CN102164956A - Novel antibodies against cancer target block tumor growth, angiogenesis and metastasis - Google Patents

Abstract

The present invention provides antibodies that target the first-third domains of N-cadherein and the fourth domain of N-cadherin, for diagnosis and therapy of cancers related to N-cadherein. Methods of diagnosis and treatment utilizing these antibodies are also described.

Description

The novel antibody of opposing cancer takes place and shifts by blocking-up tumor growth, blood vessel

The cross reference of related application

The application requires the right of priority of U.S. Patent application of submitting on April 4th, 2,008 61/042,604 and the U.S. Patent application of submitting on November 10th, 2,008 61/113,053, includes their full content in this paper by reference.

The rights and interests statement of the invention that research and development are made for federal funding

The present invention obtains NIH/NCI SOMI Training Grant R25 CA 098010 and NIH/NCI UCLA Prostate SPORE, the part support of P50 CA 092131.Government enjoys some right of the present invention.

Background of invention

Prostate cancer is modal malignant tumour, and is the U.S. male sex's the second largest cancer cause of the death.Prostate cancer is a kind of biology and clinical heterogeneous disease.The male sex that great majority suffer from this malignant tumour has the slow growing tumors that possibility can not influence individual life span, but other people can be subjected to the attack of the metastatic tumo(u)r of growth fast.The limitation of PSA screening is to lack specificity and unpredictable which patient has the risk of suffering from the intractable metastatic disease of hormone.Nearest researchs and proposes, the low quantity that might increase prostate cancer diagnosis of PSA diagnosis threshold, thereby make the evaluation complicated (Punglia etc., N Engl J Med, 349:335-342 (2003)) more between slow progress cancer patients and the invasive cancer patient.New serum that pressing for to be associated with clinical effectiveness maybe can differentiate the patient who suffers from the potential affecting conditions and tissue markers (Welsh etc., Proc Natl Acad Sci USA, 100:3410-3415 (2003)).

Nearest expression overview research prompting, the expression characteristic (signature) of metastatic tumo(u)r and non-metastatic tumour may be present in (Ramaswamy etc., Nat Genet, 33:49-54 (2003) in the primary tumor; Sotiriou etc., Proc Natl Acad Sci USA, 100:10393-10398 (2003)).Other features that make tumour be easy to transfer to certain organs also may be present in (Kang etc., Cancer Cell, 3:537-549 (2003)) in the primary tumor with certain probability.The prompting of these nearest observationss, can the disease commitment differentiate shift before or novel markings thing before the intractable prostate cancer of hormone.These markers also play a role in the biology of transitivity or the intractable prostate cancer progress of hormone.The up-to-date example that is present in the gene in the primary tumor relevant with the result and that play a role in the biology of prostate cancer progress comprises EZH2 and lim kinase (Varambally etc., Nature, 419:624-629 (2002); Yoshioka etc., Proc Natl Acad Sci USA, 100:7247-7252 (2003)).Yet these two kinds of genes are not secretor types.

For identifying the new candidate's serum or the tissue markers of the intractable prostate cancer of hormone, we have compared the allelic expression of paired hormonal dependent and the intractable prostate cancer heterograft of hormone.The LAPC-9 heterograft builds on the scleroblast bone and shifts, and relies on and proceed to and do not rely on (Craft etc., Cancer Research send to press (In Press, 1999)) behind the castrating immunodeficient mouse from male sex hormone.Before, it has been used for identifying the candidate therapeutic target of prostate cancer.Verified difference expression gene, tested sequence homology then with respect to secretory protein or cell surface protein.The evaluation of the N-cadherin of in intractable prostate cancer of hormone and bladder cancer, expressing simultaneously, sign and initial affirmation be in the news (WO/2007/109347).

Disclose before us, we are accredited as the N-cadherin supposition diagnosis and the treatment target (WO/2007/109347) of prostate cancer and bladder cancer.The content that discloses before us confirms that this target is significantly expressed in high risk and carrying out property prostate gland and tumor of bladder, and the expression that shows this target and prognosis mala are with to make progress into androgen independence relevant.Although infer once that before the N-cadherin may be useful treatment target, the medicine of unique existence is a peptide antagonists, and it does not demonstrate any clinical preceding active for prostate cancer.Understand according to us, our invention provides effective opposing to express the monoclonal antibody of the cancer of this target first.In addition, have only this proteic first ectodomain of target of N-cadherin antagonist now.We have described the antibody of target first and the 4th ectodomain.All (antibody) all has tangible anti-tumor activity.As far as we know, this is a notion of describing target the 4th ectodomain for the first time.Our antibody can be used as single agents, is used in combination, and can be used in combination with the antagonist of parallel approach of N-cadherin or downstream pathway in theory.

The present invention includes multiple monoclonal antibody at N-cadherin first and the 4th ectodomain.These antibody are blocked tumor growth in the body inner model of prostate cancer and other cancers, blood vessel takes place and transfer.They play a role by the N-cadherin signal transduction pathway of blocking responsible tumor growth, invasion and attack, blood vessel generation and shift.Described antibody also can be used for body inner control N-cadherin positive tumor and/or is used for organizational diagnosis and prognosis.

The present invention can only use with treatment or prophylaxis of tumours growth as monospecific antibody and shift.They also can be used as adjuvant or the existing tumor treatment agent of conduct.Their uses capable of being combined are with a plurality of structural domains of blocking-up N-cadherin.They also can be used in combination with chemotherapeutics or other target on cancer medicaments, and especially the medicament with those target synergistic signal transduction pathway or those targets N-cadherin Mediated Signal Transduction downstream or upstream pathway is used in combination.

The therapy or the diagnostic method of the target N-cadherin that does not still get the Green Light at present.Unique medicine of this approach of target is not very successful in the II phase in clinical, and it is non-activity in preclinical models, and our invention is activated to this preclinical models, and this illustrates that our method and reagent have obvious superiority.

Therefore, the invention provides the composition and the method for target N-cadherin, express the cancer of N-cadherin with diagnosis, prognosis and treatment, comprising but be not limited to prostate cancer and bladder cancer.

The invention summary

On the one hand, the invention provides with the hybridoma cell line of ATCC accession number PTA-9387 preservation and the antibody that produces by this hybridoma cell line, and another kind of with the hybridoma cell line of ATCC accession number PTA-9388 preservation and the antibody that produces by this hybridoma cell line.Also provide a kind of can be in vivo or external antibody or its fragment in conjunction with N-cadherin structural domain 1-3, this structural domain 1-3 is identical with the monoclonal antibody bonded N-cadherin antigenic determinant that hybridoma cell line was produced that is preserved in American Type Culture Collection with ATCC accession number PTA-9387, or can be in vivo or external antibody or its fragment in conjunction with N-cadherin structural domain 4, this structural domain 4 is identical with the monoclonal antibody bonded N-cadherin antigenic determinant that hybridoma cell line was produced that is preserved in American Type Culture Collection with ATCC accession number PTA-9388.This antibody can be humanized or complete people; Perhaps it can be bi-specific antibody or single-chain antibody (scFv).

In second aspect, the invention provides the method that a kind of anticancer is grown in patient's body.This method may further comprise the steps: being enough to make described antibody (or its fragment) in conjunction with expressing or crossing under the condition of the cancer cells of expressing the N-cadherin, give patient's antibody of the present invention (or its fragment).Described antibody (or its fragment) is grown by following approach anticancer: (a) activation or inhibition NF κ-signal conduct and transcribe; (b) activation or the internalization of inhibition N-cadherin; (c) activation or inhibition PI3 kinases or Akt approach; (d) activation or the conduction of inhibition beta-catenin white signal; (e) different dimerization of blocking-up N-cadherin and FGFR or other tyrosine kinase receptors; Or (f) blocking-up or strengthen by ADAM10 or the cutting of other metallopeptidases.In some embodiments, described cancer cells is apparatus urogenitalis cancer cells, prostate cancer cell or transitional cell bladder carcinoma cell line.

Aspect the 3rd, the invention provides a kind of cancer patients's of treatment method.This method may further comprise the steps: (a) obtain the test organization sample from the individuality that the risk of suffering from the cancer of expressing the N-cadherin is arranged; (b) compare with the control tissue sample that is known as the cancer negative patients, whether or content the existence of determining N-cadherin in the test organization sample; Thereby diagnose the cancer of described expression N-cadherin, wherein said N-cadherin is with normal or low expression level, or expressed by cell subsets, or crosses and express; (c) the N-cadherin antibody of the present invention (or its fragment) of the individual effective dose of the risk of suffering from the cancer of expressing the N-cadherin is arranged.

In some embodiments, described tissue sample is prostate gland or bladder body.In some embodiments, described cancer is prostate cancer or bladder cancer, perhaps can be metastatic carcinoma.In some embodiments, the intractable prostate cancer of described antibody (or its fragment) blocking-up hormone, or described antibody blocking cancer stem cell.In some cases, described antibody is monoclonal antibody, scFv or bi-specific antibody.In some other embodiment, described tissue sample is prostate gland or bladder body.

Aspect the 4th, the invention provides a kind of diagnosing cancer patient's method.This method may further comprise the steps: (a) obtain the test organization sample from the individuality that the risk of suffering from the cancer of expressing the N-cadherin is arranged; (b) compare with the control tissue sample that is known as the cancer negative patients, whether or content the existence of determining N-cadherin in the test organization sample by the N-cadherin antibody of the present invention (or its fragment) that makes sample contact significant quantity; Thereby diagnose the cancer of described expression N-cadherin, wherein said N-cadherin is with normal or low expression level, or expressed by cell subsets, or crosses and express.

Aspect the 5th, the invention provides a kind of method of identifying cancer stem cell.This method may further comprise the steps: (a) obtain the test organization sample from the individuality that the risk of suffering from the cancer of expressing the N-cadherin is arranged; (b) compare with the control tissue sample that is known as the cancer negative patients, adopt existence that antibody of the present invention (or its fragment) determines cancer stem cell in the test organization sample whether; Wherein said N-cadherin is with normal or low expression level, or by the stem cell Expression of Subsets and be not expression.In some embodiments, described tissue sample is prostate gland or bladder body.In some embodiments, described cancer is prostate cancer, bladder cancer, the intractable prostate cancer of hormone or metastatic carcinoma.

Aspect the 6th, the invention provides a kind of method of identifying anti--N-cadherin antibody or suppressing the compound of growth of cancer cells in patient's body.This method may further comprise the steps: (i) make the contact of candidate compound or antibody express or cross the cell of expressing N-cadherin polypeptide; (ii) by determining whether described candidate compound or antibody have the following functional effect that is used for determining described compound or antibody to N-cadherin polypeptide: (a) activate or suppress NF κ-signal conduction and transcribe; (b) activation or the internalization of inhibition N-cadherin; (c) activation or inhibition PI3 kinases or Akt approach; (d) activation or the conduction of inhibition beta-catenin white signal; (e) different dimerization of blocking-up N-cadherin and FGFR or other tyrosine kinase receptors; Or (f) blocking-up or strengthen by ADAM10 or the cutting of other metallopeptidases.Have each described activity in (a)-(f) if candidate compound or antibody demonstrate, then this antibody or compound are considered to the compound of the anti--N-cadherin antibody or the growth of the cancer of inhibition patient expression in vivo N-cadherin.For example, suppress NF κ-signal conduction or transcribe when antibody or compound; Perhaps when antibody or compound suppress the internalization of N-cadherin, described antibody or compound are considered to anti--N-cadherin antibody or suppress the compound of growth of the cancer of patient's expression in vivo N-cadherin.

Brief Description Of Drawings

Fig. 1. the signal conduction mode of N-cadherin in the prostate cancer.

Fig. 2 .N-cadherin activation NF-γ β.

Fig. 3 .N-cadherin activation NF-γ β.

Fig. 4 .N-cadherin is at the cell surface expression of C1 cell.

Fig. 5 .NF-γ β is confined to the nucleus of N-cadherin positive cell (C1).

Fig. 6 .N-cadherin is expressed and is caused inducing IL-6, IL-8, TGF β 2 and bcl-2.

Fig. 7: induced gene is relevant with N-cadherin level.

Fig. 8: the N-cadherin strikes to subtract and causes IL-6 and IL-8 downward modulation.

NF-γ 'beta ' activity after Fig. 9 .N-cadherin antibody treatment.

N-cadherin in Figure 10 .PC3 cell: antibody was cultivated in 48 hours.

Figure 11 .N-cadherin strikes to subtract and causes activatory Akt downward modulation.

The Akt activity is reduced in the activation of Figure 12 .N-cadherin specific antibody then.

