CN101356679A - Compositions and methods for diagnosing and treating cancer - Google Patents

Abstract

An isolated antibody that specifically binds to an extracellular domain of two or more human FZD receptors and inhibits growth of tumor cells is described. Also described is a method of treating cancer comprising administering an antibody of the present disclosure in an amount effective to inhibit tumor cell growth.

Description

Be used to diagnose and treat the composition and the method for cancer

Technical field

The present invention relates to oncology and new compositions and the method that is used to diagnose and treat cancer is provided.The invention provides the antibody that is used to diagnose and treat the anticancer stem cell labeling thing of solid tumor.

Background technology

Cancer is one of main causes of death in the developed world, only in the U.S., has every year the million people of surpassing diagnosis to suffer from cancer, and 500,000 people's death are arranged.In general, just there is being more than 1 people can suffer from the cancer of some form in the middle of all one's life at them in the middle of 3 people according to estimates.Cancer has 200 kinds of different types of surpassing, four kinds-breast cancer, lung cancer, colorectal cancer and prostate cancer wherein-surpass all new discovery cases half (Jemal etc., 2003, Cancer J.Clin.53:5～26).

Breast cancer is a modal cancer among the women, according to estimates, has 12% women to have the risk of suffering from this disease in life at them.Although because the improvement of checking of doing sth. in advance and treating, the death rate is reduced, breast cancer remains middle aged women's main causes of death, and the transfer breast cancer still is the disease that can not cure.Suffer from the most of patient who shifts breast cancer and when manifesting, have only one or two tract to be affected, but, got involved in a plurality of positions along with advancing of disease.The common site that transfer is got involved is the skin of chest wall and the local recurrence in soft tissue and armpit and the supraclavicular region territory.The modal position that far-end shifts is bone (far-end shift 30%～40%), secondly is lung and liver.And, when diagnosis, have far-end and shift although 1%～5% the firm diagnosis of only having an appointment suffers from the women of breast cancer, there is 50% the patient who suffers from local disease transfer and relapse finally in 5 years, all to occur approximately.At present, far-end shifts the meta survival manifest and is about 3 years.

At present breast cancer is diagnosed and method by stages comprises that tumor nodule shifts (TNM) system, this system depends on (the American Joint Committee on Cancer:AJCC Cancer Staging Manual.Philadelphia that exists that the existence of tumour in tumor size, the lymph node and far-end shift, Pa.:Lippincott-Raven Publishers, the 5th edition, 1997, the 171～180 pages; Harris, J R: " Staging of breast carcinoma " in Harris, J.R., Hellman, S., Henderson, I.C., Kinne D.W. (volume): Breast Diseases.Philadelphia, Lippincott, 1991).These parameters are in order to provide prognosis and to select suitable therapy.Can also estimate the form outward appearance of tumour, but can show tangible clinical change owing to have the tumour of similar histopathology outward appearance, therefore this method has critical limitations.At last, the mensuration of cell surface marker thing can be in order to be divided into each subclass with some tumor type.For example, an existence that factor is ERs (ER) of in the prognosis of breast cancer and treatment, considering, negative tumour is easier replys making such as hormonotherapies such as TAM or aromatase inhibitors because the positive breast cancer of ER is usually than ER.Although these analyses are useful, can only partly predict the clinical behavior of tumor of breast, and in the breast cancer that the current diagnosis instrument can't detect and therapy can't be treated at present, also have a large amount of phenotype diversity.

Prostate cancer is the most common cancer among the male sex of developed world, estimates to account for 33% in all newfound cases of cancers of the U.S., is the frequency time high cause of death (Jemal etc., 2003, CACancer J.Clin.53:5～26).Because the introducing of prostate specific antigen (PSA) blood testing, the earlier detection of prostate cancer has significantly improved survival rate; Patient's 5 annual survival rates of suffering from regional area sexual stage prostate cancer during diagnosis are near 100%.But still have patient to develop into local terminal illness the most at last or shift disease (Muthuramalingam etc., 2004, Clin.Oncol.16:505～16) above 50%.

At present, radical prostatectomy and radiotherapy provide curative therapy for the tumor of prostate of most localization.Yet for late case, selectable therapeutic scheme is very limited.For shifting disease, it is standard care that the androgen that uses luteinizing hormone-releasing hormone (LRH) (LHRH) excitomotor separately or be used in combination luteinizing hormone-releasing hormone (LRH) (LHRH) excitomotor and antiandrogen is blocked.Although androgen is farthest blocked, disease condition is development still almost, and major part forms the androgen independence disease gradually.The prostate cancer that at present hormone is difficult to cure does not also have generally accepted treatment, and commonly used be chemotherapy (Muthuramalingam etc., 2004, Clin.Oncol.16:505～16; Trojan etc., 2005, Anticancer Res.25:551～61).

Colorectal cancer worldwide is the 3rd common cancer, and is the highest cancer mortality reason of the 4th bit frequency (Weitz etc., 2005, Lancet 365:153～65).Have 5%～10% approximately in all colorectal cancers, hereditary, one of its principal mode is familial adenomatous polyposis (FAP), this familial adenomatous polyposis is an autosomal dominant disorder, and 80% the affected individuals of wherein having an appointment contains germ line mutation in adenomatous polyposis coli (APC) gene.Colorectal cancer is by circumferential growth and local the intrusion, then spread, stride by lymph diffusion, blood are capable that peritonaeum spreads and peripheral nerve spreads and invades in other positions.The common site that lymph is got involved outward is a liver, and what the outer affected frequency of organ of belly was the highest is lung.Other positions of the capable diffusion of blood comprise bone, kidney, adrenal gland and brain.

The existing system by stages of colorectal cancer is passed having or not that the degree of intestines wall and tubercle get involved based on tumour.This system by stages is restricted to 3 main Duke classifications: Duke A level disease is limited to the submucosa of colon or rectum; Duke B level disease has invades the tumour of passing muscularis propria and may pass colon or rectal wall; Duke C level disease comprises the intestines wall intrusion that has regional lymphatic metastasis of any degree.Though the excision art is very effective for colorectal cancer, in Duke A level patient, provide 95% cure rate, but this ratio drops to 75% in Duke B level patient, and to be shown in 5 years the recurrence possibility in advance be 60% in the existence of positive lymph nodes in Duke C level disease.Adopt chemotherapeutic postoperative the course of treatment Duke C level patient to be treated and recurrence rate can be reduced to 40%～50%, this is present these patients' a nursing standard.

Lung cancer is modal in the world cancer, is the third-largest modal cancer in the cancer of being diagnosed in the U.S., is the cancer mortality reason that frequency is the highest up to now (Spiro etc., 2002, Am.J.Respir.Crit.Care Med.166:1166～96; Jemal etc., 2003, CA Cancer J.Clin.53:5～26).It is believed that the ratio estimate that the lung cancer that smoking causes accounts for all lung cancer has 87%, make lung cancer become the most fatal preventable disease.Lung cancer is divided into two kinds of main types: the ratio that small-cell carcinoma of the lung (SCLC) and non-small cell lung cancer (NSCLC), these two types of lung cancer account for all lung cancer surpasses 90%.The SCLC case accounts for 15%～20%, it is characterized in that, this class lung cancer root consists of in big central airway and histology and is close to the lamella that does not have cytoplasmic cellule.SCLC has more aggressive than NSCLC, and growth fast, shift early.NSCLC accounts for 80%～85% of all cases, and is further divided into 3 main hypotypes based on histology: gland cancer, squamous cell carcinoma (epidermoid carcinoma) and maxicell undifferentiated carcinoma.

Lung cancer manifests later usually in its course of disease, and therefore the meta survival rate after the diagnosis has only 6～12 months, and total 5 annual survival rates only have 5%～10%.Though operation provides the best chance of curing, have only the small part patient to be fit to operation, major part depends on chemotherapy and radiotherapy.Although attempt the opportunity and the dose intensity of these therapies of control, survival rate does not almost improve (Spiro etc., 2002, Am.J.Respir.Crit.Care Med.166:1166～96) in 15 years in the past.

These four kinds of cancers and other cancers show as the solid tumor of being made up of the heterologous cell mass.For example, breast cancer is the mixture of cancer cell and normal cell (comprising a matter (matrix) cell, inflammatory cell and endothelial cell).Several models of cancer provide different explanations for the existence of this heterologous.A kind of model (classical model of cancer) thinks that the visibly different cancer cell population of phenotype all has the ability of breeding and forming new tumour.In this classical model, the tumour cell heterologous stems from occurent sudden change in envirment factor and the cancer cell, thereby forms various carcinogenic cells group.This model is based on such viewpoint, that is, all colonies of tumour cell all have oncogenic potential (Pandis etc., 1998, Genes, Chromosomes﹠amp to a certain degree; Cancer 12:122～129; Kuukasjrvi etc., 1997, Cancer Res.57:1597～1604; Bonsing etc., 1993, Cancer 71:382～391; Bonsing etc., 2000, Genes Chromosomes﹠amp; Cancer 82:173～183; Beerman H etc., 1991, Cytometry 12:147～54; Aubele M and WernerM, 1999, Analyt.Cell.Path.19:53; Shen L etc., 2000, Cancer Res.60:3884).

A kind of substituting model of the solid tumor cell heterologous that is observed stems from the influence of stem cell to tumor development.According to this model, cancer results from that the control normal tissues is grown and the imbalance (Beachy etc., 2004, Nature 432:324) of the mechanism kept.In the growth course of intact animal, the overwhelming majority or cell in a organized way all come from normal precursor, promptly so-called stem cell (Morrison etc., 1997, Cell 88:287～98; Morrison etc., 1997, Curr.Opin.Immunol.9:216～21; Morrison etc., 1995, Annu.Rev.Cell.Dev.Biol.11:35～71).Stem cell is such cell: (1) has multiplication capacity widely; (2) can the asymmetry cell division to generate the filial generation that one or more multiplication potentialities and/or developmental potentiality descend; (3) the symmetry cell division can be carried out and self-regeneration or oneself keep.The best research example that is embodied as body cell regeneration by the stem cell differentiation is a hemopoietic system, wherein grows immature precursor (candidate stem cell and CFU-GM) and responds molecular signal and form various haemocytes and lymphoid cell type.Other cells (cell that comprises internal organ, latex dust system and skin) are from replenishing that the microcommunity stem cell of each self-organizing obtains continuing, and research recently thinks that there is stem cell equally in most other adult tissues (comprising brain).The tumour that stems from " solid tumor stem cell " " cancer stem cell " of solid tumor (or come from) experiences unordered growth by symmetry and asymmetry fission process subsequently.In this stem cell model, solid tumor contains obvious difference and limited (even may be rare) has the normally cell subgroup of " stem cell " character, and reason is their extensive propagation and forms other solid tumor stem cell (self-regeneration) effectively and most of oncocyte of the shortage tumorigenesis potential of formation solid tumor.In fact, the sudden change in the long-lived population of stem cells can cause the formation of cancer stem cell, the basis that cancer stem cell becomes tumor growth and keeps, and the failure of current methods of treatment is facilitated in the existence of cancer stem cell.

The stem cell character of cancer at first obtains disclosing (Lapidot etc., 1994, Nature 17:645～8) in leukemia (acute myeloid leukemia (AML)).Recently, proved that pernicious HBT also has little and visibly different cancer stem cell group similarly, it can be formed tumour by enrichment in immunodeficient mouse.Find that compare with classification tumour cell not, the ESA+ of oncogenicity cell, CD44+, CD24-/low, Lin-cell mass are by 50 times of enrichments (Al-Hajj etc., 2003, Proc.Nat ' lAcad.Sci.100:3983～8).Be expected to separate the feasible crucial physiology approach that can study the basis that becomes the oncogenicity in these cells of ability of oncogenicity cancer cell, therefore be expected to develop better diagnostic assay and treatment for the cancer patient.This present invention just at purpose.

Summary of the invention

The invention provides a kind of monoclonal antibody of separation, described monoclonal antibody specificity is in conjunction with the extracellular domain of human FZD8 acceptor, and the growth of inhibition tumour cell.The present invention also provides a kind of isolated antibody, and described antibody specificity is in conjunction with the extracellular domain of two or more human FZD acceptors, and the growth of inhibition tumour cell.The present invention further provides a kind of pharmaceutical composition, this pharmaceutical composition comprises antibody disclosed in this invention and the acceptable charge material of pharmacy (vehicle).The present invention also provides a kind of treatment method for cancer in addition, and described method comprises uses the antibody disclosed in this invention that suppresses the growth of tumour cell effective dose.

Other purpose of the present invention and advantage, a part will provide in the explanation subsequently, and a part is also understood according to described explanation or is learned by practice of the present invention.Objects and advantages of the present invention will realize and reach by the key element of pointing out in the appended claims and factor combination.Should be understood that general introduction above and detailed description hereinafter just are used for illustration and explanation, rather than be used to limit invention required for protection.The accompanying drawing that merges in this manual or constitute the part of this specification shows a plurality of execution mode of the present invention, its with described explanation in order to explain principle of the present invention.In this specification and the appended claims, " " of singulative, " a kind of " or " described " unless context offers some clarification in addition, otherwise comprise the plural form referent.

Description of drawings

Fig. 1: the analysis that anti-FZD10, anti-FZD7, anti-FZD5, anti-FZD6, anti-FZD4 and anti-FZD8 antibody combine with the specificity of their corresponding symphysis acceptors.Not with GFP cotransfection (A) or with the HEK293 cell of expression total length FZD10, FZD7, FZD5, FZD6, FZD4 and the FZD8 of GFP cotransfection (B, C, D, E and F) with anti-FZD antibody or contrast IgG incubation and pass through the FACS sorting.(A) shown for each antibody the FACs comparative analysis of the antibody of anti-FZD10 and IgG isotype negative control.Be shown as positive control (base map) with the construct of the band FLAG label of anti-FLAG antibody coupling.(B) compare with contrast IgG, the FACs of the antibody of the anti-FZD7 in the cell of expression FZD7 and GFP analyzes.Be shown as positive control (base map, rightmost) with the construct of the band FLAG label of anti-FLAG antibody coupling.(C) compare with contrast IgG, the FACs of the antibody of the anti-FZD5 in the cell of expression FZD5 and GFP analyzes.The serum of FZD5 antigen-immunized animal of hanging oneself is presented at the right base map.(D) compare with contrast IgG, the FACs of the antibody of the anti-FZD6 in the cell of expression FZD6 and GFP analyzes.Be shown as positive control (base map, the right) with the construct of the band FLAG label of anti-FLAG antibody coupling.(E) compare with contrast IgG, the FACs of the antibody of the anti-FZD4 in the cell of expression FZD4 and GFP analyzes.Be shown as positive control (base map, the right) with the construct of the band FLAG label of anti-FLAG antibody coupling.(F) FACs of the anti-FZD8 antibody in the cell of expression FZD8 and GFP analyzes.

Fig. 2: FZD Fc soluble recepter has suppressed the signal transduction of Wnt.In the presence of different Wnt parts (comprising Wnt1, Wnt2, Wnt3, Wnt3a and Wnt7b), stable transfection has HEK 293 cells and the ever-increasing FZD Fc of the concentration soluble recepter incubation of 8xTCF-luciferase reporter gene.Shown in the uciferase activity forfeiture, FZD4Fc, FZD5Fc and FZD8Fc fusion have suppressed the ligand-mediated Wnt signal transduction by all 5 Wnt.

Fig. 3: disturb the evaluation of the anti-FZD5 antibody of Wnt3a part combination.According to uciferase activity, measurement exists at the Wnt3a anti-FZD5 antibody different with 32 under (individualism (left-hand bar) or at (right side bar) in the presence of the solubility FZD5Fc), and transfection has the Wnt signal transduction in HEK 293 cells of Wnt 8xTCF-luciferase reporter gene carrier.The antibody of the combination between interference FZD5Fc and the Wnt3a causes the remarkable activation (right side bar) of Wnt signal transduction.

