CN1722953A - Methods for treating cancer by inhibiting Wnt signaling - Google Patents

Abstract

This invention relates to methods of inhibiting the growth of cancer cells that overexpress a Wnt protein. The methods comprise contacting the cell with an agent that inhibits binding of the Wnt protein to a Frizzled receptor.

Description

By suppressing Wnt signal transduction treatment method for cancer

Cross reference to related application

The application requires the U.S. Provisional Application no.60/491 of submission on July 31st, 2003, the priority of the U.S. Provisional Application no.____ that submitted on October 4th, 350 and 2002 (changeing from non-provisional application 10/264,825).Each application is merged in this paper for your guidance.

Invention field

The present invention relates to suppress express the method for the growth of cancer cells of Wnt albumen.Described method comprises described cell is contacted with the reagent of Frizzled acceptor (Frizzled receptor) combination with a kind of inhibition Wnt albumen.

Background of invention

Aptery type (Wnt) Frizzled protein receptor approach comprises the important controlling gene that carries with the relevant polymorphism of elementary tumour.In downstream signal transduction process, the accumulation of kytoplasm beta-catenin is transferred to cell nucleus, and subsequently by forming compound reinforcing gene expression (Uthoff etc., Mol Carcinog, 31:56-62 (2001)) with other transcription factors.Under the situation that lacks the Wnt signal, free beta-catenin is combined in the compound that comprises an albumen, adenomatous polyp of colon (APC) gene outcome and glycogen synthase kinase (GSK)-3 β.Axle albumen, APC and beta-catenin by GSK-3 β the time phosphorylation cause beta-catenin to enter the ubiquitin approach and by proteasome degraded (Uthoff etc., MolCarcinog, 31:56-62 (2001); Matsuzawa etc., Mol Cell, 7:915-926 (2001)).

Disheveled (Dvl) is just adjusting that is positioned at the Wnt signal transduction of Frizzled acceptor downstream, beta-catenin upstream.A plurality of albumen in the GSK-3 β phosphorylation Wnt approach also play a role in the downstream of beta-catenin is regulated.The apc gene sudden change is the initiation event that colorectal carcinoma accidental and heredity takes place.Because described paraprotein is the part of Wnt signal transduction cascade reaction, the APC mutant takes place relevant with tumour.This albumen comprises the functional domain of a plurality of conducts at β connection protein combination and degradation site.The sudden change that occurs on the β connection albumen n terminal fragment often relates to the degraded that phosphorylation relies on, that ubiquitin mediates and thus β is joined the stable of albumen.Under the situation of stable kytoplasm connection protein accumulation, this albumen is transferred to cell nucleus, and interacts with the high animal migration transcription factor combination of Tcf/Lef of regulation and control as the expression of oncogene such as c-myc there.

Known in the various kinds of cell type, Wnt/ beta-catenin signal transduction promotes cells survival (Orford etc., J CellBiol, 146:855-868 (1999); Cox etc., Genetics, 155:1725-1740 (2000); Reya etc., Immunity, 13:15-24 (2000); Satoh etc., Nat Genet, 24:245-250 (2000); Shin etc., Journal of BiologicalChemistry, 274:2780-2785 (1999); Chen etc., J Cell Biol, 152:87-96 (2001); Ioannidis etc., Nat Immunol, 2:691-697 (2001)).The Wnt signal pathway also is considered to development and/or progress relevant (Polakis etc., Genes Dev, the 14:1837-1851 (2000) with tumour; Cox etc., Genetics, 155:1725-1740 (2000); Bienz etc., Cell, 103:311-320 (2000); You etc., J Cell Biol, 157:429-440 (2002)).The Wnt signal pathway is relevant with expression excessively or the amplification of c-Myc, the abnormal activation of this approach relevant (Polakis etc., Genes Dev, 14:1837-1851 (2000) with multiple human cancer; Bienz etc., Cell, 103:311-320 (2000); Brown etc., Breast Cancer Res, 3:351-355 (2001); He etc., Science, 281:1509-1512 (1998); Miller etc., Oncogene, 18:7860-7872 (1999)).In addition, c-Myc is differentiated and is transcribed one of target gene (He etc., Science, 281:1509-1512 (1998) for beta-catenin/Tcf in the colorectal cancer cell; De La Coste etc., Proc Natl Acad Sci USA, 95:8847-8851 (1998); Miller etc., Oncogene, 18:7860-7872 (1999); You etc., J Cell Biol, 157:429-440 (2002)).

Except the Wnt part, secreting type Frizzled associated protein (secreted Frizzled-relatedproteins, sFRP) family have been separated.SFRP can by with stride film Frizzled receptor competition and combine secreting type Wnt part as the soluble endogenous regulator of Wnt signal transduction play a role (Melkonyan etc., Proc Natl Acad SciUSA, 94:13636-13641 (1997)).SFRP can be by in conjunction with Wnt albumen and hinder it near cell surface signal transduction receptor antagonist Wnt function, maybe can be by promoting that offer part to the Frizzled acceptor strengthens Wnt activity (Uthoff etc., Mol Carcinog, 31:56-62 (2001)).Find that albumen that another kind is called Dickkopf (Dkk) also disturbs the Wnt signal transduction and reduces accumulation (Fedi etc., J Biol Chem, the 274:19465-19472 (1999) of kytoplasm beta-catenin; Moon etc., Cell, 88:725-728 (1997)).Dkk-1 by with the signal (Nusse etc., Nature, 411:255-256 (2001)) that antagonism Wnt induces that combines of the ldl receptor associated protein 6 (LRP6) of contiguous Frizzled acceptor.Discoveries such as nearest H.Suzuki, sFRP is excessively methylated with high-frequency in colorectal cancer cell system, and this be associated with the shortage of sFRP primary expression (Suzuki etc., Nat Genet, 31:141-149 (2002)) that excessively methylate.The expression of crossing of Dkk-1 also is found the apoptosis (Shou etc., Oncogene, 21:878-889 (2002)) that can promote the brain tumor cell.

Although in understanding, get the Wnt signal transduction up-to-date progress, the effect of this signal pathway in tumour forms is still unclear.Thereby technology formerly can't provide the compound of this signal pathway of regulation and control to can be used in the clear and definite evidence of treatment of cancer.The present invention is devoted to address these problems and other demands.

Summary of the invention

The invention provides the method for the growth of cancer cells that suppressed to express Wnt albumen.Described method comprises described cell is contacted with the reagent of Frizzled receptors bind with a kind of Wnt of inhibition albumen.

In some embodiment, described reagent is a kind of antibody.For example, described antibody can specificity in conjunction with Wnt albumen, Wnt-1 or Wnt-2.In other embodiments, described antibody specificity is in conjunction with a kind of Frizzled acceptor, as Frizzled1, Frizzled2, Frizzled3, Frizzled4, Frizzled5, Frizzled6, Frizzled7, Frizzled8, Frizzled9, Frizzled10 acceptor.

Antibody of the present invention can be monoclone antibody and can and modify with the several different methods preparation.For example, described antibody can produce by recombination form.In some embodiment, described antibody is humanized antibody or strand Fv fragment (scFv).

The present invention also provides processing tumor treatment method.In these embodiments, described cancer cell is positioned at patient's body and contact procedure is undertaken by giving the described reagent of this patient.Described method may further include and gives patient such as chemotherapeutics or radiocurable second kind of treatment reagent.Described cancer cell can be breast cancer cell, colorectal cancer cell, lung carcinoma cell, sarcoma cell or celiothelioma cell, prostate gland cancer cell, pancreatic cancer cell, cervical cancer cell, ovarian cancer cell, stomach cancer cell, esophageal cancer cell, head and neck cancer cell, hepatoma cells, melanoma cells, neuroglial cytoma or spongioblast oncocyte.

The present invention also provides and has comprised pharmaceutically acceptable excipient and the specificity pharmaceutical composition in conjunction with the monoclone antibody of Wnt or Frizzled albumen (for example Wnt1 albumen).Described antibody can be further with such as effect component couplings such as mark, radioisotope or cell toxicant chemicals.

In yet another aspect, the invention provides a kind of reagent and carry out method for screening anticancer propagation, this method comprises described reagent is contacted with Dvl albumen or nucleic acid, measure Dvl protein active or expression, and differentiate the compound that suppresses Dvl protein active or expression, thereby differentiate the reagent of anticancer propagation.Described method may further include a kind of compound through differentiating is contacted with cancer cell, and selects the compound that suppresses described cancer cell multiplication.In some embodiment, described cancer cell is lung carcinoma cell or celiothelioma cell.

The present invention also provides a kind of method that suppressed to express the growth of cancer cells of Dvl albumen, this method comprise with described cell with suppress that Dvl expresses or the reagent of activity contacts.In some embodiment, described cancer cell is lung carcinoma cell or celiothelioma cell.Described reagent can be for example micromolecular compound or siRNA.

Definition

Term " Wnt albumen " or " Wnt part " are meant and the relevant mammalian proteins family of fruit bat segment polarity gene (aptery type).In the mankind, Wnt family gene usually coding has the hydrophobic signal sequence and the glycoprotein that is rich in cysteine of the molecular weight 38-43kDa of the oligosaccharides consensus sequence that the asparagine guarded is connected (for example can be referring to Shimizu etc., Cell Growth Differ 8:1349-1358 (1997)).Wnt family comprises at least 16 mammal members.Exemplary Wnt albumen comprises Wnt-1, Wnt-2, Wnt-3, Wnt-3A, Wnt-4, Wnt-5A, Wnt-6, Wnt-7A, Wnt-7B, Wnt-8A, Wnt-8B, Wnt-10B, Wnt-11, Wnt-13, Wnt14, Wnt15 and Wnt16.Exemplary Wnt protein sequences more of the present invention are set forth in the sequence list.In addition, the expression of crossing of specific Wnt albumen has been shown relevant with some cancer.For example, WNT-2 crosses expression (Katoh etc., Int J Oncol, 19:1003-1007 (2001)) in cancer of the stomach and colorectal cancer; Wnt-1 crosses expression in head and neck cancer, and WNT-5A and Wnt-8B cross expression (Saitoh etc., Int J Mol Med, 9:515-519 (2002) in cancer of the stomach; Saitoh etc., Int J Oncol, 20:343-348 (2002)).

Term " Frizzled albumen " or " Frizzled acceptor " are meant and the relevant mammalian proteins family of fruit bat Frizzled gene (playing a role in the development of organizing polarity).The Frizzled protein family comprises at least 10 mammalian genes.Exemplary human Frizzled acceptor comprises Frizzled1, Frizzled2, Frizzled3, Frizzled4, Frizzled5, Frizzled6, Frizzled7, Frizzled8, Frizzled9 and Frizzled10.The sequence of exemplary Frizzled acceptor is set forth in the sequence list.The mammal autoploid of fruit bat Frizzled albumen has a plurality of common structure motifs.Be positioned at behind the N end on epicyte surface is that a burst, one contain domain and various zone of height that is made of the hydrophilic amino acid of 40-100 height change that 120 amino acid of the constant style of 10 cysteine residues constitute.The hydrophobic fragment of supposing forms 7 transmembrane helix structures that connected by hydrophilic loop, finishes so that the C that is positioned at the cell membrane inboard is terminal.Described domain (CRDs) and the transmembrane segment that is rich in cysteine is high conservative, and this has illustrated the mode of operation that extracellular CRD wherein links to each other with a branch of 7 transmembrane helix structures by variable join domain.Therefore, the Frizzled protein receptor with aminoterminal ligand binding domains has the kinetic model of the transmembrane signal transduction that is similar to the G-G-protein linked receptor.Frizzled1, Frizzled2 and Frizzled7 be (Sagara etc., Commun, 252:117-122 (1998)) in lung cancer and colorectal cancer; Frizzled3 is (Kirikoshi etc., Biochem Biophys Res Commun, 271:8-14 (2000)) in the human cancer cell who comprises lung cancer, cervical carcinoma and colorectal cancer; Frizzled7 is (Kirikoshi etc., Int J Oncol, 19:111-115 (2001)) in cancer of the stomach; Frizzled10 is (Kirikoshi etc., Int J Oncol, 19:767-771 (2001) in cancer of the stomach and colorectal cancer; Terasaki etc., Int J Mol Med, 9:107-112 (2002)).

Except the Wnt part, separated secreting type Frizzled associated protein (sFRP) family.SFRP can by with stride film Frizzled receptor competition and combine secreting type Wnt part and play a role as the soluble endogenous regulator of Wnt signal transduction.Therefore, sFRP brings into play antagonism to apoptosis, regulates the susceptibility of apoptosis.SFRP can be by in conjunction with Wnt albumen and hinder it near cell surface signal transduction receptor antagonist Wnt function, maybe can be by promoting that offer part to the Frizzled acceptor strengthens the Wnt activity.Up to the present, sFRP does not still interrelate with the tumour cause.

Term " Dishevelled " or " Dvl " are meant Dishevelled protein family member, its full length sequence contains 3 conserved domains usually: a DIX domain that is present on the Wnt antagonist protein axle albumen, one participates in interactional PDZ domain and a DEP domain of finding between the albumen in the albumen of regulating Rho GTP enzyme.Dvl albumen comprises, for example Dvl-1, Dvl-2 and Dvl-3.Comprise that mouse and nucleic acid and the protein sequence of the Dvl of a plurality of species of the mankind are known.Exemplary human Dvl-1, Dvl-2 and Dvl-3 protein sequence can obtain by canonical sequence NP_004412, NP_004413 and NM_004414 respectively.

Wnt signal transduction " inhibitor " for example be meant with Wnt or Frizzled protein combination or in the detection method of known Wnt signal transduction (for example, beta-catenin horizontal survey, or by the expression of the oncogene of Tcf and Lef transcription factor regulation and control) records the compound that partially or completely hinders the Wnt signal transduction.Inhibitor comprises the Wnt or the Frizzled albumen of modified forms, and natural or synthetic part, antagonist, activator, antibody, micromolecular compound or the like.More detailed description has been made to the method that detects inhibitor of the present invention in the back.

The cancer cell of Wnt albumen " cross express " is that wherein a kind of expression of specific Wnt albumen is 2 times from expression in the normal cell of homologue at least, often is at least 5 times cancer cell.The method of measuring concrete gene expression dose is known in this area.Such method comprises RT-PCR, uses described gene outcome antibody or the like.

Here employed " antibody " relates to and concrete antigen has immunoreactive immunoglobulin molecules, comprises polyclone and monoclone antibody.This term also comprises the antibody such as chimeric antibody (as the humanization mouse antibodies) and hybrid antibody genetic engineering forms such as (as bispecific antibodies).Term " antibody " also comprises the antigen combining form of antibody, and this comprises fragment with antigen binding capacity (for example Fab ', F (ab ') ₂, Fab, Fv and rIgG, also can be referring to Pierce company (Pierce Chemical Co., Rockford, catalogue IL) and handbook, 1994-1995; Also can be referring to Kuby, J. shows " immunology " the 3rd edition (Immunology, W.H.Freeman ﹠amp; Co., New York (1998)).This term also comprises recombinant single chain Fv fragment (scFv).Term " antibody " also comprises divalence or bispecific molecule, bifunctional antibody, three function antibodies and four function antibodies.For example at Kostelny etc., (1992) J Immunol 148:1547; Pack and Pluckthun (1992) Biochemistry 31:1579; Hollinger etc., 1993; Gruber etc., (1994) J Immunol:5368; Zhu etc., (1997) Protein Sci 6:781, Hu etc., (1996) Cancer Res.56:3055; Adams etc., (1993) Cancer Res.53:4026; And McCartney etc., among (1995) Protein Eng.8:301 divalence and bispecific molecule are described.

Having immunoreactive antibody with concrete antigen can be by (can be referring to Huse etc., Science 246:1275-1281 (1989) such as the recombination methods such as recombinant antibodies library screening in phage or similar substrates; Ward etc., Nature 341:544-546 (1989); And Vaughan etc., Nature Biotech.14:309-314 (1996)), or by the dna immunization animal generation with antigen or coding for antigens.

Usually, immunoglobulin has heavy chain and light chain.Every heavy chain and light chain comprise constant region and variable region (these zones are also referred to as " domain ").Light chain and variable region of heavy chain comprise 4 " skeleton districts ", have inserted 3 hypervariable regions (being also referred to as " complementary determining region " or " CDR ") therebetween.Determined the scope of skeleton district and CDR.The skeleton region sequence of different light chain or heavy chain is conservative relatively in of the same race.Constitute the light chain of antibody and heavy chain the skeleton district in conjunction with the skeleton district that forms antibody, this skeleton district is for location in three dimensions and arrange CDR and provide support.

CDR mainly is responsible for combining with epitope.The CDR of every chain often is meant CDR1, CDR2 and CDR3, in order from the terminal open numbering of N, and is also differentiated by the chain that concrete CDR was positioned at usually.Like this, V _HCDR3 is positioned on the variable region of heavy chain of its place antibody, and V _LCDR1 is the CDR1 from the light chain variable domain of its place antibody.

" the V that mentions _H" or " VH " be meant the variable region of the heavy chain immunoglobulin of antibody, this comprises the heavy chain of Fv, scFv or Fab." the V that mentions _L" or " VL " be meant the variable region of light chain immunoglobulin, this comprises the light chain of Fv, scFv, dsFv or Fab.

Term " strand Fv " or " scFv " are meant a kind of like this antibody, and wherein the light chain of traditional double chain antibody and variable region of heavy chain have been connected to form strand.Usually, between described two chains, insert connection peptides, to make it the correct active binding site that folds and set up.

" chimeric antibody " is a kind of like this immunoglobulin molecules, wherein (a) changes, replaces or exchange constant region or its part, thereby constant regions antigen binding site (variable region) and different or the kind that changes, effector function and/or species, or the diverse molecule (for example enzyme, toxin, hormone, growth factor, medicine or the like) of giving the brand-new function of chimeric antibody is connected; Or (b) with variable region change, replacement or exchange variable region or its part with antigentic specificity different or that change.

" humanized antibody " is to contain the minimum immunoglobulin molecules that is derived from the non-human immunoglobulin sequences.Humanized antibody comprises such human immunoglobulin (receptor antibody), is wherein replaced from the residue such as the CDR of non-human kinds (donor antibody) such as mouse, rat or rabbit with required specificity, affinity and ability from the residue of the complementary determining region (CDR) of receptor antibody.In some cases, the Fv skeleton district residue of human immunoglobulin is replaced by corresponding inhuman source residue.Humanized antibody also can comprise neither be present on the receptor antibody, be not present in the CDR of introducing or the residue in the frame sequence yet.Usually, humanized antibody should comprise in fact at least one, two complete variable domains normally, wherein complete or complete in fact CDR zone is corresponding to the CDR zone of non-human immunoglobulin, and complete or complete in fact skeleton (FR) district is the skeleton district of human immunoglobulin(HIg) consensus sequence.Humanized antibody preferably also should comprise normally at least a portion of human immunoglobulin(HIg) constant region (Fc) ((Jones etc., Nature 321:522-525 (1986); Riechmann etc., Nature 332:323-329 (1988); And Presta, Curr.Op.Struct.Biol.2:593-596 (1992)).Humanization can carry out (Jones etc., Nature 321:522-525 (1986) by the corresponding sequence that replaces people's antibody with rodentine CDR or CDR sequence according to Winter and colleague's thereof method basically; Riechmann etc., Nature 332:323-327 (1988); Verhoeyen etc., Science 239:1534-1536 (1988)).Thereby such humanized antibody is chimeric antibody (U.S. Patent No. 4,816,567), and the variable domains that wherein is less than a complete people is in fact replaced by the corresponding sequence from the non-human kind.

" epi-position " or " epitope " is meant the position of binding antibody on the antigen.Epi-position can be folding and put and form by by albumen three grades of the amino acid of the amino acid of adjacency or non-adjacency.The epi-position that constitutes by the amino acid of adjacency usually with kept under the sex change solvent contacts, and under the sex change solvent is handled, can lose usually by three grades of epi-positions that are folded to form.Epi-position typically comprises at least 3 in the space conformation of a uniqueness, and more usually at least 5 or 8-10 amino acid.The method of measuring the epi-position space conformation comprises that for example, X ray crystallography and 2 is tieed up nuclear magnetic resonnance." the epitope mapping method in the molecular biology method " the 66th volume (Epitope Mapping Protocols in Methods in Molecular Biology, 1996) referring to for example Glenn E.Morris chief editor).

Here employed " biological sample " is to comprise nucleic acid or polypeptide (for example nucleic acid of Wnt albumen or polypeptide), polynucleotides or the biological tissue of transcript or the sample of body fluid.Such sample includes but not limited to separate the tissue from primate (for example people) or rodent (for example mouse and rat).Biological sample also can comprise histologic section, such as slicer and cut open inspection section sample, freezing microtome section of obtaining for the histology purpose, blood, blood plasma, serum, saliva, ight soil, tears, mucus, hair, skin or the like.Biological sample also comprises the graft that is derived from patient tissue and elementary and/or cell transformed culture.Biological sample typically derives from the eucaryon organism, most preferably mammals such as primate (for example chimpanzee or people), ox, dog, cat, rodent (for example cavy, rat, mouse), rabbit freely; Or birds; Reptile; Or fish.