Figure 13. the facs analysis of the antibody cloning of target N-cadherin first ectodomain.

Figure 14. the facs analysis of the antibody cloning of target N-cadherin the 4th ectodomain.

Figure 15. the facs analysis of the mono-clonal N-cadherin antibody cloning of purifying.

Figure 16. the external invasion and attack test of mono-clonal N-cadherin antibody cloning.

Figure 17. the growth curve of the blank tumour of handling with anti-N-cadherin antibody 1H7 and EC4 of N-cadherin (null tumors) does not demonstrate significant retarding effect (17a).The tumor growth curve of the PC3 prostate cancer cell of handling with same antibody prove growth-inhibiting effective (17b).With the big PC3 growth of tumor curve of setting up under 1H7 and the EC4 antibody treatment (17c).Long term growth curve (17d) with PC3 tumour under the EC4 antibody treatment.

Figure 18. show in the body of antibody to the influence of N-cadherin and test.

Figure 19. (a) immunohistochemical staining of androgen independence LAPC9 prostate cancer; (b) with N-cadherin Antybody therapy androgen-dependent and dependent/non-dependent LAPC-9 tumour.

Figure 20. sorting and tumor growth curve unsorted LAPC9AI cell.

The FACS result of the N-cadherin sorting tumour of Figure 21 and 22. progress.

Figure 23 .LNCaP-C1 growth of tumor curve has shown the retarding effect of N-cadherin antibody.

Figure 24 .N-cadherin antibody suppresses LAPC-9 androgen independence tumour of setting up (24a) and the big LAPC-9 androgen independence growth of tumor of setting up.

Figure 25. antibody EC4 suppresses PC3 tumor growth in the nude mice in the relevant mode of dosage.

Figure 26. in two researchs of the tumour progression that reaches 45 days (26a) and 70 days (26b) monitoring, N-cadherin antibody 1H7 and EC4 demonstrate retarding effect to LAPC-9 androgen-dependent tumor growth.

Figure 27 .N-cadherin sorting cells demonstrates growth vigor in castrating SCID mouse.

Figure 28. the dependency of expression of N-cadherin and androgen receptor level in the continuous passage of LAPC9 tumour cell.

Detailed Description Of The Invention

The present invention relates to be used for the treatment of the novel monoclonal antibody of the targeted cells surface protein N-cadherin of the cancer of expressing the N-cadherin.Described cancer can be prostate cancer, bladder cancer or express other cancers of N-cadherin.

The invention still further relates to method with the antibodies for treating cancer of target N-cadherin.The inventor finds that described antibody can play a role in the following way: activation or inhibition NF κ-signal conduct and transcribe; Activate or the internalization of inhibition N-cadherin; Activate or inhibition PI3 kinases or Akt approach; Activate or suppress the beta-catenin white signal and conduct; The different dimerization of blocking-up N-cadherin and FGFR or other tyrosine kinase receptors; Or blocking-up or strengthen by the cutting of ADAM10 or other metallopeptidases.Fig. 1 is the general introduction of N-cadherin Mediated Signal Transduction.Fig. 1 has shown the activation of NF-κ β, Akt and the white approach of beta-catenin.The evidence that the inventor proposes shows that N-cadherin antibody is by changing one or more the playing a role in these approach.

Definition

The nucleic acid that " N-cadherin and E-cadherin " expression has following characteristics (for example, gene, before-mRNA, mRNA) and polypeptide, polymorphie variant, allelotrope, mutant and plant between homologue): the aminoacid sequence that (1) is had is preferably at least about 25,50,100,200,500,1000 or more on the zone of amino acids with (for example be described in GenBank accession number NM_001792 (N-cadherin mRNA) by each reference nucleic acid encoded polypeptides or aminoacid sequence described herein, NP_001783 (N-cadherin), NM_004360 (E-cadherin mRNA) and NP_004351 (E-cadherin)) have and be higher than about 60% aminoacid sequence homogeny, 65%, 70%, 75%, 80%, 85%, 90%, preferred 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or higher aminoacid sequence homogeny; (2) specificity is incorporated into antibody (for example, polyclonal antibody), and described antibody is at the immunogen that comprises as reference amino acid sequence as described in GenBank accession number NP_001783 (N-cadherin) or the NP_004351 (E-cadherin); Its immunogenic fragments separately, with and separately conservative property modify variant; (3) under stringent hybridization condition with coding respectively as GenBank accession number NP_001783 (N-cadherin) or NP_004351 (E-cadherin) nucleic acid of described reference amino acid sequence and the modification of conservative property separately variant specific hybrid thereof; (4) nucleotide sequence that has preferably at least about 25,50,100,150,200,500,1000 or more have with reference nucleic acid sequence shown in GenBank accession number NM_001792 (N-cadherin mRNA) or NM_004360 (E-cadherin mRNA) on the zone of polynucleotide and be higher than approximately 95%, preferably is higher than about 96%, 97%, 98%, 99% or higher nucleotide sequence homogeny.Polynucleotide or peptide sequence include but not limited to usually from Mammals: primates, for example people; Rodents is as rat, mouse, hamster; Ox, pig, horse, sheep or any Mammals.Nucleic acid of the present invention and protein comprise molecule natural generation or reorganization.

" cancer " expression people's cancer, sarcoma, gland cancer, lymphoma, leukemia etc. comprise solid tumor and lymph sample cancer, kidney, mammary cancer, lung cancer, kidney, bladder cancer, colorectal carcinoma, ovarian cancer, prostate cancer, carcinoma of the pancreas, cancer of the stomach, the cancer of the brain, head and neck cancer, skin carcinoma, uterus carcinoma, carcinoma of testis, esophagus cancer, liver cancer, lymphoma (comprising non-Hodgkin lymphoma and Hodgkin lymphoma), leukemia and multiple myeloma.The human cancer of " apparatus urogenitalis cancer " expression urinary tract and germinal tissue, described tissue includes but not limited to kidney, bladder, urinary tract, urethra, prostate gland, penis, testis, vulva, vagina, uterine neck and ovary tissue.

In the literary composition, the cancer that treat can be to be the cancer of feature with N-cadherin overactivity.Perhaps, the cancer that will treat of this paper can be the N-cadherin with cancer normal or that lower level is expressed, or the cancer expressed by certain cell subsets of N-cadherin, and the N-cadherin is crossed the cancer of expressing.In an embodiment of the invention, will diagnose or whether prognostic assay is feature to express the N-cadherin with the cancer of determining the patient.The various tests of determining this amplification/expression be can consider, immunohistochemistry, FISH and outflow antigen test (shed antigen assay), southern blotting technique or round pcr comprised.In addition, available in-vivo diagnostic test, for example by give in conjunction with molecules detected and by the molecule (as antibody) of detectable label (for example, radio isotope) mark and externally scan patients estimate the expression or the amplification of N-cadherin to determine the location of mark.In some embodiments, cancer to be treated also is not invasive, but expresses the N-cadherin.

" the therapy resistance " cancer, tumour cell and tumour represent to apoptosis-mediation (for example, by the death receptor cell signaling, as, the Fas ligand receptor, TRAIL acceptor, TNF-R1, chemotherapeutics, ray) or non--(for example, poison, chemical agent) cancer therapy (comprising chemotherapy, hormonotherapy, radiotherapy and immunotherapy) of apoptosis mediation produces cancer resistance or intractable.

" cross express " expression N-cadherin, LY6-E and E-cadherin in the test organization sample RNA or protein expression apparently higher than N-cadherin, LY6-E and E-cadherin RNA in the control tissue sample or protein expression respectively.In one embodiment, described tissue sample is from body.With from the patient's who unlikely develops into transfer cancerous tissue or with normal (promptly, non-carcinous) the N-cadherin of tissue sample or LY6-E express and compare, with invasion and attack, transfer, hormonal independent (for example, androgen independence) or (for example treat carcinous test organization sample that intractable relevant or similar possibility increases, bladder, prostate gland) N-cadherin or LY6-E mRNA or protein expression are 2 times usually at least, usually up to 3,4,5,8,10 or higher multiple.When observing that roughly similarly load has the gel band of specimen and control sample, this species diversity of easier discovery.The N-cadherin of expression increment or the prostate cancer of Ly6-E more may become aggressive, transitivity or develop into androgen independence or treat intractable cancer.Because small proportion N-cadherin in the primary tumor or Ly6-E positive cell might identify the tumour that has high recurrence and shift risk, so N-cadherin or the Ly6-E positive can be relevant with various cutoff values (cutoff).Term " cross express " or " cross and express " interchangeable referring to normal cell are compared, and can detect the higher levels of gene of transcribing or translating usually in cancer cells.Therefore, cross to express and be meant that crossing of protein and RNA express (owing to transcribe increases, transcribe post-treatment, translation, translation post-treatment, stability changes and the protein degradation change), and owing to transporting pattern, protein changes that expression is crossed in part that (appraising and deciding the position increases) cause and functionally active improves (for example under the situation that the enzymic hydrolysis effect of substrate increases).Crossing expression also can be to compare normal cell or reference cell (for example, BPH cell) raising 50%, 60%, 70%, 80%, 90% or higher.

Term " is expressed the cancer of N-cadherin " and is used interchangeably with " cancer relevant with the expression of N-cadherin ", and cancer cells or the tissue according to the N-cadherin of above-mentioned definition expressed in expression.

Term " cancer associated antigens " or " tumour-specific markers thing " or " tumor marker " are used interchangeably, normal cell is compared in expression, preferentially expresses and can be used to molecule (normally protein, carbohydrate or lipid) with the preferential target cancer cell of medicament in cancer cells.Marker or antigen can be at cell surface or cell inner expressions.Usually, cancer associated antigens is to compare normal cell, or the molecule of expressing in stable condition with least degrading in cancer cells for example, compared with normal cell and to be expressed as 2 times, 3 times or higher multiple.Usually, cancer associated antigens is a undesired synthetic molecule in cancer cells, for example, compares the molecule of expressing on normal cell, the molecule that contain disappearance, adds or suddenly change.Usually, cancer associated antigens will only be expressed in cancer cells, and can not synthesize in normal cell or express.Exemplary cell surface tumor marker comprises, the albumen c-erbB-2 of mammary cancer and Human epidermal growth factor receptor (HER), the PSMA of prostate cancer, and the carbohydrate Saliva Orthana class that comprises many cancers of mammary cancer, ovarian cancer and colorectal carcinoma.Tumor marker comprises that for example, the tumor inhibitor of sudden change or cyclin are as p53 in the exemplary born of the same parents.

On the contrary, from more becoming aggressive, transitivity or developing into androgen independence or treat in the cancerous tissue sample in cancer patients's the tissue sample of intractable cancer, the E-cadherin is expressed deficiency usually.This expression deficiency can be 2 times, 3 times, 4 times or at least 5 times.When observing that roughly load has the gel band of specimen and control sample similarly, this species diversity of easier discovery.More may become aggressive, transitivity or develop into androgen independence or treat in the cancer of intractable cancer, N-cadherin/E-cadherin combined value is therefore higher, can be up at least 2 times, 3 times, 4 times, 5 times, 10 times or 20 times.Because the small proportion N-cadherin positive cell in the primary tumor might identify the tumour that has high recurrence and shift risk, so the N-cadherin positive/e-cadherin feminine gender can be relevant with various cutoff values.

" agonist " refers to be incorporated into polypeptide of the present invention or polynucleotide, and the reagent of the active or expression of stimulation, raising, activation, promotion, enhanced activity, sensitization or rise polypeptide of the present invention or polynucleotide.

" antagonist " refer to suppress polypeptide of the present invention or polynucleotide expression or in conjunction with, partially or completely blocking-up stimulates, reduces, prevents, postpones activation, deactivation, reduction susceptibility or reduces polypeptide of the present invention or the active reagent of polynucleotide.