Fig. 4: the antibody of anti-FZD6 and anti-FZD5 has slowed down tumor growth.Injection has the UM-C4 colon tumor cell and the tumor growth (mm in 8 weeks in the NOD/SCID mouse of the antibody treatment of anti-FZD6 or anti-FZD5 ³) map on the x axle.Handle with respect to PBS injection contrast (black bar) with anti-FZD6 antibody 23M2 (blank bar) and anti-FZD5 antibody 44M13 (twill bar), significantly slowed down growth of tumor.

Embodiment

Term " antibody " promptly can be by at least one the antigen recognition site identification in this immunoglobulin molecules variable region and the immunoglobulin molecules of specificity combination such as the targets such as combination of albumen, polypeptide, peptide, carbohydrate, polynucleotides, lipid or aforementioned substances in order to refer to such immunoglobulin molecules.In some embodiments, antibody of the present invention comprises antagonist antibodies, and this antagonist antibodies combines and disturb the expression of for example part combination, receptor dimerizationization, cancer stem cell label albumen and/or the downstream signal transduction of cancer stem cell label albumen with cancer stem cell label protein-specific.In some embodiments, disclosed antibody comprises agonist antibody, and this agonist antibody combines with cancer stem cell label protein-specific and promotes part for example in conjunction with, receptor dimerizationization and/or the signal transduction that undertaken by cancer stem cell label albumen.In some embodiments, disclosed antibody does not disturb or promotes the biologically active of cancer stem cell label albumen, but suppresses tumor growth by for example antibody internalization and/or by immune system recognition.Term as used herein " antibody " contains complete polyclonal antibody, complete monoclonal antibody, antibody fragment (for example Fab, Fab ', F (ab ') 2 and Fv fragment), strand Fv (scFv) mutant, multi-specificity antibody (as the bispecific antibody that is generated by at least two complete antibodies), chimeric antibody, humanized antibody, human antibodies, comprises fusion and other any modified immunoglobulin molecules that comprises antigen recognition site of the antigen deciding section of antibody, as long as described antibody demonstrates required biologically active.Antibody can belong to any one classification or its subclass (isotype) (for example IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) in 5 kinds of primary categories (being IgA, IgD, IgE, IgG and IgM) of immunoglobulin (Ig), is called α, δ, ε, γ and μ according to their character of CH.Different classes of immunoglobulin (Ig) has different known subunit structure and 3-d modelling.Antibody can be expose or with such as other molecule couplings such as toxin, radioisotopes.

Be meant the part of complete antibody at this used " antibody fragment ", also refer to the antigen decision variable region of complete antibody.The example of antibody fragment includes but not limited to Fab, Fab ', F (ab ') 2 and Fv fragment, linear antibody, single-chain antibody and the multi-specificity antibody that is formed by antibody fragment.

" Fv antibody " is meant the minimum antibody fragment that contains comlete antigen identification and antigen binding site, it can be that wherein a heavy chain and a variable region of light chain form non-covalent dimeric two strands, thus also can be wherein a heavy chain and a variable region of light chain by the covalently bound strand (scFv) that makes described two chains with similar dimeric structure associating of flexible peptide linker.In this configuration, the complementary determining region of each variable region (CDR) interacts and limits the dimeric antigen-binding specificity of this Fv.As selection, single variable region (perhaps half of Fv) can be used for discerning and conjugated antigen, but compatibility is lower usually.

" monoclonal antibody " used herein is meant the homologous antibody group with high degree of specificity identification and the former determinant of monoclonal antibody or epi-position combination.This is opposite with polyclonal antibody, and polyclonal antibody generally includes the different antibodies at anti-different antigenic determinants.Complete sum full length monoclonal antibodies and antibody fragment (for example Fab, Fab ', F (ab ') 2 and Fv), strand Fv (scFv) mutant, the fusion that comprises antibody moiety and other any modified immunoglobulin molecules that comprises antigen recognition site contained in term " monoclonal antibody ".In addition, " monoclonal antibody " refers to that also the antibody that makes by any kind of mode, described mode include but not limited to that hybridoma, bacteriophage are selected, recombinant expressed and transgenic animals.

Term used herein " humanized antibody " is meant as various forms of inhuman (for example muroid) antibody that contains minimum non-human sequence's SIG chain, gomphosis immunoglobulin or its fragment.Usually, humanized antibody is such human immunoglobulin, is wherein replaced from the residue from CDR with required specificity, compatibility and ability of inhuman species (for example mouse, rat, rabbit, hamster) from the residue of complementarity-determining region (CDR).In some cases, Fv skeleton district (FR) residue of the human immunoglobulin corresponding residue that had required specificity, compatibility and ability in the antibody from inhuman species is replaced.Humanized antibody can also by in the Fv skeleton district and/or the intra-residue extra residue of the non-human of being replaced replace and further modify, to improve and to optimize antibody specificity, compatibility and/or ability.Humanized antibody comprises the almost whole of at least one (being generally two or three) variable region usually, described variable region comprises all or nearly all CDR district corresponding with the non-human immunoglobulin (Ig), and all or nearly all FR district are the FR districts of human immunoglobulin consensus sequence.Humanized antibody also comprises at least a portion of constant region for immunoglobulin or territory (Fc), and what comprise usually is at least a portion of human immunoglobulin.At United States Patent (USP) 5,225, description arranged in 539 in order to the example of the method that generates humanized antibody.

Term used herein " human antibodies " is meant the antibody that is produced by the mankind or adopts that any technology known in the art makes has antibody with the corresponding amino acid sequence of antibody that is produced by the mankind.The definition of human antibodies comprises the fragment of complete or full length antibody, this antibody and/or comprises the antibody of at least one human heavy chain and/or light chain polypeptide, for example comprises the antibody of muroid light chain polypeptide and human heavy chain polypeptide.

" hybrid antibody " is such immunoglobulin molecules, wherein will make that two kinds of different epi-positions or two kinds of different antigens can be discerned and combination by the formed tetramer from the heavy chain light chain of antibody to fitting together with different antigenic determinants zone.

Term " chimeric antibody " is meant such antibody, and wherein the amino acid sequence of immunoglobulin molecules is from two or more species.Usually, the two variable region of light chain and heavy chain is corresponding to the variable region of the antibody with required specificity, compatibility and ability that comes from mammiferous species (for example mouse, rat, rabbit etc.), but the sequence homology in constant region and the antibody that comes from another kind of species (be generally human) is to avoid causing immune response in those species.

Term " epi-position " or " antigenic determinant " can exchange use in this article mutually, and what be meant antigen can be by the part of specific antibodies identification and specificity combination.When antigen was polypeptide, epi-position both can be formed by adjacent amino acid, also can be formed by three grades of folding and juxtaposed non-adjacent amino acid through albumen.The epi-position that is formed by adjacent amino acid is still kept when albuminous degeneration usually, and can be lost usually when the albuminous degeneration by three grades of epi-positions that are folded to form.Epi-position generally includes at least 3 (usually being at least 5 or 8～10 under the more susceptible condition) and is in the amino acid in unique steric configuration.

Antibody " selective binding " or " specificity in conjunction with " be meant antibody more continually, more promptly, more enduringly, compatibility more strongly or some above combination and with epi-position reaction or associating (than with the reaction of the substituting material that comprises incoherent albumen or unite and compare)." selective binding " or " specificity in conjunction with " for example is meant antibody with at least about 0.1mM, but more commonly at least about KD and the protein combination of 1 μ M." selective binding " or " specificity in conjunction with " is meant antibody sometimes sometimes with at least about the KD of 0.1 μ M or better KD and protein combination, other the time be meant with at least about the KD of 0.01 μ M or better KD and protein combination.Because the sequence homogeneity between the homologous protein in the different plant species, specificity is in conjunction with the specificity combination of the antibody that can comprise the cancer stem cell label albumen in the more than a kind of species of identification.

Term used herein " non-specific binding " and " background in conjunction with " when using in the interaction that is relating to antibody and albumen or peptide, be meant the existence that does not rely on ad hoc structure interaction (, general and the protein combination of antibody, rather than combine with specific structure such as epi-position).

Term " separation " or " purifying " are meant that material does not contain the composition that accompanies usually with this material in the native state basically or in fact.Purity and homogenieity adopt usually to be determined such as analysis chemical technologies such as polyacrylamide gel electrophoresis or high performance liquid chromatography.Disclosed in this invention in preparation the albumen (for example antibody) or the nucleic acid of existing main species kind passed through sufficient purifying.Particularly, the nucleic acid of separation separates with ORFs, the natural flank of this gene and the albumen that coding is different from the albumen of this coded by said gene of being positioned at of this ORFs.Isolated antibody is separated with other NIgs, and separates with the immunoglobulin (Ig) with different antigen-binding specificities.Described term refers to that also nucleic acid or albumen have at least 80% purity in some embodiments, has at least 85% purity in some embodiments, has at least 90% purity in some embodiments, have at least 95% purity in some embodiments, and have at least 99% purity in some embodiments.

Term used herein " cancer " and " cancer " are meant or describe the mammiferous physiological status that cell mass wherein has the dysregulated cellular growth feature.The example of cancer includes but not limited to cancer, lymthoma, enblastoma, sarcoma and leukaemia.The example more specifically of such cancer comprises squamous cell carcinoma, small-cell carcinoma of the lung, non-small cell lung cancer, the gland cancer of lung, the carcinoma squamosum of lung, peritoneal cancer, hepatocellular carcinoma, human primary gastrointestinal cancers, cancer of pancreas, spongioblastoma, cervix cancer, oophoroma, liver cancer, carcinoma of urinary bladder, hepatoma, breast cancer, colon cancer, colorectal cancer, the carcinoma of endometrium or the cancer of the uterus, salivary-gland carcinoma, kidney, liver cancer, prostate cancer, carcinoma of vulva, thyroid cancer, hepatocellular carcinoma and various types of Head and Neck cancer.

" proliferative disorders " used herein and " hyperplasia " be meant with such as the relevant illness of abnormal cell proliferations such as cancer.

" tumour " used herein and " knurl " are meant any piece of tissue that is caused by cell transition growth or propagation, its be optimum (non-carcinous) or pernicious (carcinous, as to comprise precancerous lesion).

" transfer " used herein is meant that cancer is sent out or transfers to other zones of health from position originally and the process of similar cancerous lesion is arranged in this new position development." metastatic " or " transfer " cell is to lose the cell that contacts and invade from initial disease sites transfer by blood flow or lymph adjacent body structure with the adhesion of flanking cell.

Term " cancer stem cell ", " tumor stem cell " or " solid tumor stem cell " can exchange use in this article mutually, and refer to the such cell mass from solid tumor: (1) has multiplication capacity widely; (2) can carry out the asymmetry cell division, generate the differone generation that one or more multiplication potentialities or developmental potentiality descend; (3) the symmetry cell division can be carried out and self-regeneration or oneself keep." cancer stem cell ", " tumor stem cell " or " solid tumor stem cell " but these character make these cancer stem cells have behind the mouse of being transplanted to immunocompromised host continuously the ability of the tumour (comparing) that forms palpation with most of tumour cells that can not form tumour.The self-regeneration and the differentiation of the no sequential mode of cancer stem cell experience, thus formation has the tumour because of the abnormal cell type that can time to time change of undergoing mutation.According to U.S. Patent No. 6,004,528 are proposed, and solid tumor stem cell is different from " cancer responsibility ".In this patent, " cancer responsibility " is defined as the slowly CFU-GM type of growth, himself rare sudden change, and because occur in cellular environment that oncogenicity changes former thereby carry out symmetry cell division rather than asymmetry cell division.Described " cancer responsibility " hypothesis thereby proposition, as the result of unusual environment, a large amount of appearance are the tumour cell of the fast breeding of sudden change highly, thereby causes normal relatively stem cell accumulation to be undergone mutation then, and this causes these cells to become tumour cell.U.S. Patent No. 6,004,528 propose, and this model can be in order to promote the diagnosis of cancer.The solid tumor stem cell model is fundamentally different than " cancer responsibility " model, the result show " cancer responsibility " model the application that can not provide.The first, solid tumor stem cell is not " sudden change lacks ".U.S. Patent No. 6,004,528 described " the cancer responsibilities that sudden change lacks " can think precancerous lesion, and the cancer cell of solid tumor stem cell to be the contain sudden change that causes oncogenicity from the cancer last stage to whole later stage cancer itself.That is, solid tumor stem cell (" cancer stem cell ") should be included in and U.S. Patent No. 6,004, in the cell of the visibly different height sudden change of 528 described " cancer responsibilities ".The second, cause the gene mutation of cancer obviously to belong to solid tumor stem cell in essence and be subjected to the influence of environment.The solid tumor stem cell model prediction; the solid tumor stem cell that separates can form other tumour (can explain transfer thus) when transplanting; and " cancer responsibility " model prediction; " cancer responsibility " cell through transplanting can not form new tumour, exactly because this is that their unusual environment is an oncogenicity.The human entity knurl stem cell transplantation that the phenotype of in fact, dissociating is separated makes the present invention obviously be different from " cancer responsibility " model to the ability that mouse (entering into and the normal very different environment of tumor environment) still can form new tumour.The 3rd, solid tumor stem cell carries out symmetry cell division and asymmetry cell division probably simultaneously, makes that symmetrical cell division is not the character that must have.The 4th, solid tumor stem cell can divide fast or slowly, and this depends on a lot of parameters, and therefore slowly growth rate is not the characteristic that truly has.

Term " cancer cell ", " tumour cell " and grammatical equivalents are meant that it comprises most of tumor cell groups and oncogenicity stem cell (cancer stem cell) derived from the total group of the cell of tumour or precancerous lesion (comprising the non-tumorigenic cell).

" oncogenicity " used herein is meant the functional character of solid tumor stem cell, comprise self-regeneration (forming other oncogenicity cancer stem cell) and generate every other tumour cell (differentiation takes place and therefore form the non-tumorigenic tumour cell) thus make solid tumor stem cell form the proliferative of tumour.But self-regeneration has in the tumour (comparing with most of tumour cells that can not form tumour after the continuous transplanting) that forms palpation behind the mouse of being transplanted to immunocompromised host continuously cancer stem cell of the present invention with fertile these character that generate every other tumour cell.Tumour cell, promptly the non-tumorigenic tumour cell is obtaining can form the limited number of times of tumour (for example once or twice) behind the tumour cell after being transplanted to the mouse of immunocompromised host from solid tumor.

Term used herein " stem cell cancer label ", " cancer stem cell label ", " tumor stem cell label " or " solid tumor stem cell label " are meant one or more gene or expressed albumen, polypeptide or the peptide of described one or more gene, described one or more expression of gene level itself or with the combination of other genes be associated with the existence of oncogenicity cancer cell (comparing) with the non-tumorigenic cell.The increase of this relevant and described gene expression or reduce relevant (being level increase or minimizing of the mRNA or the coded peptide of described gene).

Being equal to statement on term used herein " unassorted tumour cell ", " sorting before cell ", " tumour cell piece " and the syntax thereof can exchange use mutually, is meant that separation is not also expressed according to the cell surface marker thing from patient's sample (for example tumor biopsy sample or flank effluent) to separate or the tumor cell group of classification.

Being equal to statement on term used herein " non-ESA+CD44+ tumour cell ", " non-ESA+44+ ", " the non-tumorigenic tumour cell of sorting ", " non-tumorigenic tumour cell ", " non-stem cell ", " tumour cell " and the syntax thereof can exchange use mutually, is meant the tumour colony that therefrom separates or pipette cancer stem cell of the present invention based on the expression of cell surface marker thing.

Term used herein " biopsy samples " or " biopsy " are meant whether tissue sample or the fluid sample of taking from study subject contain cancerous tissue with definite this sample.In some embodiments, obtaining biopsy or fluid is to suffer from cancer because suspect study subject, whether has cancer so check described biopsy or fluid.

