditions (42 ℃ hybridize and spend the night) in 50% Methanamide, 5X SSC and 0.2%SDS.After the hybridization, following washing under 55 ℃ of conditions three times: 1) in 1X SSC/0.2% SDS, wash earlier; 2) in 0.1X SSC/0.2% SDS, wash then; And 3) wash at 0.1X SSC.

Use Molecular Dynamics Generation III dual-color laser scanning/detector scanning microarray then, detect green and red fluorescence.Use BioDiscovery Autogene software processes image, will be from the data normalization of each scanning group so that the expression ratio of relative normal value to be provided.According to United States Patent (USP) serial number 60/252, the data of 358 described Algorithm Analysis microarray experiment gained, this application is by E.J.Moler, M.A.Boyle, be filed on November 20th, 2000 with F.M.Randazzo, be entitled as " precision of cDNA microarray data and accuracy " (Precision and accuracy in cDNA microarray data), this application is incorporated herein by reference especially.

Repetition is carried out in test, specifically two kinds of probes are carried out labelling with opposite color, so just experimentize two " color direction ".Sometimes each experiment all repeats two even multi-disc (each color direction a slice) more.The fluorescence level of each sequence all is expressed as from 4 microarraies 8 and repeats a little/gene or repeat a little/ratio of the geometrical mean of gene or other arrangement from 4 of 2 microarraies on the microarray.The point sample positive control data that are positioned at each repeating part are carried out standardization, and standardized degree of accuracy is included in the significance of final each difference of determining.Also the fluorescence intensity of each point and the negative control in each repeating part can be compared, to determine which point has detectable remarkable expression in each sample.

Repeat to use a fluorescence intensity statistical analysis to every group, assessing degree of accuracy and the significance that each difference detects, the p value of gained is used for checking null hypothesis---promptly between each patient's tumor and normal specimens, there is not differential expression.Initial when analyzing microarray,, and be 1.000 to the diversity ratio calibration of those points if p＞10-3 then accepts hypothesis.All there is significant difference in other all some expression between tumor and normal specimens.Normal specimens does not have if tumor sample has detectable expression, and then ratio fixes on 1000, because the expression values in the normal specimens should be 0, and ratio can not be a mathematical virtual value (for example, infinity).Tumor sample does not have if normal specimens has detectable expression, then ratio is fixed on 0.001, because the expression values in the tumor sample should be 0, and ratio can not be a mathematical virtual value.These back two kinds of situations are referred to herein as " ON/OFF ".

Embodiment 3: the expression of TFF3 in the cell

The rise of TFF3 mRNA in the cancer

From each cancer patient's sample, gather the cancerous cell and the contiguous normal cell (source of related organization's sample is referring to embodiment 1) thereof of tumor sample with laser capture microdissection method.Prepare the probe of labelling from the RNA of each sample, and be used to detect cDNA micro-array chip (referring to embodiment 2).With each micro-array chip simultaneously with the normal and cancer probe hybridization of individual patients (normally and cancer probe with different fluorometric reagents labelling in addition).To each patient's sample, determined expression is expressed as the expression in the cancerous cell and the ratio of the expression in the normal cell.What table 3 was listed is at least 20% the patient who is detected, the expression ratio that cancerous cell and normal epithelium cell occur be higher than 2 times (＞2x), be higher than 5 times (＞5x) or be lower than half (＜0.5x) patient's percentage ratio.

Table 3: the rise of TFF3 mRNA in the cancer

Cancer	# patient	＞2x	＞5x	＜0.5x
					Prostate	102	～40％	～27％	～15％
Mammary gland	23	～40％	～27％	～30％
					Colon	77	～20％	～6％	～30％
Colon Mets	33	～22％	～2％	～20％

Normal structure is to the expression of TFF3

Determine the expression (for example, the sample of non-laser capture cutting with this represent all cells type of tissue sample) of TFF3 mRNA in full tissue in normal (N) and cancer (C) tissue with real-time quantitative PCR.The results are shown in Fig. 1, carry out standardization with the HPRT logarithm value in the full tissue.The numerical value of mRNA expression is the relative value who draws as the standardization contrast with HPRT on the Y-axis.The representative of cancer sample is from the tumor sample storehouse (source of relevant biomaterial is referring to embodiment 1) of 8 individual patients.Term " 3+3 " is meant Gleason rank 6, and " 4+3 " is meant Gleason rank 7.It is worthy of note that equally in normal lung tissue (the same with colon), TFF3 is expressed by goblet cell and secretes with mucus.(referring to, for example, Am.J.Respir.Crit.Care Med., 1999,159,1330).

The SABC of TFF3 (IHC) detects among the cancer patient

With the anti-human TFF 3 polyclonal antibody of the purified rabbit of immunoaffinity the tissue sample section (the total # of tissue) of breast carcinoma, colon cancer, ovarian cancer and carcinoma of prostate (CA) individual patients and normal structure contrast (NL) is dyeed.In addition, also the sample of several important normal organs is dyeed.Table 4 has been listed and has been existed situation to be shown as the sample number of feminine gender (-) and positive (+) to TFF3, and the percentage ratio of TFF3 positive in each type.

Table 4:IHC sums up

Types of organization	Organize total #	(-) sample	(+) sample	The % positive
					Mammary gland CA	77	21	56	72.7％
Mammary gland NL	20	15	5	25.0％
					Colon C A	45	16	29	64.4％
Colon NL	32	1	31	96.9％
					Ovary CA	31	25	6	19.4％
Ovary NL	25	25	0	0.0％
					Prostate CA	67	24	43	64.2％
Prostate NL
		18	17	1	5.6％
Adrenal gland NL						4	0	4	100.0％
	Brain NL	8	8	0	0.0％
Heart NL
		10	10	0	0.0％
Kidney NL						7	6	1	14.3％
	Liver NL	8	8	0	0.0％
Pancreas NL						7	7	0	0.0％

The expression of TFF3 in the cell line

Determine the expression of TFF3 mRNA in some cell line with real-time quantitative PCR.The results are shown in Fig. 2.The expression value is the relative value who obtains as the standardization contrast with beta-actin.Same attention, Y-axis is a logarithmic scale, the expression difference of expression TFF3 in different cell line is very big.Cell line comprises HUVEC (Human umbilical vein endothelial cells), BMEC-1 (brain micro blood vessel endothelium cell), Du145 (Human Prostate Cancer Cells), PC3 (Human Prostate Cancer Cells), LnCap (Human Prostate Cancer Cells), MDAPca2B (Human Prostate Cancer Cells), 22rV1 (Human Prostate Cancer Cells), HPV7 (Human Prostate Cancer Cells), HPV10 (Human Prostate Cancer Cells), RWPE-1 (human prostatic epithelial cell), RWPE-2 (pernicious human prostatic epithelial cell), PrEC (human prostatic epithelial cell), NIH 3T3 (fibroblast), HT29 (human colon carcinoma), SW620 (human colon carcinoma), Colo320 (human colon carcinoma), MDA231 (breast carcinoma), MDA435 (breast carcinoma), and MCF7 (breast carcinoma).