" provide a kind of biological sample " and mean a kind of biological sample that is used for the method for the invention of acquisition.The most common ground, this can finish by take out cell sample from animal, but also can by use prior isolated cells (for example, by other people, other the time, with and/or be other purpose isolated cells) or be accomplished by carrying out method of the present invention in vivo.Tissue with file of treatment or treatment results history can be to be particularly useful.

In the context of two or more nucleic acid or peptide sequence, term " identical " or " identical " percentage be meant as the BLAST that uses following default parameters or BLAST2.0 sequence comparison operation rule or by manual alignment arrange and range estimation (referring to, the website of NCBI for example Http:// www.ncbi.nlm.nih.gov/BLAST/Or other network address) record, two or more sequences or subsequence are identical or (for example have the amino acid residue of concrete homogeneity percentage or nucleotide, when doing maximum when mutual comparison and between a contrast window or designation area to response alignment arrangement, about 60% is identical on a concrete zone, preferred 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher homogeneity).Such sequence is called as " much at one " then.This definition also relates to, and maybe can be used for, and treats the evaluation of order-checking row.This definition also comprises having disappearance and/or add, and those have the sequence of displacement, and natural (for example polymorphism or allele variant) and artificial variant.As described below, preferred algorithm can be calculated breach or the like.Preferably, homogeny is present on the zone that length is at least about 25 amino acid or nucleotide, or more preferably, being present in length is on the zone of 50-100 amino acid or nucleotide.

For sequence alignment, a common sequence is as canonical sequence, sequence to be measured and its comparison.When using a kind of sequence alignment algorithm, to be measured and canonical sequence input calculator are then specified the subsequence coordinate if desired, and specified sequence algorithm routine parameter.Preferably, the program parameter of acquiescence can be used, maybe other parameter can be specified.The sequence alignment algorithm calculates the sequence homogeny percentage of sequence to be measured with respect to canonical sequence subsequently on the basis of program parameter.

Here employed " comparison window " comprises that wherein one section sequence can be compared with the canonical sequence with identical adjoining position numbering from generally including 20 to 600, often being the fragment of one of the numbering of adjoining position about 50 to 200, that more commonly select about group of 100 to 150 after two sections sequence optimizations are alignd.It is to know in this area that sequence alignment is arranged the method that is used to compare.The best alignment that is used to compare is arranged and can be passed through, for example Smith ﹠amp; The local homology degree algorithm that Waterman reports in Adv.Appl.Math.2:482 (1981), by Needleman ﹠amp; The homology permutation algorithm that Wunsch reports in J.Mol.Biol.48:443 (1970), by Pearson ﹠amp; The method of the search similitude that Lipman reports in Proc.Natl.Acad.Sci.USA 85:2444 (1988), computerization utilization (Wisconsin genetics software kit (Wisconsin GeneticsSoftware Package by these algorithms, Genetics Computer Group, 575 Science Dr., Madison, WI) GAP in, BESTFIT, FASTA and TFASTA), or by manual arrangement and range estimation (referring to, " molecular biology has experimental program now " (Current Protocols in Molecular Biology) of people such as Ausubel chief editor for example, 1995 supplementary issues.

Be suitable for determining that the preferred implementation of sequence homogeny and sequence similarity percentage comprises BLAST and BLAST2.0 algorithm, these algorithms are at Altschul etc., Nuc.Acids Res.25:3389-3402 (1977) and Altschul etc., J.Mol.Biol.215:403-410 is described in (1990).BLAST and BLAST2.0 use parameter described here to be used for determining the sequence homogeny percentage of nucleic acid of the present invention and albumen.The software that carries out the BLAST analysis opens to the public by NCBI (http://www.ncbi.nlm.nih.gov/).This algorithm comprises the length short word section W that differentiates in the search sequence, and when the alignment of the field of equal length was arranged in this short word Duan Yuyi bar database sequence, both were complementary or satisfy necessarily for threshold values T, and the sequence of at first differentiating high score thus is to (HSPs).T is as the reference (Altschul etc., the same) of contiguous field score value boundary.These initial contiguous field hits are searched the foundation that it is included in interior longer HSPs as starting.As long as the arrangement score value of accumulation can improve, the field hits extends toward both sides along every sequence.For example for nucleotide sequence, the score value operation parameter M of accumulation is (to the bonus point of the residue of a pair of coupling; Always greater than 0) and N (to the deduction of points of the residue that do not match; Always less than 0) calculate.For amino acid sequence, use the score value of score matrix computations accumulative total.The field hits interrupts in extending under the following situation on each direction: make accumulative total arrange score value from its peak that reaches drop by X quantity; Because the accumulation that one or more negative score residues are arranged causes the accumulative total score value to reach 0 or lower; Or reached the end of arbitrary sequence.Sensitivity and speed that BLAST algorithm parameter W, T and X decision alignment are arranged.BLASTN program (being used for nucleotide sequence) is used field length (W) value 11, expected value (E) 10, M=5, N=-4 as acquiescence, and two strands is compared.For amino acid sequence, the BLASTP program uses field length 3, expected value 10 and BLOSUM62 score matrix (referring to Henikoff ﹠amp as acquiescence; Henikoff, Proc.Natl.Acad.Sci.USA 89:10915 (1989)) arrangement value (B) 50, expected value 10, M=5, N=-4, and two strands compared.

The BLAST algorithm also carries out the statistical analysis of similitude between two sequences (for example referring to Karlin ﹠amp; Altschul, Proc.Nat ' l.Acad.Sci.USA 90:5873-5787 (1993)).A kind of the measuring of the similarity that is provided by the BLAST algorithm is minimum probability and (P (N)), and this provides the index of the probability of accidental coupling between a kind of two nucleotide or the amino acid sequence.For example, a nucleic acid is considered to similar to canonical sequence, if in the comparison of sequence to be measured and canonical sequence minimum probability and less than about 0.2, be more preferably less than about 0.01, most preferably less than about 0.001.Logarithm value can be big negative, for example 5,10,20,30,40,70,90,110,150,170 or the like.

Two nucleotide sequences or a polypeptide sign much at one are by the polypeptide of article one nucleic acid coding and the antibody that polypeptide produced with the second nucleic acid coding immunological cross-reaction as described below to take place.Thereby, for example when two sections peptides have only the difference of preservative replacement, polypeptide usually and the second polypeptide much at one.Two nucleotide sequences another sign much at one is described two molecules or the hybridization mutually under stringent condition as described below of its complementary strand.Two nucleotide sequences another sign much at one is to use the described sequence of identical primer amplification.

Term " separation ", " purifying " or " biologically pure " be meant with its native state under be found the composition followed usually almost or the material that separates substantially.Purity and homogeneity degree use usually such as analysis chemical technologies such as polyacrylamide gel electrophoresis or HPLC to be determined.The albumen of main kind or nucleic acid are abundant purifying in the preparation.Specifically, the nucleic acid of separation is to separate with the open reading frame that the natural side of described gene links to each other and coded albumen is different with described gene coded protein with some.In some embodiment, term " purifying " expression nucleic acid or albumen produce single band basically on running gel.Preferably, this represents described nucleic acid living protein purity at least 85%, more preferably at least 95%, most preferably at least 99%.In other embodiments, " purifying " expression is removed to less a kind of impurity from the combination species that carry out purifying.With regard to this meaning, purifying does not require that purified compound is a homogeneity, and for example 100% is pure.

Term " polypeptide ", " peptide " or " albumen " here can be used alternatingly, and are meant the polymer of amino acid residue.This term be used for wherein one or more amino acid residues be corresponding to the artificial chemical simulation thing of natural amino acid amino acid polymer and natural amino acid polymer, comprise the residue of modification and the polymer of alpha-non-natural amino acid.

Term " amino acid " comprises natural and synthetic amino acid, and function class is similar to the amino acid analogue and the amino acid analog thing of natural amino acid.Natural amino acid is meant that those are by genetic code amino acids coding and those amino acid of modifying subsequently (for example hydroxy-proline, Gla and O-Phosphoserine).Amino acid analogue comprises (for example having the basic structure identical with natural amino acid, with 1 hydrogen atom, 1 carboxyl group, the alpha-carbon atom that amine groups links to each other with 1 R group) compound, for example homoserine, nor-leucine, methionine sulphoxide, first sulfonium methionine.Such analog can have modified R group (for example nor-leucine) or modified peptide backbone, but has kept the chemical constitution as natural amino acid.The amino acid analog thing be meant have the structure that is different from the common chemical constitution of amino acid but its function class is similar to the compound of natural amino acid.

Here amino acid can be represented or represent with the single-letter mark that the IUPAC-IUB biochemical nomenclature commission is recommended with its normally used 3 alphabetical marks.Similarly, nucleotide can be with its general single-letter coded representation.

" the conservative variant of modifying " is used for amino acid and nucleotide sequence.For concrete nucleotide sequence, the conservative variant of modifying is meant those codings nucleic acid identical or essentially identical amino acid sequence, or described nucleic acid encoding amino acid sequence not, for example basic identical or relevant with natural contiguous sequence sequence.Because the merger of genetic code, the identical most of protein of nucleic acid coding on many functions.For example, codon GCA, GCC, GCG and the GCU alanine of all having encoded.Therefore, the alanine of determining by a codon on each position, this codon can change described another codon into and not change encoded polypeptide.Such nucleic acid variation is " silent variant ", is conservative a type of modifying variation.Each nucleic acid encoding sequence described here has also been described the silent variant of this nucleic acid.Being proficient in those skilled in the art will be appreciated that, every kind of codon in some context amplifying nucleic acid (except AUG unique password of methionine normally, and TGG unique password of tryptophan normally) can modify to produce identical molecule on the function.Correspondingly, have nothing to do in the actual probes sequence about expression product, described sequence generally includes the silent variant of nucleic acid encoding.

As for amino acid sequence, being proficient in those skilled in the art will be appreciated that, nucleic acid, peptide, polypeptide or protein sequence are carried out other, change in coded sequence, add or deletion single amino acids or the amino acid whose displacement of low percentage, deletion or interpolation are a kind ofly " conservatively to modify variant, if described change causes with chemically similar amino acid amino acid being replaced.It is known in this area that amino acid whose conservative substitution table similar on the function is provided.Conservative modification variant like this is to homologue and allelic additional rather than repulsion between polymorphism variant, kind among the present invention.Typically conservative substitution is mutually: 1) alanine (A) and glycine (G); 2) aspartic acid (D) and glutamic acid (E); 3) asparagine acid (N) and glutamine (Q); 4) arginine (R) and lysine (K); 5) isoleucine (I), leucine (L), methionine (M) and valine (V); 6) phenyl alanine (F), tyrosine (Y) and tryptophan (W); 7) serine (S) and threonine (T); And 8) cysteine (C) and methionine (M) (showing " protein " (Proteins, 1984)) referring to for example Creighton.

Can use the term description of the multiple level of tissue such as the macromolecular structure of polypeptide structure.This is organized comprehensive discussion of level, can be referring to " cellular elements biology " the 3rd edition of showing (Molecular Biologyof the Cell (third edition, 1994)) and Cantor such as for example Alberts; Schimmel shows " biophysical chemistry " first: " large biological molecule conformation " (Biophysical Chemistry Part I:The Conformation of BiologicalMacromolecules (1980))." primary structure " is meant the amino acid sequence of concrete peptide." secondary structure " is meant the three-dimensional structure of local order in the polypeptide.These structures are commonly referred to domain.Domain is a part that usually forms the polypeptide of compact unit, and normal length is 25 to about 500 amino acid.Typical domain is by constituting such as lower structure divisions of level of organizing such as βZhe Die and α spirals." tertiary structure " is meant the three-dimensional structure that polypeptide monomer is complete." quaternary structure " is meant usually by the non-covalent three-dimensional structure that is connected to form of three grades of units independently.Anisotropic term is also referred to as energy terms.

" mark " or " but test section " is to pass through spectrophotometric, photochemistry, biochemistry, immunochemistry, chemistry or the detected synthetic of other physical means.For example, useful mark comprises fluorescent dye, electron density reagent, enzyme (as through being usually used in ELISA), vitamin h, digoxin or hapten and protein or other reagent that can detect, for example by radioactive label is combined on the peptide, or be used to detect antibody with described peptide specific reaction.Radioisotope can be for example 3H, 14C, 32P, 35S or 125I.In some cases, particularly use under the situation at the antibody of albumen among the present invention, radioisotope is as described below as toxic moiety.Mark can be incorporated on any position of nucleic acid, protein and antibody.Any method that can be used for antibody and mark coupling known in the art all can be used, and these methods comprise by Hunter etc., Nature, 144:945 (1962); David etc., Biochemistry, 13:1014 (1974); Pain etc., J.Immunol.Meth., 40:219 (1981); And Nygren, those methods of describing among the J.Histochem.and Cytochem., 30:407 (1982).Stablize radiolabeled peptides or antibody and protect its reagent of avoiding degrading can prolong the life-span of radiolabeled peptide or radiolabeled antibody composition by adding.Can use the reagent or the agent combination of any stable radiolabeled peptides or antibody, this comprises U.S. Patent No. 5,961, disclosed those reagent in 955.

" effector " or " effect part " or " effect component " be meant with antibody by attachment or chemical bond with covalent manner or by ionic bond, Van der Waals force, electrostatic interaction or hydrogen bond with the molecule of non-covalent mode in conjunction with (or be connected, or coupling)." effector " can be multiple molecule, comprise for example comprise radioactive compound, fluorescent chemicals, enzyme or substrate, as test sections such as the label that is designated as label, toxin; Can activate part, chemotherapeutant; Lipase; Antibiotic; Or the isotopic part of emission " weight " ray (as the β ray).

When for example relating to cell, nucleic acid, protein or carrier, term " reorganization " represents that described cell, nucleic acid, protein or carrier transform by the introducing of exogenous nucleic acid or protein or the change of natural acid or protein, or described cell source is from the cell of transforming like this.Therefore, recombinant cell express cell undiscovered gene or express under natural (reorganization) state for example with other mode unconventionality expression, the low gene of expressing or not expressing fully.The term here " recombinant nucleic acid " expression at first at the nucleic acid of external formation, by the nucleic acid operation, for example by using polymerase and restriction endonuclease, exists with the non-existent form of common occurring in nature usually.Like this, but realized that different sequences connect with mode of operation.Like this, the linear nucleic acid of separation, or common unconnected dna molecular all can think to meet the recon of the object of the invention by the external expression vector that is connected to form.It will be appreciated that in case prepared recombinant nucleic acid and it is imported host cell or organism again, this nucleic acid will duplicate with non-recombination form, that is to say the cells in vivo machine rather than the external control mode that use host cell; Yet such nucleic acid is in case with the recombination form generation, though duplicate with non-recombination form subsequently, appoint the recon that is considered to be suitable for the object of the invention.Similarly, " recombinant protein " is to use the albumen of the recombinant technique manufacturing expression of aforesaid recombinant nucleic acid (promptly by).

When being used to mention the part of nucleic acid, the described nucleic acid of term " allos " expression comprises two or more subsequences, and there is not identical correlation usually under field conditions (factors) in these subsequences.For example, the described nucleic acid of two or more sequences produces with recombination form usually, for example organize from independent basis because of sequence to form new functional nucleic acid, for example, a kind of promotor of source and the code area in another source.Similarly, heterologous protein generally includes the two or more subsequences (for example fusion) that do not have correlation under the natural conditions.

When mentioning protein or peptide, phrase and antibody " specificity (or selectivity) combines " or " specificity (or selectivity) immune response " are meant in the heterogeneous population that contains protein and other biological sample the association reaction that existence determined by described albumen.Therefore, under the immune detection condition of appointment, under the immune detection condition of appointment, concrete antibody combines than the high twice of background, more typically is higher 10 to 100 times than background with the specific protein sequence.

Under such condition, combine needs and select to have specific antibody at specific protein with the specificity of antibody.For example, at specified protein, polymorphism variant, allelomorph, orthologous gene and conservative modify polyclonal antibody that variant or splice variant or its part produce can select with obtain those and Wnt or Frizzled albumen not with the immunoreactive polyclonal antibody of other protein specific.This selection can realize by the deduction and the antibody of other molecule cross reactions.The panimmunity detection mode can be used to select the antibody with specific protein generation specific immune response.For example, solid phase ELISA immune detection through be usually used in selecting with the antibody of protein generation specific immune response (as for to can being used for determining the immune detection mode of specific immune response and the description of condition, can be referring to, Harlow for example; Lane shows " antibody: laboratory manual " (Antibodies, A Laboratory Manual (1988))).

" tumour cell " comprises cell precancer, cancer cell and the normal cell in the tumour.

" cancer cell " in the tissue culture, " conversion " cell or " conversion " are meant spontaneous or that induce, the unnecessary phenotypic alternation that comprises the picked-up of new genetic stocks.Can infect and the merging of new genomic DNA or the picked-up of foreign DNA produce with transforming virus though transform, thus also can be spontaneously or contact with carcinogenic reagent and then to make the endogenous gene generation of undergoing mutation.In the present invention, conversion is expressed relevant with crossing of Wnt and/or Frizzled albumen usually.Transform with other and (show " animal cell culture: basic technology handbook the 3rd edition (Culture of Animal Cellsa Manual of Basic Technique (third edition, 1994)) referring to Freshney such as cell immortalityization, misgrowth regulation and control, non-morphological change and/or the change of pernicious isophenous.

The accompanying drawing summary

Fig. 1 explanation anti-Wnt-1 or anti-Wnt-2 antibody specificity in multiple human cancer cell line is apoptosis-induced.

Fig. 2 illustrates the part (%) of apoptotic cell death after the anti-Wnt antibody treatment.

The apoptosis of Fig. 3 A explanation anti-Wnt antibody induction in multiple cancerous cell line is expressed relevant with Wnt.The influence of the apoptosis of Fig. 3 B explanation Wnt blocking peptide antagonism Wnt antibody induction.

Fig. 4 shows the time course (Fig. 4 A) and the dosage process (Fig. 4 B) of the apoptosis of anti-Wnt antibody induction in the lung cancer cell line.

Fig. 5 shows that anti-Wnt-1 monoclone antibody is apoptosis-induced in different human cancer cell lines in the experiment in vitro.A. after control antibodies or anti-Wnt-1 monoclone antibody were handled, 0.5% crystal violet was to cancer cell MCF-7 (top two row) about 48 hours of dyeing and to about 72 hours of H460 (following two row) dyeing.Contrast or the anti-Wnt-1 antibody concentration used are respectively 0.0,1.0 and 10.0 mcg/ml from left to right.B. use the apoptosis analysis example of flow cytometer.From top to bottom, H460 uses about 72 hours of the anti-Wnt-1 antibody treatment of 5.0 mcg/ml control antibodies, 1.0 mcg/ml and 5.0 mcg/ml respectively.FL1-H represents Annexin V-FITC dyeing, and FL3-H represents propidium iodide (PI) dyeing.The dose response that c.H460 and MCF-7 cancer cell are handled monoclone antibody.H460 is hatched 72 hours, MCF-7 and is hatched after 48 hours and measure.Square frame () and circle (zero) are represented the part with cell death in the MCF-7 of anti-Wnt-1 monoclone antibody processing and the H460 cell respectively.Rhombus (◇) and triangle (△) are represented the part with cell death in the MCF-7 of control antibodies processing and the H460 cell respectively.The result is mean value ± SD (an error post).

Fig. 6 A-6C illustrates that anti-Wnt-1 monoclone antibody suppresses tumor growth in vivo.

Fig. 7 has shown monoclone antibody heavy chain and the light chain region sequence that is produced by listed peptide sequence among SEQ ID NO:2, SEQ ID NO:4 or the SEQ ID NO:9.

Fig. 8 shows that Δ PDZ-Dvl suppresses the tumour formation of celiothelioma cell in vivo.The MPM LRK1A of Δ PDZ-Dvl transfection compares with empty carrier transfection contrast and can not grow behind subcutaneous (s.c.) injection nude mice with the REN cell.The result is mean value ± SD (error post) of every group of 5 laboratory animal.

Fig. 9 shows the inhibition of Dvl siRNA to the NCI-HI703 growth.Cell (3 * 10 ⁴) be tiled on the 24-23ll flat board and with Dvl siRNA (square frame) or contrast siRNA (circle) transfection.After the transfection 72 hours, collected the cell (trypan blue exclusion test, trypan blue exclusion) of surviving by trypsinized and also count in per 24 hours.Especially, transfection is after 72 hours, and the cell growth is subjected to remarkable inhibition (P＜0.05).