Term used herein " study subject " is meant any animal (for example mammal), includes but not limited to the mankind, non-human primate and rodent etc., and described study subject is the recipient of particular procedure.Term " study subject " and " patient " can exchange use when mentioning human subject in this article usually mutually.

" pharmacy is acceptable " be meant by or can or list in American Pharmacopeia or other are subjected to universally recognized pharmacopeia and are used for animal (comprising the mankind) by federation management mechanism or state government's approval.

" the acceptable salt of pharmacy " is meant that pharmacy can accept and have the salt of compound of the required pharmacological activity of parent compound.

" the acceptable excipient of pharmacy, carrier or adjutant " is meant such excipient, carrier or adjutant, promptly can be applied to study subject with at least a antibody disclosed in this invention, not destroy the pharmacological activity of described antibody and be nontoxic when using by the dosage of the described compound that is enough to the delivering therapeutic amount.

" the acceptable charge material of pharmacy " is meant diluent, adjutant, excipient or the carrier used with at least a antibody disclosed in this invention.

" prodrug " is meant the derivative that need transform this treatment active compound that generates the treatment active compound in vivo.Prodrug can just have pharmaceutical active after being converted into the effective parent compound of treatment.

Term " treatment effective dose " is meant antibody, polypeptide, polynucleotides, organic molecule or the other drug effectively disease in " treatment " study subject or the mammal or the amount of illness.In the situation of cancer, the treatment effective dose of medicine can reduce the quantity of cancer cell; Reduce tumor size; Inhibition or prevention cancer cell infiltration comprise that to peripheral organs for example cancer is disseminated in soft tissue and the bone; Suppress and stop metastases; Suppress and stop tumor growth; Alleviate the symptom of one or more and related to cancer to a certain extent, reduce M ﹠ M; Improve quality of life; Or the combination of these effects.Reach the degree that medicine prevents the growth of existing cancer cell and/or kills existing cancer cell, it can be called and suppress cell and/or cytotoxicity.

" diagnosis is provided " used herein or " diagnostic message " are meant and can be used for determining whether the patient has disease or symptom and/or disease or symptom be classified as the phenotype classification or in any information that has any classification of meaning aspect the prognosis of disease or symptom or possible treatment (can common treatment also can the be particular treatment) reaction.Similarly, diagnosis is meant the diagnostic message that any kind is provided, include but not limited to study subject whether may have symptom (for example tumour), the information relevant such as excessive risk tumour or low-risk tumour, the information relevant with prognosis with tumor grade character and/or with can be used for selecting suitable information for the treatment of.Treatment select to comprise particular chemical therapeutic agent or other treatment form as the selection of operation or radiation or with whether stop or providing treatment relevant selection.

Term used herein " provides prognosis ", " prognosis information " or " information of forecasting " be meant provide with cancer (for example, determine by diagnostic method of the present invention) existence to study subject health in the future (for example, the possibility of cancer and the risk of transfer are suffered from expection morbidity or dead) the relevant information of influence.

Term refers to 1 simultaneously as " treatment " or " treatment " or " with treatment " or " alleviations " or " with alleviation ") cure, slow down, alleviate the symptom of pathology symptom or illness of being diagnosed and/or prevent the treatment measure and 2 of advancing of pathology symptom or the illness diagnosed) prevent and/or slow down target pathology symptom or illness development precautionary measures or prevent measure.Therefore, need those patients of treatment to comprise those patients that have illness; Those patients that tendency has illness; Those patients that need prevent with illness; If study subject demonstrates one or more following effects then this patient's the method according to this invention successfully obtains " treatment ": the minimizing of cancer cell quantity or complete obiteration; Reducing of tumor size; Suppress or the cancer cell infiltration of organ around no longer occurs, comprise for example cancer sending out in soft tissue and bone; Suppress or no longer tumorigenic transfer; Suppress or no longer occur growth of tumor; Alleviate one or more symptoms relevant with particular cancers; Reduce morbidity and dead; Improve quality of life; Or some combined effect.

Term used herein " polynucleotides " or " nucleic acid " are meant that a plurality of nucleotide units (ribonucleotide or deoxyribonucleotide or relevant structural variant) by the polymer that phosphodiester bond connects and composes, include but not limited to DNA or RNA.The sequence of any known base analogue that comprises DNA and RNA contained in this term.Term " gene " is meant and comprises nucleic acid (for example DNA) sequence that generates the required coded sequence of polypeptide, precursor or RNA (for example rRNA, tRNA).Polypeptide can be encoded by complete encoding sequence, perhaps encode by any part of this coded sequence, as long as remaining with the required activity or the functional character (for example, enzymatic activity, part combination, signal transduction, immunogenicity etc.) of this complete encoding sequence or fragment gets final product.This term is also contained the code area of structural gene and is closed on this code area and make this gene be equivalent to the sequence of full length mRNA length apart from 5 ' end and 3 ' above location of the about 1kb of any end of end.Be positioned 5 of code area ' hold and the appear at sequence on the mRNA and be meant 5 ' end non-translational region sequence.The sequence that is positioned the 3 ' end or the downstream of code area and appears on the mRNA is meant 3 ' end non-translational region sequence.The gene of cDNA and genome form contained in term " gene ".The gene of genome form or gene clone comprise the code area of being interrupted by non-coding sequence (being called " introne " or " inserting the district " or " insetion sequence ").Introne is the genetic fragment of transcribing in the nRNA (hnRNA); Introne can contain such as regulating elements such as enhancers.Introne is by from nuclear transcript or primary transcribe is removed or " cutting off "; Therefore introne does not appear in mRNA (mRNA) transcript.MRNA plays in translation process and determines new amino acid sequence or the effect in proper order that forms in the polypeptide.Except containing introne, the gene of genome form can also comprise the sequence that is positioned at 5 ' end and the 3 ' end that appears at the sequence on the rna transcription basis.These sequences are called " flank " sequence or " flank " district (these flanking sequences are positioned at 5 ' end or the 3 ' end that appears at the non-translated sequence on the rna transcription basis).5 ' distolateral pterion can contain control or influence genetic transcription such as adjusting such as promoter and enhancer sequence.3 ' distolateral pterion can comprise the sequence that instructs tanscription termination, transcribes back cutting and polyadenylation.

Employed term " reorganization " is represented described cell, nucleic acid, albumen or carrier by importing heterologous nucleic acids or albumen or changing natural acid or albumen is modified when relating to cell, nucleic acid, albumen or carrier, and perhaps described cell derives from the cell through modification like this.Therefore, for example, recombinant cell gives expression to the gene of the cell that does not see natural (non-reorganization) form or gave expression to expression or the gene of unconventionality expression (for example being expressed as fragment or shearing variant that non-natural exists)." recombinant nucleic acid " used herein is meant such nucleic acid, its initial usually by manipulation nucleic acid (for example using polymerase and restriction endonuclease) in external formation, and for not seeing natural form usually.Adopt this mode can realize not homotactic being operatively connected.Therefore, the nucleic acid of the separation of linear forms or all think to be used for the recombinant of purposes of the present invention by connecting the dna molecular that does not usually connect together at the expression vector of external formation.Should be understood that in a single day recombinant nucleic acid makes and import host cell or organism, it duplicates non-reorganization ground (promptly utilizing the cells in vivo device rather than the external manipulation of host cell); Yet such nucleic acid though duplicate, still thinks to be used for the recombinant of purposes of the present invention in case reorganization makes with carrying out non-reorganization subsequently.Similarly, " recombinant protein " is to utilize the prepared albumen of the recombinant technique expression of above-described recombinant nucleic acid (promptly by).

Term used herein " heterologous gene " is meant the gene that is not in its natural surroundings.For example, heterologous gene comprise from a certain species but be directed to the gene of another kind of species.Heterologous gene comprises that also natural origin is in a certain organism but altered in some aspects (for example, that suddenlyd change, that added multicopy, be connected to non-natural and regulate on the sequence, or the like) gene.Heterologous gene obviously is different from endogenous gene, reason be the heterologous gene sequence be typically connected to not find with chromosome in gene order natural relevant or with the undiscovered chromosomal each several part related DNA sequence of nature (for example, expressed common its of Escherichia coli (coli) do not have the gene of expression).

Term used herein " carrier " is used in reference to the nucleic acid molecules of one or more dna fragmentation from a cell transfer to another cell.Term " charge material " exchanges mutually with " carrier " sometimes and uses.Carrier comes from plasmid, bacteriophage or plant virus or animal virus usually.

" connection " is meant the process that forms diester linkage between double stranded nucleic acid fragment.Except as otherwise noted, otherwise connect and to use the condition of known buffer solution and 10 T4DNA of unit ligase (" ligase ")/0.5 μ g (about equimolar amounts) dna fragmentation to be connected to finish.The connection of nucleic acid can be used in framework two albumen being linked together to generate single albumen or fusion.

Term used herein " gene expression " be meant by gene " transcribing " (for example, enzyme effect by RNA polymerase) hereditary information coded in the gene is converted into RNA (for example mRNA, rRNA, tRNA or snRNA), and is the process of albumen with this genetic transformation then by mRNA " translation " for protein coding gene.Gene expression can be regulated in a lot of stages in this process." rise " or " activation " is meant and improves the adjusting that gene expression product (for example, RNA or albumen) generates, and " downward modulation " or " inhibition " is meant the adjusting that reduces generation.The molecule (for example, transcription factor) that participates in raising or reducing usually is called " activator " and " inhibitor ".

Term " polypeptide ", " peptide ", " albumen " and " protein fragments " can exchange use in this article mutually, refer to the polymer of amino acid residue.This term is applicable to that wherein one or more amino acid residues are amino acid polymers of corresponding naturally occurring amino acid whose artificial chemical simulation thing, also is applicable to the amino acid polymer that naturally occurring amino acid polymer and non-natural exist.

Term " amino acid " is meant naturally occurring amino acid and synthetic amino acid acid and has amino acid analogue and amino acid analog thing with function like the naturally occurring amino acids.Naturally occurring amino acid is those amino acid of being modified by genetic codon amino acids coding and those postmenstruations, for example hydroxy-proline, Gla and O-phosphoserines.Amino acid analogue is meant the compound with basic chemical structure identical with naturally occurring amino acid, and for example α carbon is connected to the amino acid of hydrogen, carboxyl, amino and R group, for example homoserine, nor-leucine, methionine sulfoxide, methionine methyl sulfonium.Described analog can have modified R group (for example nor-leucine) or modified peptide backbone but still keep the basic chemical structure identical with naturally occurring amino acid.But the amino acid analog thing is meant to have and is different from amino acid whose general chemistry structure has compound with function like the naturally occurring amino acids.

" the conservative variant of modifying " is applicable to amino acid and nucleotide sequence." amino acid variant " is meant amino acid sequence.For specific nucleotide sequence, conservative modify those nucleotide sequences that variant is meant the identical or essentially identical amino acid sequence of coding, perhaps in nucleic acid (for example natural closing on) sequence that the situation middle finger of encoding amino acid sequence is basic identical or not relevant.Because the degeneracy of genetic codon, most of albumen are all by the identical nucleic acid coding of a large amount of functions.For example, codon GCA, GCC, GCG and GCU amino acids coding all are alanine.Therefore, in each position of the alanine of being stipulated by codon, this codon can be changed into another corresponding described codon and not change encoded polypeptide.Such nucleic acid variation is " silent variant ", is a kind of conservative modification variation.Each nucleotide sequence of coded polypeptide has also been described the silent variant of nucleic acid herein.Those skilled in the art should know, in some cases, also can to each codon in the nucleic acid (except generally be methionine unique password AUG with generally be the TGG of unique password of tryptophan) modified to obtain the molecule that function is watched mutually.Therefore, the silent variant of nucleic acid encoding lies in the described sequence about expression product, but does not lie in the described sequence about the actual probes sequence.For amino acid sequence, those skilled in the art will know that, amino acid whose peptide, polypeptide or the protein sequence of the replacement of nucleic acid, disappearance or increase, change in encoded sequence, increase or disappearance single amino acids or small percentage is " the conservative variant of modifying ", comprises that the result of change causes amino acid by the situation of the similar amino acid replacement of chemistry.The form that provides intimate amino acid whose conservative replacement is known in this area.Except that comprising so conservative modification variant, do not get rid of homologue and allele between polymorphism variant of the present invention, kind.Usually, conservative replacement comprises: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) N (N), glutaminase (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), bright amino acid (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); With 8) cysteine (C), methionine (M) (referring to, Creighton for example, Proteins (1984)).

Term used herein " band epi-position label " is meant and comprises chimeric polyeptides cancer stem cell label albumen or its territory sequence or part, that merge with " epi-position label ".Epi-position label polypeptide comprises enough amino acid residues so that the epi-position by antibody recognition to be provided, but still enough weak points make it not influence the activity of cancer stem cell label albumen.Suitable epi-position label has at least 6 amino acid residues usually, generally between about 8～about 50 amino acid residues, sometimes between about 10～about 20 residues.Epi-position label commonly used comprises Fc, HA, His and FLAG label.

The invention provides a kind of isolated antibody, described antibody combines with the extracellular domain specificity of human FZD8 acceptor, and suppresses the growth of tumour cell.In some embodiments, described antibody is monoclonal antibody.In some embodiments, described antibody is chimeric antibody.In some embodiments, described antibody is humanized antibody.In some embodiments, described antibody is human antibodies.

The present invention also provides a kind of isolated antibody, and described antibody combines with the extracellular domain specificity of two or more human FZD acceptor, and suppresses the growth of tumour cell.In some embodiments, described antibody combines with the extracellular domain specificity of human FZD2 and FZD6.In some embodiments, described antibody combines with the extracellular domain specificity of human FZD7 and FZD10.In some embodiments, described antibody combines with the extracellular domain specificity of human FZD4 and FZD5.In some embodiments, described antibody combines with the extracellular domain specificity of human FZD4 and FZD8.In some embodiments, described antibody combines with the extracellular domain specificity of human FZD5 and FZD8.In some embodiments, described antibody is monoclonal antibody.In some embodiments, described antibody is chimeric antibody.In some embodiments, described antibody is humanized antibody.In some embodiments, described antibody is human antibodies.

The present invention also provides a kind of isolated antibody, and described antibody combines with the extracellular domain specificity of human FZD acceptor more than three kinds.In some embodiments, described antibody combines with the extracellular domain specificity of human FZD4, FZD5 and FZD8.In some embodiments, described antibody is monoclonal antibody.In some embodiments, described antibody is chimeric antibody.In some embodiments, described antibody is humanized antibody.In some embodiments, described antibody is human antibodies.

The present invention also provides a kind of pharmaceutical composition, and this pharmaceutical composition comprises antibody disclosed in this invention and the acceptable charge material of pharmacy.

The present invention also provides a kind of hybridoma that produces antibody disclosed in this invention.

The present invention further provides a kind of treatment method for cancer, described method comprises uses antibody disclosed in this invention or the pharmaceutical composition that suppresses the growth of tumour cell effective dose.In some embodiments, described antibody and cytotoxicity have partly been carried out coupling.In some embodiments, described method further comprises and uses at least a additional therapeutic agent that is suitable for carrying out conjoint therapy.In some embodiments, described tumour cell is selected from tumor of breast, colorectum tumour, lung neoplasm, tumor of prostate, pancreatic neoplasm and head and neck neoplasm.