Embodiment 4: the antisense regulation and control of gene expression

By the gene of the differential expression of polynucleotide representative in the cancerous cell, it is expressed the available antisense technology of knocking out and analyzes, in determining that tumor takes place, and for example under the situation that promotes metastatic phenotype, the effect of gene outcome and function.

Can design a lot of different complementary oligonucleotide of mRNA that produce with difference expression gene as herein described,, and measure the ability of their inhibition of gene expression as antisense oligonucleotide.Use the polynucleotide sequence and the software program HYBsimulator the 4th edition that answer with the gene pairs of differential expression (to can be used for Windows95/Windows NT or Power Macintosh, RNAture, Inc.1003 Health Sciences Road, West, Irvine, CA 92612 U.S.) each candidate target is designed specific antisense oligonucleotide group.The factor of considering when the design antisense oligonucleotide comprises: the 1) secondary structure of oligonucleotide; 2) secondary structure of target gene; 3) specificity does not have or only has minimum crisscrossing with other expressing gene; 4) stability; 5) length and 6) terminal GC content.The design antisense oligonucleotide just can make them hybridize with its target sequence under the condition of physiological temp (for example, cultured cell provides the optimum temperature of hybridization in the cell, for example about 37 ℃) and highly strictness like this, and the homodimer that forms is minimum.

Use oligomer group and HYBsimulator program, design 3 to 10 antisense oligonucleotides and their reverse contrast thereof, and each candidate mRNA transcript synthesized, these transcripies from the corresponding gene of interested herbicide-tolerant polynucleotide sequence.In case synthetic and quantitative, in a series of cancerous cell lines, oligomer is screened, screen its efficient that knocks out to transcript.The level of using photoperiod meter (lightcycler) quantitative analysis mRNA is to determine to knock out efficient.Selecting transcript to knock out the highest oligomer of level is used in during proliferation test, on-fixed dependency growth test (anchorage independent growthassay) and apoptosis based on cell test, wherein the level of knocking out is at least about 50%, preferred about 80-90% is up to 95% or more up to can not detecting mRNA.

Can be by being transfected into LNCaP, PC3,22Rvl, MDA-PCA-2b, or the DU145 prostate gland cancer cell is measured the ability of the antisense oligonucleotide inhibition of gene expression of each design.For each transfection mixture, carrier molecule (as lipid, lipid derivant, lipoid molecule, cholesterol, cholesterol derivative or class cholesterol molecule) is prepared into the working concentration of 0.5mM in water, the solution of ultrasonic formation homogeneous, and filtered with the pvdf membrane of 0.45 μ m.In sterilization Millipore water, antisense or control oligonucleotide are prepared as the working concentration of 100 μ M then.Oligonucleotide is further used OptiMEMTM (Gibco/BRL) dilution, in microcentrifugal tube, be diluted to 2 μ M, or about 20 μ g oligonucleotide/ml OptiMEMTM.In another microcentrifugal tube, with carrier molecule, consumption is about 1.5-2nmol carrier/μ g antisense oligonucleotide normally, is diluted in and dilutes among the OptiMEMTM of the used equal volume of oligonucleotide.The antisense oligonucleotide of dilution is added to rapidly in the carrier of dilution, and pressure-vaccum mixes up and down.The oligonucleotide ultimate density that adds cell is 30nM.

Use Roche LightCycler PCR in real time instrument or PerkinElmer GeneAmp instrument that the level of the pairing target mRNA of interested target gene in the transfectional cell in the cancerous cell line is carried out quantitatively.Standardization is carried out in the value relative interior contrast (for example, beta-actin) of target mRNA.For the reaction system of each 20 μ l, the RNA that extracts (usually 0.2-1 μ g) is altogether placed in 0.5 or the 1.5ml microcentrifugal tube of sterilization, add water to cumulative volume and reach 12.5 μ l.Buffer/enzyme the mixed liquor that adds 7.5 μ l in every pipe, the preparation method of this mixed liquor is as follows, (in order) mix 2.5 μ l H2O, 2.0 μ l 10X reaction buffers, 10 μ l oligomerization dT (20pmol), 1.0 μ l dNTP mixture (every kind of 10mM), 0.5 μ l RNAsin (20u) (Ambion, Inc., Hialeah, FL) and 0.5 μ l MMLV reverse transcriptase (50u) (Ambion, Inc.).Pressure-vaccum to be mixing inclusions up and down, and under 42 ℃ of conditions incubation reaction mixed liquor 1 hour.The inclusions of each pipe is earlier centrifugal before amplification.

Be mixed with amplification mixture with following order: 1X PCR buffer II, 3mM MgCl2, every kind of dNTP of 140 μ M, 0.175pmol every kind of oligomer, 1: 50 of SYBR Green, 000 diluent, 0.25mg/ml BSA, 1 Taq of unit polymerase and H2O to 20 μ l.(concentration of PCR buffer II is 10X, available from Perkin-Elmer, and Norwalk, CT).In the concentration of 1X, it contains 10mM Tris pH 8.3 and 50mM KCl.(Molecular Probes, Eugene OR) are a kind of dyestuff that sends fluorescence when combining with double-stranded DNA to SYBR Green.In amplification procedure, produce double-stranded PCR product, promptly strengthen from the fluorescence of SYBR Green.For the amplification mixture of each 20 μ l amount, add the template RT of 2 μ l, and increase according to standard scheme.The result is expressed as with respect to non-transfected cells, only use carrier transfection (blank transfection) cell or with reverse control oligonucleotide cells transfected, the percentage ratio that corresponding gene product expression amount reduces.

The antisense regulation and control that embodiment 5:TFF3 expresses

Antisense oligonucleotide

Designed multiple antisense (AS) oligonucleotide according to the step of listing among the embodiment 4, prepared and measured them and in SW620 cell (colon carcinoma cell line of a kind of high level expression TFF3), regulated and control the ability of TFF3 mRNA level.Top table 1 has been listed oligonucleotide.Have the active antisense oligonucleotide of higher relatively reduction and comprise having SEQ ID NO:9,10,15,16,17 and 18 oligonucleotide.For wherein each, the oligonucleotide that has all synthesized reverse sequence is with comparing (RC) with expression antisense specificity.

Use AS to reduce TFF3 mRNA

The level that each oligonucleotide is transfected into the SW620 cell and used real-time quantitative PCR to detect residue TFF3 mRNA in about 36 hours after transfection is measured the effectiveness of TFF3 antisense oligonucleotide with this.

Fig. 3 has described the result who originally screens corresponding to the AS oligonucleotide gained of SEQ ID NO:9-18.AS oligonucleotide corresponding to SEQ ID NO:9 and 10 shows the most effective.Design in contrast 1 and contrast 2 AS oligonucleotide represent incoherent AS sequence, and with comparing the specificity reduction of assessing TFF3 mRNA.

Figure 4 and 5 have been described will be corresponding to SEQ ID NO:9, and 10 and 15 AS oligonucleotide and their reverse contrast (RC) are to the result who compares gained.As seen, the AS oligonucleotide demonstrates the specificity of expection.