Figure 10 illustrates that the mistake of Wnt signal antagonist FRP or DKK is expressed in the cancer cell apoptosis-induced.

Detailed Description Of The Invention

The present invention is based upon the Wnt-Fz signal pathway and plays a role in tumour forms on this discovery basis. Know that Wnt albumen often has high expression level in tumour. Yet, about cell death machine in cancer The Wnt-Fz signal transduction is modified and is known little about it. The present invention openly provides the Wnt signal transduction inhibitor can be multiple Induce the evidence of remarkable apoptosis in the cancer cell. The present invention can be used for any wherein Wnt-Fz signal transduction affects cancer cell The cancer of growth or existence. The present invention can be used for treatment such as breast cancer, colorectal cancer, lung cancer, sarcoma, Rind gall, prostate cancer, cancer of pancreas, cervical carcinoma, oophoroma, cancer of the stomach, cancer of the esophagus, head and neck cancer, hepatocellular carcinoma, The cancers such as melanoma, glioma or spongioblastoma.

Here show that sealing Wnt signal transduction causes the downward modulation of Wnt-Fz approach downstream composition, especially Dishevelled (Dvl) and β connection albumen. Here the evidence that provides also illustrates, the apoptosis of antibody induction by activate JNK, Discharging Smac/Diablo and cromoci from mitochondria to cytoplasm is taken place. Cromoci deactivation survival Albumen (survivin, a kind of inhibitors of apoptosis), this causes the activation of Guang winter enzyme. The present invention openly further provides The anti-Wnt-1 antibody of monoclonal can suppress the evidence of tumor growth in vivo.

WNT and Frizzled protein antibodies

As mentioned above, the invention provides the method for Wnt signal transduction in the inhibition cancer cell. In some reality of the present invention Execute in the mode, use antibody to hinder the combination between Wnt part and the Frizzled acceptor. Can produce for Wnt Or the antibody of Frizzled albumen.

For being proficient in those skilled in the art, the method for preparing polyclonal antibody be known (for example, Coligan, the same; Harlow ﹠ Lane, the same). Polyclonal antibody can produce in mammal, for example, By one or many injecting immune reagent and (if needs) adjuvant. Typically, immunoreagent and/or adjuvant can lead to Cross repeatedly subcutaneous or the intraperitoneal injection mode is injected into mammal. Immunoreagent can comprise by the nucleic acid institute that estimates Albumen or its fragment or its fusion of coding. Described immunoreagent and knownly in the mammal of immunity, have Immunogenic protein coupling may be useful. The example of such immunogenic protein includes but not limited to key Hole worm relative hemocyanin, seralbumin, bovine thyroglobulin and soybean trypsin inhibitor. Can make With the example of adjuvant comprise Freund's complete adjuvant and MPL-TDM adjuvant (MPL, synthetic trehalose Two diphtheria bacterium salt (trehalose dicorynomycolate). Immunization protocol can need not by being proficient in those skilled in the art Undue experimentation is selected.

In addition, antibody can be monoclonal antibody. Monoclonal antibody can be used the Milstein such as Kohler ﹠, Nature Hybridoma method preparation described in the 256:495 (1975). In hybridoma method, mouse, hamster or other close Suitable host animal usually with the immunoreagent immunity to induce generation maybe can produce with the immunoreagent specific binding The lymphocyte of antibody. In addition, described lymphocyte can carry out immunity external. Described immunoreagent typically Should comprise polypeptide or its fragment or its fusion by the nucleic acid coding of table 1-16. Usually, use the peripheral blood lymph Cell (PBL) if need human archeocyte, or uses splenocyte or LNC, if the inhuman source of needs The mammal source. Described lymphocyte passes through to use suitable fusion agent (such as polyethylene glycol) with immortal cell line subsequently (Goding is shown " monoclonal antibody: principle and put into practice " (Monoclonal to form hybridoma in fusion Antibodies:Principles and Practice), 59-103 page or leaf, 1986). The lactation that immortalized cells normally transforms The myeloma cell in zooblast, especially rodent, ox and people source. Often adopt the myeloma of rat or mouse Clone. Hybridoma can suitable, preferably comprise one or more and suppress the immortalized cells that do not merge Growth or the culture medium of the reagent of existence in cultivate. For example, if parental cell lacks hypoxanthine guanine phosphorus Ribosyltransferase (HGPRT or HPRT), the hybridoma culture medium should comprise hypoxanthine, ammonia petrin and thymidine usually (" HAT " culture medium), these reagent hinder the growth of HGPRT disappearance cell.

In some embodiments, used monoclonal antibody. Preferred embodiment be that combination is as among the embodiment 11 The monoclonal antibody of the identical epi-position of described monoclonal antibody. Specific antibodies and the identical epi-position of another kind of antibody recognition The ability that ability normally is combined with antigen by a kind of antibody competition inhibition second antibody is determined. Any one Kind of competition can be used for be measured between two kinds of antibody competition to same antigen in conjunction with method of testing. For example, for this purpose Can use the ELISA sandwich method. This is undertaken by being coated with the reaction tank surface with a kind of capture antibody. Exist subsequently Catch the upper labelled antigen that adds sub-saturated concentration in surface. This albumen can pass through specific antibody/epi-position and interact Be combined with antibody. After the washing, but in ELISA, add second with test section (for example, HRP, mark Antibody is defined as detecting antibody) covalently bound second antibody. If this antibody recognition is identical with capture antibody Epi-position because this defined epitope is not used further to combination, it can not be in conjunction with target protein. If second antibody is known Different epi-position on the other target protein, it should be able in conjunction with and can by use related substrates to activity level (by combination Antibody produce) quantitatively detect this combination. Background is by using not only as capture antibody but also as detecting antibody Single antibody defines, and wherein peak signal can be by catching with antigen-specific antibodies and using for antigen The antibody test of upper label is established. By using background and peak signal as reference, antibody can be with in pairs Mode assesses to determine epitope specificity.

First kind of antibody is considered to the competitive combination that suppresses second antibody, if use above-mentioned any detection method, Under the condition that first kind of antibody exists, the combination of second antibody and antigen has reduced at least 30%, usually at least About 40%, 50%, 60% or 75%, and often at least about 90%.

In some embodiment, the amino acid 201-212 of the anti-Wnt-1 antibody of the monoclonal among the present invention and people Wnt-1 (HNNEAGRTTVFS), the amino acid 39-52 (NVASSTNLLTDSKS) of people Wnt-1 or people Wnt-2 Amino acid 49-63 (SSQRQLCHRHPDVMR) combination. For example, such monoclonal antibody may have such as figure The binding specificity of the antibody of VH shown in 7 and VL chain (that is to say in this context, to have identical CDR or almost identical CDR). Antibody among the present invention thereby can contain as shown in Figure 7 VH or VL sequence CDR and in addition can with described VH or VL sequence at least 80% identical, preferred 85%, 90% or 95 % is identical. For example, in specific embodiment, the CDR that antibody can contain the VH of Fig. 7 and VL sequence with And human skeleton's region sequence.

In some embodiment, Wnt or Frizzled protein antibodies are chimeric humanized antibodies. As mentioned above, The antibody of humanization form is chimeric immunoglobulin (Ig), wherein from complementary determining region (CDR) residue of human antibodies Come the CDR's with specificity, affinity and ability of needing of inhuman kinds such as mouse, rat or rabbit freely Residue replaces.

That people's antibody can use is known in the art, comprise phage display library (Hoogenboom ﹠ Winter, J. Mol.Biol.227:381 (1991); Marks etc., J.Mol.Biol.222:581 (1991)) multiple technologies production. The described technology such as Cole etc. and Boerner also can be used for the preparation (" Dan Ke that Cole etc. show of human monoclonal antibodies Grand antibody and treatment of cancer " the 77th page (Monoclonal Antibodies and Cancer Therapy, p.77 (1985)) And Boerner etc., J.Immunol.147 (1): 86-95 (1991)). Similarly, people's antibody can be by turning to base Because importing people's immunity in the animal (for example wherein endogenous immunoglobulin gene by the mouse of deactivation partially or completely) The globulin seat produces. Face of Challenge is observed the people's antibody that comprises gene rearrangement, assembling and antibody deposit and is produced, This is very similar to the esoteric process of people. This method is at for example United States Patent (USP) 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016 and following scientific publications: Marks etc., Bio/Technology 10:779-783 (1992); Lonberg etc., Nature 368:856-859 (1994); Morrison, Nature 368:812-13 (1994); Fishwild etc., Nature Biotechnology 14:845-51 (1996); Neuberger, Nature Biotechnology 14:826 (1 996); Lonberg ﹠ Huszar, Intern.Rev.Immunol. 13:65-93 is described in (1995).

In some embodiments, described antibody is scFv (scFv). The VH of scFv antibody and VL district comprise through Be folded to form a strand in the antigenic structure territory that is similar to seen in double-chain antibody. In case folding, non-covalent Interact and stablize single-chain antibody. Although the VH of some antibody embodiment and VL district can directly link together, Being proficient in those skilled in the art can recognize, described zone can be by the peptide attachment by one or more Amino acid profiles Separate. Peptide attachment and use thereof are known in this area. Referring to, such as Huston etc., Proc.Natl Acad. Sci.USA 8:5879 (1988); Bird etc., Science 242:4236 (1988); Glockshuber etc., Biochemistry 29:1362 (1990); United States Patent (USP) 4,946,778 and 5,132,405 and Stemmer etc., Biotechniques 14:256-265 (1993). Described peptide attachment is except connecting described zone or keeping some minimums between VH and the VL Distance or other spatial relationships can not have special BA outward usually. Yet, can select to consist of peptide and connect The amino acid that connects thing is with some character of affecting this a part (such as folding, net charge or hydrophobicity). ScFv (scFv) Antibody optionally comprises no more than 50 amino acid of length, common no more than 40 amino acid, preferred few In 30 amino acid, more preferably no more than 20 amino acid whose peptide attachments. In some embodiment, peptide connects Connecing thing is the concatermer of sequence Gly-Gly-Gly-Gly-Ser, preferably 2,3,4,5,6 such sequences. Yet, need to recognize, can carry out some replacement of amino acid in attachment inside. For example, valine can Be replaced as glycine.

The existing description of method for preparing scFv antibody. Referring to Huse etc., the same; Ward etc., the same; And Vaughan Deng, the same. In brief, separate from the mRNA of the B cell of immune animal and prepare cDNA. Use pin To the described cDNA of the primer amplification of heavy chain immunoglobulin and variable region of light chain. Purified pcr product also connects nuclear Acid sequence. If need the peptide attachment, between heavy chain and light chain nucleic acid sequence, insert the nucleotide sequence of encoded peptide. The nucleic acid insertion vector of coding scFv is also expressed in suitable host cell. The scFv of the required antigen of specific binding Usually find by the screening of phage display library. Screening can be by any the carrying out in the several method. Screening can be expediently by using at the cell of the required antigen of its surface expression or using with required antigen coated consolidating Carry out the surface. Easily, described surface can be a kind of magnetic bead. Unconjugated bacteriophage is washed from the surface of solids Go the also bacteriophage of elution of bound.

The screening technique of no matter selecting can by genotype and the contact of the physics between the phenotype that phage display provides Be used for the combination of test each member of cDNA library and antigen, even if for huge clone library.

In some embodiment, described antibody is bispecific antibody. Bispecific antibody is to have at least two The binding specificity of synantigen or have monoclonal for the binding specificity of two epi-positions on the same antigen kind not , preferably people source or humanized antibody. In one embodiment, one of described binding specificity be for Wnt or Frizzled albumen, another kind of for another kind of cancer antigen. In addition, tetramer class technology can create many Valency reagent.

In some embodiment, described antibody and the coupling of effect part. The effect part can be any amount of molecule, Comprising such as radioactive label or fluorescently-labeled mark part, maybe can be the treatment part. If effect is partly controlled Treating part, can be cytotoxic reagent usually. In this method, cause to cancer cell guiding cytotoxic reagent The orientation of target cell is killed and wounded. This embodiment uses the antibody of Frizzled acceptor to realize usually. Cell toxicant reagent is Changeable and include but not limited to the active fragment of cell toxicity medicament or toxin or such toxin. Suitable toxin reaches Its respective segments comprises diphtheria toxin A chain, exotoxin A chain, ricin A chain, abrin A chain, rushes down Fruit element, crotin, phenomycin, enomycin, auristatin etc. Cell toxicant reagent also comprises and passes through radioactivity Isotope and Wnt or the coupling of Frizzled protein antibodies or radionuclide and the chelating of covalently bound antibody Agent is in conjunction with the radioactive compound of preparation.

The binding affinity of antibody among the present invention

The binding affinity of target antigen is typically by such as Biacore competition detection, saturation detection or such as ELISA Or the conventional antibody such as the immune detection such as RIA-antigen detection method is measured or is determined.

Such method of testing can be used for measuring the dissociation constant of antibody. Phrase " dissociation constant " refers to that antibody is to antigen Affinity. If the specificity that has combination between antibody and the antigen is the dissociation constant (K of antibody_D=1/K, wherein K Affinity costant)＜1 μ M, preferred＜100nM, most preferably＜0.1nM. Antibody molecule should have in the low interval usually K_DValue. K_D=[Ab-Ag]/(Aberle etc., EMBO Journal, 16:3797-3804 (1997)), wherein [Ab] Be the equilibrium concentration of antibody, [Ag] is the equilibrium concentration of antigen, and [Ab-Ag] is the equilibrium concentration of Antibody-antigen complex. Typically, the binding interactions between antigen and the antibody comprises reversible non-covalent combination, for example electrostatic attraction, Van der Waals force and hydrogen bond.

Antibody specific binding Wnt of the present invention or Frizzled albumen. Here " specific binding " means antibody With at least about 0.1mM, more frequently at least about 1 μ M, preferably at least about 0.1 μ M or lower, most preferably with 0.01 μ M Or lower K_DValue and protein combination.

The diagnostic test method

The present invention also provides the diagnostic test method for detection of Wnt or Frizzled protein overexpression. As mentioned above, The expression of crossing of these genes can be used for differentiating cancer cell. In preferred embodiment, interested Wnt or Frizzled The activity of gene is produced by protein quantity or the measurement gene of measuring gene transcripts (for example mRNA), measurement translation The activity of thing is determined.

Use the method for nucleic acid hybridization technique detection and/or quantitate gene transcript (mRNA or cDNA) for being proficient in this The personnel in field are known. For example, the transfer of Northern trace is to estimate mRNA existence, disappearance or right A kind of method that it is quantitative.

Probe can be the total length nucleotide sequence of coded protein or less than the nucleotide sequence of total length. Than short probe experience Its specificity of property ground test. Preferred nucleic acid probe length is 20 bases or longer. The development of hybridization portion allows Existence or disappearance to mRNA are quantitatively determined.

In another preferred embodiment, transcript (for example mRNA) can use as mentioned above based on amplification (example Such as PCR) thus method measure and directly to estimate the DNA copy number. A kind of preferred embodiment in, transcript Level is by using reverse transcription PCR (RT-PCR) estimation.

" activity " of Wnt or Frizzled gene also can detect by the polypeptide of detection or quantitative expression and be quantitative. Polypeptide can detect with quantitative by known any method for being proficient in those skilled in the art. This can Comprise such as electrophoresis, Capillary Electrophoresis, high performance liquid chromatography (HPLC), thin-layer chromatography (TLC), super diffusion chromatography etc. The analytical biochemistry method. The protein that separates also can check order to differentiate polymorphism according to routine techniques.

Use any immunity of knowing in conjunction with method of testing (referring to, for example United States Patent (USP) 4,366,241; 4,376,110; 4,517,288 and 4,837,168), antibody of the present invention also can be for detection of Wnt or Frizzled albumen, or express Their cell. As for the review of general immunity test, also can be referring to Asai chief editor's " cell biology method " The 37th volume (Methods in Cell Biology, volume 37, Asai compiles Academic Press, Inc.New York (1993)) With Stites ﹠ Terr chief editor " basis and clinical immunology " the 7th edition (Basic and Clinical Immunology, The 7th edition, Stites ﹠ Terr compiles, (1991)).

Like this, the invention provides the method that the cell of excessively expressing Wnt or Frizzled albumen is detected. One In the kind method, the tissue that the experimenter is carried out slicer and collects in vitro detection. Tissue or come self-organizing Cell contacts with anti-Wnt of the present invention or anti-Frizzled protein antibodies subsequently. The immune complex table of any formation The bright target protein that in biopsy samples, exists. For the ease of such detection, described antibody can the radioactivity mark Note or with effector molecule coupling as detectable label (such as radioactive label). In another approach, use the typical case Imaging system can detect in vivo described cell. Subsequently, the location of label is by any known detection mark The method of note thing is determined. Can use a kind of conventional method that manifests diagnosing image. For example, the paramagnetism radioactivity together The position element can be used for Magnetic resonance imaging. The internalization of antibody may be passed through outside the born of the same parents in organism relatively for extending it In conjunction with the life-span that provides be important, what the latter can be to by the mediation of the exoenzyme environment that links to each other with loop cleaning is clear Except responsive.

The discriminating of WNT signal transduction inhibitor

In the drug screening method of testing, can use Wnt or Frizzled albumen (or express their cell) or Wnt letter The member (for example Dvl) of number approach is to differentiate the reagent that suppresses the Wnt signal transduction. Thereby the invention provides screening and press down The new method of the composition of cancer processed.

The method of testing of Wnt signal transduction can be designed to detect and/or the quantitative any part of Wnt signal pathway. For example Can measure β connection protein level in a kind of agents influence cell or apoptosis-induced ability in target cell. The following describes Be suitable for the method for testing of these purposes.

Method of testing can comprise those be used for test and Wnt part, Frizzled acceptor or Wnt signal cascade another Member (for example Dvl) is in conjunction with active method. These methods of testing are particularly useful in the reagent of differentiating regulation and control Wnt activity. In fact in such method of testing, can detect any reagent. Such reagent includes but not limited to natural or synthetic Polypeptide, antibody, natural or synthetic little organic molecule, nucleic acid etc.

As mentioned above, secreting type Frizzled GAP-associated protein GAP (sFRP) family is by dividing with Frizzled acceptor competition combination Secreting type Wnt part plays a role as solvable endogenous regulation of Wnt signal transduction. Thereby, in some form On, wear car reagent based on the native ligand (for example, Wnt part or sFRP) of Frizzled acceptor.

The method of testing of any detection Wnt signal transduction satisfies high flux screening. The high flux method of testing, in conjunction with method of testing and The reporter method of testing is known equally. Therefore, for example, United States Patent (USP) 5,559,410 disclose the height of protein The flux screening method, United States Patent (USP) 5,585,639 disclose the high throughput method (being array) of screening nucleic acid combination, and United States Patent (USP) 5,576,220 and 5,541,061 discloses the high throughput method of screening part/antibody combination.

In addition, high throughput screening system can by commercial the acquisition (visible Zymark Corp. for example, Hopkinton, MA; Air Technical Industries, Mentor, OH; Beckman Instruments, Inc.Fullerton, CA; Precision Systems, Inc., Natick, MA etc.). These systems realize comprising whole samples usually Distribute, regularly hatch and finally the reading of microplate in being suitable for the detector of method of testing with the move liquid, liquid of reagent Number is in interior whole step automation mechanized operation. These configurable systems provide high flux and have started fast and height Adaptability and the customization of degree. The manufacturer of such system provides the detailed protocol of multiple high throughput system. Cause And for example, Zymark Corp. provides the screening system of describing for detection of gene transcription regulation, ligand binding etc. The technical bulletin of system.

Can be used for other methods of testing of the present invention is those methods that design for the tumor phenotypes that detects cancer cell. These Method of testing is included on the cell soft agar grows; Anchorage dependence; Contact inhibition and the limitations in density of growth; Cell Propagation; Cell death (apoptosis); Cell transformation; Growth factor or serum rely on; Tomour specific sign level; Restructuring Matrigel (Matrigel) invasion; Tumor growth in vivo and transfer; MRNA and protein expression in the cell that experience shifts And other cancer cell features.

The cytostatic ability of test agent also can be by importing test agent animal disease model and assessment Growth of cancer cells is assessed in the body. For example, human tumor cells can be imported as immune infringement animals such as " nude mices ". Test agent (for example little molecule or antibody) is formed the ability of tumour (by moving to animals administer and with tumour cell The quantity of the tumour that forms in the thing is assessed with size) with the control-animal that does not contact this reagent in tumor growth Compare.