Solid tumor is similar to it and comes source tissue, is made up of the allos cell mass.Most of these cells lack oncogenicity, this show the development of solid tumor and keep also depend on microcommunity stem cell (promptly, the oncogenicity cancer cell), these stem cells have propagation and form other tumor stem cell (self-regeneration) effectively simultaneously and tumour cell (that is non-tumorigenic cancer cell) most more differentiation, that lack tumorigenesis potential.The cancer stem cell notion is introduced after finding HSC soon first, and obtains test conclusive evidence (Park etc., 1971, J Natl.Cancer Inst.46:411～22 in acute myeloid leukaemia (AML); Lapidot etc., 1994, Nature 367:645～8; Bonnet and Dick, 1997, Nat.Med.3:730～7; Hope etc., 2004, Nat.Immunol.5:738～43).From the stem cell of solid tumor recently based on they the cell surface receptor uniqueness expression pattern with the evaluation of their self-regeneration and propagation performances in culture and xenograft animal model is separated.Find that ESA+, CD44+, CD24-/low pedigree colony has the ability that forms tumour and enrichment surpasses 50 times (Al-Hajj etc., 2003, Proc.Nat ' l.Acad.Sci.100:3983～8) of non-graded tumour cell.The ability of isolating the oncogenicity cancer stem cell from a large amount of non-tumorigenic tumour cells makes can be used microarray analysis to identify the cancer stem cell label, have the gene of differential expression with respect to non-tumorigenic tumour cell or normal breast epithelium in cancer stem cell.Cancer is diagnosed and treated to utilization of the present invention to the understanding of the cancer stem cell label that these are identified.

Cancer stem cell label of the present invention relates to human FZD acceptor, for example comprise human FZD4, FZD5 and FZD8 as the cancer stem cell label, this receptor relates to the Wnt signal transduction pathway of cancer stem cell in keeping, and by eliminating these oncogenicity cells as the treatment of cancer target.The Wnt signal transduction pathway is that the tissue behind the formation of embryo's pattern, the embryo is kept one of a plurality of crucial adjusting factor of learning with stem cell biological.More particularly, the Wnt signal transduction plays an important role in the generation of cell polarity and cell fate specialization (self-regeneration that comprises population of stem cells).The activation out of control of Wnt approach is relevant with a large amount of human cancers, and wherein this approach can change the growth destiny of tumour cell and make them maintain not differentiation and vegetative state.Therefore, carcinogenesis can be carried out (Reya and Clevers, 2005, Nature 434:843 by the homeostatic mechanism of the tissue repair usurping the control normal development and undertaken by stem cell; Beachy etc., 2004, the summary among the Nature 432:324).

The Wnt signal transduction pathway is set forth in fruit bat (Drosophila) first and grows mutant Wingless (wg) and stem from muroid proto-oncogene int-1 (being called Wnt1 at present) (Nusse and Varmus, 1982, Cell 31:99～109; Van Ooyen and Nusse, 1984, Cell 39:233～40; Cabrera etc., 1987, Cell 50:659～63; Rijsewijk etc., 1987, Cell50:649～57).The glycoprotein of the fat modification of Wnt gene code secretion, this glycoprotein has identified 19 in mammal.These oozy parts activate the receptor complex by Frizzled (Fzd) receptor family member and low-density lipoprotein (LDL) RAP 5 or 6 (LPR5/6) formation.The Fzd acceptor is the seven-transmembrane territory albumen of g protein coupled receptor (GPCR) superfamily, contains with the outer N end of the big born of the same parents of 10 conservative cysteines ligand binding domain (be called and be rich in domain cysteine (CRD) or Fri territory).Human FZD acceptor has 10: FZD1～10.Different Fzd CRD have different binding affinities (Wu and Nusse to specific Wnt, 2002, J.Biol.Chem.277:41762～9), and the Fzd acceptor is divided into the acceptor of the white approach of standard beta-catenin that activates the following stated and activates the acceptor (Miller etc. of non-standard approach, 1999, Oncogene 18:7860～72).In order to form receptor complex in conjunction with the FZD part, FZD acceptor and LRP5/6 (single channel transmembrane protein, have by 6 YWTD amino acid and repeat the outer EGF sample territory of 4 born of the same parents that thing separates) (Johnson etc., 2004, J.Bone Mineral Res.19:1749) interacts.

The standard Wnt signal transduction pathway that receptors bind activates mediate by the cytoplasmic protein Dishevelled (Dsh) with Fzd acceptor direct interaction, and causes kytoplasm to be stablized with beta-catenin accumulating in vain.When not having the Wnt signal, beta-catenin is positioned to comprise the kytoplasm destruction compound of tumor inhibitor colonic adenoma polyposis (APC) albumen and auxin in vain.These albumen play glycogen synthase kinase (GSK)-3 β combination and the white crucial support effect of phosphorylation beta-catenin of allowing, and make it can pass through ubiquitin/protease approach degraded.The activation of Dsh causes the phosphorylation of GSK3 β and dissociating of described destruction compound.The kytoplasm beta-catenin of accumulation is transported in the nuclear in vain then, and the activated transcription with the DNA binding protein interactions of Tcf/Lef family there.

Except the signals specification transduction pathway, the Wnt part also activates the white non-dependence approach of beta-catenin (Veeman etc., 2003, Dev.Cell 5:367～77).Non-standard Wnt signal transduction participates in a lot of processes, but be sure of that great majority are to participate in primitive gut by the mechanism that is similar to fruit bat plane cell polarity (PCP) approach to form activity.Other of non-standard Wnt signal transduction may mechanism comprise calcium flux, JNK and small G-protein and different trimerization G albumen.Often observe the antagonism between normative approach and the non-standard approach, have some evidences to show, the non-standard signal transduction can suppress cancer and form (Olson and Gibo, 1998, Exp.Cell Res.241:134; Topol etc., 2003, J.Cell Biol.162:899～908).Therefore in some cases, the Fzd acceptor plays the negative modulator effect of standard Wnt signal transduction pathway.For example, FZD6 suppresses the signals specification transduction (Golan etc., 2004, JBC279:14879～88) that Wnt-3a induces by the TAKl-NLK approach with the FZD1 coexpression time.Similarly, Fzd2 comes antagonism standard Wnt signal transduction (Ishitani etc., 2003, Mol.Cell.Biol.23:131～9) by the TAK1-NLKMAPK cascade under the situation that Wnt-5a exists.

Candidate stem cell (HSC) is to understand best stem cell in the body, and the Wnt signal transduction participated in simultaneously normally keeping of they with leukaemia transform (Reya and Clevers, 2005, Nature434:843).HSC is the rare cell group of the aperture tabernacle (stomal niche) that is arranged in adult marrow.These cells are characterised in that unique gene expression pattern and continue to produce the CFU-GM of differentiation more to build the ability of whole hemopoietic system again.HSC and matrix microenvironment cell thereof are all expressed the Wnt part, and the Wnt receptor activation is present among the interior HSC of body.And, beta-catenin Wnt3A white and purifying has promoted the self-regeneration that muroid HSC is external, strengthened the ability of building the blood system in their bodies again, and Wnt5A has promoted the external expansion of human HPC and the (Reya etc. that troop again in the NOD-SCID xenograft models, 2003, Nature 423:409～14; Willert etc., 2003, Nature 423:448～52; Van Den Berg etc., 1998, Blood 92:3189～202; Murdoch etc., 2003, Proc.Nat ' l Acad.Sci.100:3422～7).

Recently find that the Wnt signal transduction all plays a role in the oncogenicity growth of medullary system pedigree and lymph sample pedigree.For example, demonstrate the Wnt signal transduction (Jamieson etc., 2004, N.Engl.J, Med.351:657～67) of their growths and regeneration dependence activation from the GM CFU-GM (GMP) of chronic myelogenous leukemia.As if though leukaemia does not comprise the sudden change in the Wnt approach, autocrine and/or paracrine Wnt signal transduction can be kept carcinous self-regeneration (Reya and Clevers 2005, Nature 434:843).

Standard Wnt signal transduction pathway also plays central role in the keeping of the population of stem cells of small intestine and colon, outstanding effect (Reya and Clevers, 2005, Nature 434:843) is brought into play in the improper activation of this approach in colorectal cancer.Absorbefacient enteric epithelium is distributed in fine hair and the crypts.Stem cell is arranged in crypts and slowly division and generate the cell of fast breeding, produces thus that to move to crypts outer and occupy all differentiated cell populations of intestinal villus.Wnt signal transduction level is associated in control and brings into play leading role in the cell fate of crypts-fine hair axle, and is necessary for keeping of population of stem cells.Cause the gene delection (Korinek etc. of Tcf7/2 by homologous recombination, 1998, Nat.Genet.19:379) or Dickkopf-1 (Dkk1) (a kind of Wnt antagonist of effective secretion) cross to express (Pinto etc., 2003, Genes Dev.17:1709～13; Kuhnert etc., 2004, Proc.Nat ' l Acad.Sci.101:266～71) interrupt the Wnt signal transduction, can cause the exhaustion of intestines population of stem cells.

Colorectal cancer is modal to be to cause by the sudden change that activates in the cascade of Wnt signal transduction.Have 5%～10% approximately in all colorectal cancers, hereditary, one of its principal mode is familial adenomatous polyposis (FAP), this familial adenomatous polyposis is an autosomal dominant disorder, and 80% the affected individuals of wherein having an appointment contains germ line mutation in adenomatous polyposis coli (APC) gene.In comprising auxin and beta-catenin other components of Wnt approach in vain, also identified sudden change.Individual adenoma is to contain second nonactivated allelic epithelial clone's outgrowth, and a large amount of FAP adenomas causes the growth of gland cancer inevitably by the interpolation sudden change of oncogene and/or tumor inhibitor gene.And the activation of Wnt signal transduction pathway comprises APC and the beta-catenin function gain mutation in white, hyperplasia sexual development and tumor growth (Oshima etc., 1997, Cancer Res.57:1644～9 in can the inducing mouse model; Harada etc., 1999, EMBOJ.18:5931～42).

The effect of Wnt signal transduction in cancer is owing near the oncogene that Wnt1 (being initially int1) inserts being accredited as in the mammal tumor that muroid virus transforms is disclosed (Nusse and Varmus, 1982, Cell 31:99～109) first.From then on other evidences of the effect of Wnt signal transduction in breast cancer have been accumulated.For example, the white transgenosis of the beta-catenin in the mammal body of gland is crossed to express and is caused hyperplasia disease and gland cancer (Imbert etc., 2001, J.Cell Biol.153:555～68; Michaelson and Leder, 2001, Oncogene 20:5093～9), and the disappearance of Wnt signal transduction has been destroyed growth (Tepera etc., 2003, J.Cell Sc.116:1137～49 of normal mammalian body of gland; Hatsell etc., 2003, J.Mammary Gland Biol.Neoplasia 8:145～58).Recently, proved that mammalian stem cell is activated (Liu etc., 2004, Proc.Nat ' l Acad.Sci.101:4158) by the Wnt signal transduction.In human breast cancer, beta-catenin accumulates hint in vain to be had above the Wnt signal transduction that occurs being activated in 50% the cancer, although also do not identify concrete sudden change, but rise (Brennan and the Brown of Frizzled expression of receptor have been observed, 2004, J Mammary Gland Neoplasia 9:119～31; Malovanovic etc., 2004, Int.J.Oncol.25:1337～42).

FZD10, FZD8, FZD7, FZD4 and FZD5 are 5 in 10 human Wnt acceptors that identified.In mice embryonic, Fzd10 expresses (Nunnally and Parr together at nerve channel, appendage bud and Miao Le Guan Zhongyu Wnt7a, 2004, Dev.Genes Evol.214:144～8), and in the appendage bud growth course, play the effect (Kawakami etc. of Wnt-7a acceptor, 2000, Dev.Growth Differ.42:561～9).FZD10 in lung with the Wnt7b coexpression, and cell transfecting studies show that, the FZD10/LRP5 co-receptor activates the standard Wnt signal transduction pathway (Wang etc., 2005, Mol.Cell Biol.25:5022～30) of response Wnt7b.FZD10mRNA is subjected to raising (Saitoh etc. in many cancerous cell lines (comprising cervical cell system, gastric cells system and spongioblastoma clone) and primary cancer (comprising about 40% primary gastric cancer, colon cancer and synovial sarcoma), 2002, Int.J.Oncol.20:117～20; Terasaki etc., 2002, Int.J.Mol.Med.9:107～12; Nagayama etc., 2005, Oncogene 1～12).FZD8 is subjected to raising (Saitoh etc., 2001, Int.J.Oncol.18:991～96 in several human cancer cell lines, primary gastric cancer and the carcinoma of the rectum; Kirikoshi etc., 2001, Int.J.Oncol.19:111～5; Janssens etc., 2004, Tumor Biol.25:161～71).FZD7 expresses in whole gastric tube, and has sixth to be subjected to raising (Kirikoshi etc., 2001, Int.J.Oncol.19:111～5) in human primary gastric cancer case.The induced expression morphological change of the FZD7 extracellular domain of colon carcinoma cell line is also slowed down tumor growth (Vincan etc., 2005, Differentiation 73:142～53) in the xenogenesis explant model.FZD5 brings into play necessary effect (Ishikawa etc. in yolk bag and placenta angiogenesis, 2001, Dev.128:25～33), and in the kidney relevant, be subjected to raising (Janssens etc. with the transduction of Wnt/ beta-catenin white signal, 2004, Tumor Biology 25:161～71).FZD4 highly expresses in the intestinal crypts epithelial cell, and is to demonstrate one of several factors of differential expression (Gregorieff etc., 2005, Gastroenterology 129:626～38) in normal tissues and tumor tissues.Therefore the FZD acceptor makes these albumen become the desirable target of treatment of cancer as the evaluation of cancer stem cell label.

The invention provides a kind of cancer stem cell label, the expression of described cancer stem cell label can be used for analyzing, with diagnosis or the monitoring disease relevant with the cancer stem cell marker representation.In some embodiments, the expression of cancer stem cell label is for example expressed to determine by the polynucleotides (for example mRNA) of the described cancer stem cell label of coding.Can be by any one detects and quantitative described polynucleotides in numerous means known in the art.In some embodiments, adopt the mRNA that detects coding cancer stem cell label from for example in situ hybridization of the histotomy of patient's biopsy samples.In some embodiments, RNA is separated from tissue and detect by for example Northern trace, quantitative RT-PCR or microarray etc.For example can from tissue sample, extract total RNA, and can utilize RT-PCR, use the specific hybrid and the primer of amplification cancer stem cell label to detect the expression of cancer stem cell label polynucleotides.

In some embodiments, can be by detecting the expression that corresponding polypeptide detects the cancer stem cell label.Can detect and quantitative described polypeptide by in numerous means known in the art any one.In some embodiments, for example the operational analysis biochemical method for example electrophoresis, Capillary Electrophoresis, high performance liquid chromatography (HPLC) or thin-layer chromatography (TLC) detect cancer stem cell label polypeptide.Can also check order to the polypeptide that is separated according to standard techniques.In some embodiments, utilize immunofluorescence or immunohistochemistry on the histotomy for example, the antibody that produces with anticancer stem cell labeling albumen detects described albumen.As selection, use for example ELISA, FACS, Western trace, immunoprecipitation or arrays of immobilized protein, express with the antibody test of anticancer stem cell labeling thing.For example, can from patient's biopsy samples, isolate cancer stem cell, utilize FACS to detect the expression of cancer stem cell label albumen with FLA.In another approach, can utilize labelled antibody in typical imaging system, to detect the cell of expressing the cancer stem cell mark in the body.For example, can use the isotope-labeled antibody of paramagnetism to carry out magnetic resonance imaging (MRI).

In some embodiments of the present invention, diagnostic assay comprises and for example uses whether immunohistochemistry, in situ hybridization or RT-PCR determine the expression of cancer stem cell label in tumour cell.In other execution mode, diagnostic assay comprises the expression that uses quantitative RT-PCR for example to determine the cancer stem cell label.In some embodiments, diagnostic assay for example also comprise with control tissue for example normal epithelial organize the expression that relatively comes to determine the cancer stem cell label.