Fig. 6 has further described the NO:10 corresponding to SEQ ID, and 15,17 and 18 AS oligonucleotide is reducing the effectiveness that TFF3 mRNA expresses.The SW620 of term " UT " expression untransfected.

Embodiment 6: express the effect to propagation

Can be in malignant galactophore cancerous cell line (MDA-MB-231 (" 231 ")); The SW620 colorectal cancer cell; SKOV3 cell (a kind of Proliferation of Human Ovarian Cell system); Or LNCaP, PC3,22Rvl, MDA-PCA-2b, or assess gene expression in the DU145 prostate gland cancer cell to suppressing the effect of cell proliferation.

Cell bed board in 96 orifice plates is converged (confluence) to about 60-80%.Antisense or reverse control oligonucleotide are diluted to 2 μ M in OptiMEMTM.Then, oligonucleotide-OptiMEMTM can be added and send carrier, the carrier that send of selection can reach optimum efficiency to the particular cell types of using in the test.Then, further with oligomer/send the carrier mixture diluted to advance in the culture medium that contains serum on the cell.The ultimate density that is used for the oligonucleotide of all experiments can be about 300nM.

With method for preparing antisense oligonucleotide (referring to embodiment 4 and 5).Transfectional cell spends the night under 37 ℃ of conditions, and second day morning, the reuse fresh culture was replaced transfection mixture.Implement transfection as above-mentioned embodiment 4 and 5 described methods.

Those genes that can suppress the antisense oligonucleotide correspondence of SW620 cell proliferation are working aspect the cancerous phenotype of the colon cell that produces and keep canceration.Those genes that can suppress the antisense oligonucleotide correspondence of SKOV3 cell proliferation are working aspect the cancerous phenotype of the mammary glandular cell that produces and keep canceration.Those genes that can suppress the antisense oligonucleotide correspondence of MDA-MB-231 cell proliferation are working aspect the cancerous phenotype of the gonad cell that produces and keep canceration.Those can suppress LNCaP, PC3, and 22Rvl, the gene of the antisense oligonucleotide correspondence of MDA-PCA-2b or DU145 cell proliferation is working aspect the cancerous phenotype of the prostatic cell that produces and keep canceration.

Embodiment 7: thus exhaust inducing that the death of polypeptide pair cell carries out by exhausting mRNA

In order to assess the effect that exhausts the death of target mRNA pair cell, can transfection LNCaP, PC3,22Rvl, MDA-PCA-2b or DU145 cell, or other cell that derives from cancer interested is used for proliferation test.Cytotoxic effect for existing under cisplatin (cis) situation uses identical scheme, and just cell is present in the culture medium that contains 2 μ M medicines.Every day, because damaging the quantity that is discharged into the LDH enzyme in the culture medium, cell membrane detects cytotoxicity by measuring.The cytotoxicity detection kit of using Roche Molecular Biochemicals to provide is measured the activity of LDH.The gained data are expressed as the LDH and the ratio (rLDH/tLDH) that is present in the total LDH in the hole that is discharged in the culture medium under identical time point and identical treatment conditions.Comprise the antisense that uses BCL2 (a known anti-apoptotic genes expression) and oppositely control oligonucleotide as positive control; With respect to control oligonucleotide (because background cytotoxicity that transfection causes), the loss of BCL2 mRNA causes the increase of cell death.

The cytotoxicity and the anti-proliferative effect of embodiment 8:TFF3 antisense oligonucleotide

Effect in cancerous cell

With the different time points after AS and RC oligonucleotide (the embodiment 4 and 5) transfection, in the SW620 cell, measure the cytotoxicity and the anti-proliferative effect of TFF3 antisense oligonucleotide according to the step of embodiment 6 and 7.The LDH (lactic acid dehydrogenase) that measurement is discharged in the culture medium determines cytotoxicity with the ratio of total LDH.LDH level in the complete attached cell is then represented the situation of breeding.The same specific Antisense OligodeoxynucleotideTransfection Transfection cell of Bcl-2 of using is as positive control.Bcl-2 is known anti-apoptotic proteins, blocks this expression of gene apoptosis is increased.The result is shown among Fig. 7.

Observation of cell toxic effect in the prostate gland cancer cell (Pca2B) of different condition of culture also.The results are shown among Fig. 8.Prostate gland cancer cell CU145 and 22Rvl are obtained similar effects.In PC3 and LNCaP cell, observe more weak effect.Be used as contrast with the known antisense oligonucleotide and the reverse sequence thereof that can suppress the TFF3 expression.

Normal cell

Measure the non-specific cell toxic effect of AS oligonucleotide in contrast with normal fibroblast of not expressing TFF3 (MRC9) and normal breast epithelial cell (184B5).The results are shown among Fig. 9.These results show that TFF3 AS has special effect to the cell of expressing TFF3, rather than because the overall toxicity that the AS oligonucleotide brings.

Embodiment 9: the influence of gene expression on cell migration

Can be at LNCaP, PC3,22Rvl, MDA-PCA-2b, or the inhibition effect of assessment gene expression on cell migration in the DU145 prostate gland cancer cell, use static endotheliocyte in conjunction with the endotheliocyte of test, non-static state in conjunction with test and migration test.

In conjunction with test, (referring to embodiment 4 and 5) as indicated above prepares antisense oligonucleotide for static endotheliocyte.Use a few days ago, with prostate gland cancer cell (CaP) bed board, and carry out transfection with antisense oligonucleotide with method mentioned above (referring to embodiment 4 and 5).Use the previous day, replace culture medium with fresh culture, using the same day, (Molecular Probes, fresh culture Inc.) is replaced culture medium, and incubated cell 30min with containing 2 μ M CellTracker green CMFDA.After hatching, replace CaP culture medium, incubated cell 30-60min again with fresh culture (not containing CMFDA).Use CMF PBS/2.5mM EDTA or trypsin to make cell take off wall, centrifugal and be suspended in again among the DMEM/1%BSA/10mMHEPES of pH 7.0.At last, the CaP cell is counted, and suspended again with the concentration of 1 * 106 cell/ml.

Use first three day, (EC) is taped against on 96 orifice plates with endotheliocyte, reaches 40-50% and converges (confluence).Use the same day, wash 1 time, and in each hole, add the DMDM/1%BSA/10mM HEPES of 50 λ pH 7 with PBS.Then, in each hole, add 50K (50 λ) CaP cell among the DMEM/1%BSA/10mMHEPES of pH 7.Hatch culture plate 30min again, and wash with the 5X PBS that contains Ca++ and Mg++.After the last washing, in each hole, add 100 μ L PBS, and read to read in the plate instrument fluorescence intensity (Ab492/Em 516nm) at fluorescence.