The inhibitor of gene expression

One aspect of the present invention, the inhibitor of Wnt signal pathway (for example Dvl inhibitor) can comprise that inhibition is described The hit nucleic acid molecules of protein expression of approach. Can use conventional virus or nonviral gene transfer method to lactation Zooblast or destination organization import the nucleic acid or in addition of the polypeptide (for example dominant negative form of this albumen) of code Design Outer nucleic acid (such as the inhibitor of the expression such as the target protein such as siRNA or antisense RNA). Non-virus carrier transhipment system Turnkey draw together DNA plasmid, naked DNA and with the nucleic acid compound such as transfer tools such as liposomes. The viral vectors transhipment System comprises DNA and RNA virus, and they have genome free or that integrate after in transporte to cells. As for the review of gene therapy process, referring to Anderson, Science 256:808-813 (1992); Nabel ﹠ Feigner, TIBTECH 11:211-217 (1993); Mitani ﹠ Caskey, TIBTECH 11:162-166 (1993); Dillon, TIBTECH 11:167-175 (1993); Miller, Nature 357:455-460 (1992); Van Brunt, Biotechnology 6 (10): 1149-1154 (1988); Vigne, Restorative Neurology and Neuroscience 8:35-36 (1995); Kremer ﹠ Perricaudet, British Medical Bulletin 51 (1): 31-44 (1995); " microbiology and immunology have theme now " (Current Topics in Microbiology of Doerfler and Bohm chief editor And Immunology) in the book Haddada etc. the chapters and sections of showing (1995) and Yuetal, Gene Therapy 1:13-26 (1994).

In some embodiment, used small molecules interference RNA. In mammalian cell, long two strands The importing of RNA (＞30 nucleotides) often causes effective antiviral response, shows as synthetic non-specific pressing down of albumen System and RNA degraded. The RNA interference phenomenon is at for example Bass, Nature 411:428-29 (2001); Elbahir etc., Nature 411:494-98 (2001); And Fire etc., Nature 391:806-l1 is described and begs in (1998) Opinion has wherein also been inquired into the method for preparing RNA interfering. The siRNA inhibitor is less than 100 base-pairs, usually 30bp Or shorter, and by means preparation known in the art. Can have directly according to exemplary siRNA of the present invention To 29bp, 25bp, 22bp, 21bp, 20bp, 15bp, 5bp or length in its vicinity any or between it.

Non-viral conveyer method

The non-viral conveyer method of the nucleic acid of the polypeptide of code Design of the present invention comprises liposome transfection, microinjection, base Because of rifle, virion, liposome, immunoliposome, polycation or lipid nucleic acid zygosome, naked DNA, people The antigen of worker's virion and DNA strengthens picked-up. Fat transfection (Lipofection) United States Patent (USP) 5,049,386 for example, Describe to some extent in 4,946,787 and 4,897,355, and lipofectin reagent can (for example obtain by commercial Transfectam^TMAnd Lipofectin^TM). Be suitable for polynucleotides the transfection of effective Receptor recognition fat cation and in The property lipid comprise Felgner WO 91/17424 and WO 91/16024 described those. Can (exsomatize to cell Administration) or destination organization (vivo medicine-feeding) transhipment.

Comprise the lipid such as immune lipid complex: the preparation of nucleic acid complexes for being proficient in those skilled in the art is Know (referring to for example Crystal, Science 270:404-410 (1995); Blaese etc., Cancer Gene Ther. 2:291-297 (1995); Behr etc., Bioconjugate Chem.5:382-389 (1994); Remy etc., Bioconjugate Chem.5:647-654 (1994); Gao etc., Gene Therapy 2:710-722 (1995); Ahmad etc., Cancer Res.52:4817-4820 (1992); United States Patent (USP) 4,186,183,4,217,344,4,235,871,4,261,975, 4,485,054,4,501,728,4,774,085,4,837,028 and 4,946,787).

The virus conveyer method

Transporting target Wnt pathway protein (for example Dvl) inhibitor with RNA or dna virus system is this area Interior known. The conventional viral system of transporting such nucleic acid inhibitor can comprise the reverse transcription for transgenosis Virus, slow virus (lentivirus), adenovirus, adeno-associated virus and herpes simplex virus vector.

In many gene therapies are used, wish that gene therapy vector is transported to particular tissue type (example with high specific Such as cancerous lung tissue). Viral vectors is usually transformed and is made it to merge by the envelope protein on expression and the viral outer surface Part have specificity for given cell type. Selection has and known depositing on the cells of interest type The part of acceptor with affinity. For example, reported in PNAS92:9141-9151 (1995) can for Han etc. Transfer albumen (heregulin) to transform MLS to express the people of merging with gp70, and the recombinant virus sense Dye the human breast cancer cell that some expresses EGF-R ELISA. This principle can extend to other and express part The virus of fusion and the target cell of expressed receptor pair. For example, can transform filobactivirus and have pin with displaying Antibody fragment (for example Fab or Fv) to the cell receptor specific binding affinity of in fact any selection. Although Top description is mainly used in viral vectors, and identical principle can be used for non-virus carrier. Can transform such Carrier is thought and is helped special picked-up sequence by concrete target cell picked-up to comprise.

Gene therapy vector can by to independent patient's administration, as described belowly typically carry out transhipment in the body by whole body administration (for example in the intravenous, peritonaeum, intramuscular, subcutaneous or encephalic are inculcated) or topical application.In addition, carrier can exsomatize and be transitted to cell, such as the cell of transplanting from independent patient.

For being proficient in those skilled in the art, be used to diagnose, the cells in-vitro transfection (for example, by transfectional cell being re-entered host's organism) of research or gene therapy knows.In some embodiment, cell separates from experimenter's body, also fails back experimenter's body (for example patient) again with the inhibitor nucleic acids transfection.Multiple be suitable for exsomatizing the cells transfected type for be proficient in those skilled in the art be know (referring to, " the animal cell culture: basic technology handbook (Culture of Animal Cells of being shown such as Freshney for example, A Manual of Basic Technique), the 3rd edition, 1994)) and for explanation how from the patient separates and cultured cell is quoted list of references).

Be transduction in the body of realizing cell, the carrier (for example, reverse transcription albumen, adenovirus, liposome or the like) that contains therapeutic nucleic acids also can be directly to the organism administration.In addition, can give naked DNA.Be generally used for importing the administration of molecule by any finally to contact with blood or histocyte.For being proficient in those skilled in the art, the proper method that gives such nucleic acid is available and knows, and though number of ways can be used for the administration of particular composition, particular approach often provides faster, more effective reaction than other approach.

Pharmaceutically acceptable carrier part is by the particular composition of administration and the ad hoc approach decision that is used to give said composition.Correspondingly, as described below, exist pharmaceutical composition of the present invention multiple suitable formulation (referring to, for example, " Remington's Pharmaceutical Science " (Remington ' sd Pharmaceutical Sciences), the 17th edition, 1989)).

Kit in diagnosis, research and therapeutic are used uses

As mentioned above, the invention provides in some cancer specific Wnt or Frizzled albumen and cross the evidence of expression.Thereby, can use kit to detect specific nucleic acid disclosed herein or protein.In diagnosis and research application, such kit can comprise any or all of following composition: detectable, buffer solution, Wnt specificity or Frizzled protein-specific nucleic acid or antibody, hybridization probe and/or primer or the like.Therapeutic products can comprise aseptic salt solution or other pharmaceutically acceptable emulsions and suspension basis.

In addition, described kit can comprise the guiding material that contains the specification (promptly using scheme) of putting into practice method of the present invention.Though guiding material typically comprises written or printed material, is not limited only to this.Anyly can store such specification and can all comprise in the present invention with the medium that the end user links up.Such medium includes but not limited to electronic storage medium (for example disk, tape, chip), light medium (for example CD-ROM) or the like.Such medium can comprise the address of the Internet address of the guiding material that provides such.

The present invention also provides the kit of the inhibitor that is used to screen the Wnt signal transduction.Such kit can be prepared by material that is easy to obtain and reagent.For example, such kit can comprise one or more following material: Wnt or Frizzled polypeptide or polynucleotides, reaction tube and test the required Wnt signal transduction functionality specification of (for example β joins protein level).

Methods of treatment

The inhibitor administration

The reagent (for example antibody) that suppresses the Wnt signal transduction can pass through the several different methods administration, and these methods include but not limited to (for example intravenous, intramuscular, intracutaneous, endoperitoneal and subcutaneous route), local, the oral or cutaneous penetration of parenteral.These methods can be used for preventative and/or therapeutic treatment.

As mentioned above, inhibitor of the present invention can be used for the treatment of the cancer relevant with the Wnt signal transduction.Composition in order to administration generally can comprise the inhibitor that is dissolved in pharmaceutically acceptable carrier (preferably aqueous carrier).Can use multiple aqueous carrier, for example buffer salt solution or the like.These solution are aseptic and do not contain unwanted reagent usually.That these compositions can comprise is pharmaceutically acceptable, near the required auxiliary reagent of physiological condition, regulate reagent or the like such as pH regulator and buffering reagent, toxicity, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate or the like.The concentration of active agent can alter a great deal in these formulations, and according to mode of administration and the patient demand selected, mainly selects on bases such as liquid volume, viscosity, body weight.

Thereby the typical pharmaceutical composition that is used for intravenous administration should be about 0.1 to 10 milligram of every patient every day.Every patient can give 0.1 every day to about 100 milligrams dosage, especially when the concealed location administration is not entered under the situation of blood flow, such as to body cavity or organ lumen administration.In local application, can use higher dosage.For being proficient in those skilled in the art, the practical methods of the composition of preparation parenteral administration is known or conspicuous, and as " Remington's Pharmaceutical Science " the 15th edition (Remington ' s Pharmaceutical Science, the 15th edition, Mack Publishing Company, Easton, Pennsylvania (1980)) etc. on the publication more detailed description is arranged.

Depend on medication, described pharmaceutical composition can be with multiple unit dosage forms administration.For example, the unit dosage forms that is suitable for oral administration includes but not limited to powder, tablet, ball, capsule and lozenge.Need recognize that antibody should be protected to exempt from digestion under the oral administration condition.This tolerates acid and enzymatic hydrolysis by described molecule and compound the making it of composition usually, or realizes by described molecule being packaged in the suitable tolerance carrier (as liposome or protection barrier).The method that protection reagent is avoided digesting is to know in this area.

The composition (for example antibody) that contains inhibitor of the present invention can be therapeutic or prophylactic treatment administration.In therapeutic is used, with the patient medication of composition that is enough to treat or stop the amount of disease and complication thereof to tormented by disease (for example breast cancer) to small part.Be enough to realize that the amount of this purpose is defined as " treatment effective dose ".The effective dose of this use depends on severity of disease and patient's healthy integrated status.Depend on the dosage and the frequency of needs of patients and tolerance, composition can be in single or divided doses.Under any circumstance, described composition should provide the reagent of the present invention of quantity sufficient so that the patient is effectively treated.The inhibitor quantity that can prevent or delay cancer development among the patient is called " preventative effective dose ".The needed given dose of prophylactic treatment can depend on medical condition and patient's medical history, the particular cancers that need prevent and as other factors such as age, body weight, sex, method of administration, validity.Such prophylactic treatment can be used for, and for example previous patient that must cross cancer is preventing the generation once more of cancer, or the patient with important possibility of developing cancer under a cloud.

For purpose of the present invention, " patient " comprises people and other animals, especially mammal.Like this, described method can be used for human treatment and veterinary applications.In preferred embodiment, the patient is mammal, primate preferably, and the patient is the people in most preferred embodiments.

Other known cancer methods of treatments can be used in combination with method of the present invention.For example, the inhibitor of Wnt signal transduction also can be used for to other cancer therapeutic agents (as 5FU, vincaleukoblastinum, radiating streptozotocin D, cis-platinum, ninopterin or the like) guiding or sensitized cell.In other embodiments, method of the present invention can be used with radiotherapy or the like.

In some cases, described antibody belongs to when activating the hypotype of SC with the transmembrane protein compound tense, thus the cytotoxicity (ADCC) that mediated cell toxicity or antigen rely on.Like this, the antibody of the Frizzled albumen by giving the anticancer cell surface of patient can be treated cancer.Antibody labeling can activate auxiliary toxin, gathering toxin useful load or the method for local ablation cell otherwise is provided.In these embodiments, described antibody and the coupling of effect part.The effect part can be any amount of molecule, comprises maybe can being the therapeutic part, as cell toxicant reagent such as radioactive label or fluorescently-labeled mark part.

Wnt or Frizzled polypeptide are used as vaccine

Except using the administration of Wnt signal transduction inhibitor, Wnt or Frizzled albumen or its immunogenic fragments can be used as the vaccine combination administration to activate the antibody response of HTL, CTL and anti-intrinsic protein.Such vaccine combination can comprise, for example, the fat peptide is (referring to for example Vitiello etc., (1995) J.Clin.Invest.95:341-349), pack into poly-third peptide combinations of handing over fat-glycolide (PLG) microballoon capsule (referring to, for example, Eldridge etc., (1991) Molec.Immunol.28:287-294; Alonso etc. (1994) Vaccine 12:299-306; Jones etc. (1995) Vaccine 13:675-681), be included in the peptide combinations (ISCOMS in the immune activation complex; Referring to, for example, Takahashi etc. (1990) Nature 344:873-875; Hu etc. (1998) Clin.Exp.Immunol.113:235-243), (MAPs of multiple antigenic peptide system; Referring to for example Tarn (1988) Proc.Nat ' l Acad Set USA85:5409-5413; Tam (1996) J.Immunol.Methods 196:17-32)) the viral transport vehicle (content that 379 pages of Perkus etc. are shown in " vaccine development notion " (Concepts in Vaccine Development de Gruyter, 1996) of Kaufmann chief editor; Chakrabarti etc. (1986) Nature 320:535-537; Hu etc. (1986) Nature 320:537-540; Kieny etc. (1986) AIDS Bio/Technology 4:790-795; Top etc. (1971) J.Infect.Dis.124:148-154; Chanda etc. (1990) Virology 175:535-547), the particle in virus or synthetic source is (referring to, (1996) J.Immunol.Methods 192:25-35 such as Kofler for example; Eldridge etc. (1993) Sem.HematoL 30:16-24; Falo etc. (1995) Nature Med.7:649-653).

Vaccine combination often comprises adjuvant.Many adjuvants comprise and are used to protect antigen to avoid quick catabolic reagent, such as aluminium hydroxide or mineral oil, and the immune response activator, such as the albumen in lipid A, CBP or Mycobacterium tuberculosis source.Some adjuvant has and can obtain by commercial; as for example incomplete Freund and Freund's complete adjuvant (Difco Laboratories; Detroit; MI), Merck adjuvant 65 (Merck and Company; Inc.; Rahway; NJ), AS-2 (SmithKline Beecham; Philadelphia, PA), as polysaccharide, poly-phosphatide, biodegradable microspheres, monophosphoryl lipid A and the quil A of soluble suspension, acidylate sugar, cation or the anionic derivativeization of aluminium salt, calcium salt, molysite or the zinc salt of alumine hydroxide colloid or aluminium phosphate, acidylate tyrosine.Such as GM-CSF, interleukin-22,7,12 and the cell factor of other analog growth factors also can be used as adjuvant.

Vaccine can be used as the nucleic acid compositions administration, wherein gives DNA or RNA or its fragment that the patient encodes Wnt or Frizzled polypeptide.Referring to (1990) Science 247:1465-1468 such as for example Wolff; United States Patent (USP) 5,580,859,5,589,466,5,804,566,5,739,118,5,736,524,5,679,647; And WO 98/04720.Example based on the transhipment technology of DNA comprises " naked DNA ", promotes that (particle gun) that (Bupivacaine, polymer, peptide-mediation) transhipment, cation lipid compound and particle mediate or pressure-mediated transhipment are (referring to for example United States Patent (USP) 5,922,687).

Use resembles the method for the gene of dna vaccination to be known, and comprises the gene of hope or its part placed under regulatable promotor or the tissue-specific promoter's control and make it the expression in vivo the patient.As the gene of dna vaccination can encode total length Wnt or Frizzled albumen, or a part that can encoding said proteins.

In some embodiment, dna vaccination comprises the gene and the dna vaccination of coding adjuvant molecule.Such adjuvant molecule comprises the cell factor of raising at the immunogenic response of the coded polypeptide of dna vaccination.

For therapeutic or preventative immune purpose, peptide of the present invention can be expressed with virus or bacteria carrier.The example of expression vector comprises the attenuated virus host, as cowpox or bird acne.This method comprises uses vaccinia virus, for example, and as the carrier of the nucleotide sequence of expressing coding Wnt or Frizzled polypeptide or polypeptide fragment.Import after the host, recombined vaccinia virus is expressed immunogenic peptide, and causes immune response thus.For example at United States Patent (USP) 4,722, cowpox carrier and the method that is used for immunization protocol described in 848.Another kind of carrier is BCG.In (1991) Nature 351:456-460 such as Stover, the BCG carrier has been described.Multiple other can be used for the carrier of therapeutic administration or immunity, and for example adenovirus and gland relevant viral vector, retroviral vector, salmonella typhi carrier, detoxification anthrax toxin carrier or the like can be conspicuous.Referring to (2000) Mol.Med.Today 6:66-71 such as for example Shata; Shedlock etc. (2000) J.Leukoc.Biol.68:793-806; And (2000) In Vivo 14:571-85 such as Hipp.

Embodiment

Following examples are used to illustrate, rather than restriction the present invention.

Material and method

Cell-line

People's non-small cell lung cancer (NSCLC) cell-line ((NCI-H460, NCI-H838 and NCI-A549), normal lung cell-line (CCL-75, fibroblast), MCF-7 (MCF-7 and SKBR-3), CCL188 SW480 and people's celiothelioma cancerous cell line H28 derive from American Type Culture Collection (ATCC) (Manassas, VA).Other human celiothelioma cancerous cell line NCI-H290 derive from NTH (Frederick, MD) and REN (be so kind as to give in Steven doctor's Albelda PA) laboratory for University of Pennsylvania, Philadelphia by Pennsylvania university.Normal mesothelial cell is that LP-9 derives from Harvard University (Harvard University, Boston, cell culture core institution MA).Human osteosarcoma cancerous cell line Saos-2 derives from the cell culture mechanism of UCSF.Mouse cell line of mammary gland: be so kind as to give by Frank doctor's McCormick of UCSF Cancer center laboratory with empty carrier (C57MG) with the C57MG of Wnt-1 (C57Wnt-1) transfection.Except CCL-75, LP-9 and Saos-2, these cells are cultivated in the RPMI1640 that adds 10% hyclone, 100 international unit/milliliters and 100 mcg/ml.CCL-75 cultivates in interpolation contains the MEM of Earle BSS of 2mM L-glutamic acid, 1.0mM Sodium Pyruvate, the nonessential amino acid of 0.1mM, 1.5 grams per liter sodium bicarbonates and 10% hyclone.LP-9 adds that 10 nanograms/milliliter EGF add among the M199 of 0.4 mcg/ml HC and cultivates containing 15%CS.Saos-2 cultivates in having replenished the McCoy ' s 5a medium of 2mM L-glutamic acid and 15% hyclone.Normal people's tracheole epithelial cell (SAEC) and bronchial epithelial cell (BEC) derive from Clonetics, and (Walkersville is MD) and at CloneticsSAGM ^TMCultivate in the Bullet kit.All cells is at 5%CO ₂Humidified incubator in 37 ℃ of cultivations.

Antibody and cell are hatched

Experiment cell the previous day is tiled on the 6 hole flat boards.Replace normal medium with the medium that contains multiple concentration antibody subsequently and at 5%CO ₂Humidified incubator in 37 ℃ of cultured cells.Use the conventional scheme collecting cell to be used for further analysis in different time points.The anti-Wnt-1 of purifying and anti-Wnt-2 polyclonal antibody (sheep IgG) derive from SantaCruz Biotechnology (Santa Cruz, CA).In contrast, (sheep IgG also derives from Santa CruzBiotechnology (Santa Cruz, CA)) to have used anti-SOCS-3 (SOCS-3 the is a kind of cytoplasmic protein) polyclonal antibody of purifying in parallel laboratory test.

The Western trace

Used conventional scheme as mentioned previously (Yoshikawa etc., Nat Genet, 28:29-35 (2001)).Anti-Dvl3, anti-survivin and anti-Bcl-2 antibody derive from Santa Cruz Biotechnology (Santa Cruz, CA).Anti-Guang winter enzyme 3, anti-Guang winter enzyme 9 antibody derive from Oncogene (Cambridge, MA).Anti-β actin, anti-Smac/Diablo and anti-β connection protein antibodies derive from Cell Signaling Technology, and Inc. (Beverly, MA).Anti-cell pigment C antibody derives from BD Biosciences.Anti-Active -JNK antibody derive from Promega (Madison, WI).For detecting the change of β connection albumen, kytoplasm extract and as discussed previously testing (Wang etc., Mol Cell Biol, 19:5923-5929 (1999)) have been prepared.