Then, the detection of cancer stem cell marker representation can be in order to provide prognosis and to select therapy.Prognosis can be expressed based on any known risk of cancer stem cell label indication.And the detection of cancer stem cell label can comprise for example with the treatment that antibody carried out that resists detected cancer stem cell label albumen in order to select suitable therapy.In some embodiments, described antibody specificity is in conjunction with the extracellular domain such as cancer stem cell label albumen such as human FZD acceptors.

Within the scope of the invention, suitable antibody is to have for example material of following one or more effects: disturb the expression of cancer stem cell label; Disturb the activation of cancer stem cell signal transduction pathway by for example spatially suppressing interaction between cancer stem cell label and its part, acceptor or the co-receptor; Activate the cancer stem cell signal transduction pathway by for example combination as part or promotion endogenic ligand; Or in conjunction with the cancer stem cell label and suppress tumor cell proliferation.

In some embodiments, the antibody of anticancer stem cell labeling thing plays the effect that born of the same parents regulate cancer stem cell label protein function outward.In some embodiments, at the born of the same parents of the antibody of cancer stem cell label outer in conjunction with intrinsic activation (for example kinase activity) that can be by for example suppressing the cancer stem cell label and/or by spatially suppress for example cancer stem cell label and its part, and its acceptor, and co-receptor or and extracellular matrix between interaction suppress the signal transduction of cancer stem cell label albumen.In some embodiments, the born of the same parents of the antibody of for example anticancer stem cell labeling thing are outer in conjunction with transporting the cell surface expression of reducing the cancer stem cell label by the internalization of for example cancer stem cell label albumen or the cell surface of minimizing cancer stem cell label.In some embodiments, the outer combination of the born of the same parents of the antibody of anticancer stem cell labeling thing can be by for example bringing into play as the effect of bait part or the signal transduction of increase part combination promotion cancer stem cell label albumen.

In some embodiments, the antibody of anticancer stem cell labeling thing is attached on the cancer stem cell label albumen and has following one or more effects: suppress the propagation of tumour cell, trigger the cell death of tumour cell or prevent the transfer of tumour cell.In some embodiments, trigger cell death at the antibody of cancer stem cell mark toxin, chemotherapeutics, radioisotope or other similar reagents by institute's coupling.For example, at the antibody of cancer stem cell label with in the tumour cell of expressing this cancer stem cell label by the albumen internalization be activated toxin conjugated.In some embodiments, express the cell death of the cell of described cancer stem cell label albumen at the antibody of cancer stem cell label by ADCC (ADCC) mediation.ADCC gets involved the cracking of cell by the Fc effector cell partly that can discern antibody.Many lymphocytes, monocyte, tissue macrophages, granulocyte and acidophic cell for example have the Fc acceptor and can the mediated cell cracking (Dillman, 1994, J.Clin.Oncol.12:1497).

In some embodiments, trigger the cell death of the cell of expressing cancer stem cell label albumen by activating complement dependent cellular cytotoxicity (CDC) at the antibody of cancer stem cell label.CDC participates in the combining and the activation of complement protein cascade subsequently of Fc part of SC and antibody, thereby causes cell membrane destruction, and finally causes the death of cell.The biologic activity of antibody is known to a great extent by the constant region of antibody molecule or Fc district decision (Uananue and Benacerraf, Textbook of Immunology, the 2nd edition, Williams and Wilkins, the 218th page (1984)).As belong to identical subclass but come from the antibody of different plant species, belonging to a different category with the antibody of subclass is different in this respect.As if in human antibodies, IgM combines the most effective antibody classification with complement, secondly be IgG1, IgG3 and IgG2, but IgG4 lacks (Dillman, 1994, J.Clin.Oncol.12:1497 very much in the activating complement cascade; Jefferis etc., 1998, Immunol.Rev.163:59～76).According to the present invention, can prepare that antibody-like with required biologic activity.

Can measure the ability that mediates the target cell cracking at any specific antibodies of cancer stem cell by complement activation and/or ADCC.Make interested cell at growth in vitro and mark; With antibody with SC or can be added in the cell culture by the immunocyte that antigen antibody complex activates.By for example detecting the lysis of target cell from the cell lysis release mark.In fact, can use patient's oneself serum to come examination antibody as complement source and/or immunocyte source.Then can with can be in testing in vitro the antibody of activating complement or mediation ADCC be used for the treatment of this particular patient.

In some embodiments, can trigger cell death, thereby suppress angiogenesis at the antibody of cancer stem cell label.Angiogenesis is such process, that is, by angiogenesis, by the new blood vessel of the vascularization of preexist, and angiogenesis is the required basic process of normal growth in for example embryonic development, wound healing and the reaction of laying eggs.Greater than 1mm ²～2mm ²Solid tumor growth also need angiogenesis to supplement the nutrients and oxygen, if there is not angiogenesis, tumour cell will be dead.In some embodiments, express the vascular cell of this cancer stem cell label, comprise for example required component of blood vessel assembling of endothelial cell, smooth muscle cell or extracellular matrix at the antibody target of cancer stem cell label.In some embodiments, the antibody at the cancer stem cell label suppresses the growth factor signal transduction that vascular cell is raised, assembles, kept or survives required.

Antibody at the cancer stem cell label can be used for diagnosis as herein described and methods of treatment.In some embodiments, antibody of the present invention is for example in order to the imitate expression of the cancer stem cell label albumen in product such as patient tissue biopsy samples, pleural effusion or the blood sample of detection of biological.The biopsy sample can be made section and for example use immunofluorescence or immunohistochemistry detects albumen.In addition, can separate from the individual cells of sample and by facs analysis and detect through fixing cell or living cells.In some embodiments, can on protein arrays, use antibody to come the expression of the cancer stem cell label on test example such as the tumour cell, in the cell lysate or in other protein samples.In some embodiments, antibody of the present invention is by with antibody growth in order to the inhibition tumour cell with contacting based on the tumour cell in the animal model in the mensuration of cell in vitro or the body etc.In some embodiments, the antibody at the cancer stem cell label by the administering therapeutic effective dose uses described antibody to treat cancer among the patient.

Can prepare polyclonal antibody by any known method.Polyclonal antibody can come immune animal (for example rabbit, rat, mouse, donkey etc.) to produce by MSI or intraperitoneal injection related antigen (fragments of peptides of purifying, total length recombinant protein, fusion etc.), and described antigen can be chosen and be diluted in coupling such as keyhole limpet hemocyanin (KLH) in the Sterile Saline, seralbumin wantonly and unite to form stable emulsion with adjuvant (for example complete Freund's adjuvant or incomplete Freund's adjuvant).Then from recovery polyclonal antibody through the blood of like this animal of immunity and ascites etc.Allow collected blood clotting, decant go out serum and make its clarification and measure the albumen titre by centrifugal.Can comprise that affinity chromatography, ion-exchange chromatography, gel electrophoresis, dialysis etc. are purified into polyclonal antibody from serum or ascites according to this area standard method.

Monoclonal antibody for example can adopt, and Kohler and the described hybridoma method of Milstein (1975, Nature 256:495) prepare.Adopt hybridoma method, generate the antibody that combines with the immunity antigentic specificity to cause lymphocyte according to as mentioned above mouse, hamster or other suitable host animals being carried out immunity.Can also carry out immunity to lymphocyte external.After the immunity, isolated lymphocytes, and for example use polyethylene glycol that itself and suitable myeloma cell line are merged, can never merge the hybridoma of selecting among lymphocyte and the myeloma cell subsequently thereby form.Then according to immunoprecipitation, Western blotting and external in conjunction with measuring (radioimmunoassay (RIA) for example; Enzyme linked immunosorbent assay (ELISA) (ELISA)) hybridoma of the monoclonal antibody of the anti-selected antigen of the generation specificity of Que Dinging uses standard method (Goding, Monoclonal Antibodies:Principles andPractice, Academic Press, 1986) external breeding or in culture as breeding in the animal ascites tumour body.Then from as above at being purified into monoclonal antibody described culture medium of polyclonal antibody or the ascites fluid.

As selection, can also use as United States Patent (USP) 4,816,567 described recombinant DNA methods prepare monoclonal antibody.Use can specific amplification the Oligonucleolide primers of gene of the heavy chain of coding monoclonal antibody and light chain, isolate the polynucleotides of encoding said antibody by for example RT-PCR from mature B cell or hybridoma, and use conventional program to determine their sequence.Then the polynucleotides of institute's separated coding heavy chain and light chain are cloned in the suitable expression, when with this expression vector transfection to such as Escherichia coli (E.coli) cell, ape COS cell, Chinese hamster ovary (CHO) cell or myeloma cell etc. otherwise do not generate in the host cell of immunoglobulin (Ig), produce monoclonal antibody by this host cell.In addition, recombinant monoclonal antibodies or its fragment (McCafferty etc., 1990, Nature, 348:552～554 that can from the phage display library of the CDR that expresses described required species, isolate required species; Clackson etc., 1991, Nature, 352:624～628; With Marks etc., 1991, J.Mol.Biol, 222:581～597).

Can use recombinant DNA technology, further modify the polynucleotides of coding monoclonal antibody in many different modes, to produce substituting antibody.In some embodiments, the light chain of for example mouse monoclonal antibody and the constant region of heavy chain can be substituted by 1) constant region of human antibodies for example, to produce chimeric antibody or 2) the NIg polypeptide, merge antibody to produce.In some embodiments, block or remove described constant region to produce the required antibody fragment of monoclonal antibody.Can use the directed mutagenesis of variable region or specificity that monoclonal antibody is optimized in high density mutagenesis, compatibility etc.

In some embodiments of the present invention, be humanized antibody at the monoclonal antibody of cancer stem cell label.Humanized antibody is the antibody that contains in the variable region from the minimum sequence of non-human (for example muroid) antibody.Such antibody reduces antigenicity when being used to be applied to human subject in treatment and HAMA (human anti-mouse antibody) replys.In practice, humanized antibody normally has human antibodies minimum or that do not have the non-human sequence.Human antibodies is the antibody that is produced by the mankind or has amino acid sequence corresponding to the antibody that is produced by the mankind.

Can use various techniques known in the art to prepare humanized antibody.Can use the CDR of the CDR replacement human antibodies of the non-human antibody (for example mouse, rat, rabbit, hamster etc.) with required specificity, compatibility and ability to come antagonist to carry out humanization (Jones etc., 1986, Nature, 321:522～525; Riechmann etc., 1988, Nature, 332:323～327; Verhoeyen etc., 1988, Science, 239:1534～1536).Can by in the Fv skeleton district and/or the replacement of the intra-residue extra residue of non-human of being replaced further modify humanized antibody, to improve and to optimize antibody specificity, compatibility and/or ability.

Can use various techniques known in the art directly to prepare humanized antibody.Can produce through the human bone-marrow-derived lymphocyte of the immortalization of external immunity or from the immune body that can generate the antibody that is used for anti-target antigen separate the human bone-marrow-derived lymphocyte of the immortalization that obtains (referring to, Cole etc. for example, Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, the 77th page (1985); Boemer etc., 1991, J.Immunol., 147 (1): 86～95; With United States Patent (USP) 5,750,373).And, can from the phage library of express human antibody wherein, select human antibodies (Vaughan etc., 1996, Nat.Biotech., 14:309～314; Sheets etc., 1998, Proc.Nat ' l.Acad.Sci., 95:6157～6162; Hoogenboom and Winter, 1991, J.Mol.Biol., 227:381; Marks etc., 1991, J.Mol.Biol., 222:581).Can also make humanized antibody in the transgenic mice that contains the human immunoglobulin gene seat, described human immunoglobulin gene seat can generate all forming members of human antibodies after immunity under the situation that does not have endogenous immunoglobulin to generate.This method is in United States Patent (USP) 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; With 5,661, description is arranged in 016.

The bispecific antibody of specific recognition cancer stem cell label is also contained in the present invention.Bispecific antibody is can specific recognition and in conjunction with the antibody of at least two kinds of different epi-positions.Described different epi-position (for example can be in same a part, same cancer stem cell label polypeptide) in or be in the different molecules, for example make these two antibody can specific recognition and in conjunction with cancer stem cell label and for example 1) such as effector molecule on the leucocytes such as TXi Baoshouti (for example CD3) or Fc acceptor (for example CD64, CD32 or CD16), perhaps 2) cytotoxic agent that will be explained below.Bispecific antibody can be complete antibody or antibody fragment.

Exemplary bispecific antibody can be in conjunction with two kinds of different epi-positions, and wherein at least one epi-position comes from polypeptide of the present invention.As selection, can with the antigen arm of immunoglobulin molecules with can with such as the triggering molecule on the leucocytes such as TXi Baoshouti molecule (for example, CD2, CD3, CD28 or B7) or the arm combination of the Fc receptors bind of IgG, with the cytophylaxis mechanism of the cell that is conceived to express specific antigen.Can also use bispecific antibody cytotoxic agent to be imported the cell of expressing specific antigen.These antibody have antigen combination arm and can be in conjunction with the arm of cytotoxic agent or radionuclide chelators such as EOTUBE, DPTA, DOTA or TETA.The technology of preparation bispecific antibody is common (Millstein etc., 1983, Nature 305:537～539 in this area; Brennan etc., 1985, Science 229:81; Suresh etc., 1986, Methods inEnzymol.121:120; Traunecker etc., 1991, EMBO is J.10:3655～3659; Shalaby etc., 1992, J.Exp.Med.175:217～225; Kostelny etc., 1992, J.Immunol.148:1547～1553; Gruber etc., 1994, J.Immunol.152:5368; With United States Patent (USP) 5,731,168).Also imagination has the antibody more than two valencys.For example, can prepare three-specific antibody (Tutt etc., J.Immunol.147:60 (1991)).

In some embodiments, provide antibody fragment for example to improve the tumour penetrability.Known have a multiple technology that is used to generate antibody fragment.Traditionally, these fragments obtain (for example, Morimoto etc., 1993, Journal of Biochemical andBiophysical Methods 24:107～117 by the proteolytic digestion of complete antibody; Brennan etc., 1985, Science, 229:81).In some embodiments, antibody fragment produces by reorganization.Fab, Fv and scFv antibody fragment can express in Escherichia coli or other host cells and secrete, thereby can generate these a large amount of fragments.Can also from antibody phage discussed above library, isolate described antibody fragment.Antibody fragment can also be a United States Patent (USP) 5,641 for example, the linear antibody described in 870, and can be monospecific antibody or bispecific antibody.Those skilled in the art know that the other technologies that generate antibody fragment.

According to the present invention, there are various technology to be applicable to that generation has specific single-chain antibody (referring to U.S. Patent No. 4,946,778) to polypeptide of the present invention.In addition, also there is the whole bag of tricks to be applicable to and makes up Fab expression library (Huse etc., Science 246:1275～1281 (1989)), fast and effeciently to identify FZD acceptor or derivatives thereof, fragment, analog or homologue had required specific monoclonal antibody Fab fragment.Can generate the antibody fragment of the idiotype contain polypeptide of the present invention by art technology, described antibody fragment includes but not limited to: (a) F (ab ') 2 fragments that generate of the pepsin digestion by antibody molecule; (b) the Fab fragment that produces of the disulphide bridges by reduction F (ab ') 2 fragments; (c) by using papain and reducing agent to handle the Fab fragment that antibody molecule produces; (d) Fv fragment.

Can also be as required, particularly under the situation of antibody fragment, antagonist is modified to prolong its serum half-life.This can realize by for example following method: will remedy the receptors bind epi-position by the sudden change of appropriate area in the antibody fragment and be incorporated in the antibody fragment, perhaps described epi-position is incorporated in the peptide tag, then peptide tag is fused to the centre (for example synthetic) of the arbitrary end or the antibody fragment of antibody fragment by DNA or peptide.