For non-static endotheliocyte in conjunction with test, preparation CaP as indicated above.Use first three day, EC is taped against on 24 orifice plates, reach 30-40% and converge (confluence).Use the same day, handled a part of cell 6 hours, reuse 2X PBS washing with cytokine.Then, in each hole, be added in the 150K-200K CaP cell of pH 7DMEM/1%BSA/10mM HEPES.Culture plate was placed on the shaking table (70RPM) 30 minutes, and reuse contains the 3X PBS washing of Ca++ and Mg++.After the last washing, in each hole, add 500 μ L PBS, and read to read in the plate instrument fluorescence intensity (Ab492/Em516nm) at fluorescence.

For shifting test, preparation CaP as indicated above has following change.Use the same day, (Molecular Probes, fresh culture Inc.) is replaced the CaP culture medium, and incubated cell 30min with containing 5 μ M CellTracker green CMFDA.After hatching, replace CaP culture medium, incubated cell 30-60min again with fresh culture (not containing CMFDA).Use CMF PBS/2.5mM EDTA or trypsin to make the CaP cell take off wall, centrifugal and be suspended in again in the EGM-2-MV culture medium.At last, the CaP cell is counted, and suspended again with the concentration of 1 * 106 cell/ml.

Used preceding 5-7 days, EC is taped against on the Fluorblok transwell, reach 30-40% and converge (confluence).Use first three day and use the same day, replace culture medium with fresh culture.Then, the CaP cell that in each transwell, adds the 50K labelling.30min before the fluorescence reading for the first time adds the FITC-glucosan (10K MW) of 10 μ g on the filter membrane that is covered with EC.Read to read in the plate instrument fluorescence intensity (Ab492/Em 516nm) at a plurality of time points at fluorescence then.

Those can suppress LNCaP, and the antisense oligonucleotide that PC3,22Rvl, MDA-PCA-2b or DU145 prostate gland cancer cell are attached to endotheliocyte shows that its corresponding gene may work aspect the cancerous phenotype of the prostatic cell that produces and keep canceration.Those can suppress LNCaP, and PC3,22Rvl, the antisense oligonucleotide of MDA-PCA-2b or DU145 migration of prostate cancer cells show that its corresponding gene may work aspect the cancerous phenotype of the prostatic cell that produces and keep canceration.

Embodiment 10: the effect that gene expression forms colony

Can in the soft agar test, measure gene expression, SKOV3 cell, MD-MBA-231 cell, LNCaP cell, PC3 cell, 22Rvl cell, the effect that the colony of MDA-PCA-2b cell and DU145 cell forms to the SW620 cell.The operational approach of soft agar test is as follows: at first, in several hours of cell shop layer, 0.6% agar of shop one deck 2ml is dissolved in the bottom of culture medium.Cellular layer is formed on the bottom, with method mentioned above use 0.05% trypsin and in culture medium washed twice to remove cells transfected from culture dish.Count at Coulter enumerator pair cell, and be suspended in again in the culture medium, concentration is every ml 106.The liquid of 10 μ l is added (to count with the WST1 check) in 96 orifice plates with culture medium, or further dilution is used for the soft agar test.2000 cells adding on 0.6% the bottom-layer agar in the agar of 800 μ 1 0.4% are provided with repeating hole.After cellular layer solidifies, drip the 2ml culture medium in the above, and do not use and transport carrier and add antisense or oppositely contrast oligomer.(with the preparation of method described in the embodiment 3) added fresh culture and oligomer in every 3-4 days.To 3 weeks, formed colony in 10 days.With the naked eye the colony zone is counted.The Wst-1 metabolic cost can be used for compensating the tiny difference of initiator cell number.Can scan bigger zone and write down vision difference.

The antisense oligonucleotide that those colonies that can suppress the SW620 cell form shows that its corresponding gene is working aspect the cancerous phenotype of the colon cell that produces and keep canceration.The antisense oligonucleotide that those colonies that can suppress the SKOV3 cell form shows that its corresponding gene is working aspect the cancerous phenotype of the mammary glandular cell that produces and keep canceration.The antisense oligonucleotide that those colonies that can suppress the MDA-MB-231 cell form shows that its corresponding gene is working aspect the cancerous phenotype of the gonad cell that produces and keep canceration.Those can suppress LNCaP, PC3, and 22Rvl, MDA-PCA-2b, or the antisense oligonucleotide that the colony of DU145 cell forms shows that its corresponding gene is working aspect the cancerous phenotype of the prostatic cell that produces and keep canceration.

Embodiment 11: antisense oligonucleotide is to the inhibition of the on-fixed dependency growth of cancerous cell

With TFF3 Antisense OligodeoxynucleotideTransfection Transfection prostate cancer cell line MDA Pca-2b (as shown in figure 10; The preparation of relevant antisense oligonucleotide is referring to embodiment 4 and 5) in 96 orifice plates, cultivate every hole 1000 or 500 cells (as shown in figure 10), and in containing the culture medium of soft agar suspension growth 7 days.Detect the growth of soft agar cell colony according to the Alamar indigo plant of cell absorption.The result shows that contrast AS (C79-7) and TFF3 AS oligonucleotide (but not being independent RC oligonucleotide) suppress the on-fixed dependency growth of prostate cancer cell line significantly.Prostate cancer cell line PC3 has also been obtained similar result.

Embodiment 12: the functional analysis of check product differential expression in the carcinoma of prostate among the patient

The gene outcome (as TFF3) that can further analyze difference expression gene sequence in the cancerous cell for example, promotes or suppresses the development of canceration phenotype to determine the effect and the function of gene outcome in tumor takes place.For example, can assess function with the corresponding gene outcome of this paper genes identified by the function of blocking gene product in cell.For example, gene outcome be secreting type or with situation that cell membrane combines under, can produce blocking antibody and add cell to detect the effect of its pair cell phenotype, be under the situation of cancerous tumor cell as cell transformation, the phenotype of canceration especially.In order to produce antibody, select clone, and obtain to represent the part or all of sequence of complete encoding sequence, and produce antibody corresponding to selected gene outcome.Then, antibody is combined with cell, and assess it tumorigenic effect.

Gene when the differential expression that this paper identified, when the albumen of its gene outcome and known function shows sequence homology (for example with specific kinases or protease) and/or when going out homology (for example containing the domain or other consensus sequence that are present in protease family or kinases family) with the protein family phenotype of known function, can use the micromolecule that suppresses or strengthen corresponding albumen or protein family function detect this gene outcome in tumor takes place effect and the activity of this gene outcome.

Other function test includes, but not limited to those tests of analyzing corresponding gene expression cell cycle and cell migration effect.The method of implementing these tests is known in the art.

Embodiment 13: contig comparison (contig assembly) and other gene characterize

The sequence of polynucleotide provided by the invention (for example TFF3) can be used for expanding the pairing gene order information of polynucleotide (for example, have gene of polynucleotide sequence described herein or by the mRNA of gene code).The sequence information of this expansion can be used for further characterizing pairing gene conversely, and this provides the extraneous information (for example, the normal function of gene outcome) of correlation gene product essence again conversely.These extraneous informations can provide extra evidence for gene outcome is used as the treatment target position again, and provide further guidance for regulating and control its active preparation type.