Apoptosis is analyzed

According to manufacturer's scheme, (Oncogene, Cambridge MA) dye by the trypsinization collecting cell and with Annexin V FITC apoptosis detection kit.Subsequently, the cell of dyeing is immediately by flow cytometer (FACScan; Decton Dickinson, Franklin Lake NJ) analyzes.Has the phosphatidylserine of exposure but the viable apoptotic cell of intact cell film combines with Annexin V-FITC and repels propidium iodide.But the cell that is in necrosis or apoptosis later stage all marks of Annexin V-FITC and propidium iodide.

The RNA interference analysis

Test and with not containing antibiotic fresh culture cell was tiled in 6 hole flat boards in preceding 24 hours.The siRNA of ion exchange HPLC purifying (siRNA of Wnt-1 siRNA and non-silence contrast, purity＞97%) available from Qiagen-Xeragon (Germantown, Maryland).The siRNAs of freeze-drying be dissolved in annealing buffer and be heated to once more 95 ℃ 1 minute, 37 ℃ of incubations are 1 hour subsequently.SiRNA analyzes as ((Elbashir etc., Methods 26,199-213,2002) carry out some improvement and carry out to previous described scheme.After the siRNA transfection, before further analyzing, hatched 3 to 5 days for dull and stereotyped 37 ℃.

In-vivo tumour suppresses research

As described in a preceding part, cultivate people NSCLC cell-line H460 and MCF-7 MCF-7.To 4 * 10 of dorsal area volume injected 100 microlitres of the female nude mice in age in 5-10 week ⁶Individual tumour cell.Subsequently with the monoclonal anti Wnt-1 antibody of 100 microlitre volumes, contrast monoclone antibody or PBS buffer solution intraperitoneal injection animal.The injected dose of monoclonal anti Wnt-1 antibody and contrast monoclone antibody is 50 micrograms.Every kind of injection is weekly.Form by 5 mouse for every group.The tumour size is determined with weekly interval according to routine techniques.

Statistical analysis

Data presented represent mean value (+S.E.M.).Be more different processing modes and cell-line, used the unpaired T check in the Excel program.

The result

Embodiment 1: anti-Wnt antibody specificity is induced the apoptosis in the multiple different people cancer cell

We have checked by using in the anti-Wnt antibody and whether the Wnt signal transduction can suppress cells survival in these cancers.When we hatch about 32 hours (we have checked 3 strain NSCLC, 2 strain breast cancer, 2 strain colorectal cancers, 1 strain sarcoma and 2 strain celiothelioma cell-lines) with multiple cancerous cell line and anti-Wnt-1 or Wnt-2 antibody (10 mcg/ml), we find that two kinds of antibody all can cause significant cell death (from 30% to 97%) (Fig. 1) except 1 strain colorectal cancer cell is SW480 (only 4-8%).On the contrary, anti-cytoplasmic protein (SOCS3) antibody (10 mcg/ml) does not show significant cytotoxicity (from 4% to 45%) (Fig. 1) in these cell-lines of the overwhelming majority.Be not have a kind of antibody that the two strain normal cells system (strain is normal lung fibroblast (CCL-75), and another strain is a normal mesothelial cell system (LP-9)) of our check is had appreciable impact (from 2% to 8%) (Fig. 1) meaningly.

In order to determine that whether the antibody-mediated cell death of anti-Wnt is because the change of apoptosis, cell with Annexin V-FITC and iodate third ingot (PI) dyeing 32 hours, carries out the apoptosis analysis with flow cytometer subsequently after antibody treatment.As shown in Figure 2, we find that most cell death is by apoptosis (from 28% to 91%) in the cancerous cell line of our check.In addition, in two strain normal cells systems, do not detect apoptosis (only 2% to 6%) behind the antibody incubation (Fig. 2).These results show, by use anti-Wnt antibody hinder the Wnt signal transduction can the specificity inducing cancer cell in but be not apoptosis in the normal cell.

Embodiment 2: the apoptosis of anti-Wnt antibody induction is expressed relevant with this Wnt

For whether the apoptotic effect of studying anti-Wnt antibody induction is relevant with the situation of this Wnt albumen, we have checked the expression of the Wnt in the cell-line that we tested.As shown in Figure 3A, we find that Wnt-1 has high level expression in the cancerous cell line of antagonism Wnt-1 antibody treatment sensitivity.Yet, handle (see figure 1) among insensitive normal lung cell-line CCL-75 at antagonist, only detect micro-Wnt-1 and express.At two kinds of elementary normal lung cells (tracheole epithelial cell (SAEC) and bronchial epithelial cell (BEC)) (Fig. 3) and the expression that does not detect Wnt-1 in normal mesothelial cell system (LP-9) (undisclosed data).Wnt-2 expresses and has obtained the similar viewing result relatively.

In contrast, we have checked the apoptosis induction that anti-Wnt antibody and anti-Wnt antibody blocking peptide are hatched altogether in the NSCLC cell-line.After hatching about 24 hours, we find that the apoptosis of anti-Wnt antibody induction can significantly be suppressed (P＜0.01) by its blocking peptide.Generally speaking, these results represent, this Wnt expresses relevant in the cell of the apoptosis of anti-Wnt antibody induction and our check.

Embodiment 3: the apoptosis of anti-Wnt-1 antibody induction is a quick process and is dose-dependent

To two kinds of NSCLC cell-line: H838 and A549 (Fig. 4 A and Fig. 4 B), we have carried out dosage and time course experiment.The flow cytometry analysis explanation of anti-Wnt antibody incubation after about 32 hours, the antibody of 1 mcg/ml can be apoptosis-induced.Arbitrary antibody of 20 mcg/ml concentration causes significant apoptotic cell death.The apoptosis that anti-Wnt-1 antibody (with the concentration of 8 mcg/ml) is induced can detect after hatching in 6 hours, after hatching in 50 hours, finds that nearly all cell stands apoptosis or necrosis.On the contrary, in parallel laboratory test, the anti-SOCS3 antibody of reference is invalid to those cancer cells.Anti-Wnt-1 antibody and normal lung cell-line (CCL-75) are hatched also insensitive to time or dosage.

Embodiment 4: the apoptosis of anti-Wnt antibody induction is relevant with the downward modulation of Dvl-3 and kytoplasm β connection albumen

The Wnt signal transduction has proved by Dvl and has activated transcribing of β connection albumen/Tcf mediation.The Wnt signal transduction is also stablized kytoplasm β connection albumen.Like this, whether our apoptosis of having measured anti-Wnt antibody induction depends on the instability of Dvl and kytoplasm β connection albumen.We find in the cancerous cell line that we checked, all significantly downward modulations of Dvl and kytoplasm β connection protein level after the anti-Wnt antibody treatment.On the contrary, in normal cell, do not find the variation of Dvl and kytoplasm β connection protein level after the anti-Wnt antibody treatment.We have also detected with the apoptosis behind the Apigenin processing cancer cell that hinders CK-1 activity (causing suppressing the Dvl activity).Apigenin handles downward modulation kytoplasm β connection protein level.These presentation of results, the apoptosis of anti-Wnt antibody induction are by suppressing the function of Wnt/Frizzled signal pathway downstream components D vl/ β connection albumen at least in part.

Embodiment 5: anti-Wnt antibody is apoptosis-induced by survivin expression downward modulation and Guang winter enzyme-3 activation subsequently

Next, we have checked the molecular mechanism of the apoptosis of this special anti-Wnt antibody induction in the cancer cell.Have been found that the activation unlatching apoptosis pathway of Guang winter enzyme-9 and the Guang winter enzyme-9 of activation pass through cutting and activate as the downstream of Guang winter enzyme-3 to carry out Guang winter enzyme amplification apoptosis pathway.Survivin (one of inhibitors of apoptosis IAP family member) plays a significant role in the activation that suppresses Guang winter enzyme-3 and Guang winter enzyme-9.In the cancer cell of antagonism Wnt antibody treatment sensitivity, (activation) the Guang winter enzyme-3 and the Guang winter enzyme-9 of cutting form are all raised.We find that also survivin expression is significantly reduced in these cells.On the contrary, be among the CCL-75 at the insensitive normal cell of antagonism Wnt antibody, we do not detect the downward modulation of going up the mediation survivin expression of the Guang winter zymoprotein of two kinds of cutting forms.These results show, anti-Wnt antibody by suppress inhibitors of apoptosis-survivin and activate Guang winter enzyme-9 and Guang winter enzyme-3 apoptosis-induced.

Embodiment 6: the apoptosis of anti-Wnt antibody induction with to cytoplasm, discharges Smac/Diablo and carefully from mitochondria Born of the same parents' pigment C and JNK activation are relevant

In apoptotic process, Smac/Diablo (the Guang winter enzyme with low pI value in the 2nd the mitochondria source/direct protein-bonded activator of IAP) function is that the Guang winter enzyme of removing the IAP mediation suppresses.The apoptosis activation causes that Smac/Diablo and cromoci are discharged into cytoplasm from mitochondrial subintimal space.Cromoci directly activates Apaf-1 and Guang winter enzyme-9, and Smac/Diablo and multiple IAP interact with the startup Guang winter enzyme of eliminating the IAP mediation and the inhibition of effect Guang winter enzyme.Consistent with above result (wherein in cancer cell but be not that Guang winter enzyme-3 is active in normal cell improve), after we find anti-Wnt antibody treatment, in the cytoplasm of cancer cell but be not that the level of Smac/Diablo and cromoci in normal cell cytoplasm improves.Our result represents that the direct activation of inhibition that Smac/Diablo and cromoci may be by eliminating survivin and/or other IAP mediation respectively and Guang winter enzyme participates in the apoptosis of anti-Wnt antibody induction.

How to regulate and control for the apoptosis of further determining this special anti-Wnt antibody induction, we have checked other compositions of apoptosis pathway.Surprisingly, the JNK activity significantly improves in the cancer cell after we find to handle.On the contrary, be the raising that does not detect the JNK activity among the CCL-75 at the insensitive normal cell of antagonism Wnt antibody.We have also found the activity of down-regulated expression JNK excessively of Dvl in normal mesothelial cell is.In addition, by using Apigenin also can improve the JNK activity with the inhibition of the Dvl of sealing CK-1 activity.In sum, relate to may be by the raising of the JNK behind the sealing Wnt signal pathway of deactivation Dvl activation and JNK activity for the apoptosis of anti-Wnt antibody induction.

Anti-Wnt antibody specificity is apoptosis-induced in the mouse mammary glandular cell of embodiment 7:Wnt-1 transfection

As reference, because these cells have been described its free β connection protein pool, we have compared the apoptotic effect that anti-Wnt antibody incubation is induced in the C57MG cell of mouse C57MG and Wnt-1 transfection.Known Wnt-1 signal transduction unlatching in the C57MG of Wnt-1 transfection cell, but in the C57GM cell of untransfected or empty carrier transfection, close.Anti-Wnt-1 antibody incubation after 42 hours flow cytometry show in the C57GM cell of untransfected or empty carrier transfection, there be not obviously to act on (being less than 10% cell death after hatching).Yet, in the C57MG of Wnt-1 transfection cell, observe significant cell death (greater than 85% cell death, P＜0.001).

The apoptosis that anti-Wnt-1 induces in the C57MG cell of Wnt-1 transfection may be also relevant with the downward modulation of kytoplasm β connection albumen with Dvl-3, and by survivin expression mediation Guang winter enzyme-3 activation subsequently down, and be discharged into the kytoplasm and the JNK activation from mitochondria by Smac/Diablo and cromoci.The C57MG cell-line of Wnt-1 transfection is as the desirable reference model of our invention, and these data provide more supports for our discovery in human cancer cell.

Embodiment 8: anti-Wnt-1 monoclone antibody is in external apoptosis-induced and inhibition in vivo in the different people cancer cell Tumor growth

Generation is at the antibody of the peptide that is derived from people Wnt-1.Especially produce hybridoma cell line with sequence numbering 2 and sequence numbering 4.A kind of monoclone antibody produces at the synthetic peptide corresponding to the 201-212 amino acid (acetate-HNNEAGRTTVFS-acid amides) of people Wnt-1.Antibody uses the a-protein affinity purification.Use this monoclone antibody to assess the expression of Wnt-1 in the multiple human cell-line.Cell-line has comprised 3 strain breast cancer cell lines (HuL100, MCF-7 and SKBR-3), 5 strain malignant mesothelioma cell-line (REN, H513, H290, MS-1, and H28), 4 strain non-small cell lung cancer (NSCLC) cell-line (A549, H460, H838, and HI703), 2 strain sarcoma cell lines (MES-SA and Saos-2), 1 strain colon carcinoma cell line Sw480 and 4 strain normal cells (tracheole epithelial cell (SAEC) and normal person's bronchial epithelial cell (NHBE), LP-9 and CCL-75).We find except not expressing or the A549 of minimum expression Wnt-1, MES-SA, and H513, outside SKBR-3 and the SW480, the Wnt-1 expression is higher in most of these cell-lines.Not observing Wnt-1 in two kinds of elementary normal lung cells (SAEC and NHBE) expresses.The Wnt-1 that only detects floor level in normal lung fibroblast CCL-75 and normal mesothelial cell system (LP-9) expresses.

As with reference to experiment, uses identical monoclone antibody we at the mouse mammary glandular cell (C57Wnt-1) of Wnt-1 transfection but less than in the empty carrier cells transfected (C57mv7) found the Wnt-1 expression.

For test described anti-Wnt-1 monoclone antibody whether can specificity in conjunction with the Wnt-1 of the native form in the cultured cells, we use monoclone antibody separately or have carried out the co-immunoprecipitation experiment by the monoclone antibody of hatching sealing with blocking peptide (30 times to the antibody amount) in advance in the cell extract of various kinds of cell system.C57Wnt-1 and C57mv7 cell are respectively as the positive and negative control.NSCLC (H460) and breast cancer (MCF-7) cell-line are also tested.Wnt-1 albumen in C57Wnt-1, H460 and the MCF-7 cell and anti-Wnt-1 monoclone antibody precipitation.On the contrary, when anti-Wnt-1 monoclone antibody and blocking peptide were hatched in advance, the ability of antibody described in these cells and the co-precipitation of Wnt-1 albumen was hindered.In negative control, there are not Wnt-1 albumen and independent anti-Wnt-1 antibody or the monoclone antibody co-precipitation of hatching in advance with blocking peptide.These data declarations, described anti-Wnt-1 monoclone antibody specificity is in conjunction with the Wnt-1 albumen of native form.

Secondly, we have handled NSCLC cell-line H460 and breast cancer cell line MCF-7 with this monoclone antibody.After hatching about 48-72 hour, we have all found tangible cell death (under 10 mcg/ml concentration greater than 60% cell death, P＜0.001) (Fig. 5 A) in these two kinds of cell-lines.Yet all do not see obvious effect in our these two kinds of cell-lines after handling with the contrast monoclone antibody.Cellular damage is because apoptosis induced (Fig. 5 B) to a great extent.The apoptosis of inducing by this monoclone antibody be dosage and time dependent (among the H460 10 mcg/ml antibody incubations after about 72 hours greater than 60% cell death, among the MCF-7 10 mcg/ml antibody incubations after about 48 hours greater than 40% cell death) (Fig. 5 C).We have also handled other and have crossed the cancerous cell line of expressing Wnt-1, comprise breast cancer HuL100, NSCLC HI703, celiothelioma H28 and REN, and sarcoma Saos-2.We have found similar result.

As the specificity contrast, we have checked among H460, MCF-7 and the HI703 by using the apoptosis induction of the monoclone antibody of sealing through blocking peptide (30 times to the antibody amount) night incubation.After hatching 48 hours, we find that the apoptosis of anti-Wnt-1 antibody induction can obviously be suppressed (P＜0.003) by its blocking peptide.The blocking peptide of same dose does not influence the existence (8.0 mcg/ml 48 hours) of these cells separately.As negative control, we use the A549 cell that does not have Wnt-1 significantly to express.Separately with monoclone antibody (8.0 mcg/ml) or use through blocking peptide (30 times to antibody) and hatch in advance and after the monoclone antibody of sealing handles about 48 hours, do not detect significant apoptosis induction.This result is consistent with the Wnt-1 expression situation of A549 cell.

Described anti-Wnt-1 monoclone antibody suppresses Wnt/ β connection protein signal approach and also activates apoptosis-induced by the Guang winter enzyme-3 that release cromoci, survivin expression are in harmonious proportion down subsequently

We find that in the cancer cell of check, anti-Wnt-1 monoclone antibody is handled back Dvl-3 and kytoplasm β connection albumen and Cyclin D1 horizontal down-regulation.We have also carried out the TOP/FOP test and have found in these cells, and after monoclone antibody was handled, the transcriptional activity that TCF relies on descended.On the contrary, in the cancer cell of normal cell or shortage (or having minimum) Wnt-1 expression, after handling, anti-Wnt-1 monoclone antibody do not find the change of the transcriptional activity that Dvl, kytoplasm β connection protein level or TCF rely on.These presentation of results, apoptosis to the small part that anti-Wnt-1 monoclone antibody is induced are by suppressing transcriptive intermediate that Dvl/ β connection albumen relies on.

In the apoptosis-induced H460 cell of anti-therein Wnt-1 monoclone antibody, we find that the Guang winter enzyme-3 of cutting (activity) form raises.Active consistent with Guang winter enzyme-3, we handle in the H460 cell matter of back in anti-Wnt-1 monoclone antibody and have detected the cromoci level that increases.In addition, we find, survivin expression downward modulation in these H460 cells after the antibody treatment.

Other people show that the Wnt-1 signal transduction is opened in the C57Wnt-1 cell, but close in the C57mv7 cell.In contrast, we have detected anti-Wnt-1 monoclone antibody and whether can suppress in the C57Wnt-1 cell transduction of Wnt/ β connection protein signal.The Western engram analysis of C57mv7 shows that anti-Wnt-1 monoclone antibody is handled kytoplasm β connection albumen and Cyclin D1 horizontal down-regulation in the C57Wnt-1 cell of (8.0 mcg/ml 48 hours) back, does not express but detect Cyclin D1 in the C57mv7 cell.After anti-Wnt-1 monoclone antibody was handled, kytoplasm β connection protein level also remained unchanged among the C57mv7.As one man, the transcriptional activity that the TCF that records by the TOP/FOP test in the C57Wnt-1 cell relies on also reduces, but remains unchanged in the C57mv7 cell.These data show, the Wnt/ β connection protein signal transduction in the cell-line that anti-Wnt-1 monoclone antibody suppresses to check.

RNA disturbs

We study the effect that reticent Wnt-1 expresses according to the scheme (Elbashir etc., Methods 26,199-213,2002) that people such as Elbashir describe by using RNAi.With described monoclonal anti Wnt-1 antibody class seemingly, with Wnt-1 siRNA handle 3-5 days apoptosis-induced in the cancerous cell line (as the MCF-7 cell) of expressing Wnt-1.100nMWnt-1 siRNA induces obvious apoptosis, but non-reticent siRNA contrast (100nM) or transfection reagent all can not be apoptosis-induced.We confirm that Wnt-1siRNA handles the silence that back (100nM 72 hours) Wnt-1 expresses by Western engram analysis (non-reticent siRNA is (100nM 72 hours) in contrast).In order to confirm whether apoptotic effect is relevant with the inhibition of Wnt-1 signal transduction, we have illustrated that also Wnt-1 siRNA handles back Dvl-3, kytoplasm β connection albumen and survivin expression horizontal down-regulation.

The inhibition of cancer growth in the body

We have tested described monoclonal anti Wnt-1 antibody and whether can suppress tumor growth in vivo subsequently.We inject nude mice respectively with H460 and MCF-7 cell.Animal is accepted 50 microgram monoclonal anti Wnt-1 antibody, contrast monoclone antibody or PBS by weekly intraperitoneal injection subsequently.Fig. 6 A shows that though control antibodies does not obviously suppress, the monoclonal anti Wnt-1 antibody of dosage significantly suppresses described two kinds of growth of tumor (P＜0.001) like this.Not only under the situation that begins the injection of monoclonal anti Wnt-1 antibody behind the tumor cell inoculation immediately (Fig. 6 A), and after setting up, tumour begins all can observe the inhibition (P＜0.005) (Fig. 6 B) of tumor growth under the situation of antibody treatment (week behind the tumor cell inoculation).In the research of using the MCF-7 cell (Fig. 6 C), handled for 3 weeks with anti-Wnt-1 monoclone antibody and contrast monoclone antibody after, the measurement gross tumor volume.5 animals in every group.Do not develop with the animal of anti-Wnt-1 monoclone antibody injection treatment and tumour.Yet, have 3 to develop and tumour in 5 control-animals.(injection is carried out once in a week in MCF-7 cell inoculation one all posterior peritoneums).

The VH and the VL region sequence of the anti-Wnt-1 monoclone antibody of using in the above-mentioned research have been measured.CDR and skeleton district from hybridoma cell line analyze by the RT-PCR amplification and by agarose gel.Described VH and VL region sequence have been shown among Fig. 7.