Expression or active " inhibitor ", " activator " and " conditioning agent " are used for respectively representing with inhibition, activation or adjusting molecule external and expression in vivo or activity test evaluation, for example, part, antagonist, agonist and their homologue and stand-in.Term " conditioning agent " comprises inhibitor and activator.Inhibitor is, for example, suppress polypeptide of the present invention or polynucleotide and express, perhaps in conjunction with, partially or completely block stimulation or enzymic activity, reduce, prevent, postpone activation, deactivation, desensitization or downward modulation polypeptide of the present invention or the active material of polynucleotide, as antagonist.Activator is, for example, induce or activate the expression of polypeptide of the present invention or polynucleotide, perhaps in conjunction with, stimulate, improve, open, activate, promote, strengthen activation or enzymic activity, sensitization or rise polypeptide of the present invention or the active material of polynucleotide, as agonist.Conditioning agent comprise natural generation with synthetic part, antagonist, agonist, chemical small molecules etc.The experiment of identifying inhibitor and activator for example comprises, existing or not existing under the condition of polypeptide of the present invention or polynucleotide, the conditioning agent compound of inferring is put on cell, measures it then to polypeptide of the present invention or the active functional impact of polynucleotide.Sample that comprises polypeptide of the present invention or polynucleotide or the trier that to handle with potential activator, inhibitor or conditioning agent with do not have the control sample of inhibitor, activator or conditioning agent to make comparisons, with the detection influence degree.The relative reactivity value of control sample (not handling with conditioning agent) is appointed as 100%.With respect to contrast, the activity value of polypeptide of the present invention or polynucleotide is about 80%, and optional is 50% or during 25-1%, has realized inhibition.With respect to contrast, the activity value of polypeptide of the present invention or polynucleotide is 110%, and optional is 150%, optionally is 200-500%, or 1000-3000% or when higher, has realized activation.

Term used herein " test compounds " or " drug candidate " or " conditioning agent " or its grammer equivalents have been described natural generation or any molecule of synthetic, for example protein, oligopeptides are (for example, be about about 25 amino acid of 5-, preferably be about 10-20 or 12-18 amino acid, preferably long 12,15 or 18 amino acid), organic molecule, polysaccharide, lipid, lipid acid, polynucleotide, RNAi, siRNA, antibody, oligonucleotide etc.Test compounds can be the form in test compounds library, and enough multifarious combinatorial library or random library for example are provided.Test compounds randomly is connected in fusion partners, as target compound, rescue compound, dimerization compound, stable compound, addressable compound and other functional moiety.Generally can be tested and appraised have some desired characteristic or activity (for example suppressing active) test compounds (being called " lead compound "), produce the variant of this lead compound and assess the characteristic and the active new chemical entities that produces of those variant compounds with useful property.Usually adopt high flux screening (HTS) method in this analysis.

" organic molecule " refers to natural generation or synthetic organic molecule, its molecular weight is greater than about 50 dalton and less than about 2500 dalton, preferably less than about 2000 dalton, preferably between about 1000 dalton of about 100-, more preferably between about 500 dalton of about 200-.

Cytotoxic agent comprises " cell cycle specific " or " antimitotic " or " cytoskeleton interaction " medicament.These terms are used interchangeably, any medicament of the cell in the expression blocking-up mitotic division.This medicament can be used for chemotherapy.Usually, cell cycle specific agents is in conjunction with the cytoskeletal protein tubulin and block tubulin polymerization formation microtubule, thereby causes cell fission to rest on mid-term.Exemplary cell cycle specific agents comprises vinca alkaloids, taxanes, colchicine and podophyllotoxin.Exemplary vinca alkaloids comprises vinealeucoblastine(VLB), vincristine(VCR), vindesine and vinorelbine.Exemplary taxanes comprises taxol and Docetaxel.Other examples of cytoskeleton interaction medicine comprise the 2-methoxyestradiol.

" siRNA " or " RNAi " expression forms the nucleic acid of double-stranded RNA, and as siRNA during at the cell inner expression identical with certain gene or target gene, this double-stranded RNA can reduce or suppress described gene or target gene expression.The double-stranded RNA that " siRNA " or " RNAi " so expression are formed by complementary strand.The complementary portion of the siRNA of hybridization formation duplex molecule is basic identical or identical usually.In one embodiment, siRNA represents basic identical or identical and form the nucleic acid of double-stranded siRNA with target gene.Usually, the length of siRNA at least about 15-50 Nucleotide (for example, the length of every complementary sequence of double-stranded siRNA is 15-50 Nucleotide, and the length of double-stranded siRNA is about 15-50 base pair, preferred about 20-30 nucleotide base, preferred length is about 20-25 or about 24-29 Nucleotide, and for example length is 20,21,22,23,24,25,26,27,28,29 or 30 Nucleotide.

The design of siRNA molecule and carrier and manufacturing are that those of ordinary skills know.For example, the effective ways that design suitable siRNA be from the mRNA transcript the AUG initiator codon (for example, referring to, Fig. 5) and scan A A dinucleotides sequence (referring to EMBO J 20:6877-6888 (2001) such as Elbashir.Each AA and 3 ' adjacent nucleotide are potential siRNA target sites.The length of adjacent site sequence will determine the length of siRNA.For example, 19 adjacent sites will obtain the long siRNA of 21 Nucleotide, and the siRNA with 3 ' overhang UU dinucleotides is normally the most effective.This method is also with transcribe hair clip siRNA with RNA pol III consistent.RNA pol III locates to stop transcribing at 4-6 Nucleotide poly (T), thereby forms the RNA molecule with short poly (U) tail.Yet the siRNA with other 3 ' terminal dinucleotides overhangs also can effectively produce RNAi, can rule of thumb select this sequence.When selecting, can avoid adjoining the target sequence of base pair (referring to the American National biotechnology (NCBI of information center above 16-17 by the BLAST retrieval with the homology of other encoding sequences, National Center for Biotechnology Information), network address: ncbi.nlm.nih.gov/BLAST).

Can directly use siRNA, or induce RNAi, its standard difference with the siRNA expression vector.Carrier can be inserted with by short intervening sequence and separate and end at two inverted repeats that are used to a string T of stopping transcribing.Estimate that the rna transcription thing of expressing will be folded into bob folder siRNA.For the length and the composition of the intervening sequence of the ring of the order of the length of the inverted repeats of the stem of the hair clip of siRNA target sequence, coding hypothesis, inverted repeats, coding hair clip and whether to have the selection of 5 ' overhang be variable.The preferred sequence of siRNA expression cassette is sense strand, at interval short and antisense strand.(for example, hair clip siRNA 15-30) is suitable for to have these different stem length.The length that connects the ring of the sense strand of hair clip siRNA and antisense strand is variable (for example, 3-9 Nucleotide, or longer).Can comprise other controlling elements that promotor and expression enhanser or operability are connected in the nucleotide sequence of coding siRNA in the carrier.Statement " control sequence " is meant that the encoding sequence that can be operatively connected is at the necessary dna sequence dna of the organic expression in vivo of specific host.Be applicable to the control sequence of prokaryotic cell prokaryocyte, for example, comprise promotor, optional operon sequence and ribosome bind site.Known promotor, polyadenylic acid signal and the enhanser of utilizing of eukaryotic cell.These controlling elementss can be designed to allow the clinician by adding or controlling and closed or open genetic expression by the external factor that these controlling elementss responded.

Structure has adopted standard well known in the art to be connected when containing the suitable carrier of required therapeutic gene coding and control sequence and restriction technologies (referring to Maniatis etc., " molecular cloning laboratory manual " (Molecular Cloning:A Laboratory Manual), the cold spring port, New York (1982)).Isolating plasmid, dna sequence dna or synthetic oligonucleotide can be cut, repair or reconnect into desired form.

When nucleic acid and another kind of nucleotide sequence were in the functional mutual relationship, then this nucleic acid was " can be operatively connected ".For example, the DNA of current sequence or secretion conductor is expressed as when participating in before the polypeptide excretory albumen, and then this DNA is operably connected with the DNA of polypeptide; When promotor or enhanser can influence transcribing of encoding sequence, then this promotor or enhanser were operably connected with this encoding sequence; Perhaps when the residing position of ribosome bind site can promote translation, then this ribosome bind site was operably connected with encoding sequence.Generally speaking, " can be operatively connected " means that connected dna sequence dna is close mutually, and adjoins in the secretion leader sequence, and is in the reading frame.Yet enhanser is successive not necessarily.Can finish connection by ligation on the restriction enzyme site easily.If such site does not exist, so can be according to conventional practice synthetic oligonucleotide joint or connexon.

" measurement function effect " expression for example, is measured physics and chemistry or phenotypic effect to raising or reducing the mensuration of the compound that is subjected to the parameter that polynucleotide of the present invention or polypeptide influence indirectly or directly.This functional effect can be measured by any method known to those skilled in the art, for example, and proteinic beam split (for example, fluorescence, absorbancy, specific refractory power), hydrokinetics (for example, shape), chromatogram or deliquescent change; Measurement can induce marker or protein to transcribe activity; Measure in conjunction with active or combination test, for example, with combining of antibody; Measure the change of ligand binding affinity; Measure calcium current; The accumulation of the enzyme product of measurement polypeptide of the present invention or the consumption of substrate; Change enzymic activity, for example, kinase activity, the variation of measuring polypeptide protein level of the present invention; Measure R NA stability; The G protein binding; G protein coupled receptor (GPCR) phosphorylation or dephosphorylation; Signal transduction, for example, receptor-ligand binding, second messenger's concentration (for example, Ca in cAMP, IP3 or the born of the same parents ²⁺); Identify the expression of downstream or reporter gene (CAT, luciferase, β-gal, GFP etc.), for example, by chemoluminescence, fluorescence, colorimetric reaction, antibodies, can induce marker and part in conjunction with test.

To comprise nucleic acid of the present invention or proteinic sample or trier and not have the control sample of inhibitor, activator or conditioning agent to make comparisons with what potential activator, inhibitor or conditioning agent were handled, to detect the inhibition degree.The relative protein-active value of control sample (not handling with inhibitor) is appointed as 100%.With respect to contrast, activity value is about 80%, and is preferred 50%, more preferably during 25-0%, can realize suppressing.When activity value is 110% with respect to contrast (not handling with activator), more preferably 150%, the more preferably 200-500% 2-5 of contrast (promptly be doubly) more preferably during 1000-3000%, can realize activation.

" biological sample " comprises tissue slice, for example biopsy and postmortem sample, and the freezing microtome section of obtaining for the histology purpose.This sample comprises blood and blood constitutent or product (for example, serum, blood plasma, thrombocyte, red corpuscle etc.), phlegm, tissue, culturing cell, for example primary culture, explant and transformant, ight soil, urine etc.Biological sample is usually available from most eukaryotes, Mammals most preferably, and primate for example is as chimpanzee or people; Ox; Dog; Cat; Rodent is as cavy, rat, mouse; Rabbit; Or birds; Reptilia; Or fish.

" biopsy " refers to take out the process of tissue sample for diagnosis or prognosis evaluation, also refers to tissue samples self.Any biopsy technology known in the art can be applied to diagnosis of the present invention and method of prognosis.Applied biopsy technology depends on the size and the type factors such as (being (that is, blood or ascites) entity or that suspend) of types of organization to be assessed (that is, prostate gland, lymphoglandula, liver, marrow, hemocyte), tumour usually.Representational biopsy technology comprises excision biopsy, incisional biopsy, needle biopsy, operation biopsy and bone marrow biopsy." incisional biopsy " expression cuts off with whole tumour body and around its very thin one deck healthy tissues." cut formula biopsy " expression cuts a wedge shape tissue that comprises the square section diameter of tumour.Diagnosis or the prognosis made by splanchnoscopy or fluorescent microscopy can require the tumour entity is carried out " biopsy of core pin " or " fine needle aspiration biopsy ", and it will obtain the suspension of tumour entity inner cell usually.About the discussion of biopsy technology can referring to, Harrison ' s Principles of Internal Medicine (the gloomy principle of internal medicine Harry) for example, volumes such as Kasper, the 16th edition, 2005, the 70 chapters, and whole V part.

With regard to two or more nucleic acid or peptide sequence, term " identical " or " homogeny " per-cent refer to utilize default parameters BLAST as follows or BLAST 2.0 sequence comparison algorithms, or by manual comparison and visual observations (referring to, NCBI network address ncbi.nlm.nih.gov/BLAST etc. for example) it is identical or have the same amino acid residue of particular percentile or Nucleotide (promptly to be measured to two or more sequences or subsequence, when the comparison of carrying out maximum correspondence in comparison window or designated area and comparison, 60% homogeny of on the specific region, having an appointment, preferred 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher homogeny).Claim these sequences " substantially the same " then.This definition also refers to or applicable to the complementation (compliment) of cycle tests.This definition also comprises the sequence that has disappearance and/or add, and those sequences with replacement.As described below, preferred algorithm can illustrate breach etc.Preferably at least about 25 amino acid or length of nucleotides, or more preferably has homogeny in the zone of 50-100 amino acid or length of nucleotides.

Sequence relatively in, generally with a kind of sequence as the reference sequence of comparing with cycle tests.When using sequence comparison algorithm, will test with reference sequence and all import computer, if necessary then specify the subsequence coordinate, and specified sequence algorithm routine parameter.Preferably use the program parameter of acquiescence, perhaps can specify the parameter of alternative.Then, this sequence comparison algorithm calculates the sequence homogeny percentage ratio of cycle tests with respect to reference sequence according to program parameter.