In one embodiment, use a sequence that is present in the polynucleotide of the present invention among the clone to carry out the contig comparison." contig " is the continuous sequence of the nucleotide of comparison gained the nucleotide sequence of eclipsed from having (for example total or similar substantially) sequence information.The synthetic public ESTs that gets of Chiron (expressed sequence labelling) sequence can be used in the contig comparison with the sequence that comes from the various clones in a plurality of cDNA library.

Use software program Sequencher, 4.05 editions, carry out the contig comparison according to manufacturer's guidance, produced the overall comparison of contig ed sequence with this.Then, can obtain to derive from the common sequences of contig with the sequence information of gained in the contig comparison with the Sequencher program.Common sequences as in TeraBLASTN, search DGTI Double Twist Gene Index (Double Twist, Inc., Oakland, search sequence CA), the latter has comprised all EST and the nonredundancy sequence in the public database.

Compare and use homology to search software program by contig, sequence information provided herein can be increased the gene with polynucleotide sequence of the present invention definite or definite prediction at an easy rate.The information of gained can further be used for identifying the function of the gene outcome of polynucleotide correspondence described herein.Although for operation of the present invention is not necessary, can in regulating and control the design of gene therapy of its active and regulation and control cancerous phenotype (for example suppressing canceration, propagation or the like), targeting provide guidance for the evaluation of pairing gene.

Detect in the body of embodiment 14:TFF3 nertralizer

Can detect the anti-tumor activity of TFF3 nertralizer in animal model according to the clinical preceding appraisal procedure of drug candidate known in the art.Suitable animal model comprises TRAMP mice (available from NCI-Frederick Mouse Models of Human Cancer Consortium Repository or The Jackson Laboratory) that is used for carcinoma of prostate and the Min mice (available from The Jackson Laboratory) that is used for colon cancer.The animal model that is used for other cancer is known in the art.

Embodiment 15:TFF3 epi-position

Can identify the TFF3 linear epitope that is used for antibody recognition and generation with in the several different methods known in the art any.Some methods as embodiment comprise the antibody binding capacity of surveying the peptide that derives from the antigen aminoacid sequence.Can use BIACORE or ELISA method to assess in conjunction with situation.Other technology is included in plane solid support (" chip ") and goes up peptide library is exposed to antibody, and with any the detection in conjunction with situation in the employed several different methods in solid phase screening field.In addition, phage display can be used for screening peptide library to choose epi-position several after taking turns biological elimination (biopanning).Suitable antibody nertralizer according to the present invention can be discerned the epi-position of linearity or conformation, or its combination.

Following table 5 provides the TFF3 position that is accredited as linear epitope, and it is fit to the identification of anti-TFF3 antibody.

Table 5

The ECD name	Through localized aminoacid sequence position	Through localized epi-position position	Length	SEQ ID NO：	Sequence
						TFF3#
1	27-34	27-33	8-mer	20	AVPAKDRV
						TFF3#
1	27-34	28-34	8-mer	21	VPAKDRVD
						TFF3#
1	27-34	27-34	9-mer	22	AVPAKDRVD
						TFF3#
2	36-44	36-43	8-mer	23	GYPHVTPK
						TFF3#
2	36-44	37-44	8-mer	24	YPHVTPKE
						TFF3#
2	36-44	36-44	9-mer	25	GYPHVTPKE
						TFF3#3	63-71	63-70	8-mer	26	FKPLQEAE
TFF3#3	63-71	64-71	8-mer	27	KPLQEAEC
						TFF3#3	63-71	63-71	9-mer	28	FKPLQEAEC

Embodiment 16: the growth of the polyclonal antibody inhibition tumor cell of anti-TFF3

A. the production of polyclonal antibody

Anaesthetize New Zealand white rabbit and used the DNA expression vector of coding human TFF 3 to carry out immunity at the 0th day and the 28th day.Particularly, for each immunity, to the DNA expression vector of injection 0.6mg in every rabbit muscle, and the injection site is used gentle electric current momently, and muscle cell absorbs DNA in this position to stimulate.Then, gave the interior injection of rabbit muscle recombined human TFF3 albumen with booster immunization in every month, recombiant protein emulsifying in the MF-59 adjuvant or in incomplete Freund's adjuvant.Back 14 days blood sample collections of each immunity, the serum that is produced by blood sample with the ELISA test determination is with the titre of the antibody response determining reorganization human TFF 3 albumen is produced.Then, the method that adopts chromatography is by protein A post separation of serum component, thus in each sample the IgG purification antibody component.

The B.SW620 proliferation test

Can influence the survival of cancerous cell in order to measure rabbit polyclonal antibody, the serial dilutions that will take from the IgG purification s of immunize rabbit adds cancerous cell line (SW620), and Western hybridization shows these cancerous cell line secretions TFF3 albumen.Be used for this test, earlier the concentration with 600 cells/well adds the SW620 cell in 96 orifice plates, and cell is grown in the growth medium that contains 10% hyclone (FBS).(the 0th day) discards culture medium after 24 hours, adds then to contain 1%FBS and the variable concentrations fresh growth medium from the IgG of immunity or pre-immunize rabbit.The concentration of every kind of IgG is all measured in four repeating holes.At the 4th day, discard culture medium in the slave plate again, Xiang Kongzhong adds the part that contains 1%FBS and each IgG component.The 0th, 1,4,5,6 and 7 days, use " cell titer list solution cell proliferation test " (Promega) to measure the relative number of cell in each hole.

For IgG antibody from a kind of immunize rabbit (rabbit 707), the results are shown among Figure 11 of such test.This figure is compared the propagation quantity in the 7th day detected IgG of containing hole, the hole (before the immunity) of contained IgG being taken from rabbit before the immunity for the first time with take from several (" immunity day 156 ") of taking turns after the immunity and compare.Contain the propagation that the hole of " immunity day 156 " IgG demonstrates and significantly be lower than the hole of containing " before the immunity " IgG.

Although the present invention is described with reference to its concrete embodiment, those skilled in the art should be understood that and can make a lot of changes to it under the prerequisite that does not deviate from spirit of the present invention and scope, also can be replaced with equivalent.In addition, can make a lot of modifications to adapt to specific situation, material, material composition, process, program step or each step to purpose of the present invention, spirit and scope.All such modifications all within the scope of the appended claims.

Sequence table

＜110〉Chiron Corp (CHIRON CORPORATION)

M.J Jia Natapu (Janatpour, Mary, J.)