The apoptosis that embodiment 9:Wnt-2 expresses and the Wnt-2 monoclone antibody is induced

Analyzed Wnt-2 gene expression in multiple human tumor and the corresponding nonneoplastic tissue sample.(Inc.) hybridize by BD Biosciences with containing the cancer scanning array II of 19 kinds of dissimilar human tumors with the nonneoplastic tissue sample that matches for radiolabeled Wnt-2 cDNA probe.Compare with its normal counter pair, Wnt-2 mainly crosses in colon, stomach, rectum and thyroid tumors and expresses.

Produced at monoclone antibody corresponding to the synthetic peptide of the 49-63 amino acid (SSQRQLCHRHPDVMR) of people Wnt-2.Antibody uses a-protein to carry out affinity purification.In human melanoma FEMX and LOX cell, measured the Wnt-2 monoclone antibody to effect of apoptosis.The result shows that anti-Wnt-2 monoclone antibody is apoptosis-induced in FEMX and LOX human melanoma cell.As the anti-Wnt-1 monoclone antibody among the embodiment 8, described antibody is also induced the apoptosis in human colon carcinoma HCT-116 and the SW480 cell.

The VH and the VL region sequence of the anti-Wnt-2 monoclone antibody used in the above-mentioned research have been determined.CDR and skeleton district (FR) from hybridoma cell line analyze by the RT-PCR amplification and by Ago-Gel.The sequence that has shown VH and VL district among Fig. 7.

Embodiment 10: celiothelioma has β connection albumen by the Dvl activation and crosses expression, and the transcriptional activity of β connection albumen Form relevant with tumour

We have further studied the effect of Wnt signal transduction in celiothelioma.We find that most of celiothelioma cell is crossed expression Dvl-3.Use the western trace to study the expression of Dvl-3 and kytoplasm β connection albumen in the celiothelioma cell.The Western engram analysis shows, compares with the normal pleura contrast that its homology matches, and the kytoplasm β that 8 mistakes in 10 fresh malignant mesothelioma tissues are expressed Dvl-3 albumen and had a raising joins albumen.And the additional malignant mesothelioma cell of 5 kinds of detections (two kinds elementary MPM cultured cell with three kinds of cell-line: LRKIA, REN and H513) is compared with normal pleura contrast, has high-caliber Dvl-3 and kytoplasm β connection albumen.The immunohistochemical analysis of kinds of tumor cells has proved that kytoplasm, nuclear and film join albumen in conjunction with β.Do not have to suddenly change in the exon 3 of we find (to comprise the case and the pernicious cases of oozing out of two examples that detect with the western trace) in 13 kinds of celiothelioma tissues β connection albumen.Selecting exon 3 to carry out mutation analysis is because the NH of its beta-catenin of having encoded ₂-terminal adjustment structure territory found in the past that this domain contained the activation sudden change.In addition, do not detecting sudden change in our the complete code area in the three strain celiothelioma cell-lines (LRK1A, REN and H513) at beta-catenin.

Use the luciferase reporter gene of Tcf dependence, also checked the transcriptional activity of beta-catenin.Used the western engram analysis to confirm the expression of APC, GSK-3 β and Tcf4 in all tumours of research.By the compound-mediated transcription activity determining of Tcf-beta-catenin is that remarkable mistake is expressed in the celiothelioma cell-line of Dvl and kytoplasm beta-catenin the ratio with reporter gene activity.Cell comprises wild type or the mutant Tcf binding motif and the pRL-TK internal reference report member that contains sea cucumber luciferase (Renilla Luciferase) cDNA of polymerization with pTOPFLASH or pFOPFLASH report member transient transfection, these members in firefly luciferase cDNA upstream.PTOPFLSH after 24 hours is passed through in the genetic transcription of Tcf mediation: the ratio of pFOPFLLASH uciferase activity determines that wherein every kind of activity is proofreaied and correct at the uciferase activity of pRL-TK reporter.

Comprise that the celiothelioma cell with high-level kytoplasm beta-catenin of cell, LREL1A, REN and H513 cell-line from the MPM exudate shows the remarkable multiplication of the genetic transcription activity (pTOPFLASH/pFOPFLASH) of beta-catenin Tcf mediation (1.5-2.4 times, P＜0.01).On the contrary, the normal mesothelial cell of the minimum expression of kytoplasm beta-catenin does not show difference.

Transcriptional activity (the report detection confirms under by Gal4-beta-catenin Expression of Fusion Protein situation) according to beta-catenin in the report detection of Tcf-beta-catenin mediation with the celiothelioma cytological classification, is divided into the positive or negative transcriptional activity.In the celiothelioma cell, the kytoplasm of beta-catenin is expressed and to be added apigenin and to suppress but strengthened by wild type Dvl, and apigenin can be by the degraded of casein kinase i I and the PDZ-Dvl Dvl that degrades.In addition, PDZ-Dvl suppresses the transcriptional activity that Tcf relies on.The colony that the stably express of PDZ-Dvl suppresses to have the celiothelioma cell of the positive transcriptional activity of beta-catenin forms, and the celiothelioma cell with the negative transcriptional activity of beta-catenin shows stable colony and forms.We have confirmed that Tcf relies on transcribing and Gal4-beta-catenin fusion and celiothelioma cell in the result of tumour formation well relevant.

Dvl-3 stablizes the kytoplasm beta-catenin in the celiothelioma cell.We are verified by the Western engram analysis, show the expression of expressing with the kytoplasm beta-catenin of crossing of Dvl-3 albumen from the fresh malignant mesothelioma cell of pleural exudate.Except containing the H28 cell-line of beta-catenin district homozygous deletion, the malignant cell of the detection of the every other Dvl-3 of having high expressed shows recently and expresses from the significantly higher kytoplasm beta-catenin of the cell of normal pleura.These presentation of results, in the celiothelioma cell activation of Dvl-3 with the kytoplasm beta-catenin from film transporte to cells matter and cell nucleus.

The Tcf dependent form that beta-catenin activates in the celiothelioma cell is transcribed.Lacked beta-catenin with another kind of because homozygous deletion and express but express reporter analysis in the celiothelioma cell-line (H28) of Dvl and determine and compare by having significantly to cross in the celiothelioma cell of expressing Dvl and kytoplasm beta-catenin by the compound-mediated transcriptional activation of Tcf-beta-catenin.Cell comprises wild type or the mutant Tcf binding motif and the pRL-TK internal reference report member that contains sea cucumber luciferase (Renilla Luciferase) cDNA of polymerization with pTOPFLASH or pFOPFLASH report member transient transfection, these members in firefly luciferase cDNA upstream.PTOPFLSH after 24 hours is passed through in the genetic transcription of Tcf mediation: the ratio of pFOPFLLASH uciferase activity determines that wherein every kind of activity is proofreaied and correct at the uciferase activity of pRL-TK reporter.The celiothelioma cell of high expressed kytoplasm beta-catenin, celiothelioma patient's pernicious exudate, LRKIA and REN show 1.8-2.4 raising doubly in the transcriptional activity of pTOPFLASH reporter, H290 and H513 show 1.4-1.5 raising doubly.On the contrary, H28 and the normal mesothelial cell who does not express or express on a small quantity the kytoplasm beta-catenin do not show difference between pTOPFLASH and pFOPFLASH activity.Such result shows that beta-catenins a large amount of in the celiothelioma cell can be used as mediator.

The Gal4-beta-catenin activates pG5 in the celiothelioma cell.In addition, use Gal4-beta-catenin member to measure the transcriptional activity of beta-catenin regulation and control in the celiothelioma cell to get rid of the possibility that celiothelioma cell-line lacks required transcriptional machinery.Cotransfection is transcribed after the pSG424-Gal4-beta-catenin of Gal4-beta-catenin fusion and pG5 (CAT reports member), has measured the genetic transcription of Gal4-beta-catenin mediation.These activity are carried out standardization to get rid of the background level of activation according to the CAT activity of pG5 report member.Determine that by the Western engram analysis that uses tag antibody the Gal4-beta-catenin expresses in the celiothelioma of all transfections.LRKIA, REN and H28 show that activity has improved 10-25 doubly after the pG5 contrast transfection after with pSG424-Gal4-beta-catenin and pG5 cotransfection.The HeLa cell shows 25 times raising.On the contrary, H513 shows the raising of several times, and H290 only shows background activity.These high activities confirm, in the celiothelioma of a large amount of beta-catenins help transcriptional activity among LRKIA and the REN, but this is impossible at H290, even if it has the more overactivity that Tcf dependent form is transcribed.

Apigenin induces the degraded of Dvl, and this causes the stable of kytoplasm beta-catenin.Apigenin is by promoting the degraded of Dvl and beta-catenin to the inhibition of casein kinase i I in the galactophore epithelial cell, this causes cell inhibitory effect.By in medium, adding Apigenin has suppressed LRKIA, REN and H290 during the processing of 48 hours degraded Dvl and kytoplasm beta-catenin growth.These presentation of results are partly regulated and control the transfer of beta-catenin in the celiothelioma cell by the activation of the Dvl of casein kinase i I mediation.

PDZ-Dvl suppresses the function of endogenous Dvl in the celiothelioma cell and the stability of kytoplasm beta-catenin.Dvl albumen has 3 conserved domains: a dix domain that is present in the Wnt antagonist protein axle albumen; A DEP domain that participates in interactional PDZ domain between the albumen and in the protein of regulating Rho GTP enzyme, find.The transcriptional activation of going up mediation LEF-1 mediation of beta-catenin needs the function of three conserved domains in the mammal.According to other researchers' discovery, pCS-mouse Dvl-1 transfection 293T cell causes the genetic transcription activity of the Tcf mediation of beta-catenin to improve 15 times.This activity is suppressed by the pCS-cDNA cotransfection of pCS-mouse Dvl-1 member by coded delta PDZDvl-1.And, the transcriptional activity of the beta-catenin that Tcf relies among the LRKIA is reduced (from 2.1 times to 1.3 times by the transfection of pCS-Δ PDZ-Dvl-1, P＜0.05), yet the transfection of pCS-Dvl-1 has strengthened the transcriptional activity of the beta-catenin that Tcf relies on (from 2.1 times to 3.8 times, P＜0.05), this show beta-catenin in these cells Tcf mediation transcribe obviously to be regulated and control by Dvl.b.

In order to check additional Wnt pathway activation in the MPM, we distinguish transfection LRK1A, REN and H513 cell-line with retrovirus with Δ PDZ-Dvl-1 and wild type Dvl-1.The expression of Δ PDZ-Dvl-1 albumen is induced in the retrovirus transfection of pLXN-Δ PDZ-Dvl-1, and this albumen is obviously compared according to the expression (P＜0.05) that has reduced the kytoplasm beta-catenin in the cell of all detections.These presentation of results, Dvl regulates the kytoplasm beta-catenin in the celiothelioma cell.

Use Atlas human cancer 1.2 arrays to show that the c-myc expression is reduced by Δ PDZ-Dvl-1 transfection among the REN.On the other hand, use the Western engram analysis, verified is that the COX-2 of a target gene of Wnt/ beta-catenin approach is reduced by Δ PDZ-Dvl-1 transfection.

Δ PDZ-Dvl transfection suppresses celiothelioma cell-line in soft agar and athymic mouse tumour forms

We checked Dvl/ beta-catenin approach with MPM cell-line in effect in the relation of cell growth.We induce Δ PDZ-Dvl-1 to express in LRK1A, REN and H513 by the retrovirus transfection, use empty carrier in contrast.Select the back cell to be tiled on 0.35% the soft agar and colony count after 28 days.Compared with the control, LRK1A and the colony among the REN with Δ PDZ-Dvl-1 transfection forms remarkable decline (P＜0.01).H513 can not grow on soft agar.In addition, compared with the control, growth in the body of LRK1A and REN hypodermic tumour in the athymic mouse (P＜0.05 and P＜0.005 have respectively significantly been suppressed with Δ PDZ-Dvl-1 transfection with mutant; Fig. 8).

The effect of embodiment 11:Dvl activation in non-small cell lung cancer

We have checked the effect of Dvl activation in non-small cell lung cancer (NSCLC) subsequently.Present embodiment explanation, Dvl-3 crosses in the NSCLC cell-line of the NSCLC of fresh excision and foundation and expresses.It is the additional evidence that is caused by upstream incident (expressing as Dvl) that embodiment also provides the Wnt signal transduction by the beta-catenin approach of standard.

In order to estimate the function of Wnt signal transduction among the NSCLC, we have analyzed Dvl and have expressed and function.The normal lung tissue of fresh tumour of 8 routine NSCLC (4 routine squamous cell carcinomas and 4 routine gland cancer) and homology coupling thereof derives from and accepts the patient of tumor resection as the part that early stage I phase NSCLC is treated.The patient does not accept any previous tretament (for example chemotherapy).The Western engram analysis of these samples shows that 75% (3 examples in example of 3 in the 4 routine squamous cell carcinomas and the 4 routine gland cancer) of the cancer cell of all detections are crossed expression Dvl-3, and the normal lung tissue of corresponding coupling does not show the expression of Dvl-3.And, by the Western engram analysis, 6 examples cross 5 examples in the NSCLC tumour express Dvl-3 show Wnt-1 or Wnt-2 than high expressed.Do not detect the expression of Dvl-1 or Dvl-2.

Be further check Dvl function, we have synthesized the siRNA (siRNA) that can suppress Dvl-1 ,-2 and-3 Dvl.We have detected the function of Dvl in lung cancer cell line HI703 by handling with Dvl siRNA and contrast siRNA.We have selected HI703 to be because its transcriptional activity of expressing Dvl-3 and showing the beta-catenin of Tcf dependence.After siRNA handled, Dvl-3 expressed and is suppressed, and Dvl-1 and Dvl-2 still do not express simultaneously.It may be noted that in the cell of handling be accompanied by the remarkable reduction (P＜0.05) of the transcriptional activity of Tcf dependence, beta-catenin is expressed corresponding reduction.Finally, the siRNA of Dvls has obviously suppressed H1703 cell growth (P＜0.05) (Fig. 9) in 24 orifice plates.In addition, the colony in the 100mm plate forms also and is obviously suppressed (P＜0.05).In compare other cell-lines (such as A549 (lung cancer cell line) and SW480 (because the APC sudden change makes the colon carcinoma cell line of Wnt signal pathway abnormal activation)) that have lower level Dvl and express with H1703, the cell growth is not subjected to the influence of Dvl siRNA.

Discuss

As mentioned above, the effect in human tumor forms is known little about the Wnt part.Here the data declaration that is represented, Wnt signal in the human cancer cell, bring into play the cause effect from but the treatment of cancer target spot.

The data declaration that provides above, anti-Wnt-1 and anti-Wnt-2 antibody can be induced the apoptosis among the human cancer cell.And our data representation, antitumor action are that the sealing by the Wnt signal pathway causes.The apoptotic cell death of anti-Wnt antibody induction is not only relevant with the Wnt protein expression, and consistent with Dvl and kytoplasm beta-catenin expression decreased in the human tumor cells that detects.On the contrary, in normal cell system, Dvl and kytoplasm beta-catenin are kept par after the anti-Wnt antibody treatment.Antibody does not show detectable effect to normal cell-line, and this illustrates in anti-Wnt-1 or the anti-Wnt-2 antibody capable specificity inducing cancer cell but is not apoptosis in the normal cell.In view of polyclonal antibody may produce non-specific action, we have used anti-Wnt-1 monoclone antibody further to study the specificity of anti-Wnt antibody effect.Anti-Wnt-1 monoclone antibody can be apoptosis-induced in crossing the human cancer cell line (for example Human Lung Cancer cell-line H460 and human breast cancer cell line MCF-7) who expresses Wnt-1 albumen.Similar with the result who obtains from polyclonal antibody research, in these tumour cells, after anti-Wnt-1 monoclone antibody was handled, Dvl and kytoplasm beta-catenin reduced.Yet, anti-Wnt-1 monoclone antibody shows much higher specificity than anti-Wnt-1 polyclonal antibody, for example, anti-Wnt-1 monoclone antibody is only apoptosis-induced in crossing the tumour cell (H460 and MCF-7) of expressing Wnt-1 albumen, and does not have detectable effect in the tumour cell of expressing Wnt-2 albumen; Anti-Wnt-1 polyclonal antibody is apoptosis-induced cell death in crossing the tumour cell of expressing Wnt-1 or Wnt-2.In sum, these data show, anti-Wnt antibody treatment can the inducing tumor-specific apoptosis and reduced Wnt-Dvl-β connection protein signal approach in the human cancer cell.

By Frizzled acceptor and Dvl albumen, two different approaches of Wnt signal activation: normative approach (being β connection albumen approach) and JNK approach.Dvl albumen has the domain of 3 high conservatives: DIX, PDZ and DEP.Wherein, DIX and PDZ domain are that normative approach is necessary, and the DEP domain is important for the activation of JNK approach.Shown that JNK activates play a crucial role (Wang etc., Mol Cell Biol, 19:5923-5929 (1999)) in starting apoptosis.Recently, proofs such as Chen, Wnt-1 transcribes inhibition apoptosis (Chen etc., J Cell Biol, 152:87-96 (2001)) by activating β connection albumen and TCF.In this research, after the anti-Wnt antibody treatment, both observed β connection albumen and crossed expression, by having observed the JNK activity that improves, this explanation normative approach and JNK approach have all participated in the apoptosis of anti-Wnt antibody induction.In addition, the Dvl JNK activity of having crossed down-regulated expression in the normal mesothelial cell system, and by using Apigenin to improve the JNK activity with the inhibition of the Dvl of sealing CK-1 activity.Probably, after the anti-Wnt antibody treatment activation of JNK by Dvl.

In addition, the Tcf that the inhibition that Dvl expresses in the NSCLC cell of siRNA mediation has reduced the beta-catenin mediation transcribes, and this further is supported in Dvl in some lung carcinoma cell, and to cross expression be important for the Wnt/ beta-catenin approach of standard.Cell growth and colony that Dvl suppresses also to have suppressed in the NSCLC cell form, and this illustrates that upstream incident unusual in the Wnt signal transduction forms relevant with tumour among the NSCLC.

By the Dvl degraded of siRNA mediation caused H1703 but be not the A549 cell growth inhibition.The both is the pinacocyte lung cancer cell line, and A549 has wild type p53 but H1703 has the p53 of sudden change deactivation.Like this, the state of p53 at least can the pinacocyte lung cancer cell line of these two kinds of processing of partial interpretation between the difference of Dvl function.

In order to further specify anti-Wnt antibody induction mechanism of apoptosis in the human cancer cell, we have checked other possible compositions in the apoptosis pathway.For example, having detected Smac/Diablo in these tumour cells with the Wnt antibody treatment is discharged in the cytoplasm.Smac/Diablo (the Guang winter enzyme with low pI value in the 2nd kind of mitochondria source/direct IAP is in conjunction with the albumen activity factor) (Du etc., Cell, 102:33-42 (2000); Verhagen etc., Cell, 102:43-53 (2000)) suppress to work by the Guang winter enzyme that discharges the IAP mediation.The activation of apoptosis causes that Smac/Diablo is discharged into cytoplasm together with cromoci from the mitochondrial inner membrane space.Cromoci directly activate Apaf-1 and Guang winter enzyme-9 and Smac/Diablo and a plurality of IAPs interact with remove the IAP mediation to starting and inhibition (Chai etc., Nature, the 406:855-862 (2000) of effect Guang winter enzyme; Srinivasula etc., JBiol Chem, 275:36152-36157 (2000)).With in the above-mentioned cancer cell but be not that the active result who improves of Guang winter enzyme-3 is consistent in the normal cell, we find after the anti-Wnt antibody treatment at cancer cell but are not the raisings of the level of Smac/Diablo and cromoci in the Normocellular cytoplasm.Our result shows, reaches by the inhibition of removing survivin and/or other IAPs mediation respectively and directly activates Guang winter enzyme, and Smac/Diablo and cromoci all may participate in the apoptosis of this anti-Wnt antibody induction.

More than find explanation, Wnt antibody may not only directly be induced the apoptosis in the cancer cell of expressing Wnt albumen, and removed possible pesticide resistance by the normal apoptotic machine that recovers these tumour cells.The most likely destruction of apoptosis mechanism of drug-fast basis in the tumour cell.Expressing excessively of inhibitors of apoptosis survivin is the common characteristic of most of human cancer.Verified, the targeting of survivin is improved the sensitivity of tumour cell pair cell cytotoxic drug and antisense survivin is enough to cause apoptosis in people's celiothelioma cell.And, also reported the synergistic effect between antisense survivin and the chemotherapy.

We are verified, and the Wnt antibody treatment significantly reduces the level of survivin.In sum, the Wnt antibody capable strengthens and works in coordination with the effect of conventional chemotherapy among the reinforcement human cancer cell.