Allos coupling antibody is also located within the scope of the present invention.Allos coupling antibody is made of two covalently bound antibody.Proposed for example to utilize such antibody that immunocyte is targeted on harmful cell (U.S. Patent No. 4,676,980).What also expect is, can use method known in the synthetic proteins chemistry, comprises that those relate to the described antibody of the external preparation of the method for using crosslinking agent.For example, can use disulfide exchange reaction or make up immunotoxin by forming thioether bond.The example that is used for the suitable reagent of this purpose comprises imino group mercaptides (iminothiolate) and methyl-4-sulfydryl fourth imino-ester (methy-4-mercaptobutyrimidate).

Should be appreciated that and be that the object of the invention, the antibody of being modified can comprise the variable region that makes any kind that described antibody and human FZD acceptor be associated.In this respect, described variable region can comprise or come from and can be induced and strengthen humoral response and produce the mammal of any kind of the immunoglobulin (Ig) of anti-required tumor associated antigen.Like this, the variable region of institute's modified antibodies can for example derive from the mankind, muroid, non-human primate (for example machin, stump-tailed macaque etc.) or wolf.In some embodiments, the variable region of the immunoglobulin (Ig) of being modified and constant region all are human variable region and constant regions.In other embodiments, the variable region of compatibility antibody (generally come from non-human source) can transform or the specificity customization to improve binding ability or to reduce the immunogenicity of this molecule.In this respect, can carry out humanization or change to the useful variable region of the present invention by the amino acid sequence that comprises introducing.

Replace the variable domain that changes simultaneously in heavy chain and the light chain by at least a portion of one or more CDR, and if desired, can carry out described change by displacement of part skeleton district and sequence variation.Although CDR can come from skeleton district source antibody classification or or even the identical antibody of subclass, what also expect is that CDR can come from different classes of antibody, preferably comes from the antibody of different plant species.May not need the whole CDR that are used for from the donor variable region to replace all CDR so that the antigen binding capacity of a variable region is transferred to another variable region.More suitably be only to need to shift to keeping active necessary those residues of antigen binding site.In U.S. Patent No. 5,585, provided explanation in 089,5,693,761 and 5,693,762.Those skilled in the art have the ability fully by implementing conventional test or test by trial-and-error method to obtain the functional antibodies that immunogenicity reduces.

Although change variable region, but those skilled in the art are to be understood that, modified antibody of the present invention will comprise such antibody or its immunoreactivity fragment, wherein, when with have the approximate identical immunogenic antibody that comprises natural or unaltered constant region when comparing, the at least a portion in the one or more constant zones in the described antibody is deleted or thereby the alternate manner change provides described biochemical characteristic, as the tumor-localizing of enhancing or the serum half-life of shortening.In some embodiments, the constant region of modified antibody comprises human constant region.The modification that the constant region compatible with the present invention carried out is included in one or more amino acid whose interpolations, disappearance or replacement in one or more domains.That is, modified antibody disclosed herein can comprise one or more constant regions of three CH (CH1, CH2 or CH3) and/or change or the modification that constant region of light chain (CL) is carried out.In some embodiments of the present invention, expected excalation wherein or lacked the modified constant region of one or more domains fully.In some embodiments, modified antibody comprises domain disappearance construct or the variant (Δ CH2 construct) of wherein having removed whole C H2 territory.In some embodiments, the constant zone of being saved will can be provided usually the short amino acid introns (for example 10 residues) of some molecular flexibility of being given by the disappearance constant region to be replaced.

Except their configuration, known in the art is that constant region mediates several effector functions.For example, the activating complement system that combines of the C1 component of complement and antibody.The conditioning and the cracking of complement activation pair cell pathogen are extremely important.Complement activation also stimulates inflammatory reaction, and can participate in the autoimmunity hypersensitivity.In addition, antibody combines with cell by the Fc district, and wherein the Fc acceptor site in the antibody Fc district is attached to the Fc acceptor (FcR) on the cell.Have in a large number, comprise that IgG (γ acceptor), IgE (η acceptor), IgA (α acceptor) and IgM (μ acceptor) have specific Fc acceptor different classes of antibody.Antibody can trigger many important and various biological answer-replies with the combining of Fc acceptor on the cell surface, comprise the antibody sandwich particle engulf and the removing of destruction, immune complex, the cracking (cytotoxicity that is called the antibody dependent cellular mediation, or ADCC) of antibody sandwich target cell that killer cell is implemented, the release of inflammatory mediator, the placenta that immunoglobulin (Ig) generates shift and control.Although the existing research to a certain degree of various Fc acceptors and acceptor site also has a lot of unknown parts to their location, 26S Proteasome Structure and Function.

Though be not to limit the scope of the invention, it is believed that the antibody that comprises by the constant region of modification described herein can provide altered effector function, this has influenced the biological profile of institute's administration of antibodies conversely again.For example, the disappearance in constant zone or inactivation (by point mutation or other modes) can reduce combining of modified antibody and Fc acceptor in the circulation, strengthen the location of tumour thus.In other situation, with the present invention coincide be, modify constant region regulated complement in conjunction with and shorten cytotoxic non-specific connection of serum half-life and coupling thus.But, other that can use constant region are modified and are eliminated disulfide bond or oligosaccharide part, thereby make and can improve antigentic specificity or antibody flexibility, therefore can add strong fix.Similarly, can use biochemistry well known to those skilled in the art or molecular engineering technology easily to realize the modification of constant region being carried out according to the present invention.

Should be noted in the discussion above that modified antibody can directly be fused to the CH3 territory hinge area of corresponding modified antibody by transformation.In other construct, may wish provides the peptide introns between hinge area and modified CH2 and/or CH3 territory.For example, can express the compatibility construct, wherein CH2 lacks, and remaining CH3 territory (modification or unmodified) is connected on the hinge area by 5～20 amino acid introns.Can increase that such introns for example keep free with the regulating element of guaranteeing constant domain and be come-at-able or make hinge area keep flexible.Yet, should be noted in the discussion above that in some cases, can confirm the unwanted immune response that the amino acid introns have immunogenicity and can bring out anti-described construct.Therefore, any introns that are added to described construct are non-immunogenicity relatively, if perhaps can keep the required biochemistry quality of the antibody modified, even can save introns fully.

Except lacking whole constant zone, should be understood that, can by excalation or a small amount of or or even the replacement of single amino acids antibody of the present invention is provided.For example, the sudden change of the monamino acid in institute's favored area in CH2 territory may be enough to significantly reduce Fc in conjunction with the location of also strengthening tumour thus.Similarly, may wish to lack simply the part in one or more constant zones of controlling effector function to be regulated (for example complement CLQ combination).The selected characteristics (serum half-life) that this excalation of constant region can be improved antibody keeps the integrality of other relevant required functions of the constant zone of study subject simultaneously.And, mention one or more amino acid whose sudden change that can be by can improving gained construct overview or replace the constant region of modifying disclosed antibody as mentioned in passing.In this respect, when the activity that provides by conservative binding site (for example Fc combination) might be able to be provided, the configuration and the immunogenicity characteristic of the antibody that basic maintenance is modified.Some execution mode can comprise one or more amino acid of adding constant region to the function that strengthens desirable characteristics such as effector or provide more cytotoxin or carbohydrate absorption.In such execution mode, may wish to insert or duplicate specific sequence from selected constant zone.

The present invention also comprises chimeric antibody, humanized antibody and human antibodies or similar variant and the equivalent of their antibody fragment basic and that this paper proposes.These variants or equivalent can contain for example conservative sudden change that replaces, and promptly one or more amino acid are replaced by similar amino acid.For example, conservative replacement is meant that an amino acid is replaced by another amino acid in the same big class, for example an acidic amino acid is replaced by another acidic amino acid, and a basic amino acid is replaced by another basic amino acid, and perhaps a neutral amino acid is replaced by another neutral amino acid.It is known that conserved amino acid is substituted in this area.

The invention still further relates to the immune conjugate that comprises the antibody that is coupled to cytotoxic agent.Cytotoxic agent comprises chemotherapeutics, growth inhibitor, toxin (for example enzyme activity toxin of bacterium, fungi, plant or animal origin or its fragment), radioactive isotope (that is radioactivity conjugate) etc.The chemotherapeutics that can be used for producing described immune conjugate comprises for example methotrexate (MTX), adriamycin, Doxorubicin, melphalan, mitomycin C, Chlorambucil, daunorubicin or other intercalators.Available enzyme activity toxin and fragment thereof comprise diphtheria A chain, the non-binding active fragment of diphtheria toxin, the exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, α-Zhou Qujunsu (α-sarcin), tung oil tree albumen (Aleurites fordii protein), the carnation toxalbumin, pokeroot (Phytolaca americana) albumen (PAPI, PAPII and PAP-S), momordica charantia inhibitor (momordica charantia inhibitor), curcin, crotin, Saponaria officinalis inhibitor (sapaonaria officinalis inhibitor), white tree toxalbumin, silk splits albumen (mitogellin), the limitation rhzomorph, phenomycin, enomycin, and trichothecene (tricothecenes).Various radionuclides can be used to prepare the antibody of radioactivity coupling, comprise ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y and ¹⁸⁶Re.Can use following material to prepare the conjugate of antibody and cytotoxic agent: the agent of various difunctionality albumen coupling such as N-succinimido-3-2-(pyridine two mercaptan) propionic ester (SPDP); imido thia pentane (IT); the difunctionality derivative of imines ester (as own diimine dimethyl ester HCL); active ester (as disuccinimidyl suberate); aldehyde (as glutaraldehyde); double azido compound (as two (to the azido benzoyl base) hexamethylene diamine); two-tetroxide derivative (as two-(pyrazine formoxyl)-ethylenediamines); vulcabond is (as methylene phenyl 2; the 6-vulcabond) and two-active fluorine compounds (as 1; 5-two fluoro-2, the 4-dinitro benzene).Can also use the conjugate of antibody and one or more micromolecule toxin, described micromolecule toxin for example is the derivative with toxin activity of calicheamycin, maytansinol (maytansinoid), single-ended born of the same parents' verticillium toxin (trichothene) and CC1065 and these toxin.

Conjugate antibody is made of two covalently bound antibody.Proposed for example to utilize such antibody that immunocyte is targeted to harmful cell (U.S. Patent No. 4,676,980).What expect is to use method known in the synthetic proteins chemistry (comprising those methods that relate to crosslinking agent) the described antibody of external preparation.For example, can use disulfide exchange reaction or make up immunotoxin by forming thioether bond.The example that is used for the suitable reagent of this purpose comprises imino group mercaptides (iminothiolate) and methyl-4-sulfydryl fourth imino-ester (methyl-4-mercaptobutyrimidate).

How useful no matter obtain amount, antibody of the present invention can be with any one use in numerous coupling forms (that is immune conjugate) or the not coupling form.As selection, antibody of the present invention can use natural immunology defense with the control study subject with non-coupling form or " exposing " form, comprises that CDC (CDC) and ADCC (ADCC) eliminate malignant cell.In some embodiments, can use in numerous known chelating agents any one or direct mark, with antibody be coupled to radiation isotope as ⁹⁰Y, ¹²⁵I, ¹³¹I, ¹²³I, ¹¹¹In, ¹⁰⁵Rh, ¹⁵³Sm, ⁶⁷Cu, ⁶⁷Ga, ¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁸⁶Re and ¹⁸⁸Re.In other execution mode, disclosed composition can comprise and is coupled to medicine, prodrug or biological answer-reply dressing agent, as methotrexate (MTX), adriamycin and lymphokine (as interferon).Other execution modes of the present invention comprise the application with the antibody of particular organisms toxin such as couplings such as ricin or diphtheria toxin.In other execution modes, modified antibody can be compound with other immunocompetence parts (for example antibody or its fragment), and wherein the gained molecule can be attached on oncocyte and effector cell such as the T cell simultaneously.Selection uses the modified antibodies of coupling or not coupling will depend on type and residing stage, the use of supplemental treatment (for example chemotherapy or external radiation) and patient's the situation of cancer.Should be understood that those skilled in the art can easily make such selection according to the instruction of this paper.

Can measure the immunologic opsonin combination of antibody of the present invention by any means known in the art.Operable immunoassay includes but not limited to use such as the BIAcore analytic approach, the facs analysis method, immunofluorescence technique, immunocytochemical method, the Western blotting, radioimmunoassay, the ELISA method, " sandwich " immunoassay, immunoprecipitation assay, the precipitin reaction method, the GDP reaction method, immunity diffusion measurement method, agglutination assay, the complement fixation determination method, immunoradiometry, the competitiveness of fluorescence immunoassay and albumin A immunoassay etc. and noncompetitive are measured system.Such determination method is conventional and known (referring to volumes such as for example Ausubel, 1994, Current Protocols in Molecular Biology in this area, the 1st volume, JohnWiley and Sons, Inc., New York, in this mode by reference with its whole introducings).

In some embodiments, use ELISA determines the immunologic opsonin at the antibody of cancer stem cell label.The ELISA determination method comprises preparation antigen, with the plate hole of antigen coated 96 hole titer plates, but add the antibody at the cancer stem cell label be coupled to detection compound such as zymolyte (for example horseradish peroxidase or alkaline phosphatase) in plate hole, incubation a period of time is also detected the existence of antigen.In some embodiments, but at the antibody of cancer stem cell label not with the detection compound coupling, but in plate hole, add alternatively and can discern this second coupling antibody at the antibody of cancer stem cell label.In some embodiments, need not antigen coated plate hole, but but the available second antibody that adds behind the antigen with the detection compound coupling that adds at the antibody sandwich plate hole of cancer stem cell label and in the plate hole of Xiang Jingbao quilt., those skilled in the art strengthen the parameter of detected signal and ELISA variable known in the art but should knowing change (referring to volumes such as for example Ausubel, 1994, Current Protocols in Molecular Biology, the 1st volume, John Wiley and Sons, Inc., New York is in 11.2.1).

Can determine antibody and the binding affinity of cancer stem cell label antigen and the dissociation rate of antibody-AI by the competitive binding assay method.An example of competitive binding assay method is a radioimmunoassay, and this method is included under the situation that has the ever-increasing unlabelled antigen of content, will be through the antigen of mark (for example ³H or ¹²⁵I) or its fragment or variant and interested antibody incubation, detect the antibody that is attached to through the antigen of mark then.Can determine at the compatibility of the antibody of cancer stem cell label with in conjunction with dissociation rate according to scatchard mapping analysis data.In some embodiments, the combination and the speed of dissociating at the antibody of cancer stem cell label is determined in use BIAcore dynamic analysis.The BIAcore dynamic analysis comprises analyzes combining and dissociating of the chip that is fixed with cancer stem cell label antigen on antibody and the surface.

In some embodiments, the polynucleotides of separation are contained in the present invention, and this polynucleotide encoding contains the antibody of anti-human FZD acceptor or the polypeptide of its fragment.Therefore, term " polynucleotides of coded polypeptide " is contained the polynucleotides that comprise the coded sequence of this polypeptide only and is comprised the extra coded sequence and/or the polynucleotides of non-coding sequence.Polynucleotides of the present invention can be rna form or dna form.DNA comprises cDNA, genomic DNA and synthetic DNA; And can be two strands or strand, if strand, it can be coding strand or noncoding strand (antisense strand).

The invention still further relates to the above for example variant of the polynucleotides of fragment, analog and derivative of encoding.The variant of polynucleotides can be the variant that the non-natural of the naturally occurring allele variant of these polynucleotides or these polynucleotides exists.In some embodiments, polynucleotides can have such coded sequence, and this coded sequence is the naturally occurring allele variant of the coded sequence of disclosed polypeptide.As known in the art, allele variant is the alternative form that for example replacement, disappearance or the interpolation of one or more nucleotides still do not have obviously to change the function of coded polypeptide that has of polynucleotide sequence.