C. Reinhard (Reinhard, Christoph)

P. add the West Asia (Garcia, Pablo)

＜120〉as the trefoil factor 3 (TFF3) of target for anti-cancer therapy

<130>CHIR0003-500(19154.005)

<150>US 60/493,173

<151>2003-08-07

<150>US 60/498,438

<151>2003-08-28

<160>28

<170>PatentIn version 3.2

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

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Gly Cys Cys Phe Asp Ser Arg Ile Pro Gly Val Pro Trp Cys Phe Lys

50 55 60

Pro Leu Thr Arg Lys Thr Glu Cys Thr Phe

65 70

<210>2

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＜213〉homo sapiens (Homo sapiens)

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Met Leu Gly Leu Val Leu Ala Leu Leu Ser Ser Ser Ser Ala Glu Glu

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Tyr Val Gly Leu Ser Ala Asn Gln Cys Ala Val Pro Ala Lys Asp Arg

20 25 30

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

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Gly Cys Cys Phe Asp Ser Arg Ile Pro Gly Val Pro Trp Cys Phe Lys

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Pro Leu Gln Glu Ala Glu Cys Thr Phe

65 70

<210>3

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＜213〉homo sapiens (Homo sapiens)

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Met Ala Ala Arg Ala Leu Cys Met Leu Gly Leu Val Leu Ala Leu Leu

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Ala Val Pro Ala Lys Asp Arg Val Asp Cys Gly Tyr Pro His Val Thr

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＜213〉homo sapiens (Homo sapiens)

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Ile Pro Gly Val Pro Trp Cys Phe Lys Pro Leu Gln Glu Ala Glu Cys

115 120 125

Thr Phe

130

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＜213〉homo sapiens (Homo sapiens)

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cacccccaag gagtgcaaca accggggctg ctgctttgac tccaggatcc ctggagtgcc 180

ttggtgtttc aagcccctga ctaggaagac agaatgcacc ttctgaggca cctccagctg 240

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ctcagttttt ctgtcccttt gctcccggca agctttctgc tgaaagttca tatctggagc 360

ctgatgtctt aacgaataaa ggtcccatgc tccacccg 398

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＜213〉homo sapiens (Homo sapiens)

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caagcaaaca atccagagca gctgtgcaaa caacggtgca taaatgaggc ctcctggacc 180

atgaagcgag tcctgagctg cgtcccggag cccacggtgg tcatggctgc cagagcgctc 240

tgcatgctgg ggctggtcct ggccttgctg tcctccagct ctgctgagga gtacgtgggc 300

ctgtctgcaa accagtgtgc cgtgccagcc aaggacaggg tggactgcgg ctacccccat 360

gtcaccccca aggagtgcaa caaccggggc tgctgctttg actccaggat ccctggagtg 420

ccttggtgtt tcaagcccct gcaggaagca gaatgcacct tctgaggcac ctccagctgc 480

ccccggccgg gggatgcgag gctcggagca cccttgcccg gctgtgattg ctgccaggca 540

ctgttcatct cagcttttct gtccctttgc tcccggcaag cgcttctgct gaaagttcat 600

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gtgcctgaaa aaaaaaaaaa aaaaa 685

<210>7

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<212>DNA

＜213〉homo sapiens (Homo sapiens)

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gcaaaccagt gtgccgtgcc agccaaggac agggtggact gcggctaccc ccatgtcacc 180

cccaaggagt gcaacaaccg gggctgctgc tttgactcca ggatccctgg agtgccttgg 240

tgtttcaagc ccctgcagga agcagaatgc accttctgag gcacctccag ctgcccccgg 300

ccgggggatg cgaggctcgg agcacccttg cccggctgtg attgctgcca ggcactgttc 360

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agcctgatgt cttaacgaat aaaggtccca tgctccaccc taaaaaaaaa aaaaaaaaaa 480

aaaaaaaaaa a 491

<210>8

<211>432

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>8

cgctccccag tagaggaccc ggaaccagaa ctggaatccg cccttaccgc ttgctgccaa 60

aacagtgggg gctgaactga cctctcccct ttgggagaga aaaactgtct gggagcttga 120

caaaggcatg caggagagaa caggagcagc cacagccagg agggagagcc ttccccaagc 180

aaacaatcca gagcagctgt gcaaacaacg gtgcataaat gaggcctcct ggaccatgaa 240

gcgagtcctg agctgcgtcc cggagcccac ggtggtcatg gctgccagag cgctctgcat 300

gctggggctg gtcctggcct tgctgtcctc cagctctgct gaggagtacg tgggcctgtc 360

tgcaaaccag tgtgccgtgc cagccaagga cagggtggac tgcggctacc cccatgtcac 420

ccccaaggag tg 432

<210>9

<211>22

<212>DNA

＜213〉artificial sequence

<220>

＜223〉TFF3 antisense oligonucleotide

<400>9

tccttggctg gcacggcaca ct 22

<210>10

<211>23

<212>DNA

＜213〉artificial sequence

<220>

＜223〉TFF3 antisense oligonucleotide

<400>10

cgggagcaaa gggacagaaa agc 23

<210>11

<211>23

<212>DNA

＜213〉artificial sequence

<220>

＜223〉TFF3 antisense oligonucleotide

<400>11

gaagaactgt cctcgggtgg agc 23

<210>12

<211>25

<212>DNA

＜213〉artificial sequence

<220>

＜223〉TFF3 antisense oligonucleotide

<400>12

tcagaaagtc tcaggcacga agaac 25

<210>13

<211>25

<212>DNA

＜213〉artificial sequence

<220>

＜223〉TFF3 antisense oligonucleotide

<400>13

gcagcagaaa taaagcacaa cctca 25

<210>14

<211>25

<212>DNA

＜213〉artificial sequence

<220>

＜223〉TFF3 antisense oligonucleotide

<400>14

aacagtagcg agagtggttg tgaaa 25

<210>15

<211>22

<212>DNA

＜213〉artificial sequence

<220>

＜223〉TFF3 antisense oligonucleotide

<400>15

cggcacggca cactggtttg ca 22

<210>16

<211>25

<212>DNA

＜213〉artificial sequence

<220>

＜223〉TFF3 antisense oligonucleotide

<400>16

ggtgcattct gtcttcctag tcagg 25

<210>17

<211>25

<212>DNA

＜213〉artificial sequence

<220>

＜223〉TFF3 antisense oligonucleotide

<400>17

ggctccagat atgaactttc agcag 25

<210>18

<211>25

<212>DNA

＜213〉artificial sequence

<220>

＜223〉TFF3 antisense oligonucleotide

<400>18

ggtggagcat gggaccttta ttcgt 25

<210>19

<211>22

<212>DNA

＜213〉artificial sequence

<220>

＜223〉TFF3 antisense oligonucleotide

<400>19

tggcacggca cactggtttg ca 22

<210>20

<211>8

<212>PRT

＜213〉artificial sequence

<220>

＜223〉peptide of chemosynthesis

<400>20

Ala Val Pro Ala Lys Asp Arg Val

1 5

<210>21

<211>8

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＜213〉artificial sequence

<220>

＜223〉peptide of chemosynthesis

<400>21

Val Pro Ala Lys Asp Arg Val Asp

1 5

<210>22

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉peptide of chemosynthesis

<400>22

Ala Val Pro Ala Lys Asp Arg Val Asp

1 5

<210>23

<211>8

<212>PRT

＜213〉artificial sequence

<220>

＜223〉peptide of chemosynthesis

<400>23

Gly Tyr Pro His Val Thr Pro Lys

1 5

<210>24

<211>8

<212>PRT

＜213〉artificial sequence

<220>

＜223〉peptide of chemosynthesis

<400>24

Tyr Pro His Val Thr Pro Lys Glu

1 5

<210>25

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉peptide of chemosynthesis

<400>25

Gly Tyr Pro His Val Thr Pro Lys Glu

1 5

<210>26

<211>8

<212>PRT

＜213〉artificial sequence

<220>

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<400>26

Phe Lys Pro Leu Gln Glu Ala Glu

1 5

<210>27

<211>8

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＜213〉artificial sequence

<220>

＜223〉peptide of chemosynthesis

<400>27

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1 5

<210>28

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉peptide of chemosynthesis

<400>28

Phe Lys Pro Leu Gln Glu Ala Glu Cys

1 5

Claims (93)

1.TFF3 nertralizer preparation be used for the treatment of or the medicine of prophylaxis of cancer in purposes.