Other antagonists of Wnt signal or Frizzled acceptor also can be apoptosis-induced by Dvl.For example, sFRP as the solubility regulatory factor of Wnt signal transduction by combine secreting type Wnt part performance function (Melkonyan etc., Proc Natl Acad Sci USA, 94:13636-13641 (1997)) with the Frizzled receptor competition.Specifically, sFRP can be by with the Wnt protein combination and hinder the function that it comes antagonism Wnt near cell surface signal transduction acceptor, maybe can be by promoting that offering part to the Frizzled acceptor strengthens Wnt activity (Uthoff etc., Int J Oncol, 19:803-810 (2001)).Frizzled receptor antagonist (for example, at the specific antibody of its ectodomain or at the little molecule of specificity of born of the same parents' intracellular domain) can be induced the apoptosis among the human cancer cell who expresses Wnt/Frizzled albumen.Really, Figure 10 shows that the mistake of Wnt signal antagonist FRP or DKK is expressed in the cancer cell apoptosis-induced.Thereby, such antagonist also can be used for the treatment of cancer, for example lung cancer, celiothelioma, breast cancer, colorectal cancer, cervical carcinoma, oophoroma, prostate cancer, cancer of pancreas, cancer of the stomach, cancer of the esophagus, head and neck cancer, hepatocellular carcinoma, melanoma, glioma, spongioblastoma, leukemia or lymphoma.

In a word, our result shows that the Wnt monoclone antibody may can be induced the tumor specificity antiapoptotic among the human cancer cell by normative approach and JNK approach.Our data declaration, for treatment of cancer, Wnt/Frizzled is useful treatment target spot, and shows that from the result of heterograft mouse model the Wnt monoclone antibody is the good candidate reagent of cancer target treatment of cancer.

The reticent mechanism analysis of embodiment 12:Dachsous (ds) gene and Fat gene and in the human cancer tissue The adjusting of wnt-frizzled signal pathway

Adjusting machine about Wnt-Fz signal transduction in the cancerous tissue is known little.Dachsous (Ds) and Fat albumen are two members (Mahoney etc., Cell 67:853-868,1991 of cadherins (cadherin) superfamily; Clark, H.F etc., Genes Dev, 9:1530-1542,1995).Data show that they have participated in the developmental Fz signal transduction of fruit bat (Yang etc., Cell, 108:675-688,2002).The report that in cancerous tissue, acts on about Ds and Fat not.

In the present embodiment, we have illustrated Fat up-regulated and Ds down-regulated expression in fresh human cancer tissue (comprising lung cancer and celiothelioma) and human cell system (comprising breast cancer, colon cancer, lung cancer and celiothelioma).We have also differentiated the abnormal methylation in the CpG island of the Ds promoter region relevant with Ds Transcriptional Silencing in the human cancer tissue.In addition, we find, active rise and the downward modulation of Ds with Fat of Fz is relevant.The recovery of Ds and the sealing of Fat can be regulated the active of Fz signal pathway and anticancer growth.

The present invention greatly helps the therapeutic strategy for the treatment of human cancer.For example, use said method, can seal the Fat activity.Such method comprises, for example, seals Fat transcribes and/or it is active antisense oligonucleotides or little compound molecule.In addition, use known gene therapy method, can recover the Ds activity.In addition, the present invention can be used as diagnostic tool.Methylating is one of early stage incident of cancer formation.The technology that use is known (for example, methylation status of PTEN promoter) can be used for early diagnosis of cancer to the detection that methylates on the CpG island of Ds promoter region.

The hereditary change of embodiment 13:Frizzled (fz) gene and LRP (LDL associated protein) gene and in cancer, making With distinct methods the mutant and/or the clipped form of these acceptors carried out target

LRPs (LDL associated protein; LRP-1 is to-5) be the co-receptor of Wnt part.Show that Wnt, Fz and LRP albumen is high expressed (Liu etc., Cancer Res, 60:1961-1967,2000 in multiple cancerous tissue (comprising breast cancer, colon cancer, lung cancer or the like); Laurencot etc., Int J Cancer, 72:1021-1026,1997; Berger, W etc., Int J Cancer, 88:293-300,2000; Schneider etc., Breast Cancer Res, 3:183-191,2001; Schneider etc., Anticancer Res, 20:4373-4377,2000).In addition.This signal is considered to composing type unlatching downstream transcriptional activity in cancer.Yet it is unknown that the signal transduction mechanism that this composing type is opened in the cancer remains.

In the present embodiment, we have illustrated that the hereditary change in frizzled (fz) gene and/or LRP (LDL associated protein) gene causes all Fz acceptors and/or the mutant of LRP co-receptor (domain that born of the same parents are outer, that stride film and/or born of the same parents are interior) and/or the formation of clipped form in the cancerous tissue.The cancer type that we detect comprises breast cancer, colon cancer, prostate cancer, lung cancer, celiothelioma and sarcoma.Above-mentioned hereditary change comprises that chromosome deletion (isozygoty or heterozygosis), chromosome translocation, chromosomal break, chromosome inversion, inside lack on a small quantity, insertion and point mutation.The sudden change of these Fz acceptors and/or LRP co-receptor and/or clipped form cause the composing type signal transduction regardless of the existence of Wnt part, and this has caused the downstream transcriptional activity of composing type in the cancerous tissue successively.On the contrary, the mutant form that in normal structure/cell, does not have Fz acceptor and/or LRP co-receptor.

The present invention shows that the Fz acceptor of Wnt signal pathway and/or the sudden change of LRP co-receptor and/or clipped form are that cancer is specific.They have the very big possibility as exploitation curative drug (for example, aforesaid little molecule, compound, antibody, antisense oligonucleotides or RNAi) target spot.These medicines are target cancerous tissue rather than normal cell only.Thereby the present invention can have very great help to treating the multiple treatment for cancer strategy tool of colon cancer, breast cancer, lung cancer (as NSCLC), celiothelioma and sarcoma or the like that comprises as mentioned above.

Need to understand, embodiment described here and embodiment are proficient within the spirit and scope and appended claims scope that multiple transformation that those skilled in the art propose in view of the above or change be included in the application only for purposes of illustration.All publications, patent and the patent application of being quoted here by with reference to intactly, all sidedly in conjunction with in this application.