" comparison window " used herein comprises with reference to being selected from 20-600, common about 50-200, the section that adjoins the position of arbitrary quantity among more common about 100-150, two sequences are carried out optimum comparison after, the reference sequence that the sequence in this section and equal amts can be adjoined the position is made comparisons.The method that aligned sequences compares is well known in the art.Can pass through, for example following method is carried out the optimal sequence comparison so that relatively: local homology's algorithm of Smith and Waterman, Adv.Appl.Math.2:482 (1981), the homology alignment algorithm of Needleman and Wunsch, J.Mol.Biol.48:443 (1970), the similarity searching method of Pearson and Lipman, Proc.Nat ' l.Acad.Sci.USA 85:2444 (1988), these algorithms (GAP in the Wisconsin genetics software package of genetics calculating group (Genetics Computer Group) (Wisconsin Genetics Software Package) is carried out in computerize, BESTFIT, FASTA and TFASTA, 575 Doctor of Sciences (Science Dr.), state of Wisconsin Madison (Madison, WI)), or manual comparison and range estimation are (referring to for example, " newly organized molecular biology experiment guide " (Current Protocols in Molecular Biology) (volume such as Ausubel, 1995 supplementary issues)).

The preferred example that is fit to the algorithm of mensuration sequence homogeny and sequence similarity percentage ratio is respectively BLAST and BLAST 2.0 algorithms, referring to Altschul etc., Nuc.Acids Res.25:3389-3402 (1977) and Altschul etc., J.Mol.Biol.215:403-410 (1990).Use BLAST and BLAST 2.0, set parameter as herein described, to determine nucleic acid of the present invention and proteinic sequence homogeny percentage ratio.The software that carries out the BLAST analysis can be obtained by NCBI (NCBI) website from public's channel.This algorithm comprises at first by the short word (short words) of identifying length W in search sequence identifies the higher assessment sub-sequence to (HSP), when the word of equal length is compared in this short word and the database sequence, mates or satisfies some on the occasion of the threshold value T that marks.T is called adjacent words scoring threshold value (Altschul etc., the same).With hitting of these initial adjacent words as starting the seed that the search discovery contains their longer HSP.This word hits along extending on the both direction of each sequence, marks up to increasing the cumulative comparison.For nucleotide sequence, utilize parameter M (the award scoring of a pair of coupling residue; Always＞0) and the N (point penalty of mispairing residue; Always＜0) calculate the accumulation scoring.For aminoacid sequence, calculate the accumulation scoring with rating matrix.When following situation occurred, the termination word hit the extension on all directions: accumulation comparison scoring reduces X than maximum acquisition value; Because the accumulation of one or more negative scoring residue comparisons is below the accumulation scoring vanishing or zero; Perhaps reach the end of each sequence.BLAST algorithm parameter W, T and X have determined the sensitivity and the speed of comparison.The default value that BLASTN program (for nucleotide sequence) adopts is as follows: word length (W) 11, and expected value (E) 10, M=5, N=-4, and compare two chains.For aminoacid sequence, the default value that the BLASTP program is used is: word length 3, expected value (E) 10, the BLOSUM62 rating matrix is (referring to Henikoff and Henikoff, Proc.Natl.Acad.Sci.USA 89:10915 (1989)) comparison (B) 50, expected value (E) 10, M=5, N=-4, and compare two chains.

" nucleic acid " refers to deoxyribonucleotide or ribonucleotide and their polymkeric substance and the complement thereof of strand or double chain form.This term comprises the main chain residue that contains known nucleotide analogue or modification or the nucleic acid of connection, they be synthesize, the producing of natural generation with non-natural, the binding characteristic of they and reference nucleic acid is similar, the metabolic way of they and reference Nucleotide is similar.The example of this class analogue includes but not limited to: thiophosphatephosphorothioate, phosphoramidate (phosphoramidates), methyl-phosphonate, chirality-methyl-phosphonate, 2-O-methyl ribonucleotides, peptide-nucleic acid (PNA).

Show unless have in addition, specific nucleic acid sequence is also implicit comprise its conservative modify variant (as, degenerate codon replaces) and complementary sequence, and the sequence that spells out.Specifically, the sequence that the 3rd mixed base in position of (or all) codon that can be by producing one or more selections and/or Hypoxanthine deoxyriboside residue replace obtains degenerate codon and replaces (Batzer etc., Nucleic Acid Res.19:5081 (1991); Ohtsuka etc., J.Biol.Chem.260:2605-2608 (1985); Rossolini etc., Mol.Cell.Probes 8:91-98 (1994)).Term nucleic acid and gene, cDNA, mRNA, oligonucleotide and polynucleotide are used interchangeably.

Also implicit comprise " splice variant " of specific nucleotide sequence.Similarly, the implicit any protein that comprises the splice variant coding of this nucleic acid of the specified protein of nucleic acid encoding.Hint that as its title " splice variant " is the alternative splicing product of gene.After transcribing, but montage original nucleic acid transcript, thereby different (variable) nucleic acid montage products not homopolypeptide of encoding.Produce the machine-processed different of splice variant, but comprise the alternative splicing of exon.This definition also comprises the variable polypeptide (alternate polypeptide) that is derived from same nucleic acid by read-through transcription.This definition comprises the spawn of montage reaction, comprises the montage product of recombinant forms.The example of potassium channel splice variant is described in Leicher etc., J.Biol.Chem.273 (52): 35095-35101 (1998).

Term " polypeptide ", " peptide " and " protein " are used interchangeably at this paper, refer to the polymkeric substance of amino-acid residue.This term can be applicable to the aminoacid polymers that one or more amino-acid residues are the amino acid whose artificial chemical simulation things of corresponding natural generation, and the aminoacid polymers of the aminoacid polymers of natural generation and non-natural generation.

Term " amino acid " refers to natural generation and synthetic amino acid and amino acid analogue and amino acid analog thing by working with the similar mode of natural generation amino acid.The amino acid of natural generation is the genetic code amino acids coding, and the amino acid of modifying subsequently, as oxyproline, Gla and O-phosphoserine.Amino acid analogue refers to have with the amino acid of natural generation the compound of identical basic chemical structure, promptly with hydrogen, carboxyl, amino and R base bonded α carbon, as homoserine, nor-leucine, methionine sulfoxide, methionine(Met) methyl sulfonium.This analogue has the R base (as nor-leucine) of modification or the peptide main chain of modifying, but keeps identical with the amino acid whose basic chemical structure of natural generation.The amino acid analog thing refers to that structure is different from amino acid whose general chemistry structure, but the mode of action is similar to the amino acid whose chemical compound of natural generation.

In this article, amino acid can be represented with general trigram code name or single-letter code name that the IUPAC-IUB biochemical nomenclature commission is recommended.Equally, Nucleotide can be with its generally accepted single-letter coded representation.

" the conservative variant of modifying " is applicable to amino acid and nucleotide sequence.For specific nucleotide sequence, conservative variant refer to the to encode nucleic acid of identical or essentially identical aminoacid sequence of modifying perhaps when nucleic acid not during encoding amino acid sequence, refers to essentially identical sequence.Since the degeneracy of genetic code, a large amount of identical any given protein of nucleic acid encoding of function.For example, codon GCA, GCC, GCG and the GCU L-Ala of all encoding.Therefore, be defined as at codon on each position of L-Ala, codon can be changed into described any corresponding codon and do not change encoded polypeptides.The nucleic acid variation is " silent variant ", and it is conservative a kind of of variation that modify.Every kind of nucleotide sequence of coded polypeptide as herein described is also described every kind of possible silent variant of this nucleic acid.Those skilled in the art will recognize that, but each codon in the modification of nucleic acids (except AUG and TGG, AUG is unique password of methionine(Met) normally, and TGG is unique password of tryptophane normally), to produce the identical molecule of function.Therefore, with regard to expression product but not at regard to the actual probes sequence, in described each sequence, hinted each silent variant of nucleic acid encoding.

As for aminoacid sequence, those skilled in the art will recognize that, nucleic acid, peptide, polypeptide or protein sequence are carried out single replacement, disappearance or adding, changing in encoding sequence, to add or to delete an amino acid or a small amount of amino acid is " the conservative variant of modifying ", this change makes certain monoamino-acid be replaced by amino acid like the chemofacies.It is well known in the art that intimate amino acid whose conservative replacement table is provided.The conservative variant of modifying of this class comprises does not get rid of analogue and allelotrope between polymorphism variant of the present invention, kind.

Below eight groups contain separately and can guard the amino acid that replaces mutually: 1) L-Ala (A), glycine (G); 2) aspartic acid (D), L-glutamic acid (E); 3) l-asparagine (N), glutamine (Q); 4) arginine (R), Methionin (K); 5) Isoleucine (I), leucine (L), methionine(Met) (M), Xie Ansuan (V); 6) phenylalanine (F), tyrosine (Y), tryptophane (W); 7) Serine (S), Threonine (T); With 8) halfcystine (C), methionine(Met) (M) (referring to for example, Creighton, Proteins (1984)).

" mark " or " but test section " is to pass through the constituent that spectrophotometric method, photochemical method, biochemical method, immuno-chemical method, chemical process or other physical method detect.For example, useful marker comprises ³²P, fluorescence dye, electron density reagent, enzyme (as through being usually used in ELISA), vitamin H, digoxin, maybe can detect or can be used for detecting protein and haptens with the antibody of peptide specific reaction by for example radioactively labelled substance being mixed in the peptide.

When for example addressing cell, nucleic acid, protein or carrier, term " reorganization " is represented this cell, nucleic acid, protein or carrier by introducing heterologous nucleic acids or protein or change natural acid or protein has been done modification, or this cell source is from the cell of so modifying.Therefore, for example reconstitution cell is expressed in the cell of natural (non-reorganization) form undiscovered gene or expresses unconventionality expression, the low natural gene of expressing or not expressing in other cases.

When being used for nucleic acid moiety, term " allos " refers to that this nucleic acid is included in the two or more subsequences that are not present under the natural situation in the same relation.For example, nucleic acid generally is that reorganization produces, have two or more from independent basis because of sequence, they are through rearranging new functional nucleic acid, for example promotor is from a source and originate from another in the coding region.Similarly, heterologous protein represents that this albumen comprises the two or more subsequences (as fusion rotein) that are not present under the natural situation in the same relation.

Phrase " stringent hybridization condition " refers to probe and its target sequence (usually in the complex mixture of nucleic acid) hybridization, but not with the condition of other sequence hybridization.Stringent condition is a sequence dependent, can be variant in varying environment.Long sequence specific hybrid under comparatively high temps.The detailed guidance of nucleic acid hybridization is referring to Tijssen, Techniques in Biochemistry and Molecular Biology--Hybridization with Nucleic Probes (biological chemistry and Protocols in Molecular Biology-use nucleic acid probe hybridization), " Overview of principles of hybridization and the strategy of nucleic acid assays (hybridization principle and the general introduction of nucleic acid testing program) " (1993).Stringent condition is selected usually than the pyrolysis chain temperature (T of concrete sequence under regulation ionic strength pH _m) low about 5-10 ℃.T _mBe under ionic strength, pH and the nucleic acid concentration of regulation, 50% with target complementary probe and target sequence hybridization balance the time temperature (because the target sequence of existence is excessive, T _mThe time, when balance, have 50% probe to be occupied).Also can enter destabilizing agent, for example methane amide obtains stringent condition.For selectivity or specific hybrid, positive signal is the twice at least of background, preferably 10 of background hybridization times.Exemplary rigorous hybridization conditions can be as described below: 50% methane amide, 5 * SSC and 1%SDS, 42 ℃ of cultivations, perhaps 5 * SSC, 1%SDS, 65 ℃ of cultivations, with 0.2 * SSC and 0.1%SDS 65 ℃ of washings.

If encoded polypeptides is basic identical, the nucleic acid of phase mutual cross is still not basic identical under stringent condition so.For example, when copying with the maximum codon degeneracy generation nucleic acid that genetic code allowed, this thing happens for possibility.In this case, nucleic acid is generally hybridized under the hybridization conditions of medium strictness.Exemplary " medium stringent hybridization condition " comprises 37 ℃, in the damping fluid of 40% methane amide, 1M NaCl, 1%SDS, hybridize, and 45 ℃, 1 * SSC washing.Positive hybridization is the twice of background at least.Those of ordinary skills are not difficult to recognize, can utilize other hybridization and wash conditions that the condition of similar severity is provided.Many reference provide other guidance of definite hybridization parameter, for example " newly organized molecular biology experiment guide " (Current Protocols in Molecular Biology), volumes such as Ausubel, (the John Wiley﹠amp of John Willie father and son company; Sons).