2. purposes as claimed in claim 1 is characterized in that, described TFF3 nertralizer comprises nucleic acid.

3. as each described purposes in claim 1 and 2, it is characterized in that described cancer is breast carcinoma, colon cancer, carcinoma of prostate, ovarian cancer or gastric cancer.

4. as each described purposes among the claim 1-3, it is characterized in that TFF3 is differential expression in the cell of described cancer.

5. purposes as claimed in claim 1 is characterized in that described nertralizer comprises antisense molecule.

6. purposes as claimed in claim 5 is characterized in that, described antisense molecule comprise or overlapping SEQ IDNO:5-19 in any one sequence.

7. purposes as claimed in claim 1 is characterized in that described nertralizer comprises the RNAi molecule.

8. purposes as claimed in claim 7 is characterized in that, described RNAi molecule comprises or be overlapping corresponding to any one sequence among the SEQ ID NO:5-19.

9. purposes as claimed in claim 1 is characterized in that, described TFF3 nertralizer comprises antibody, and described antibody specificity ground combines with TFF3.

10. purposes as claimed in claim 1 is characterized in that, described cancer is not colon cancer or carcinoma of prostate.

11.TFF3 nertralizer preparation be used for the treatment of or the medicine of prophylaxis of cancer in purposes, use is united in described medicine and conventional treatment of cancer.

12. purposes as claimed in claim 11 is characterized in that, the treatment of cancer of described routine is a chemotherapy.

13. purposes as claimed in claim 11 is characterized in that, the treatment of cancer of described routine is the hormone ablation.

14. purposes as claimed in claim 13 is characterized in that, described hormone is an androgen.

15.TFF3 the purposes in the medicine of nertralizer apoptosis in the preparation regulating cell.

16. purposes as claimed in claim 15 is characterized in that, described cell is mammiferous cell.

17. purposes as claimed in claim 15 is characterized in that, described cell is a cancerous cell.

18. purposes as claimed in claim 15 is characterized in that, described cell is the cell of mammary gland, prostate, colon, ovary or stomach.

19.TFF3 the purposes of nertralizer in preparing the medicine that suppresses tumor growth or reduce gross tumor volume.

20. method as claimed in claim 19 is characterized in that, described tumor comprises the TFF3 cell of differential expression therein.

21.TFF3 nertralizer is used for purposes in the medicine that regulating cell and TFF3 express relevant at least a physiological effect in preparation.

22. purposes as claimed in claim 21 is characterized in that, described physiological effect is to improve cell movement ability or anti-apoptosis.

23.TFF3 nertralizer is used for suppressing the purposes of the medicine of cell migration, adhesion or propagation in preparation.

24.TFF3 nertralizer is used for reducing the purposes of the medicine of cancerous cell infecting potential in preparation.

25.TFF3 nertralizer is used for purposes in the medicine that regulating cell TFF3 expresses in preparation.

26. a method that detects TFF3 in biological sample, described method comprises: described sample is contacted with the TFF3 nertralizer, and detect the combination between TFF3 in described nertralizer and the described sample.

27. method that in biological sample, detects the existence of cancer, described method comprises: described biological sample is contacted with the TFF3 nertralizer, and detect the sign of TFF3 differential expression in the described biological sample, wherein the sign of TFF3 differential expression has been indicated the existence of cancer.

28. method as claimed in claim 27 is characterized in that, described detection comprises the result of described contact and contrast is compared.

29. a method that detects the patient to TFF3 nertralizer sensitivity, described method comprises: detect the differential expression of TFF3 in described patient's cancer sample, wherein the sign of TFF3 differential expression has indicated this patient to described TFF3 nertralizer sensitivity.

30. method as claimed in claim 28 is characterized in that, the sign of the differential expression of described TFF3 is the rise of TFF3 in patient's cancer sample.

31. method as claimed in claim 28 is characterized in that, described patient's cancer sample comes from mammary gland, prostate, colon, ovary or gastric tissue.

32. the method for cancer progression in the assess patient, described method comprises: in the time of will putting the very first time among the patient when expression of TFF3 and second time point expression of TFF3 compare, wherein the expression ratio very first time of TFF3 increases the progress of having indicated described cancer during point to some extent during second time point.

33. method as claimed in claim 30 is characterized in that, the described TFF3 that has improved expresses the expression that has improved at least about 25%.

34. one kind is detected the method that the cancer metastasis risk increases among the patient, described method comprises: in the time of will putting the very first time among the patient when expression of TFF3 and second time point expression of TFF3 compare, wherein the expression of TFF3 increases to some extent during point with respect to the very first time and has indicated the described increase that shifts risk during second time point.

35., it is characterized in that described TFF3 nertralizer comprises detectable labelling as each described method among the claim 1-34.

36., it is characterized in that described TFF3 nertralizer comprises radioactive label as each described method among the claim 1-35.

37., it is characterized in that described TFF3 nertralizer comprises antibody, nucleic acid, antisense molecule or RNAi molecule as each described purposes among the claim 11-36.

38. an antisense molecule, the expression of described antisense molecule regulation and control TFF3.

39. the described antisense molecule of claim 38 is characterized in that, described antisense molecule comprise or overlapping SEQID NO:5-19 in arbitrary sequence.

40. the described antisense molecule of claim 38 is characterized in that, described antisense molecule comprises any sequence among the SEQ ID NO:5-19.

41. a RNAi molecule, the expression of described RNAi molecular regulation TFF3.

42. RNAi molecule as claimed in claim 41 is characterized in that, described RNAi molecule comprise or or overlapping SEQ ID NO:5-19 in arbitrary sequence.

43. a compositions, described compositions contain described antisense molecule of claim 38 and pharmaceutically acceptable carrier.

44. a compositions, described compositions contain described RNAi molecule of claim 43 and pharmaceutically acceptable carrier.

45. an isolating anti-TFF3 antibody is characterized in that, described antibody recognition is corresponding at least one zone of SEQ ID NO:20,21,22,23,24,25,26,27 or 28 TFF3 sequence.