Sequence table

Sequence table

Seq ID No:1People Wnt-1 peptide sequence #1

MGLWALLPGWVSATLLLALAALPAALAANSSGRWWGIVNVASSTNLLTDSKSLQLVLEPS

LQLLSRKQRRLIRQNPGILHSVSGGLQSAVRECKWQFRNRRWNCPGAPGPHLFGKIVNRG

CRETAFIFAITSAGVTHSVARSCSEGSIESCTCDYRRRGPGGPDWHWGGCSDNIDFGRLF

GREFVDSGEKGRDLRFLMNLHNNEAGRTTVFSEMRQECKCHGMSGSCTVRTCWMRLPTLR

AVGDVLRDRFDGASRVLYGNRGSNRASRAELLRLEPEDPAHKPPSPHDLVYFEKSPNFCT

YSGRLGTAGTAGRACNSSSPALDGCELLCCGRGHRTRTQRVTERCNCTFHWCCHVSCRNC

THTRVLHECL

Seq ID No:2People Wnt-1 peptide sequence #2

39?NVASSTNLLTDSKS(C)52

Seq ID No:3People Wnt-1 peptide sequence #3

131?SAGVTHSVARSC?142

Seq ID No:4People Wnt-1 peptide sequence #4

200?HNNEAGRTTVFS(C)212

Seq ID No:5People Wnt-1 peptide sequence #5

274?LEPEDPAHKPPSP(C)286

Seq ID No:6People Wnt-1 peptide sequence #6

332?DGCELLCCGRGHRTRTQVTERC347

Seq ID No:7People Wnt-1 peptide sequence #7

354?HVSCRNCTHTRVLHHECL?370

Seq ID No:8People Wnt-2 peptide sequence #1

MNAPLGGIWLWLPLLLTWLTPEVNSSWWYMRATGGSSRVMCDNVPGLVSSQRQLCHRHPD

VMRAISQGVAEWTAECQHQFRQHRWNCNTLDRDHSLFGRVLLRSSRESAFVYAISSAGVV

FAITRACSQGEVKSCSCDPKKMGSAKDSKGIFDWGGCSDNIDYGIKFARAFVDAKERKGK

DARALMNLHNNRAGRKAVKRFLKQECKCHGVSGSCTLRTCWLAMADFRKTGDYLWRKYNG

AIQVVMNQDGTGFTVANERFKKPTKNDLVYFENSPDYCIRDREAGSLGTAGRVCNLTSRG

MDSCEVMCCGRGYDTSHVTRMTKCGCKFHWCCAVRCQDCLEALDVHTCKAPKNADWTTAT

Seq ID No:9People Wnt-2 peptide sequence #2

49?SSQRQLCHRHPDVMR?63

Seq ID No:10People Wnt-2 peptide sequence #3

137?CDPKKMGSAKDSKG150

Seq ID No:11People Wnt-2 peptide sequence #4

171?VDAKERKGKDAR(C)183

Seq ID No:12People Wnt-2 peptide sequence #5

344?DVHTCKAPKNADWTTAT(C)360

Seq ID No:13People Wnt-3 peptide sequence #1

MEPHLLGLLLGLLLGGTRVLAGYPIWWSLALGQQYTSLGSQPLLCGSIPGLVPKQLRFCR

NYIEIMPSVAEGVKLGIQECQHQFRGRRWNCTTIDDSLAIFGPVLDKATRESAFVHALAS

AGVAFAVTRSCAEGTSTICGCDSHHKGPPGEGWKWGGCSEDADFGVLVSREFADARENRP

DARSAMNKHNNEAGRTTILDHMHLKCKCHGLSGSCEVKTCWWAQPDFRAIGDFLKDKYDS

ASEMVVEKHRESRGWVETLRAKYSLFKPPTERDLVYYENSPNFCEPNPETGSFGTRDRTC

NVTSHGIDGCDLLCCGRGHNTRTEKRKEKCHCIFHWCCYVSCQECIPJYDVHTCK

Seq ID No:14People Wnt-3A peptide sequence #1

MAPLGYFLLLCSLKQALGSYPIWWSLAVGPQYSSLGSQPILCASIPGIVPKQLRFCRNYV

EIMPSVAEGIKIGIQECQHQFRGRRWNCTTVHDSLAIFGPVLDKATRFSAFVHAIASAGV

AFAVTRSCAEGTAAICGCSSRHQGSPGKGWKWGGCSEDIEFGGMVSREFADARENRPDAR

SAMNRHNNEAGRQAIASHMHLKCKCHGLSGSCEVKTCWWSQPDFPAIGDFLKDKYDSASE

MVVEKHRESRGWVETLRPRYTYFKVPTERDLVYYEASPNFCEPNPETGSFGTRDRTCNVS

SHGIDGCDLLCCGRGHNARAERRREKCRCVFHWCCYVSCQECTRVVDVHTCK

Seq ID No:15People Wnt-4 peptide sequence

MSPRSCLRSLRLLVFAVFSAAASNWLYLAKLSSVGSISEEETCEKLKGLIQRQVQMCKRN

LEVMDSVRRGAQLAIEECQYQFRNRRWNCSILDSLPVFGKVVTQGTREAAFVYAISSAGV

AFAVTRACSSGELEKCGCDRTVHGVSPQGFQWSGCSDNIAYGVAFSQSFVDVRERSKGAS

SSRaLMNLHNNEAGRKAILTHMRVECKCHGVSGSCEVKTCWRAVPPFRQVGHALKEKFDG

ATEVEPRRVGSSRALVPRNAQFKPHTDEDLVYLEPSPDFCEQDMRSGVLGTRGRTCNKTS

KAIDGCELLCCGRGFHTAQVELAERCSCKFHWCCFVKCRQCQRLYELHTCR

Seq ID No:16People Wnt-5 peptide sequence

MAGSAMSSKFFLVALAIFFSFAQVVIEANSWWSLGMNNPVQMSEVYIIGAQPLCSQLAGL

SQGQKKLCHLYQDHMQYIGEGAKTGIKECQYQFRHRRWNCSTVDNTSVFGRVMQIGSRET

AFTYAVSAAGVVNAMSRACREGELSTCGCSRAARPKDLPRDWLWGGCGDNIDYGYRFAKE

FVDARERERIHAKGSYESARILMNLHNNEAGRRTVYNLADVACKCHGVSGSCSLKTCWLQ

LADFRKVGDALKEKYDSAAAMRLNSRGKLVQVNSRFNSPTTQDLVYIDPSPDYCVRNEST

GSLGTQGRLCNKTSEGMDGCELMCCGRGYDQFKTVQTERCHCKFEWCCYVKCKKCTEIVD

QFVCK

Seq ID No:17People Wnt-5B peptide sequence

MPSLLLLFTAALLSSWAQLLTDANSWWSLALNPVQRPEMFIIGAQPVCSQLPGLSPGQRK

LCQLYQEHMAYIGEGAKTGIKECQHQFRQRRWNCSTADNASVFGRVMQIGSRETAFTHAV

SAAGVVNAISRACREGELSTCGCSRTARPKDLPRDWLWGGCGDNVEYGYRFAKEFVDARE

REKNFAKGSEEQGRVLMNLQNNEAGRRAVYKMADVACKCHGVSGSCSLKTCWLQLAEFRK

VGDRLKEKYDSAAAMRVTRKGRLELVNSRFTQPTPEDLVYVDPSPDYCLRNESTGSLGTQ

GRLCNKTSEGMDGCELMCCGRGYNQFKSVQVERCHCKFHWCCFVRCKKCTEIVDQYICK

Seq ID No:18People Wnt-6 peptide sequence

MLPPLPSRLGLLLLLLLCPAHVGGLWWAVGSPLVMDPTSICRKARRLAGRQAELCQAEPE

VVAELARGARLGVRECQFQFRFRRWNCSSHSKAFGRILQQDIRETAFVFAITAAGASHAV

TQACSMGELLQCGCQAPRGRAPPRPSGLPGTPGPPGPAGSPEGSAAWEWGGCGDDVDFGD

EKSRLFMDARHKRGRGDIRALVQLHNNEAGRLAVRSHTRTECKCHGLSGSCALRTCWQKL

PPFREVGARLLERFHGASRVMGTNDGKALLPAVRTLKPPGRADLLYAADSPDFCAPNRRT

GSPGTRGRACNSSAPDLSGCDLLCCGRGHRQESVQLEENCLCRFHWCCVVQCHRCRVRKE

LSLCL

Seq ID No:19People Wnt-7A peptide sequence

MNRKALRCLGHLFLSLGMVCLRIGGFSSVVALGATIICNKIPGLAPRQRAICQSRPDAII

VIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSRDGAFTYAIIAAGVAHAIT

AACTHGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLM

NLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGVVLKDKYNEAVHVEP

VRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQ

ASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK

Seq ID No:20People Wnt-7B peptide sequence

MHRNFRKWIFYVFLCFGVLYVKLGALSSVVALGANIICNKIPGLAPRQRAICQSRPDAII

VIGEGAQMGINECQYQFRFGRWNCSALGEKTVFGQELRVGSREAAFTYAITAAGVAHAVT

AACSQGNLSNCGCDREKQGYYNQAEGWKWGGCSADVRYGIDFSRRFVDAREIKKNARRLM

NLHNNEAGRKVLEDRMQLECKCHGVSGSCTTKTCWTTLPKFREVGBLLKEKYNAAVQVEV

VRASRLRQPTFLRIKQLRSYQKPMETDLVYIEKSPNYCEEDAATGSVGTQGRLCNRTSPG

ADGCDTMCCGRGYNTHQYTKVWQCNCKFHWCCFVKCNTCSERTEVFTCK

Seq ID No:21People Wnt-8A peptide sequence

MGNLFMLWAALGICCAAFSASAWSVNNFLITGPKAYLTYTTSVALGAQSGIEECKFQFAW

ERWNCPENALQLSTHNRLRSATRETSFIHAISSAGVMYIITKNCSMGDFENCGCDGSNNG

KTGGHGWIWGGCSDNVEFGERISKLFVDSLEKGKDARALMNLHNNRAGRLAVRATMKRTC

KCHGISGSCSIQTCWLQLAEFREMGDDYLAKYDQALKIEMDKRQLRAGNSAEGHWVPAEA

FLPSAEAELIFLEESPDYCTCNSSLGIYGTEGRECLQNSHNTSRWERRSCGRLCTECGLQ

VEERKTEVLSSCNCKFQWCCTVKCDQCRHVVSKYYCARSPGSAQSLGRVWFGVYI

Seq IS No:22People Wnt-8B peptide sequence

MFLSKPSVYICLFTCVLQLSHSWSVNNFLMTGPKAYLIYSSSVAAGAQSGIEECKYQFAW

DRWNCPERALQLSSHGGLRSANRETAFVHAISSAGVMYTLTRNCSIGDFDNCGCDDSRNG

QLGGQGWLWGGCSDNVGFGEAISKQFVDALETGQDARAAMNLHNNEAGRKAVKGTMKRTC

KCHGVSGSCTTQTCWLQLPEFREVGAHLKEKYHAALKVDLLQGAGNSAAARGAIADTFRS

ISTRELVHLEDSPDYCLENKTLGLLGTEGRECLRRGRALGRWELRSCRRLCGDCGLAVEERRAETVSSC

NCKFHWCCAVRCEQCRRRVTKYFCSRAERPRGGAAHKPGRKP

Seq ID No:23People Wnt-10A peptide sequence

MGSAHPRPWLRLRPQPQPRPALWVLLFFLLLLAAAMPRSAPNDILDLRLPPEPVLNANTV

CLTLPGLSRRQMEVCVRHPDVAASAIQGIQIAIHECQHQFRDQRWNCSSLETRNKIPYES

PIFSRGFRESAFAYAIAAAGVVHAVSNACALGKLKACGCDASRRGDEEAFRRKLHRLQLD

ALQRRGKGLSHGVPEHPALPTASPGLQDSWEWGGCSPDMGFGERFSKDFDSREPHRDIHA

RMRLHNNRVGRQAVMENMRRKCKCHGTSGSCQLKTCWQVTPEFRTVGALLRSRFHRATLI

RPHNRNGGQLEPGPAGAPSPAPGAPGPRRRASPADLVYFEKSPDFCEREPRLDSAGTVGR

LCNKSSAGSDGCGSMCCGRGHNILRQTRSERCHCRFHWCCFVVCEECRITEWVSVCK

Seq ID No:24People Wnt-10B peptide sequence

MLEEPRPRPPPSGLAGLLFLALCSRALSNEILGLKLPGEPPLTANTVCLTLSGLSKRQLG

LCLRNPDVTASALQGLHIAVHECQHQLRDQRWNCSALEGGGRLPHHSAILKRGFRESAFS

FSMLAAGVMHAVATACSLGKLVSCGCGWKGSGEQDRLRAKLLQLQALSRGKSFPHSLPSP

GPGSSPSPGPQDTWEWGGCNHDMDFGEKFSRDFLDSREAPRDIQARMRIHNNRVGRQVVT

ENLKRKCKCHGTSGSCQFKTCWRAAPEFRAVGAALRERLGRAIFIDTHNRNSGAFQPRLR

PRRLSGELVYFEKSPDFCERDPTMGSPGTRGRACNKTSRLLDGCGSLCCGRGHNVLRQTR

VERCHCRFHWCCYVLCDECKVTEWVNVCK

Seq ID No:25People Wnt-11 peptide sequence

MRARPQVCEALLFALALQTGVCYGIKWLALSKTPSALALNQTQHCKQLEGLVSAQVQLCR

SNLELMHTVVHAAREVMKACRRAFADMRWNCSSIELAPNYLLDLERGTRESAFVYALSAA

TISHAIARACTSGDLPGCSCGPVPGEPPGPGNRWGRCADNLSYGLLMGAKFSDAPMKVKK

TGSQANKLMRLHNSEVGRQALRASLEMKCKCHGVSGSCSIRTCWKGLQELQDVAADLKTR

YLSATKVVHRPMGTRKHLVPKDLDIRPVKDWELVYLQSSPDFCMKNEKVGSHGTQDRQCN

KTSNGSDSCDLMCCGRGYNPYTDRVVERCHCKYHWCCYVTCRRCRTVERYVCK

Seq ID No:26People Wnt-12 peptide sequence

MLEEPRPRPPPSGLAGLLFLALCSRALSNEILGLKLPGEPPLTANTVCLTLSGLSKRQLG

LCLRNPDVTASALQGLHIAVHECQHQLRDQRWNCSALEGGGRLPHHSAILKRGFRESAFS

FSMLAAGVMHAVATACSLGKLVSCGCGWKGSGEQDRLRAKLLQLQALSRGKSFPHSLPSP

GPGSSPSPGPQDTWEWGGCNHDMDFGEKFSRDFLDSREAPRDIQARMRIHNNRVGRQVVT

ENLKRKCKCHGTSGSCQFKTCWRAAPEFRAVGAALRERLGRAIFIDTHNRNSGAFQPRLR

PRRLSGELVYFEKSPDFCERDPTMGSPGTRGRACNKTSRLLDGCGSLCCGRGHNVLRQTR

VERCHCRFHWCCYVLCDECKVTEWVNVCK

Seq ID No:27People Wnt-13 peptide sequence

MLRPGGAEEAAQLPLRRASAPVPVPSPAAPDGSRASARLGLACLLLLLLLTLPARVDTSW

WYTGALGARVICDNIPGLVSRQRQLCQRYPDIMRSVGEGAREWIRECQHQFRHHRWNCTT

LDRDHTVFGRVMLRSSREAAFVYAISSAGVVHAITRACSQGELSVCSCDPYTRGRHHDQR

GDFDWGGCSDNIHYGVRFAKAFVDAKEKRLKDARALMNLHNNRCGRTAVRRFLKLECKCH

GVSGSCTLRTCWRALSDFRRTGDYLRRRYDGAVQVMATQDGANFTAARQGYRRATRTDLV

YFDNSPDYCVLDKAAGSLGTAGRVCSKTSKGTDGCEIMCCGRGYDTTRVTRVTQCECKFH

WCCAVRCKECRNTVDVHTCKAPKKAEWLDQT

Seq ID No:28People Wnt-14 peptide sequence

MLDGSPLARWLAAAFGLTLLLAALRPSAAYFGLTGSEPLTILPLTLEPEAAAQAHYKACD

RLKLERKQRRMCRRDPGVAETLVEAVSMSALECQFQFRFERWNCTLEGRYRASLLKRGFK

ETAFLYAISSAGLTHALAKACSAGRMERCTCDEAPDLENREAWQWGGCGDNLKYSSKFVK

EFLGRRSSKDLRARVDFHNNLVGVKVIKAGVETTCKCHGVSGSCTVRTCWRQLAPFHEVG

KHLKHKYETALKVGSTTNEAAGEAGAISPPRGRASGAGGSDPLPRTPELVHLDDSPSFCL

AGRFSPGTAGRRCHREKNCESICCGRGHNTQSRVVTRPCQCQVRWCCYVECRQCTQREEV

YTCKG

Seq ID No:29People Wnt-15 peptide sequence

MRPPPALALAGLCLLALPAAAASYFGLTGREVLTPFPGLGTAAAPAQGGAHLKQCDLLKL

SRRQKQLCRREPGLAETLRDAAHLGLLECQFQFRHERWNCSLEGRTGLLKRGFKETAFLY

AVSSAALTHTLARACSAGRMERCTCDDSPGLESRQAWQWGVCGDNLKYSTKFLSNFLGSK

RGNKDLRARADAHNTHVGIKAVKSGLRTTCKCHGVSGSCAVRTCWKQLSPFRETGQVLKL

RYDSAVKVSSATNEALGRLELWAPARQGSLTKGLAPRSGDLVYMEDSPSFCRPSKYSPGT

AGRVCSREASCSSLCCGRGYDTQSRLVAFSCHCQVQWCCYVECQQCVQEELVYTCKH

Seq ID No:30People Wnt-16 peptide sequence

MERHPPMQLTTCLRETLFTGASQKTSLWWLGIASFGVPEKLGCANLPLNSRQKELCKRKP

YLLPSIREGARLGIQECRSQFRHERWNCMITAAATTAPMGASPLFGYELSSGTKETAFIY

AVMAAGLVHSVTRSCSAGNMTECSCDTTLQNGGSASEGWHWGGCSDDVQYGMWFSRKFLD

FPIGNTTGKENKVLLAMNLHNNEAGRQAVAKLMSVDCRCHGVSGSCAVKTCWKTMSSFEK

IGHLLKDKYENSIQISDKIKRKMRRREKDQRKIPIHKDDLLYVNKSPNYCVEDKKLGIPG

TQGRECNRTSEGADGCNLLCCGRGYNTHVVRHVERCECKFIWCCYVRCRRCESMTDVHTCK

Seq ID No:31People Frizzled-1 peptide sequence (born of the same parents are rich in cysteine structure territory outward)

MAEEEAPKKSRAAGGGASWELCAGALSARLAEEGSGDAGGRRRPPVDPRRLARQLLLLLW

LLEAPLLLGVRAQAAGQGPGQGPGPGQQPPPPPPQQQQSGQQYNGERGISVPDHGY CQPI

SIPLCTDIAYNQTIMPNLLGHTNQEDAGLEVHQFYPLVKVQCSAELKFFLCSMYAPVCTV

LEQALPPCRSLCERARQGCEALMNKFGFQWPDTLKCEKFPVHGAGELCVGQNTSDKGTPT

PSLLPEFWTSNPQHGGGGHRGGFPGGAGASERGKFSCPRALKVPSYLNYHFLGEKDCGAP

CEPTKVYGLMYFGPEELR

Seq ID No:32People Frizzled-2 peptide sequence (born of the same parents are rich in cysteine structure territory outward)

MRPRSALPRLLLPLLLLPAAGPAQFHGEKGISIPDHGF CQPLSIPLCTDIAYNQTLMPNL

LGHTNQEDAGLEVHQFVPLVKVQCSPELRFFLCSMYAPVCTVLEQAIPPCRSICERARQG

CEALMNKFGFQWPERLRCEHFPRHGAEQICVGQNHSEDGAPALLTTAPPPGLQPGAGGTP

GGPGGGGAPPRYATLEHPFHCPRVLKVPSYLSYKFLGERDCAAPCEPARPDGSMFFSQEE

TR

Seq ID No:33People Frizzled-3 peptide sequence

MAMTWIVFSLWPLTVFMGHIGGHSLFSCEPITLRMCQDLPYNTTFMPNLLNHYDQQTAAL

AMEPFHPMVNLDCSRDFRPFLCALYAPICMEYGRVTLPCRRLCQRAYSECSKLMEMFGVP

WPEDMECSRFPDCDEPYPRLVDLNLAGEPTEGAPVAVQRDYGFWCPRELKISPDLGYSFL

HVRDCSPPCPNMYFRREELS

Seq ID No:34People Frizzled-4 peptide sequence

MAWRGAGPSVPGAPGGVGLSLGLLLQLLLLLGPARGFGDEEERRCDPIRISMCQNLGYNV

TKMPNLVGHELQTDAELQLTTFTPLIQYGCSSQLQFFLCSVYVPMCTEKINIPIGPCGGM

CLSVKRRCEPVLKEFGFAWPESLNCSKFPPQNDHNHMCMEGPGDEEVPLPHKTPIQPGEE

CHSVGTNSDQYIWVKRSLNCVLKCGYDAGLYSRSAKE

Seq ID No:35People Frizzled-5 peptide sequence

MARPDPSAPPSLLLLLLAQLVGRAAAASKAPVCQEITVPMCRGIGYNLTHMPNQFNHDTQ

DEAGLEVHQFWPLVEIQCSPDLRFFLCTMYTPICLPDYHKPLPPCRSVCERAKAGCSPLM

RQYGFAWPERMSCDRLPVLGRDAEVLCMDYNRSEATTAPPRPFPAKPTLPGPPGAPASGG

ECPAGGPFVCKCREPFVPILKESHPLYNKVRTGQVPNCAVPCYQPSFSADERT

Seq ID No:36People Frizzled-6 peptide sequence

MEMFTFLLTCIFLPLLRGHSLFTCEPITVPRCMKMAYNMTFFPNLMGHYDQSLAAVBMEH

FLPLANLECSPNIETFLCKAFVPTCIEQIHVVPPCRKLCEKVYSDCKKLIDTFGIRWPEE

LECDRLQYCDETVPVTFDPHTEFLGPQKKTEQVQRDIGFWCPRHLKTSGGQGYKFLGIDQ

CAPPCPNMYFKSDELE

Seq ID No:37People Frizzled-7 peptide sequence

MRDPGAAVPLSSLGFCALVLALLGALSAGAGAQPYHGEKGISVPDHGFCQPISIPLCTDI

AYNQTILPNLLGHTNQEDAGLEVHQFYPLVKVQCSPELRFFLCSMYAPVCTVLDQAIPPC

RSLCERARQGCEALMNKFGFQWPERLRCENFPVHGAGEICVGQNISDGSGGPGGGPTAYP

TAPYLPDLPFTALPPGASDGKGRPAFPFSCPRQLKVPPYLGYRFLGERDCGAPCEPGRAN

GLMYFKEEERR

Seq ID No:38People Frizzled-8 peptide sequence

MEWGYLLEVTSLLAALALLQRSSGAAAASAKELACQEITVPLCKGIGYNYTYMPNQFNHD

TQDEAGLEVHQFWPLVEIQCSPDLKFFLCSMYTPICLEDYKKPLPPCRSVCERAKAGCAP

LMRQYGFAWPDRMRCDRLPEQGNPDTLCMDYNRTDLTTAAPSPPRRLPPPPPGEQPPSGS

GHGRPPGARPPHRGGGRGGGGDAAAPPARGGGGGKARPPGGGAAPCEPGCQCRAPMVSVSSERH

PLYNRVKTGQIANCALPCHNPFFSQDERA

Seq ID No:39People Frizzled-9 peptide sequence

MAVAPLRGALLLWQLLAAGGAALEIGRFDPERGRGAAPCQAVEIPMCRGIGYNLTRMPNL

LGHTSQGEAAAELAEFAPLVQYGCHSHLRFFLCSLYAPMCTDQVSTPIPACRPMCEQARL

RCAPIMEQFNFGWPDSLDCARLPTRNDPHALCMEAPENATAGPAEPHKGLGMLPVAPRPA

RPPGDLGPGAGGSGTCENPEKFQYVEKSRSCAPRCGPGVEVFWSRRDKD

Seq ID No:40People Frizzled-10 peptide sequence

MQRPGPRLWLVLQVMGSCAAISSMDMERPGDGKCQPIEIPMCKDIGYNMTRMPNLMGHEN

QREAAIQLHEFAPLVEYGCHGHLRFFLCSLYAPMCTEQVSTPIPACRVMCEQARLKCSPI

MEQFNFKWPDSLDCRKLPNKNDPNYLCEAPNNGSDEPTRGSGEFPPLFRPQRPHSAQEHPLKDGGPG

RGGCDNPGKFHHVEKSASCAPLCTPGVDVYWSREDKR

Seq ID No:41People Dvl-3 amino acid sequence

MGETKIIYHL?DGQETPYLVK?LPLPAERVTL?ADFKGVLQRP?SYKFFFKSMD?DDFGVVKEEI

SDDNAKLPCF?NGRVVYWLVS?AEGSHPDPAP?FCADNPSELP?PPMERTGGIG?DSRPPSFHPH

AGGGSQENLD?NDTETDSLVS?AQRERPRRRD?GPEHATRLNG?TAKGERRREP?GGYDSSSTLM

SSELETTSFF?DSDEDDSTSR?FSSSTEQSSA?SRLMRRHKRR?RRKQKVSRIE?RSSSFSSTTD

STMSLNITTV?TLNMEKYNFL?GISIVGQSNE?RGDGGIYIGS?IMKGGAVAAD?GRIEPGDMLL

QVNEINFENM?SNDDAVRVLR?EIVHKPGPIT?LTVAKCWDPS?PRGCFTLPRS?EPIRPIDPAA

WVSHTAAMTG?TFPAYGMSPS?LSTITSTSSS?ITSSIPDTER?LDDFHLSIHS?DMAAIVKAMA

SPESGLEVRD?RMWLKITIPN?AFIGSDVVDW?LYHNVEGFTD?RREARLYASN?LLKAGFIRHT

VNKITFSEQC?YYIFGDLCGN?MANLSLHDHD?GSSGASDQDT?LAPLPHPGAA?PWPMAFPYQY

PPPPHPYNPH?PGFPELGYSY?GGGSASSQHS?EGSRSSGSNR?SGSDRRKEKD?PKAGDSKSGG

SGSESDHTTR?SSLRGPRERA?PSERSGPAAS?EHSHRSHHSL?ASSLRSHHTH?PSYGPPGVPP

LYGPPMLMMP?PPPAAMGPPG?APPGRDLASV?PPELTASRQS?FRMAMGNPSE?FFVDVM

Seq ID No:42People Dvl-1 amino acid sequence

MAETKIIYHM?DEEETPYLVK?LPVAPERVTL?ADFKNVLSNR?PVHAYKFFFK?SMDQDFGVVK

EEIFDDNAKL?PCFNGRVVSW?LVLAEGAHSD?AGSQGTDSHT?DLPPPLERTG?GIGDSRPPSF

HPNVASSRDG?MDNETGTESM?VSHRRERARR?RNREEAARTN?GHPRGDRRRD?VGLPPDSAST

ALSSELESSS?FVDSDEDGST?SRLSSSTEQS?TSSRLIRKHK?RRRRKQRLRQ?ADRASSFSSI

TDSTMSLNIV?TVTLNMERHH?FLGISIVGQS?NDRGDGGIYI?GSIMKGGAVA?ADGRIEPGDM

LLQVNDVNFE?NMSNDDAVRV?LREIVSQTGP?ISLTVAKCWD?PTPRSYFTVP?RADPVRPIDP

AAWLSHTAAL?TGALPRYELE?EAPLTVKSDM?SAVVRVMQLP?DSGLEIRDRM?WLKITIANAV

IGADVVDWLY?THVEGFKKER?EARKYASSLL?KHGFLRHTVN?KITFSEQCYY?VFGDLCSNLA

TLNLNSGSSG?TSDQDTLAPL?PHPAAPWPLG?QGYPYQYPGP?PPCFPPAYQD?PGFSYGSGST

GSQQSEGSKS?SGSTRSSRRA?PGREKERRAA?GAGGSGSESD?HTAPSGVGSS?WRERPAGQLS

RGSSPRSQAS?ATAPGLPPPH?PTTKAYTVVG?GPPGGPPVRE?LAAVPPELTG?SRQSFQKAMG

NPCEFFVDIM

Seq ID No:43People Dvl-2 amino acid sequence

MAGSSTGGGG?VGETKVIYHL?DEEETPYLVK?IPVPAERITL?GDFKSVLQRP?AGAKYFFKSM

DQDFGVVKEE?ISDDNARLPC?FNGRVVSWLV?SSDNPQPEMA?PPVHEPRAEL?APPAPPLPPL

PPERTSGIGD?SRPPSFHPNV?SSSHENLEPE?TETESVVSLR?RERPRRRDSS?EHGAGGHRTG

GPSRLERHLA?GYESSSTLMT?SELESTSLGD?SDEEDTMSRF?SSSTEQSSAS?RLLKRHRRRR

KQRPPRLERT?SSFSSVTDST?MSLNIITVTL?NMEKYNFLGI?SIVGQSNERG?DGGIYIGSIM

KGGAVAADGR?IEPGDMLLQV?NDMNFENMSN?DDAVRVLRDI?VHKPGPIVLT?VAKCWDPSPQ

AYFTLPRNEP?IQPIDPAAWV?SHSAALTGTF?PAYPGSSSMS?TTTSGSSLPD?GCEGRGLSVH

TDMASVTKAM?AAPESGLEVR?DRMWLKITIP?NAFLGSDVVD?WLYHHVEGFP?ERREARKYAS

GLLKAGLIRH?TVNKITFSEQ?CYYVFGDLSG?GCESYLVNLS?LNDNDGSSGA?SDQDTLAPLP

GATPWPLLPT?FSYQYPAPHP?YSPQPPPYHE?LSSYTYGGGS?ASSQHSEGSR?SSGSTRSDGG

AGRTGRPEER?APESKSGSGS?ESEPSSRGGS?LRRGGEASGT?SDGGPPPSRG?STGGAPNLRA

HPGLHPYGPP?PGMALPYNPM?MVVMMPPPPP?PVPPAVQPPG?APPVRDLGSV?PPELTASRQS

FHMAMGNPSE?FFVDVM

Claims (35)

1. a method that suppressed to express the growth of cancer cells of Wnt albumen is characterized in that, described method comprises described cell is contacted with the reagent of Frizzled receptors bind with the described Wnt albumen of inhibition.

2. the method for claim 1 is characterized in that, described reagent is a kind of antibody.

3. method as claimed in claim 2 is characterized in that, described antibody specificity is in conjunction with described Wnt albumen.

4. method as claimed in claim 3 is characterized in that, described Wnt albumen is Wnt-1.

5. method as claimed in claim 3 is characterized in that, described Wnt albumen is Wnt-2.

6. method as claimed in claim 2 is characterized in that, described antibody specificity is in conjunction with a kind of Frizzled acceptor.

7. method as claimed in claim 6, it is characterized in that described Frizzled acceptor is Frizzled1, Frizzled2, Frizzled3, Frizzled4, Frizzled5, Frizzled6, Frizzled7, Frizzled8, Frizzled9 and Frizzled10 acceptor.

8. method as claimed in claim 2 is characterized in that, described antibody is a kind of monoclone antibody.

9. method as claimed in claim 8 is characterized in that, described antibody is that recombination form produces.

10. method as claimed in claim 8 is characterized in that, described antibody is a kind of humanized antibody.

11. method as claimed in claim 8 is characterized in that, described antibody is a kind of strand Fv fragment (scFv).

12. the method for claim 1 is characterized in that, described cancer cell is positioned at patient's body and contact procedure is undertaken by giving the described reagent of patient.

13. method as claimed in claim 12 is characterized in that, described reagent is a kind of antibody.

14. method as claimed in claim 12 is characterized in that, described method further comprises and gives the patient second kind of therapeutic agent.

15. method as claimed in claim 14 is characterized in that, described second kind of therapeutic agent is chemotherapeutics.

16. method as claimed in claim 14 is characterized in that, described second kind of therapeutic agent is radiotherapy.

17. the method for claim 1, it is characterized in that described cancer cell is breast cancer cell, colorectal cancer cell, lung carcinoma cell, sarcoma cell, celiothelioma cell, cervical cancer cell, ovarian cancer cell, prostate gland cancer cell, pancreatic cancer cell, stomach cancer cell, esophageal cancer cell, head and neck cancer cell, hepatocellular carcinoma cells, melanoma cells, neuroglial cytoma, spongioblast oncocyte, leukaemia or lymphoma cell.

18. an anti-Wnt monoclone antibody is characterized in that, described antibody specificity is in conjunction with the peptide of SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:9.

19. monoclone antibody as claimed in claim 18 is characterized in that, described antibody comprises V shown in Figure 7 _HOr V _L

20. monoclone antibody as claimed in claim 18 is characterized in that, described V _HComprise V shown in Figure 7 _H1 CDR of chain.

21. monoclone antibody as claimed in claim 20 is characterized in that, described V _HComprise V shown in Figure 7 _HWhole 3 CDR of chain.

22. monoclone antibody as claimed in claim 18 is characterized in that, described V _LComprise V shown in Figure 7 _L1 CDR in district.

23. monoclone antibody as claimed in claim 22 is characterized in that, described VL comprises V shown in Figure 7 _LWhole 3 CDR in district.

24. a pharmaceutical composition is characterized in that, described pharmaceutical composition comprises pharmaceutically acceptable excipient and the specificity monoclone antibody in conjunction with Wnt-1 or Wnt-2.

25. pharmaceutical composition as claimed in claim 24 is characterized in that, described antibody further combines with a kind of effect component.

26. pharmaceutical composition as claimed in claim 24 is characterized in that, described effect component is a kind of fluorescent marker.

27. pharmaceutical composition as claimed in claim 24 is characterized in that, described effect component is a kind of radioisotope or a kind of cell toxicant chemicals.

28. compositions and methods of screening anticancer propagation, it is characterized in that described method comprises this reagent is contacted with Dvl albumen, measure the active of Dvl albumen or express, and differentiate and suppress Dvl albumen or active compound, thereby differentiate the reagent that a kind of anticancer is bred.

29. method as claimed in claim 28 is characterized in that, described method further comprises the compound through differentiating contact with cancer cell, and the selection anticancer compound of breeding.

30. method as claimed in claim 28 is characterized in that, described cancer cell is a lung carcinoma cell.

31. a method that suppressed to express the growth of cancer cells of Dvl albumen is characterized in that, described method comprises to be expressed described cell or the reagent of activity contacts with a kind of Dvl of inhibition.

32. method as claimed in claim 31 is characterized in that, described cancer cell is a lung carcinoma cell.

33. method as claimed in claim 31 is characterized in that, described reagent is a kind of little molecule.

34. method as claimed in claim 31 is characterized in that, described reagent is a kind of siRNA.

35. method that suppressed the growth of cancer cells of expression wnt or Frizzled albumen, it is characterized in that, described method comprises described cell contacted with a kind of reagent in conjunction with Frizzled acceptor born of the same parents intracellular domain, thereby suppresses combining of this Frizzled acceptor and intracellular protein.

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