For PCR, normally about 36 ℃ of the temperature of low severity amplification is not though annealing temperature can wait between about 32 ℃ and 48 ℃ according to primer length.For high severity pcr amplification, typical temperature is about 62 ℃, though high severity annealing temperature can not wait between about 50 ℃ and about 65 ℃ according to primer length and specificity.The typical recycling condition of high and low severity amplification comprises 90 ℃-95 ℃, 30 seconds-2 minutes sex change stage, the annealing stage that continues 30 seconds-2 minutes and about 72 ℃, 1-2 minute extension stage.Scheme and guidance low and high severity amplified reaction see, Innis etc. for example, (1990) PCR Protocols, AGuide to Methods and Applications (PCR scheme, methods and applications guide), (the Academic Press of academic press company, Inc.), New York).

" antibody " refers to comprise the polypeptide of the framework region of immunoglobulin gene, or its specificity combination and the antigenic fragment of identification.The immunoglobulin gene of generally acknowledging comprises κ, λ, α, γ, δ, ε and μ constant region gene and a large amount of immune globulin variable region gene.Light chain is categorized as κ or λ.Heavy chain is categorized as γ, μ, α, δ or ε, and then defines immunoglobulin class respectively, IgG, IgM, IgA, IgD and IgE.Usually, the antigen binding domain of antibody is most important for bonded specificity and avidity.

Exemplary immunization sphaeroprotein (antibody) structure unit comprises the tetramer.Each tetramer is made of identical two pairs of polypeptide chains, and each is to having one " light chain " (about 25kDa) and one " heavy chain " (about 50-70kDa).The N-end of each chain defines main about 100-110 or more a plurality of amino acid whose variable region of being responsible for antigen recognition.Variable light chain (the V of term _L) and variable heavy chain (V _H) refer to these light chains and heavy chain respectively.

The antibody that exists can be, for example complete immunoglobulin (Ig) or with the fragment of many abundant signs of various peptide enzymic digestions generations.Therefore, for example, the disulfide linkage under the pepsin digested antibody hinge region is to produce F (ab) ' ₂, i.e. the dimer of Fab, Fab itself is connected in V by disulfide linkage _H-C _H1 light chain.F (ab) ' ₂Can under mild conditions, reduce opening the disulfide linkage of hinge region, thereby with F (ab) ' ₂Dimer changes into Fab ' monomer.Fab ' monomer comes down to have the Fab of hinge region part (referring to Fundamental Immunology (basic immunology) (Paul volume, the 3rd edition, 1993).Though the digestion according to complete antibody has defined various antibody fragments, the technician should know, can by chemical process or these fragments of employing recombinant DNA technology de novo synthesis.Therefore, term used herein " antibody " also comprises by modifying the antibody fragment that complete antibody produces, or adopt the recombinant DNA method de novo synthesis those (for example, strand Fv) or utilize that phage display library identifies those (referring to, McCafferty etc. for example, Nature 348:552-554 (1990)).

In order to prepare suitable antibody of the present invention and for application of the present invention, as recombinant chou, mono-clonal or polyclonal antibody, applicable multiple technologies known in the art (referring to, for example, Kohler﹠amp; Milstein, Nature 256:495-497 (1975); Kozbor etc., Immunology Today 4:72 (1983); Cole etc., 77-96 page or leaf, " monoclonal antibody and cancer therapy " (Monoclonal Antibodies and Cancer Therapy), Alan R.Liss, Inc. (1985); Coligan, " the up-to-date experiment guide of immunology " (Current Protocols in Immunology) (1991); Harlow﹠amp; Lane, " antibody laboratory manual " (Antibodies, A Laboratory Manual) (1988); And Goding, " monoclonal antibody principle with put into practice " (Monoclonal Antibodies:Principles and Practice) (second edition, 1986)).Encoding the gene of heavy chain of antibody interested and light chain can be from cell clone, and for example, the gene of coding monoclonal antibody can and be used for making recombinant monoclonal antibodies from the hybridoma clone.The gene library of coding monoclonal antibody heavy chain and light chain also can be from hybridoma or plasma cell manufacturing.The combination at random of heavy chain and light chain gene product will produce the antibody that has different antigen-specifiies in a large number (referring to, for example, Kuby, Immunology (third edition, 1997)).The technology (United States Patent (USP) 4,946,778, United States Patent (USP) 4,816,567) of making single-chain antibody or recombinant antibodies also is applicable to the antibody of making polypeptide of the present invention.Simultaneously, transgenic mice or other biological (as other Mammalss) also can be used to express humanized antibody or people's antibody (referring to, for example, United States Patent (USP) 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, 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 (1996); And Lonberg﹠amp; Huszar, Intern.Rev.Immunol.13:65-93 (1995)).Perhaps, display technique of bacteriophage can be used to identify specificity in conjunction with selected antigenic antibody and different poly-Fab fragment (referring to, for example, McCafferty etc., Nature348:552-554 (1990); Marks etc., Biotechnology 10:779-783 (1992)).Antibody can be made into two special, promptly can discern two kinds not synantigen (referring to, for example, WO 93/08829, Traunecker etc., EMBO are (1991) J.10:3655-3659; With Suresh etc., " Enzymology method " (Methods in Enzymology) 121:210 (1986)).Antibody also can be the allos conjugate, for example, and two covalently bound antibody, or immunotoxin (referring to, for example, United States Patent (USP) 4,676,980, WO 91/00360; WO92/200373; With EP 03089).

Humanization or primate non-human antibody's method is well known in the art.Usually, humanized antibody contains from inhuman source and introduces one or more amino-acid residue.These inhuman amino-acid residues often are called input residue (import residue), and they take from the input variable region usually.Humanization in fact can according to Winter and colleague thereof (referring to, for example, Jones etc., Nature 321:522-525 (1986); Riechmann etc., Nature 332:323-327 (1988); Verhoeyen etc., Science 239:1534-1536 (1988) and Presta, Curr.Op.Struct.Biol.2:593-596 (1992)) method, replace the corresponding sequence of people's antibody with rodents CDR or CDR sequence.Therefore, this humanized antibody is chimeric antibody (U.S. Patent number 4,816,567), wherein is shorter than complete people variable region basically and is replaced by the corresponding sequence of inhuman kind.In practice, humanized antibody generally is a number of C DR residue, may be that some FR residues are by people's antibody that residue replaced in similar site in the rodent animal antibody.

" chimeric antibody " is the antibody molecule that meets the following conditions, wherein: (a) constant region, or its part, be that change, that replace or exchange, thereby antigen binding site (variable region) is connected in the constant region that classification, effector function and/or kind are different or change, or be connected in the diverse molecule that to give the chimeric antibody new features, for example enzyme, toxin, hormone, somatomedin, medicine etc.; Or (b) variable region, or its part be that change, that replace or exchange, thereby the variable region has antigen-specific different or that change.Preferred antibody of the present invention and be preferred for antibody of the present invention and comprise humanized antibody and/or chimeric mAb.

In the embodiment, described antibody coupling is in " effector " part.The effector part can be any amount of molecule, comprises the marker part, and for example radioactively labelled substance or fluorescent marker perhaps can be the treatment parts.On the one hand, antibody is regulated activity of proteins.This effector partly includes, but not limited to antitumour drug, toxin, radiotherapeutic agents, cytokine, second antibody or enzyme.In addition, in the embodiment provided by the invention, antibody of the present invention is connected in the enzyme that prodrug is changed into cytotoxic agent.

Immune conjugate can be used to effector part target N-cadherin positive cell is especially expressed the proteic cell of N-cadherin or Ly6.By observing load similarly the gel band of specimen and control sample is arranged, be very easy to find this species diversity.The example of cytotoxic agent includes, but are not limited to: ricin, Dx, daunorubicin, taxol, ethidium bromide, mitomycin, etoposide, Vumon, vincristine(VCR), vinealeucoblastine(VLB), colchicine, dihydroxyl anthracin diketone, dactinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, toxalbumin and glucocorticosteroid and other chemotherapeutics, and radio isotope.Suitable certification mark thing includes, but not limited to radio isotope, fluorescent chemicals, bioluminescent compound, chemiluminescence compound, metal chelator or enzyme.

In the part embodiment, the invention provides the antibody of anti-N-cadherin.But N-cadherin antibody whole body uses with treatment cancer (for example, prostate gland or bladder cancer), can use separately or with the coupling of effector part.Be coupled to the N-cadherin antibody of toxic agent (as ricin) and not link coupled antibody can be the useful therapeutical agent that natural target has the prostate cancer cell of N-cadherin.This antibody can be used for the blocking-up invasion and attack.Be applicable to that N-cadherin antibody of the present invention includes, but not limited to GC4,1H7,1F12 and 2B3.

In addition, the recombinant protein of the present invention that comprises the antigen binding domain of any monoclonal antibody of the present invention can be used to treat cancer.In this case, the antigen binding domain of recombinant protein is incorporated into second proteic at least one functionally active part with therapeutic activity.Described second albumen can include, but are not limited to: enzyme, lymphokine, oncostatin or toxin.Suitable toxin comprises Dx, daunorubicin, taxol, ethidium bromide, mitomycin, etoposide, Vumon, vincristine(VCR), vinealeucoblastine(VLB), colchicine, dihydroxyl anthracin diketone, dactinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, ricin, toxalbumin, glucocorticosteroid and radio isotope.

The technology that therapeutical agent is coupled to antibody is known, referring to for example, Arnon etc., " in cancer therapy, use the monoclonal antibody of the immune target of medicine " (Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy), publish in " monoclonal antibody and cancer therapy " (Monoclonal Antibodies And Cancer Therapy), Reisfeld etc. (volume), 243-56 page or leaf (Arl Inc. (Alan R.Liss, Inc.) 1985); Hellstrom etc., " be used for the antibody that medicine is sent " (Antibodies For Drug Delivery), publish in " medicine of control is sent " (Controlled Drug Delivery) (the 2nd edition), Robinson etc. (volume), 623-53 page or leaf (MD company (Marcel Dekker, Inc) .1987); Thorpe, " antibody carrier of cytotoxic agent in the cancer therapy: summary " (Antibody Carriers Of Cytotoxic Agents In Cancer Therapy:A Review), publish in " monoclonal antibody ' 84: biology and clinical application " (Monoclonal Antibodies ' 84:Biological and Clinical Applications), Pinchera etc. (volume), 475-506 page or leaf (1985); With Thorpe etc., " preparation of antibody-toxin conjugated thing and cell toxicant characteristic " (The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates), Immunol.Rev., 62:119-58 (1982)).

When relating to protein or peptide, term " specificity (or selectivity) in conjunction with " in antibody or " with ... specificity (or selectivity) immune response takes place " refer in the heterogeneous population of protein and other biological substance, to determine the association reaction of this proteinic existence.Therefore, under specified immunity test condition, this specific antibody and combining of specified protein are at least the twice of background, the 10-100 that is more typically background doubly more than.Under these conditions, the specificity of antibody is in conjunction with the antibody that need select according to the specificity of itself and specified protein.For example, optional majority clonal antibody so that only obtain with selected antigen not with the polyclonal antibody of other protein generation specific immune responses.Can with other molecules interactional antibody take place and realize this selection by deducting.Can utilize the antibody of various immunoassay forms selections and specified protein generation specific immune response.For example, usually with solid phase ELISA immunoassay select with the antibody of protein generation specific immune response (referring to, for example, Harlow﹠amp; Lane, " the immunoassay form and the condition that are used for determining specific immune response have been described in the antibody application experiment chamber handbook (Using Antibodies, A Laboratory Manual) (1998)).

" treatment significant quantity or dosage " expression of this paper produces the dosage of the required effect of administration.Accurately dosage and preparation will depend on therapeutic purpose, and will by those skilled in the art adopt known technology determine (referring to, for example, Lieberman, " pharmaceutical dosage form " (Pharmaceutical Dosage Forms) (1-3 volume, 1992); Lloyd, " pharmaceutical technology, science and technology " (The Art, Science and Technology of Pharmaceutical Compounding) (1999); " Lei Mingdun pharmaceutical science with put into practice " (Remington:The Science and Practice of Pharmacy), the 20th edition, Gennaro compiles (2003); And Pickar, " Rapid Dose Calculation " (Dosage Calculations) (1999)).