In some embodiments, polynucleotides comprise the coded sequence of mature polypeptide, and this coded sequence merges in same reading frame and for example helps to express in host cell and the polynucleotides of secrete polypeptide (for example targeting sequencing of the secretion sequence of transporting from transit cell as the control polypeptide).Polypeptide with targeting sequencing is a kind of precursor protein, can have the targeting sequencing that excised by host cell to form the polypeptide of mature form.Polynucleotides such precursor protein of can also encoding, this precursor protein is that maturation protein adds extra 5 ' terminal amino acid residue.Maturation protein with precursor sequence is a kind of precursor protein, and is a kind of albumen of inactive form.After the excision precursor sequence, what stay promptly is to have active maturation protein.

In some embodiments, polynucleotides comprise the coded sequence of mature polypeptide, and this coded sequence merges to have in same reading frame and can be used for for example flag sequence of the coded polypeptide of purifying.For example, in the situation of bacterial host, this flag sequence can be the maturation protein that is merged with purifying and this mark by the hexahistidine tag that the pQE-9 carrier provides, perhaps, when using mammalian hosts (for example COS-7 cell), this flag sequence can be hemagglutinin (HA) label that stems from influenza hemagglutinin protein.

In some embodiments, the invention provides isolated nucleic acid molecule, the polynucleotides that this nucleic acid molecules and coding contain the polypeptide of the antibody of anti-human FZD acceptor or its fragment have at least 80% homogeneity, at least 85% homogeneity, at least 90% homogeneity, at least 95% homogeneity, in some embodiments, has at least 96%, 97%, 98% or 99% homogeneity.

Have with " homogeneity " of contrast nucleotide sequence polynucleotides and be meant for for example at least 95% nucleotide sequence, except polynucleotide sequence can comprise at the most the nucleotides of 5 point mutation/100 contrast nucleotide sequence, the nucleotide sequence of polynucleotides was identical with control sequence.In other words, in order to obtain to have and the homogeneity of contrast nucleotide sequence polynucleotides at least 95% nucleotide sequence, have 5% nucleotides to lack in the control sequence at the most or replaced by other nucleotides, the nucleotides that perhaps accounts for 5% the quantity at the most of total nucleotide in the control sequence can be inserted in the control sequence.These sudden changes of control sequence can occur in the amino terminal position or the carboxyl terminal position of contrast nucleotide sequence, the perhaps optional position between these terminal positions, perhaps individually be dispersed among the nucleotides of control sequence, perhaps be dispersed in the control sequence with one or more adjacent groups.

As a kind of actual conditions, can use known computer program such as Bestfit program (Wisconsin Sequence Analysis Package, the 8th version that is used for Unix, GeneticsComputer Group, University Research Park, 575 Science Drive, Madison, WI 53711), determine according to a conventional method whether any specific nucleotide molecule has at least 80% homogeneity with control sequence, at least 85% homogeneity, at least 90% homogeneity, in some embodiments, whether have at least 95%, 96%, 97%, 98% or 99% homogeneity.Local homology's algorithm of Bestfit use Smith and Waterman (Advances in Applied Mathematics 2:482 489 (1981)) finds the optimum homology fragment between the two sequences.When using Bestfit or other sequence alignment program determines that whether a specific sequence for example has 95% homogeneity with control sequence of the present invention arbitrarily, parameter is arranged so that 5% the homology breach at the most that can list calculating homogeneity percentage at the contrast nucleotides sequence of total length and allow the nucleotides sum that accounts for control sequence.

The polynucleotides variant can contain in code area and/or noncoding region and changes.In some embodiments, the polynucleotides variant contains performance or the active variation that produces reticent replacement, interpolation or disappearance but do not change coded polypeptide.In some embodiments, nucleotide variants utilizes the degeneracy of genetic codon to replace generation by silence.Can for example optimize the codon expression (human mRNA's codon being changed into those codons of bacterial host such as Escherichia coli preference) that is used for specific host and produce the polynucleotides variant according to various reasons.

Polypeptide of the present invention can be to comprise the antibody of anti-human FZD acceptor or recombinant polypeptide, natural polypeptides or the synthetic polypeptide of its fragment.What should know in this area is, can change some amino acid sequence of the present invention and does not have obviously to influence the 26S Proteasome Structure and Function of albumen.Therefore, the present invention also comprises the polypeptide variants that demonstrates primary activity or comprises the antibody of anti-human FZD receptor protein or the polypeptide variants in the zone of its fragment.Such mutant comprises disappearance, insertion, inversion, repetition and various types of replacement.

Polypeptide and analog can further be modified to contain extra chemical part (the non-common part of albumen).These derivative moieties can improve solubility, biological half life or the absorbability of albumen.Described part can also reduce or eliminate any required side effect of albumen etc.The summary of relevant these parts can be at REMINGTON ' S PHARMACEUTICAL SCIENCES, and the 20th edition, Mack Publishing Co., Easton, PA (2000) finds.

The polypeptide of separation as herein described can be by suitable arbitrarily method preparation known in the art.Described method comprises from direct albumen synthetic method and even the dna sequence dna and these sequences are expressed in the suitable conversion host that makes up the peptide sequence of this separation of coding to be expressed.In some embodiments, by or the dna sequence dna of the interested wild-type protein of composite coding, utilize recombinant technique constructed dna sequence.Optionally, can come the functional analogue (referring to for example Zoeller etc., Proc.Nat ' l.Acad.Sci.USA 81:5662～5066 (1984) and U.S. Patent No. 4,588,585) of the described sequence of mutagenesis by direct mutagenesis so that this sequence to be provided.

In some embodiments, use oligonucleotide synthesizer to make up the dna sequence dna of the interested polypeptide of coding by chemical synthesis.Such oligonucleotides can design and select those codons in order to the host cell preference that generates interested recombinant polypeptide based on required amino acid sequence of polypeptide.The polynucleotide sequence of separation that can the application standard method comes the polypeptide of the interested separation of composite coding.For example, can use complete amino acid sequence to make up the gene (back-translated gene) of anti-translation.The DNA oligomer that can synthesize in addition, the nucleotide sequence that contains the specific polypeptide that is useful on the coding separation.For example, several short oligonucleotides that can synthesize the each several part of the required polypeptide that is used to encode connect then.Each bar oligonucleotides contains usually and is useful on 5 of complementary assembling ' or 3 ' protruding terminus.

In case assembling (by synthetic, site-directed mutagenesis or additive method), the polynucleotide sequence of specific polypeptide of interested separation of encoding just is inserted in the expression vector, and operably is connected to is applicable to the expression control sequenc that described albumen is expressed in required host.Can confirm the correctness of assembling by nucleotide sequencing, estriction map, the expression of biologically active polypeptide in suitable host.As known in the art, in order in the host, to obtain the high expression level of rotaring redyeing gene, this gene must operably be connected in selected expressive host, have function transcribe with the accurate translation control sequence on.

The DNA that uses recombinant expression carrier to increase and express coding cancer stem cell label polypeptide fusion.Recombinant expression carrier is reproducible DNA construct, it has the synthetic DNA fragment of coding cancer stem cell label polypeptide fusion or bioequivalence analog or the dna fragmentation in cDNA source, and described fusion or bioequivalence analog operably are connected to suitable the transcribing or translation adjusting element that comes from mammal, microbe, virus or insect genes.Transcriptional units generally comprises the assembly (1) of following material has regulating action in gene expression one or more gene elements, for example transcripting promoter or enhancer, (2) be transcribed into mRNA and structure or the coded sequence of translating into albumen, (3) suitable transcribe and beginning and terminator sequence, sequence described in detail are as mentioned opened in translation.Such regulating element can comprise the operon sequence that control is transcribed.Ability (generally providing by origin of replication) of duplicating among the host and the selection gene of being convenient to discern transformant can additionally be provided.When each DNA district is interrelated on function, they are operably connected.For example, when participating in the precursor expression of polypeptide secretion, the DNA of signal peptide sequence (secretion property targeting sequencing) operably is connected on the DNA of polypeptide at it; When the promoter control sequence was transcribed, promoter operably was connected on the coded sequence; Perhaps when the ribose binding site was positioned with the permission translation, the ribose binding site operably was connected on the coded sequence.Usually, operably connect to mean it is adjacent, and in the situation of secretion property targeting sequencing, mean adjacent and be positioned at and read frame.Plan is used in structural detail in the yeast expression system and comprises and can make host cell carry out the targeting sequencing of the exocytosis of the albumen translated.As selection, need not under the situation of targeting sequencing or transit sequence at express recombinant protein, described structural detail can comprise the terminal methionine residues of N-.This residue can be chosen wantonly from expressed recombinant protein excision subsequently so that final product to be provided.

Host's selection is depended in the selection of expression control sequenc and expression vector.Can adopt a lot of different expressive host and/or carrier combinations.The useful expression vector that is used for eucaryon host comprises the carrier that for example contains from the expression control sequenc of SV40, bovine papilloma virus, adenovirus and cytomegalovirus.The useful viral vectors that is used for bacterial host comprises known bacterial plasmid (Tathagata comprises pCR 1, pBR322, pMB9 and derivative thereof from the plasmid of Escherichia coli), host range plasmid (as M13) and thread single stranded DNA bacteriophage on a large scale.

Be applicable to that the host cell of expressing cancer stem cell label albumen is included in prokaryotes, yeast and insect or the senior eukaryotic cells under the suitable promoter control.Prokaryotes comprise gram negative organism or gram-positive organism, for example Escherichia coli or Bacillus bacteria (bacilli).Senior eukaryotic cells comprises the clone of having established in mammal source as mentioned below.Also can adopt cell free translation system.Clone that bacterium, fungi, yeast and mammalian cell host are suitable for and expression vector are described carriers such as Pouwels (Cloning Vectors:ALaboratory Manual, Elsevier, N.Y, 1985), introduce its relevant disclosure in this mode by reference.

Can advantageously adopt various mammals or insect cell culture systems to come express recombinant protein.Can be in mammalian cell express recombinant protein because such albumen generally can correctly fold, suitable modification and have complete function.The example of suitable mammalian host cell line comprises (the Cell 23:175 as Gluzman, 1981) the COS-7 clone of described MK cells and can express other clones of suitable carrier, for example L cell, C127,3T3, Chinese hamster ovary (CHO), HeLa and bhk cell system.Mammalian expression vector can comprise and is connected to the non-transcribed element for the treatment of expressing gene such as origin of replication, suitable promoter and enhancer and other 5 ' or 3 ' flank non-transcribed sequence and 5 ' or 3 ' non-translated sequence, as essential ribosome bind site, polyadenylation site, shear donor and acceptor site and transcription terminator.Be used for being summarized by Luckow and Summers (Bio/Technology 6:47 (1988)) at the rhabdovirus system of insect cell generation heterologous protein.

Can be according to suitable arbitrarily method purifying by transforming the albumen that the host produces.Such standard method comprises chromatography (for example ion-exchange chromatography, affinity chromatography and fractionation column chromatography), centrifugal process, difference solubility method or is used for any other standard methods of protein purification.Can will be connected on the albumen to be easy to carrying out purifying in conjunction with territory, influenza tunicle sequence (influenza coat sequence) and glutathione-S-transferase such as six histidines, maltose by suitable affinity column.The technology such as proteolysis, nulcear magnetic resonance (NMR) and x ray Laue method that can also be suitable for are carried out physics to the albumen that is separated and are characterized.

For example, for example Amicon or Millipore Pellicon ultra filtration unit concentrate from the supernatant that recombinant protein is secreted into the system in the culture medium albumen thickening filtration device that at first can use commercially available acquisition.After concentration step, concentrate can be added in the suitable purification media.As selection, can adopt anion exchange resin, for example outstanding matrix or base material with diethylamino ethyl (DEAE) group.Matrix can be acrylamide, agarose, glucan, cellulose or in protein purification other types commonly used.As selection, can adopt cation-exchange step.Suitable cation-exchanger comprises the various insoluble matrixs that contain sulfo group propyl group or carboxyl methyl.That at last, can adopt one or more RPLC steps of using hydrophobicity RPLC (RP-HPLC) medium (for example having the outstanding attached methyl or the silica gel of other aliphatic groups) is further purified cancer stem cell albumen-Fc composition.Also can adopt the various combinations of some or all aforementioned purification steps that the homology recombinant protein is provided.

Initial extraction that can be by from cell precipitation is carried out then one or morely concentrating, is saltoutd, water-based ion-exchange or size exclusion chromatography step are come the recombinant protein that generates in the separation of bacterial culture.High performance liquid chromatography (HPLC) can be used for carrying out last purification step.The used microbial cell of express recombinant protein can come broken by any conventional method, and described method comprises circulating freezing resistance method, sonicated method, mechanical crushing method or uses the method for lysis agent.

The invention provides the method for the growth of the oncogenicity cell that suppresses expression cancer stem cell label, this method is used the antibody of anti-cancer stem cell label as herein described.In some embodiments, the described method of growth that suppress to express the oncogenicity cell of cancer stem cell label is included in and external described cell is contacted with the antibody of anticancer stem cell labeling thing.For example, the immortalized cell line or the cancerous cell line of expressing the cancer stem cell label are cultivated in culture medium, this culture medium is added with the antibody of anti-expressed cancer stem cell label to suppress the growth of cell.In some embodiments, from for example isolating the tumour cell that contains tumor stem cell patient's samples such as biopsy sample, pleural effusion or blood sample, and it is cultivated in being added with at the culture medium of the antibody of cancer stem cell label to suppress the cell growth.

In some embodiments, the method for growth that suppress to express the oncogenicity cell of cancer stem cell label comprises described cell is contacted with antibody at the cancer stem cell label.In some embodiments, the oncogenicity cell is contacted with antibody at the cancer stem cell label.For example, the xenogenesis explant of expressing the cancer stem cell label is grown in the mouse (for example NOD/SCID mouse) of immunocompromised host, this mouse is applied antibody at the cancer stem cell label to suppress growth of tumor.In some embodiments, from for example isolating the cancer stem cell of expressing the cancer stem cell label patient's samples such as biopsy sample, pleural effusion or blood sample, and it is expelled in the mouse of immunocompromised host, then this mouse is used antibody at the cancer stem cell label to suppress the growth of tumour cell.In some embodiments, use antibody at the cancer stem cell label when the oncogenicity cell being imported in the animal or in the short time afterwards to prevent growth of tumor.In some embodiments, after having grown into certain size, uses in the oncogenicity cell antibody at the cancer stem cell label as therapeutant.

The present invention also provides the pharmaceutical composition of the antibody that comprises target cancer stem cell label.Described pharmaceutical composition can be used for suppressing the cancer of growth of tumour cell and treatment human patients.

By being prepared, the combination of antibody purification of the present invention and the acceptable charge material of pharmacy (for example carrier, excipient) is used to the preparation (Remington that preserves and use, The Science and Practiceof Pharmacy (the 20th edition) Mack Publishing, 2000).The acceptable charge material of suitable pharmacy includes but not limited to non-toxicity buffer, as phosphate, citrate and other organic acids; Such as salt such as sodium chloride; Antioxidant comprises ascorbic acid and methionine; Anticorrisive agent (stearyl dimethyl benzyl ammonium chloride for example; Chloor-hexaviet; Benzalkonium chloride; Benzethonium chloride; Phenol, butanols or phenmethylol; Alkyl paraben is as methyl p-hydroxybenzoate or propylparaben; Catechol; Resorcinol; Cyclohexanol; The 3-amylalcohol; And metacresol); Low molecular weight polypeptide (for example less than about 10 amino acid residues); Albumen is as seralbumin, gelatin or immunoglobulin (Ig); Hydrophilic polymer is as polyvinylpyrrolidone; Amino acid is as glycine, glutamine, asparagine, histidine, arginine or lysine; Carbohydrate is as monose, disaccharides, glucose, mannose or dextrin; Chelating agent is as EDTA; Sugar is as sucrose, maltitol, trehalose or D-sorbite; The salify counter ion counterionsl gegenions are as sodium; Metal complex (for example Zn-protein complex); And non-ionic surface active agent, as TWEEN or polyethylene glycol (PEG).