46. antibody as claimed in claim 47 is characterized in that described antibody produces by the method that comprises following step:

A) synthetic antibody library on phage;

B) by this phage and compositions are contacted the screening of sample being carried out the storehouse, said composition comprises at least one zone corresponding to SEQ ID NO:20,21,22,23,24,25,26,27 or 28 TFF3 sequence;

C) separate and the bonded phage of described compositions, being characterized as it and having the ability that combines with at least one zone corresponding to SEQID NO:20,21,22,23,24,25,26,27 or 28 TFF3 sequence of this antibody wherein, its binding affinity is at least 10 ⁸1/; With

D) analyze this isolating phage to determine to be combined with at least one regional amino acid coding corresponding to SEQ ID NO:20,21,22,23,24,25,26,27 or 28 TFF3 sequence.

47. antibody as claimed in claim 45 is characterized in that, described antibody is monoclonal antibody antibody.

48. antibody as claimed in claim 45 is characterized in that, described antibody is polyclonal antibody antibody.

49. antibody as claimed in claim 45 is characterized in that, described antibody is chimeric antibody.

50. antibody as claimed in claim 45 is characterized in that, described antibody is the antibody of peopleization.

51. antibody as claimed in claim 45 is characterized in that, described antibody is single-chain antibody.

52. antibody as claimed in claim 45 is characterized in that, described antibody is the Fab fragment.

53. antibody as claimed in claim 45 is characterized in that, described antibody is through labelling.

54. antibody as claimed in claim 53 is characterized in that, described labelling is enzyme, radiosiotope or fluorophor.

55. antibody as claimed in claim 45 is characterized in that, described antibody is lower than about 1 * 10 to the binding affinity of the polypeptide except that TFF3 ⁵K _a

56. a cell that has separated, described cell is used to produce the described antibody of claim 45.

57. a hybridoma, described hybridoma are used to produce the described antibody of claim 45.

58. a compositions, described compositions comprise described anti-TFF3 antibody of claim 45 and pharmaceutically acceptable carrier.

59.TFF3 the purposes of nertralizer in the medicine of preparation treatment or prophylaxis of cancer is characterized in that described nertralizer is the described antibody of claim 45.

60.TFF3 the purposes of nertralizer in the medicine of preparation treatment or prophylaxis of cancer is characterized in that described medicine is united use with conventional treatment of cancer, wherein said TFF3 nertralizer is the described antibody of claim 45.

61. purposes as claimed in claim 60 is characterized in that, the treatment of cancer of described routine is a chemotherapy.

62. purposes as claimed in claim 60 is characterized in that, the treatment of cancer of described routine is the hormone ablation.

63. purposes as claimed in claim 62 is characterized in that, described hormone is an androgen.

64.TFF3 the purposes of nertralizer in the apoptosis-induced medicine of preparation is characterized in that described nertralizer is the described antibody of claim 45.

65., it is characterized in that described cell is a mammalian cell as the described method of claim 64.

66., it is characterized in that described cell is a cancerous cell as the described purposes of claim 64.

67.TFF3 nertralizer is used for reducing gross tumor volume in preparation, prevents the purposes of the medicine of tumor growth or inhibition tumor growth, it is characterized in that described TFF3 nertralizer is the described antibody of claim 45.

68., it is characterized in that described tumor comprises the TFF3 cell of differential expression therein as the described purposes of claim 67.

69.TFF3 nertralizer is used for purposes in the medicine that regulating cell and TFF3 express relevant at least a physiological effect in preparation, it is characterized in that described TFF3 nertralizer is the described antibody of claim 45.

70., it is characterized in that described physiological effect is to improve cell movement ability or anti-apoptosis as the described purposes of claim 69.

71.TFF3 nertralizer is used for suppressing the purposes of the medicine of cell migration, adhesion or propagation in preparation, it is characterized in that described TFF3 nertralizer is the described antibody of claim 45.

72.TFF3 nertralizer is used for reducing the purposes of the medicine of cancerous cell infecting potential in preparation, it is characterized in that described TFF3 nertralizer is the described antibody of claim 45.

73. a method that detects TFF3 in biological sample, described method comprises: the described antibody of described sample and claim 45 is contacted, and detect the combination between TFF3 in described nertralizer and the described sample.

74., it is characterized in that described TFF3 nertralizer comprises detectable labelling as the described method of claim 73.

75. method that in biological sample, detects the existence of cancer, described method comprises: the described antibody of described biological sample and claim 45 is contacted, and detect the sign of TFF3 differential expression in the described biological sample, wherein the sign of TFF3 differential expression has been indicated the existence of cancer.

76., it is characterized in that described detection comprises the result of described contact and contrast are compared as the described method of claim 75.

77., it is characterized in that described TFF3 nertralizer comprises detectable labelling as the described method of claim 75.

78. an isolating polypeptide, described polypeptide comprise three or still less be selected from SEQ ID NO:20,21,22,23,24,25,26,27 or 28 aminoacid sequence.

79., it is characterized in that the length of described polypeptide is about 80 aminoacid of about 8-as the described peptide of claim 78.

80., it is characterized in that described polypeptid specificity ground and anti-TFF3 antibodies as the described peptide of claim 78.

81. a method of using TFF3 differential expression in the antibody test sample, described method comprises:

A) allowing antibody: under the condition that the TFF3 complex forms, the described antibody of claim 45 is combined with described sample;

B) amount of the described complex of mensuration; With

C) with the amount of described complex with compare the differential expression of the raising of complex level indication TFF3 in the wherein said sample.

82. the epi-position of the polypeptide of an isolating SEQ ID NO:1-4 is carried fragment.

83. carry fragment as the described epi-position of claim 82, described fragment comprises about 20 continuous amino acids of about 6-among the SEQ ID NO:1-4.

84. carry fragment as the described epi-position of claim 83, described fragment comprises about 10 continuous amino acids among the SEQ ID NO:1-4.

85. carry fragment as the described epi-position of claim 82, described fragment comprises about 20 continuous amino acids of about 6-among the SEQ ID NO:2.

86. carry fragment as the described epi-position of claim 83, described fragment comprises about 10 continuous amino acids among the SEQ ID NO:2.

87. carry fragment as the described epi-position of claim 83, described fragment comprises SEQ ID NO:20,21,22,23,24,25,26,27 or 28.

88. an isolating anti-TFF3 antibody, described antibody obtains by carry a certain object of fragment immunity with the described epi-position of claim 83.

89. a pharmaceutical composition, described compositions comprise described antibody of claim 45 and pharmaceutically acceptable carrier.

90. as the described pharmaceutical composition of claim 89, it is characterized in that, in the described antibody and TFF3.

91. a generation is used for the treatment of the method for the antibody of cancer, described method comprises: identification can in conjunction with and in and the antibody of TFF3, and in recombinant expressed host cell, express this antibody.

92. a pharmaceutical composition, described compositions comprise in the combination also and antibody and the pharmaceutically acceptable carrier of TFF3, it is characterized in that, described antibody is to use recombinant host cell to produce.

93., it is characterized in that described recombinant host cell is selected from down group as the described pharmaceutical composition of claim 92: Chinese hamster ovary cell, myeloma cell and bacterial host cell.

Images