Term " pharmacy acceptable salt " or " pharmaceutically acceptable carrier " should comprise according to the concrete substituting group of finding on the compound described herein, use the salt of the active compound of nontoxic relatively acid or alkali preparation.When The compounds of this invention contains relative tart functional group, can obtain base addition salt by the required alkali of neutral form contact capacity with this compound, described alkali can be pure alkali form or the form in suitable inert solvents.The example of pharmaceutically acceptable base addition salt comprises sodium salt, sylvite, calcium salt, ammonium salt, organic amino or magnesium salts, or class is saloid.When The compounds of this invention contains alkaline relatively functional group, can obtain acid salt by the required acid of neutral form contact capacity with this compound, described acid can be pure sour form or the form in suitable inert solvents.The example of pharmaceutically-acceptable acid addition comprises: derived from the salt of mineral acid, example hydrochloric acid salt, hydrobromate, nitrate, carbonate, supercarbonate, phosphoric acid salt, hydrophosphate, dihydrogen phosphate, vitriol, hydrosulfate, hydriodate or phosphite etc., and derived from nontoxic relatively organic acid salt, as acetate, propionic salt, isobutyrate, maleate, malonate, benzoate, succinate, suberate, fumarate, lactic acid salt, mandelate, phthalate, benzene sulfonate, p-methyl benzenesulfonic acid salt, Citrate trianion, tartrate, mesylate etc.Also comprise amino acid whose salt, as arginic acid salt etc. and organic acid salt, as the salt of glucuronic acid or galacturonic acid etc. (referring to for example, Berge etc., Journal of Pharmaceutical Science 66:1-19 (1977)).Some specific compound of the present invention contains the alkalescence and the acidic functionality that can make this compound change alkali or acid salt into.The present invention is fit to use other pharmaceutically acceptable carrier well known by persons skilled in the art.

This salt can be contacted with alkali or acid, and separate parent compound in a usual manner, thus the neutral form of this compound of regenerating.Some physical property of the parent form of this compound (for example solubleness in polar solvent) is different with various salt forms, but is equal to the parent form of this compound at this salt of others of the object of the invention.

Except that salt form, the present invention also provides the compound of prodrug forms.The prodrug of compound described herein is to provide compound of the present invention by chemically changed easily under physiological condition.In addition, under the environment that exsomatizes, prodrug can change The compounds of this invention into by chemistry or biochemical method.For example, when being placed in the percutaneous plaster bank that contains suitable enzymes or chemical reagent, prodrug can slowly change The compounds of this invention into.

Some compound of the present invention can non-solvent closes form and solvent and closes form (comprising hydrated form) and exist.Usually, the solvent form of closing is equal to non-solvent and closes form, all should fall into the scope of the invention.Some compound of the present invention can polycrystalline form or amorphous form existence.Usually, all physical form are equal in the application that the present invention considered, these physical form all should fall into the scope of the invention.

Some compound of the present invention has unsymmetrical carbon (optical center) or two key; Racemoid, diastereomer, geometrical isomer and single isomer all should fall into the scope of the invention.

Epithelium is the matter feature between matter conversion (EMT) expression epithelial tumor cell has obtained.In cancer, EMT is relevant with motor behavior (motile behavior) with invasion and attack, and may be the main process under the transfer case.EMT is relevant with prognosis mala, and is mediated by multiple transcription factor (as SNAIL, SLUG and TWIST).

The E-cadherin is a kind of cell surface protein, and it participates in epithelial cell-cell adhesion, and disappears in aggressive and metastatic solid tumors usually.

Detailed embodiment

The invention describes two kinds of generations can be in conjunction with the mouse hybridoma cell system of the monoclonal antibody of N-cadherin antigenic determinant.Described mouse hybridoma cell is with ATCC accession number PTA-9387 (1H7) and ATCC accession number PTA-9388 (EC4) preservation.

In an embodiment of the invention, antibody is to be produced by the hybridoma cell line with ATCC accession number PTA-9387 preservation.In yet another embodiment of the present invention, antibody is to be produced by the hybridoma cell line with ATCC accession number PTA-9388 preservation.

Cell with ATCC accession number PTA-9387 and ATCC accession number PTA-9388 preservation is abideed by budapest treaty, be preserved in American Type Culture Collection (ATCC) on July 23rd, 2008, the 10801 big ways for education (University Boulevard), Manassas, the Virginia, 20110.Detect and confirmed survival on August 29th, 2008.

In some embodiments of the present invention, having made can be in vivo or antibody or its fragment of the external combination N-cadherin antigenic determinant with the monoclonal antibody that hybridoma cell line was produced that is preserved in American Type Culture Collection with ATCC accession number PTA-9387 identical.In some embodiments of the present invention, having made can be in vivo or antibody or its fragment of the external combination N-cadherin antigenic determinant with the monoclonal antibody that hybridoma cell line was produced that is preserved in American Type Culture Collection with ATCC accession number PTA-9388 identical.

In some embodiments, described antibody can be in conjunction with first ectodomain of N-cadherin.In some embodiments, described antibody can be in conjunction with second ectodomain of N-cadherin.In some embodiments, described antibody can be in conjunction with three categories of overseas Chinese's outer structure territory of N-cadherin.In some embodiments, described antibody can in conjunction with the N-cadherin first to three categories of overseas Chinese's outer structure territory.In some embodiments, described antibody can be in conjunction with the 4th ectodomain of N-cadherin.

The invention still further relates to the method that suppresses the interior growth of cancer cells of patient's body or described cancer cells is killed.Described method generally includes under the following conditions, gives the patient with claim 5 or 11 described antibody or its binding fragment: be enough to make described antibody or its binding fragment in conjunction with described tumour cell or prostate cancer tumour cell; Regulate cytoactive; Suppressing the tumour cell blood vessel takes place; And causing growth of tumour cell to suppress or death, expression N-cadherin is expressed or crossed to wherein said cancer cells.

In some embodiments of the present invention, described antibody conducts by activation or inhibition NF κ-signal and transcribes and regulate cytoactive.In some embodiments of the present invention, described antibody is by activating or suppressing the internalization of N-cadherin and regulate cytoactive.In some embodiments of the present invention, described antibody is by activating or suppressing the PI3 kinases or the Akt approach is regulated cytoactive.In some embodiments of the present invention, described antibody is by activating or suppressing the conduction of beta-catenin white signal and regulate cytoactive.In some embodiments of the present invention, described antibody is regulated cytoactive by the different dimerization of blocking-up N-cadherin and FGFR or other tyrosine kinase receptors.In other embodiments more of the present invention, described antibody is regulated cytoactive by the cutting action of blocking-up or enhancing ADAM10 or other metallopeptidases.

In some embodiments, described antibody suppresses the interior apparatus urogenitalis growth of cancer cells of patient's body or described cancer cells is killed.In some embodiments, described antibody suppresses the interior prostate cancer cell growth of patient's body or described cancer cells is killed.In some other embodiments, described antibody suppresses the interior growth of bladder cancer cells of patient's body or described cancer cells is killed.

Preparation and administration

According to currently known methods will resist-N-cadherin antibody or immune conjugate give human patients, as intravenous administration, for example as injecting or by continous pouring in for some time, by in (intracerobrospinal) in intramuscular, intraperitoneal, the myelencephalon, subcutaneous, intraarticular, the synovial membrane, interior, oral, the part of sheath or inhalation route.Preferably by intravenously or the subcutaneous antibody that gives.Administration can be topical or whole body administration.

The composition that gives comprises the medicament described herein (for example, N-cadherin inhibitor, N-cadherin antibody and immune conjugate, N-cadherin siRNA and their carrier) that is dissolved in pharmaceutically acceptable carrier (preferred aqueous carrier) usually.Can use various aqueous carriers, as buffer saline or the like.These solution are aseptic and do not contain unwanted material usually.These compositions can be by conventional known sterilising technology degerming.Said composition can contain the required pharmaceutically acceptable auxiliary substance of simulation physiological condition, as pH regulator agent and buffer reagent, toxicity conditioning agent etc., for example, sodium acetate, sodium-chlor, Repone K, calcium chloride, Sodium.alpha.-hydroxypropionate etc.Surfactant concentration in these preparations can extensively change, and mainly according to factors such as liquid volume, viscosity, body weight, selects according to selected specific administration mode and needs of patients.

Therefore, typically supply the pharmaceutical composition of intravenous administration will be with medicament different.Preparation can be by the parenteral administration the practical methods of composition be well-known to those skilled in the art, more detailed description is referring to " Lei Mingdun pharmaceutical science ", the 15th edition, (the Mack Publishing Co. of Mike publishing company, Easton, PA, 1980) etc. in the publication.

Can give pharmaceutical composition with various unit dosage according to medication.For example, the unit dosage of suitable oral administration includes but not limited to: powder, tablet, pill, capsule and lozenge.Known to when the orally give antibody, tackle it and protect avoiding and digested.This is normally compound by molecule and the composition that makes molecule opposing quilt acid and enzymic hydrolysis are carried out, or realizes by molecule being packaged in the carrier (as liposome or protection barrier) with appropriate resistance.The method that the protection medicament is avoided being digested is known in the art.

Pharmaceutical preparation in particular for the pharmaceutical preparation of antibody of the present invention and immune conjugate and inhibitor, can be mixed and made into by the antibody that will have required purity and pharmaceutically acceptable carrier, vehicle or the stablizer chosen wantonly.This reagent can be freeze-dried preparation or aqueous solution.Acceptable carrier, vehicle or stablizer are nontoxic to acceptor under using dosage and concentration.Acceptable carrier, vehicle or stablizer can be acetate, phosphoric acid, citric acid and other organic acids; Antioxidant (for example, xitix) sanitas; Low molecular weight polypeptide; Protein, as serum albumin or gelatin, or hydrophilic polymer (as polyvinylpyrrolidone); And amino acid, monose, disaccharides and other carbohydrate, comprise glucose, seminose or dextrin; Sequestrant; And ion and nonionogenic tenside (for example, polysorbate); Salify counter ion such as sodium; Metal complex (for example Zn-protein complex); And/or nonionogenic tenside.The concentration of antibody can be mixed with 0.5-200mg/ml or 10-50mg/ml.

Also can provide other active compounds in the preparation, comprise: chemotherapeutics, cytotoxic agent, cytokine, growth inhibitor and hormone antagonist reagent.Activeconstituents also can be made into sustained release preparation (for example, the semi-transparent matrix of solid hydrophobic polymkeric substance (for example, polyester, hydrogel (for example, poly-(2-hydroxyethyl-methacrylic ester) or poly-(vinyl alcohol)), polylactide.Antibody and immune conjugate also can wrap in the microcapsule that prepare by (for example) condensation technique or interfacial polymerization, example is respectively Walocel MT 20.000PV or gelatin-microcapsule and poly--(methyl methacrylate) microcapsule, or wraps into colloid drug delivery system (for example liposome, albumin microsphere, micro emulsion, nano particle and Nano capsule) or wrap into thick dripping in the emulsion (macroemulsion).

Can give described composition treats or preventive treatment.In treatment is used, the composition of " treatment significant quantity " can be given ill (for example, cancer) patient.The overall health that will depend on severity of disease and patient to the effective amount of this application.Tolerance according to required dosage and frequency and patient takes single or multiple to give composition.For purposes of the present invention, " patient " or " object " comprises people and other animals, especially Mammals.Therefore, described method can be used for human treatment and animal doctor's application.In a preferred embodiment, described patient is a Mammals, preferred primates, and in most preferred embodiment, described patient is the people.Other known cancer therapies can be used in combination with the inventive method.For example, be used for composition of the present invention and also can be used to other cancer therapeutic agents of cell-targeting or make cell to other cancer therapeutic agent sensitivities, described other cancer therapeutic agent is for example 5FU, vinealeucoblastine(VLB), dactinomycin, cis-platinum, methotrexate etc.

In other embodiments, the inventive method and other cancer therapies are (for example, radical prostatectomy), radiotherapy (external beam or brachytherapy), (for example, testectomy suppresses the LHRH-analogue therapy that testosterone produces to hormonotherapy, anti--androgenotherapy), or chemotherapy is used in combination.Radical prostatectomy comprises removes complete prostate gland and some peripheral organization.When also not diffusing out this tissue, cancer adopts this processing usually.Radiotherapy is commonly used to treat the prostate cancer that still is confined in the prostate gland or has been diffused into surrounding tissue.If disease further develops, can adopt radiotherapy to dwindle gross tumor volume.Hormonotherapy is generally used for prostate cancer and has been diffused into the patient outside the prostate gland or has recurred the patient.The purpose of hormonotherapy is to reduce the androgenic level of androgen, thereby makes prostate cancer dwindle or grow slower.Luliberin (LHRH) promotes the minimizing that testosterone produces.These medicaments can every month or the longer time injection once.Two kinds of such analogues are Leuprolide and goserelin.Also can adopt androgen antagonist (for example, flutamide, bicalutamide and Nilutamide).Male sex hormone is blocked expression fully and is used in combination androgen antagonist and testectomy or LHRH analogue.Chemotherapy can be used for prostate cancer and has been diffused into the patient that prostate gland is outer and hormonotherapy is failed.It can not destroy whole cancer cells, but the tumor growth and easing the pain of slowing down.Some chemotherapeutic can be used to treat with hormonotherapy treatment back tumour recovers the prostate cancer of growth or continued growth and generation diffusion, and these chemotherapeutic are for example Dx (adriamycin), Emcyt, etoposide, mitoxantrone, vinealeucoblastine(VLB) and taxol.Two or more medicines are produced chemical sproof possibility to reducing cancer cells to chemotherapy usually together.Small cell carcinoma is a kind of rare prostate cancer, compares hormonotherapy, and it is stronger to the response of chemotherapy.

In some embodiments, " cardioprotectant " also with N-cadherin antibody, N-cadherin binding inhibitors or N-cadherin siRNA molecular combinations be used for the present invention (referring to, United States Patent (USP) 6,949,245).Cardioprotectant is a kind of preventing or the compound or the composition of the myocardial dysfunction (being myocardosis and/or congestive heart failure) that reduction is relevant with drug administration (as using anthracycline antibiotics to the patient).Cardioprotectant can, for example, blocking-up or reduce the heart toxic effect of free radical mediated and/or prevent or reduce the oxidative stress damage.The ion of the cardioprotectant that this definition comprised comprises: iron chelating agent dexrazoxane (ICRF-187) (Seifert etc., " pharmacological agent yearbook " (The Annals of Pharmacotherapy) 28:1063-1072 (1994)); Lipid lowerers and/or antioxidant are as probucol (Singal etc., J.Mol.Cell Cardiol.27:1055-1063 (1995)); Amifostine (amineothiot 2-[(3-aminopropyl) amino] sulfur alcohol-dihydrogen phosphoric acid ester, be also referred to as WR-2721, and its dephosphorylation cellular uptake form that is called WR-1065) and S-3-(3-methylamino propyl group amino) propyl group phosphorus-thioic acid sulfoacid (WR-151327), referring to Green etc., Cancer Research 54:738-741 (1994); (Bristow, M.R. are selected from " drug-induced heart trouble " (Drug-Induced Heart Disease) that Bristow M R compiles, New York: Elsevier 191-215 (1980)) to digoxin; Beta blocker is as metoprolol (Drugs 47:Suppl 4:31-9 (1994) such as Hjalmarson; With Shaddy etc., Am.Heart is (1995) J.129:197-9); Vitamin-E; Xitix (vitamins C); Free-radical scavengers, as Oleanolic Acid, ursolic acid and N-acetylcystein (NAC); The spin trapping compound is as α-phenyl-tert-butylnitrone (PBN); (Paracchini etc., Anticancer Res.13:1607-1612 (1993)); Organoselenium based compound such as P251 (Elbesen); Or the like.

Combination medicine-feeding consider to adopt independent preparation or single medicine preparation to take medicine simultaneously, and with the random order successive administration, wherein preferably over a period to come two kinds of (or all) promoting agents bring into play their biologic activity simultaneously.

The molecule of regulating expression of N-calmodulin and/or function indirectly or directly and compound through identifying can be used for treating the cancer of expressing the N-calmodulin respectively.N-cadherin conditioning agent can give separately or give with chemotherapy, radiotherapy or the immunotherapy of routine and the therapy combination of now having developed.

The preparation that is fit to oral administration comprises: (a) liquor, as be suspended in the packing nucleic acid of the significant quantity in the thinner (as water, salt solution and/or PEG 400); (b) capsule, wafer or tablet, it contains the liquid state of predetermined amount, solid-state, particulate state or gelatinous activeconstituents separately; (c) be suspended in suspension in the suitable liquid; (d) suitable emulsion.Tablet form can comprise following one or more: the carrier of lactose, sucrose, N.F,USP MANNITOL, sorbyl alcohol, calcium phosphate, W-Gum, yam starch, Microcrystalline Cellulose, gelatin, colloid silica, talcum, Magnesium Stearate, stearic acid and other vehicle, tinting material, filler, tackiness agent, thinner, buffer reagent, wetting agent, sanitas, seasonings, dyestuff, disintegrating agent and pharmaceutically compatible.Lozenge form can comprise activeconstituents in seasonings (as sucrose), simultaneously, the activeconstituents of pastille is included in the inert base, as gelatin and glycerine or sucrose and gum arabic emulsion, gel etc., wherein except comprising activeconstituents, also contain carrier known in the art.

Independent or with the selected compounds of other suitable combination things combinations, can be made into aerosol preparations (getting final product " spraying ") so that give by suction.Aerosol preparations can be put into acceptable pressurized propellant, in Refrigerant 12, propane, nitrogen etc.

The preparation that is fit to rectal administration comprises, for example, suppository, its nucleic acid and suppository base by packing constitutes.Suitable suppository base comprises natural or synthetic triglyceride level or paraffinic.In addition, also may use gelatin rectal capsule, it is formed by compound of selecting and substrate combination, and described matrix comprises, for example, and liquid triglycerides, macrogol and paraffinic hydrocarbons.

The preparation that is fit to administered parenterally (for example interior by internode (intraarticular), intravenously, intramuscular, knurl, intradermal, intraperitoneal and subcutaneous route administration) comprises water-based and nonaqueous isotonic sterile injection liquid, wherein can comprise antioxidant, buffer reagent, fungistat and make preparation and the isoosmotic solute of receptor's blood, and water-based and non-aqueous sterile suspensions, wherein can comprise suspension agent, solubilizing agent, thickening material, stablizer and sanitas.In practice of the present invention, can give composition by for example venoclysis, oral, local, intraperitoneal, intravesical or intrathecal route.Preferred medication is administered parenterally, oral administration and intravenous administration.The preparation of compound can be present in unitary dose or the multiple doses sealed vessel, as ampoule or medicine bottle.

Can be from sterilized powder, particle and tablet preparation injection solution or the suspension of describing type before.The nucleic acid cell transformed that is used to stripped therapy also can give by above-mentioned intravenously or parenteral route.

Pharmaceutical preparation is preferably taked unit dosage.In this form, goods are divided into the unitary dose that contains an amount of active ingredient.Described unit dosage can be the goods of packing, and described packing contains the preparation of independent amount, as is packaged in tablet, capsule and powder in medicine bottle or the ampoule.Simultaneously, unit dosage also can be capsule, tablet, cachet or a lozenge itself, perhaps can be the packaged form that contains suitable quantity capsule, tablet, cachet or lozenge.If desired, also can contain other compatible therapeutical agents in the composition.

Preferred pharmaceutical preparation is with one or more active N-cadherin conditioning agents, and optional and one or more chemotherapeutics or immunotherapeutic agent combination are sent with the sustained release preparation form.Usually, in treatment was used, N-cadherin conditioning agent gave as sensitiser, was used for improving the responsive type of tumour cell to other cell toxicant cancer therapies of comprising chemotherapy, radiotherapy, immunotherapy and hormonotherapy.

In treatment treatment for cancer purposes, being used for the N-cadherin conditioning agent of pharmaceutical methods of the present invention or the initial dosage of inhibitor is that every day about 0.001 is to about 1000mg/kg.To about 500mg/kg, or about 0.1mg/kg is to about 200mg/kg from about 0.01mg/kg for adoptable per daily dose scope, or about 1mg/kg about 100mg/kg extremely, or about 10mg/kg about 50mg/kg extremely.Yet formulation also can change according to patient's demand, the severity of disease of treatment and the compound that is adopted.For example, can consider cancer types and stage that particular patient is diagnosed, rule of thumb determine dosage.For the present invention, the dosage that gives the patient should be enough to produce useful therapeutic response over time.Dosage range will depend in the particular patient and give specific support or existence, the nature and extent of any adverse side effect that the transducer cell type accompanies.The doctor understands the suitable dose that how to determine to be used for particular case.Usually, use smaller dose begin treatment less than this compound optimal dosage.Afterwards, improve dosage with less amplitude, until being issued to best effect at corresponding conditions.For for simplicity, if desired, the gradation separately and in a day of total per daily dose can be given.

Be used for pharmaceutical preparation of the present invention (for example, N-cadherin siRNA, N-cadherin antibody, N-cadherin vaccine, N-cadherin inhibitor and immune conjugate) and be delivered to Mammals usually, comprise people and other non-human mammals.Non-human mammal with the inventive method treatment comprises domestic animal (being dog class, cat class, muroid, rodent class and Lagomorpha) and agricultural animal (ox, horse, sheep, pig).

Embodiment

Provide following examples, with explanation but not the present invention of requirement for restriction protection.

Embodiment 1: adopt the NF κ beta receptor experiment of TAL as negative control.

Shown in Fig. 2-3, NF κ beta receptor is active in the cell of stably express N-cadherin improves.All like this in being full of and exhausting androgenic medium.Show that also because the N-cadherin that LNCaP-C1 expresses is more than C2 and C3, NF κ 'beta ' activity is relevant with the amount of N-cadherin expression.

Embodiment 2

Shown in Fig. 4-5, the N-cadherin is at the cell surface expression of LNCaP-C1 cell, and the NF κ β that these cells have higher amount expresses, but NF κ β is activated, shown in the strong nuclear signal in the C1 cell.Control cells is also expressed NF κ β, but mainly is positioned at kytoplasm.

Embodiment 3

Fig. 6-7 shows that the N-cadherin demonstrates the rise of NF κ β target gene TGF β, IL-6, IL-8 and bcl-2 to the activation of NF κ β.

Embodiment 4

As shown in Figure 8, strike and subtract (knockdown) N-cadherin and cause NF κ β target gene such as IL-6 and IL-8 to express to reduce, show that once more the N-cadherin regulates NF κ β.

Embodiment 5

Fig. 9 has shown with the NF κ β promoter activity before and after the processing of N-cadherin targeting antibodies.Data presentation the rise immediately of these antibody-mediated NF κ β, reduced at the 7th day subsequently.These data presentation, antibody can part play a role by changing the transduction of NF κ β downstream signal.

Embodiment 6

Figure 10 has shown the dyeing of the N-cadherin of using or use the processing of N-cadherin targeting antibodies.In the control treatment cell, most of N-cadherins appear in the born of the same parents; And when having antibody, the N-cadherin is expressed and is stable at cell surface.These Notes of Key Datas are stabilized in the another kind of mechanism of action that cell surface is a N-cadherin antibody with the N-cadherin.The change that this may cause the beta-catenin white signal by metallopeptidase (as ADAM 10 etc.) cutting and the mediation of internalization N-cadherin to conduct.

Embodiment 7

As shown in Figure 11, the downward modulation of N-cadherin can cause the activation of PI3K/Akt signal transduction path to reduce.

Embodiment 8

As shown in Figure 12, handle cell with N-cadherin targeting antibodies and cause Akt phosphorylation or activation to change (go up to be in harmonious proportion and reduce), this has supported these antibody all or part of by changing the viewpoint that the PI3K-Akt signal transduction plays a role.

Embodiment 9:FACS analyzes the screening of the antibody cloning that has shown target N-cadherin first ectodomain.

Figure 13 has shown the identification of prostate cancer cell surface protein.

Embodiment 10:FACS analyzes the screening of the antibody cloning that has shown target N-cadherin the 4th ectodomain.

Figure 14 shows, the N-cadherin on clone's identification prostate cancer cell surface.

The subclone of embodiment 11:N-cadherin antibody.

Figure 15 shows, the protein on the mono-clonal recognizing cells surface of purifying.

Embodiment 12: external invasion and attack test.

Figure 16 shows, the invasiveness that monoclonal antibody 1F12,1H7 that is produced by contriver group and 2B3 suppress N-cadherin positive cancer cell.GC4 is contrast.

The transfer of embodiment 13:N-cadherin antibody blocking tumor growth and PC3 prostate cancer cell, but invalid to the blank tumour of N-cadherin.

Subcutaneous implantation PC3 cell also makes its growth.Shown in Figure 17 b,, the 15th day tumour give antibody or contrast, twice weekly (200 microgram), totally two weeks can touch the stage time when reaching.1H7 and EC4 can both slow down tumor growth.They are blocking-up transfer fully also.5/5 control mice has nodus lymphoideus transferring rate, and by contrast, 1H7 and EC4 treatment mouse are respectively 0/5 and 0/5.On the contrary, (Figure 17 a) less than influence to the blank growth of tumor of N-cadherin for 1H7 and EC4 antibody.Figure 17 c and 17d be clear shown handle the big tumour (handling) set up since the 21st day or in long-term disposal (processing lasts up to 62 days) N-cadherin antibody to the restraining effect of PC3 tumor growth.

Embodiment 14: the tumour of ex vivo experiment demonstrates the influence of antibody to the N-cadherin.

As shown in figure 18, two kinds of control tumor colors are very red, take place consistent with deep-level blood vessel.They also invest and invade local side muscle tissue deeply.On the contrary, the mouse of 1H7 and EC4 processing has pale, the limpid tumour of not invading local muscle.Tumour comes off from lower-hierarchy easily, proves that anti--N-cadherin antibody has suppressed that blood vessel takes place and the local invasion and attack of blocking-up, and this is consistent with the result that finds in the mouse that does not suffer from metastatic disease.

Embodiment 15: immunohistochemical staining

The contriver has carried out immunohistochemical staining to androgen independence LAPC 9 prostate cancers.The result shows that (Figure 19 a) only to have a little cell subset to express the N-cadherin.

Embodiment 16: androgen-dependent and dependent/non-dependent LAPC-9 tumor growth

For determine to be convenient N-cadherin when cross expressing or only expressing in cell subsets in cell, whether the N-cadherin can become treatment and diagnosis target spot, and the contriver has studied the tumor growth of androgen-dependent and dependent/non-dependent LAPC-9 tumour.Respectively with contrast PBS or N-cadherin antibody 1H7 and EC4 processing androgen-dependent and dependent/non-dependent LAPC-9 tumour.The result shows, even if the N-cadherin is only expressed, also be enough to postpone androgen independence growth of tumor and development (Figure 20) with antibody treatment in a little subgroup androgen independence cell.These results suggest, the formation of androgen independence tumour need N-cadherin cell mass, and its blocking-up is enough to postpone tumor development.These results are consistent with the explanation of N-cadherin mark androgen independence population of stem cells.The blocking-up stem cell growth is enough to block tumor growth.These results also show, antibody can act on expresses normal level or even the cell of low-level N-cadherin.

Embodiment 17: the positive and negative growth of tumour cell of sorting and unsorted N-cadherin

Be to determine of the influence of N-cadherin positive cell to growth of tumour cell, sorting the N-cadherin positive and negative cells, obtained 100% and 0% male N-cadherin cell mass respectively.Then cell is injected the castrating mouse, compare the negative colony of N-cadherin, tumour (Figure 20) is more accelerated and formed effectively to N-cadherin positive cell, and this explanation N-cadherin positive cell has growth vigor, and perhaps they have the stem cell characteristic and tumorigenicity is better than negative colony.The growth of unsorted cell is similar to N-cadherin positive cell.

Embodiment 18: from the facs analysis of the tumour of N-cadherin sorting cells

The facs analysis comparison is by the pure N-cadherin positive and negative cells growing tumors and contrast the not tumour of sorting population growth.N-cadherin positive rate from the tumour of 100%N-cadherin positive cell has only 41.25% (Figure 21 and 22), illustrates that these cells have become the blank cell of N-cadherin.This is that to become the hypothesis of stem cell of the higher N-cadherin negative cells of differentiation degree consistent with N-cadherin positive cell.Simultaneously, the tumour that the negative colony of N-cadherin produces has the 9%N-cadherin positive (Figure 21 and 22), with unsorted cell similar (Figure 21 and 22).This illustrates that the growth of these cells requires the acquisition of stem-like cell colony or raises the N-cadherin to form the androgen independence tumour.The needs that make the N-cadherin form the androgen independence tumour have caused the delay of tumorigenicity.

Embodiment 19: the LNCaP-C1 growth of tumor of foundation is suppressed by N-cadherin antibody.

As shown in Figure 23, at the 45th day to the 56th day N-cadherin antibody 1H7 and EC4 are carried the mouse of LNCaP-C1 tumour, they occur at the 72nd day the retarding effect of tumor growth.

Embodiment 20:N-cadherin antibody suppresses LAPC-9 androgen independence tumor growth.

As Figure 24 a as seen, carry the mouse (the 7th generation) of the LAPC-9 androgen independence tumour of foundation since the 15th day with N-cadherin antibody 1H7 and EC4, weekly twice, 10mg/kg.Retarding effect appears at the 30th day.1H7 and EC4 are shown in Figure 24 b to the retarding effect of the big LAPC-9 androgen independence tumour of foundation, and wherein, animal is not accepted antibody treatment in the time of the 17th day.

The dose-dependently growth-inhibiting of embodiment 21:EC4 antibody

As shown in Figure 25, the PC3 tumour is subjected to the inhibition (giving by the 27th day at the 13rd day) of EC4 antibody in the intravital growth of nude mice, observes more outstanding inhibition effect in the experiment that gives higher dosage antibody.

Embodiment 22: toxicity research

Because N-cadherin wide expression in various tissues, if will resist-N-cadherin antibody should consider its genotoxic potential as therapeutical agent.The present inventor finds, 1H7 and EC4 antibody and mouse N-cadherin cross reactivity, but in long-term or dosage escalation mice study, do not observe toxicity in vivo.

Embodiment 23:N-cadherin antibody suppresses LAPC-9 androgen-dependent tumor development and becomes the androgen independence tumour.

Shown in Figure 26 a and 26b, in the castrating mouse, N-cadherin antibody 1H7 and EC4 effectively suppress LAPC-9 androgen-dependent tumour to the androgen independence tumor development.(Figure 26 a) gave antibody from the 0th day to the 31st day, observed animal 45 days in first research.In second research (Figure 26 b), the effect of having observed long-term Antybody therapy.1H7 demonstrates moderate delay to tumor development, and EC4 demonstrates more secular effect to the blocking-up tumor development.

Embodiment 24:N-cadherin positive cell has growth vigor in the castrating mouse.

As shown in figure 27, in the SCID mouse of castrating, the positive LAPC-9 tumour of N-cadherin demonstrates above the negative LAPC-9 growth of tumor of N-cadherin advantage.On the other hand, Figure 28 shows, negative correlation between N-cadherin expression level and the expression of androgen receptor in the LAPC-9 androgen independence cell, in continuous passage, the LAPC-9 cell develops into the acquisition androgen independence, and simultaneously they increase the expression of N-cadherin and the expression of androgen receptor is reduced.

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Claims (33)

1. hybridoma cell line with ATCC accession number PTA-9387 preservation.

2. antibody that produces by the described hybridoma cell line of claim 1.

3. hybridoma cell line with ATCC accession number PTA-9388 preservation.

4. antibody that produces by the described hybridoma cell line of claim 4.

5. an antibody or its fragment, it can be in vivo or external combination and the identical N-cadherin structural domain 1-3 of monoclonal antibody bonded N-cadherin antigenic determinant that hybridoma cell line was produced that is preserved in American Type Culture Collection with ATCC accession number PTA-9387.

6. antibody as claimed in claim 5 is characterized in that, described antibody is humanized or people's antibody completely.

7. antibody as claimed in claim 5 is characterized in that, described antibody is bi-specific antibody or scFv.

8. an antibody or its fragment, it can be in vivo or external combination and the identical N-cadherin structural domain 4 of monoclonal antibody bonded N-cadherin antigenic determinant that hybridoma cell line was produced that is preserved in American Type Culture Collection with ATCC accession number PTA-9388.

9. antibody as claimed in claim 8 is characterized in that, described antibody is humanized or people's antibody completely.

10. antibody as claimed in claim 8 is characterized in that, described antibody is bi-specific antibody or scFv.

11. a method that suppresses growth of cancer cells in patient's body said method comprising the steps of:

Be enough to make as antibody as described in claim 5 or 8 or its fragment and as described under the cancer cells bonded condition, give this antibody or its fragment, wherein said cancer cells is expressed or is crossed expression N-cadherin, and wherein said antibody is grown by following approach anticancer:

(a) activation or inhibition NF κ-signal conduct and transcribe;

(b) activation or the internalization of inhibition N-cadherin;

(c) activation or inhibition PI3 kinases or Akt approach;

(d) activation or the conduction of inhibition beta-catenin white signal;

(e) different dimerization of blocking-up N-cadherin and FGFR or other tyrosine kinase receptors; Or

(f) blocking-up or enhancing are by ADAM10 or the cutting of other metallopeptidases.

12. method as claimed in claim 11 is characterized in that, described cancer cells is the apparatus urogenitalis cancer cells.

13. method as claimed in claim 11 is characterized in that, described cancer cells is a prostate cancer cell.

14. method as claimed in claim 11 is characterized in that, described cancer cells is a transitional cell bladder carcinoma cell line.

15. a method for the treatment of the cancer patients may further comprise the steps:

(a) obtain the test organization sample from the individuality that the risk of suffering from the cancer of expressing the N-cadherin is arranged;

(b) compare with know the control tissue sample for the individuality of cancer feminine gender available from oneself, whether or content the existence of determining N-cadherin in the test organization sample; Thereby diagnose the cancer of described expression N-cadherin, wherein said N-cadherin is with normal or low expression level, or expressed by cell subsets, or crosses and express; With

(c) have the risk of suffering from the cancer of expressing the N-cadherin individual effective dose as claim 5 or 8 described antibody or fragments.

16. method as claimed in claim 15 is characterized in that, described tissue sample is prostate gland or bladder body.

17. method as claimed in claim 15 is characterized in that, described cancer is a prostate cancer.

18. method as claimed in claim 15 is characterized in that, described cancer is a bladder cancer.

19. method as claimed in claim 15 is characterized in that, the intractable prostate cancer of described antibody blocking hormone.

20. method as claimed in claim 15 is characterized in that, described antibody blocking cancer stem cell.

21. method as claimed in claim 15 is characterized in that, described tissue sample is prostate gland or bladder body.

22. method as claimed in claim 15 is characterized in that, described cancer is a metastatic carcinoma.

23. method as claimed in claim 15 is characterized in that, described antibody is monoclonal antibody.

24. method as claimed in claim 15 is characterized in that, described antibody is scFv.

25. method as claimed in claim 15 is characterized in that, described antibody is bi-specific antibody.

26. a diagnosing cancer patient method may further comprise the steps:

(a) obtain the test organization sample from the individuality that the risk of suffering from the cancer of expressing the N-cadherin is arranged;

(b) compare with know the control tissue sample for the individuality of cancer feminine gender available from oneself, whether or content the existence of determining N-cadherin in the test organization sample as claim 5 or 8 described N-cadherin antibody or fragment by making sample contact significant quantity; Thereby diagnose the cancer of described expression N-cadherin, wherein said N-cadherin is with normal or low expression level, or expressed by cell subsets, or crosses and express.

27. a method of identifying cancer stem cell may further comprise the steps:

(a) obtain the test organization sample from the individuality that the risk of suffering from the cancer of expressing the N-cadherin is arranged;

(b) compare with control tissue sample, adopt existence that claim 5 or 8 described antibody determine cancer stem cell in the test organization sample whether available from the individuality that is known as the cancer feminine gender; Wherein said N-cadherin is with normal or low expression level, or by the stem cell Expression of Subsets and be not expression.

28. method as claimed in claim 27 is characterized in that, described tissue sample is prostate gland or bladder body.

29. method as claimed in claim 27 is characterized in that, described cancer is a prostate cancer.

30. method as claimed in claim 27 is characterized in that, described cancer is a bladder cancer.

31. method as claimed in claim 27 is characterized in that, described cancer is the intractable prostate cancer of hormone.

32. method as claimed in claim 27 is characterized in that, described cancer is a metastatic carcinoma.

33. the method identifying anti--N-cadherin antibody or suppress the compound of growth of cancer cells in patient's body said method comprising the steps of:

(i) make the contact of described compound or antibody express or cross the cell of expressing N-cadherin polypeptide; With

(ii) by determining whether described compound or antibody have the following functional effect that is used for determining described compound or antibody to N-cadherin polypeptide:

(a) activation or inhibition NF κ-signal conduct and transcribe;

(b) activation or the internalization of inhibition N-cadherin;

(c) activation or inhibition PI3 kinases or Akt approach;

(d) activation or the conduction of inhibition beta-catenin white signal;

(e) different dimerization of blocking-up N-cadherin and FGFR or other tyrosine kinase receptors; Or

(f) blocking-up or enhancing are by ADAM10 or the cutting of other metallopeptidases.

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