Pharmaceutical composition of the present invention can be used to be used for topical therapeutic or whole body therapeutic in any mode in numerous modes.Using can be local application (mucosal administration for example comprises that vagina is carried and rectum is carried), as the employing transdermal patch, and the using of ointment, lotion, cream, gel, drops, suppository, spray, liquor and pulvis; Pulmonary administration (for example by powder or aerocolloidal suction or be blown into, comprises and uses using of sprayer; In the tracheae, in the nose, epidermis and transdermal administration); Orally administered; Or stomach and intestine use outward, comprise that intravenous, artery are interior, subcutaneous, in the peritonaeum or intramuscular injection or infusion; Or encephalic (for example in the sheath or in the ventricles of the brain) is used.

Described treatment preparation can be a unit dosage form.Described preparation comprises and is used for oral, parenteral administration, rectal administration or the tablet of using by suction, pill, capsule, pulvis, granule, the solution in water or non-aqueous media or suspending agent or suppository.In such as solid composites such as tablets, the primary activity component can be mixed with pharmaceutical carrier.Conventional compressing tablet composition comprises cornstarch, lactose, sucrose, D-sorbite, talcum, stearic acid, dolomol, dicalcium phosphate or natural gum, and other diluents (for example water), contain preparation compositions before the solid of homogeneous mixture of The compounds of this invention or the acceptable salt of its non-toxicity pharmacy with formation.Then preparation compositions before the described solid is further divided into the unit dosage form of the above-mentioned type.Can carry out dressing to the tablet of described new compositions, pill etc., perhaps carry out the compound of other modes and the dosage form with long-acting advantage is provided.For example, described tablet or pill can comprise the inner composition by outer set subpackage quilt.And described two kinds of components can be separated by enteric layers, this enteric layers play anti-disintegration and make internal composition can be in good condition by stomach or delay to discharge.Have various materials can be used for described enteric layers or dressing, such material comprises the mixture of numerous polymeric acid and polymeric acid, and such material for example is lac, the pure and mild cellulose acetate of cetyl.

Pharmaceutical preparation comprise with liposome (Epstein, etc., 1985, Proc.Natl.Acad.Sci.USA82:3688; Hwang etc., 1980, Proc.Natl.Acad.Sci.USA 77:4030; With United States Patent (USP) 4,485,045 and 4,544,545) antibody of the present invention of complexing.Liposome with circulation timei of prolongation is disclosed in United States Patent (USP) 5,013, in 556.Some liposome can prepare by the anti-phase evaporation that use comprises the fluid composition of the phosphatidyl-ethanolamine (PEG-PE) that phosphatid ylcholine, cholesterol and PEG derive.Liposome can be extruded the liposome that obtains to have required diameter by the filter with definite aperture.

Described antibody can also wrap and be loaded in the micro-capsule.Such micro-capsule for example prepares by condensation technique or by interfacial polymerization, (for example for example be respectively at the colloid delivery system, liposome, albumin microsphere spheroid, micro emulsion, nano particle and nanocapsule) or big emulsion in hydroxy-methyl cellulose or gelatin-micro-capsule and poly-(methyl methacrylate) micro-capsule, as at Remington, described in The Science andPractice of Pharmacy (the 20th edition) the Mack Publishing (2000).

In addition, can prepare sustained release preparation.The suitable example of sustained release preparation comprises the semi permeability matrix of the solid hydrophobic polymer that contains described antibody, and described matrix is moulding product form (for example film or micro-capsule).The example of sustained-release matrix comprises polyester, hydrogel (for example poly-(methacrylic acid-2-hydroxyethyl ester) or poly-(vinyl alcohol)), polyactide (United States Patent (USP) 3,773,919), the copolymer of L-glutamic acid and 7-ethyl-L-glutamate, nondegradation ethane-acetic acid ethyenyl ester, degradability lactic acid-ethanol copolymer (as LUPRON DEPOT TM (the Injectable microspheres body of forming by lactic acid-ethanol copolymer and leuprorelin acetate)), Sucrose acetoisobutyrate and poly--D-(-)-3-hydroxybutyric acid.

In some embodiments, treatment comprises mixture co-administered of antibody of the present invention and chemotherapeutics or multiple different chemotherapeutics.The treatment that administration of antibodies is carried out can be before using chemotherapeutics, simultaneously or carry out afterwards.The chemotherapeutics that the present invention expects comprises chemical substance or medicine known in the art and can be commercially available, and for example Doxorubicin, 5 FU 5 fluorouracil, cytarabine (" Ara-C "), endoxan, plug are for group, busulfan, cytotoxin (Cytoxin), safe element, methotrexate (MTX), cis-platinum, melphalan, vincaleukoblastinum and carboplatin.Use altogether and use successively co-administered can comprising, described using altogether can be using of single pharmaceutical preparation, also can be to use using of preparation separately, described using successively can be with the using of random order, but normally uses in the regular hour so that all activating agents can be brought into play their biologically active simultaneously.The preparation of described chemotherapeutics and dosage regimen can use according to the specification of manufacturer, are perhaps rule of thumb determined by the skilled practitioner in this area.The preparation of chemotherapeutics and dosage be at Chemotherapy Service Ed., M.C.Perry, and Williams and Wilkins, Baltimore, Md. also has description in (1992).

In other execution mode, treatment comprises antibody of the present invention and radiotherapeutic co-administered.The treatment that administration of antibodies is carried out can be before using radiotherapy, simultaneously or carry out afterwards.Described radiotherapeutic any dosage regimen that can use the skilled practitioner in this area to determine.

In other execution mode, treatment can comprise other antibody co-administered of antibody of the present invention and anti-other tumor associated antigen, and described other antibody include but not limited to the antibody that combines with EGFR, HER2 and VEGF.And treatment can comprise using of one or more cell factors, and this treatment can follow the exenterate of cancer cell or treatment doctor to think necessary other treatment.

For treatment of diseases, the suitable dosage of antibody of the present invention depends on that the response of process, the disease of type, severity of disease and the disease of disease to be treated, the purpose of using this antibody are therapeutic purpose or preventative purpose, previous therapy, patient's clinical medical history etc., and all these is according to treatment doctor's judgement.Antibody can be used once, also can in continuing the serial therapy of a couple of days to several months, use, or until realizing recovery from illness or reaching subdue (for example tumor size reduces) of morbid state.Optimizing dosage can determine that the result calculates according to the drug accumulation in patient's body, and can change according to the difference of the relative effectivenes of indivedual antibody.The doctor in charge can easily determine optimal dosage, dosed administration method and repetition rate.Dosage is generally 0.01 μ g～100mg/kg body weight, and can every day, weekly, every month or use one or many every year.The doctor in charge can be according to residence time and the concentration repetition rate of estimating dosed administration of medicine in body fluid or tissue.

The invention provides and comprise antibody described herein and can be in order to implement the kit of methods described herein.In some embodiments, described kit comprises at least a antibody purification at the cancer stem cell label that is arranged in one or more containers.In some embodiments, described kit comprises and carries out detection assay all components essential and/or that be enough to carry out this detection assay, comprises all contrasts, the explanation of measuring and any necessary software that is used for interpretation of result and presents.Those skilled in the art should know easily that antibody disclosed by the invention can easily join in one of kit form of having established well known in the art.

Execution mode disclosed in this invention can be further detailed with reference to following examples, the method that described embodiment has described the preparation of antibody disclosed in this invention in detail and used antibody disclosed in this invention.One skilled in the art will appreciate that and under the situation that does not break away from the present disclosure scope, to carry out many changes material and method.Identical Reference numeral is all represented same or analogous object as much as possible in whole accompanying drawing.

Embodiment

Embodiment 1

The preparation of FZD antibody

Antigen preparation

With the recombinant polypeptide produced in fragments of the extracellular domain of human FZD acceptor is the antigen that is used for Antibody Preparation.Use the standard recombinant dna technology to separate the polynucleotides of the 1st～158 amino acid (SEQ ID NO:6) of the 1st～224 amino acid (SEQ ID NO:5) of the 1st～207 amino acid (SEQID NO:4) of the 1st～233 amino acid (SEQ ID NO.3) of the 1st～255 amino acid (SEQ ID NO:2) of the 1st～227 amino acid (SEQ ID NO:1) of the FZD10 that encodes, FZD7, FZD5, FZD6, FZD4 and FZD8.These polynucleotides with N end in the frame be connected to the human Fc label or histidine-tagged on, and be cloned in the transhipment plasmid vector, in insect cell, to carry out the expression of baculovirus mediation.The transfection of use standard, infection and cell cultivation program make the recombinant insect cell (O ' Reilley etc. that express corresponding FZD polypeptide, Baculovirusexpression vectors:A Laboratory Manual, Oxford:Oxford University Press (1994)).

The cutting of using cutting forecasting software SignalP 3.0 simulations to delineate the endogenous burst of human FZD acceptor, but cut point may be because amino acid coupling or directed and different in the actual body.The prediction cutting of FZD10 is between the 20th～21 amino acid, so the FZD10 antigen protein comprises about the 21st amino acid～227th amino acid.The prediction cutting of FZD7 is between the 31st～32 amino acid, so the FZD7 antigen protein comprises about the 32nd amino acid～255th amino acid.The prediction cutting of FZD5 is between the 26th～27 amino acid, so the FZD5 antigen protein comprises about the 27th amino acid～233rd amino acid.The prediction cutting of FZD6 is between the 17th～18 amino acid, so the FZD6 antigen protein comprises about the 18th amino acid～207th amino acid.The prediction cutting of FZD4 is between the 39th～40 amino acid, so the FZD4 antigen protein comprises about the 40th amino acid～224th amino acid.The prediction cutting of FZD8 is between the 27th～28 amino acid, so the FZD8 antigen protein comprises about the 28th amino acid～158th amino acid.

Use albumin A and Ni++ chelating agent affinity chromatography from insect cell condition medium (conditioned medium), to be purified into antigen protein.Utilize PBS (pH=7) that 1mg/ml is dialysed, is concentrated into to purified antigen protein, and at immunity filtration sterilization in the preparation.

Immunity

With purified FZD10, FZD7, FZD5, FZD6, FZD4 and FZD8 antigen protein (antibody-solutions; Mountain View is CA) according to standard techniques immune mouse (n=3).Just exempt from the back about 70 days, adopting ELISA and facs analysis to come the antigen recognizing (as detailed below) of the blood of the individual mouse of examination.Select two the highest animals of antibody titer to carry out last antigen and strengthen (antigen boost), isolate splenocyte afterwards and be used for the hybridoma preparation.Hybridoma in 96 orifice plates by 1 cells/well bed board, by at the ELISA of antigen protein and the supernatant of each plate hole of facs analysis examination.Select the high hybridoma of several antibody titers and in the bottled culture of static state, enlarge to cultivate.Use albumin A or Protein G agarose chromatography to be purified into antibody from the hybridoma supernatant.The monoclonal anti body and function FACS of purifying tests once more and carries out the isotype analysis to select IgG and IgM antibody.

Epitope mapping

In order to identify the antibody that to discern the specific region that comprises the FZD extracellular domain that is rich in domain cysteine, carry out epitope mapping.Use the standard recombinant dna technology, preparation comprises the mammal expression plasmid carrier of CMV promoter in the polynucleotides upstream of the fragment of coding FZD extracellular domain.Utilize transient transfection then, with expression of recombinant proteins in the mammalian cell of being cultivated.After the transfection 24～48 hours, harvesting, and on the SDS-PAGE acrylamide gel isolated cell crack protein so that the antibody of the mouse of the FZD antigen immune that is used for hanging oneself carries out the Western trace.Can further analyze the competitiveness combination of the antibody of the ligand binding domain of discerning FZD with Wnt albumen by ELISA.

In order to identify the defined epitope of discerning for the antibody of anti-FZD in the extracellular domain, used the SPOT system (Sigma Genosys, The Woodlands, TX).Adopt the SPOT synthetic technology, synthesize, and it is covalently bound on the cellulose membrane by amino acid a series of ten residue linear peptides overlapping and that cover whole FZD extracellular domain.Described film is at room temperature with sealing buffer solution precincubation 8 hours, and spends the night 4 ℃ of hybridization with antibody.Wash film then, (Amersham Bioscience, Piscataway, two temperature resistances NJ) educate, wash and make it visual with the signal that the contains 3-amino-9-ethyl carbazole liquid that develops the color with coupling horseradish peroxidase (HRP).Determine by the defined epitope of antibody recognition thus.

Facs analysis

In order to select monoclonal antibody, adopted the FACs analysis by the identification n cell surface FZD albumen of hybridoma clone generation.The HEK293 cell separately with the coding corresponding FZD (FZD10) full length cDNA clone the expression vector transfection or with this expression vector with the expression GFP carrier (FZD7, FZD5, FZD6, FZD4 and FZD8) cotransfection.In the situation of FZD10, FZD7, FZD6 and FZD4 expression vector, Flag epi-position label imports at amino terminal, make can the proof tape label the expression of FZD acceptor on cell surface.After the transfection 24～48 hours, with cell harvesting in suspension and on ice with anti-FZD antibody, FLAG antibody, immune serum (being used for the FZD5 express cell) or the contrast IgG incubation that combines in order to detection background antibody.Washed cell has the anti-mouse two anti-detections one of fluorescent chromophore to resist with coupling.Cell by FACS sorting institute mark then is with the anti-FZD antibody of the cell surface expression of identifying the corresponding FZD acceptor of specific recognition.Identify identification FZD10 (Figure 1A); FZD7 (Figure 1B); FZD5 (Fig. 1 C); FZD6 (Fig. 1 D); The antibody of FZD4 (Fig. 1 E) and FZD8 (Fig. 1 F).Similarly, use the outer part of born of the same parents of combination as the antigen that is used for mouse immune, generate the antibody of identification FZD1, FZD2, FZD3 and FZD9.

Chimeric antibody

After identifying the monoclonal antibody of specific recognition FZD acceptor, modify these antibody to overcome human anti-mouse antibodies (HAMA) immune response when using rodent animal antibody as therapeutic agent., and this variable region is connected in the frame respectively on IgG 1 heavy chain and κ constant region of light chain of feeding in the animal expression carrier from the variable region that hybridoma is isolated the heavy chain and the light chain of selected monoclonal antibody by RT-PCR.As selection, use such as human Ig expression vector such as TCAE 5.3 grades, this carrier contains IgG 1 heavy chain and κ constant region of light chain gene (Preston etc., 1998, the Infection﹠amp on same plasmid; Immunity 66:4137～42).The expression vector of coding chimeric heavy chain and light chain then cotransfection to Chinese hamster ovary (CHO) cell with the generation chimeric antibody.The immunoreactivity and the compatibility that compare chimeric antibody and muroid parental antibody by ELISA and FACS.

Humanized antibody

Because the chimeric antibody therapeutant still often has antigenicity, and produces human anti-chimeric antibody (HACA) immune response, the chimeric antibody that therefore can resist the FZD acceptor further carries out humanization.In order to produce humanized antibody, use recombinant DNA technology, the kind that the key position (may comprise hypervariable sequence or complementary determining region (CDR) from 3 weak points) of the specificity of antibody decision motif and/or the required skeleton district, CDR district of correctly locating aforesaid heavy chain of antibody and light chain variable territory is built into human heavy chain and light chain antibody gene respectively is in the dna sequence dna, be cloned into then in the mammalian expression vector, to be used at expressing cho cell.The immunoreactivity and the compatibility that compare humanized antibody and parent's chimeric antibody by ELISA and FACS.In addition, can use the site-directed mutagenesis of variable region or specificity that humanized antibody is optimized in high density mutagenesis, compatibility etc.

Human antibodies

In some embodiments, use display technique of bacteriophage to separate the human antibodies of the extracellular domain of specific recognition FZD acceptor.The phage displaying antibody library that examination contains the human antibodies variable region that is shown as strand Fv or is shown as the fab territory is to the recognition reaction of specific, the high-affinity of above-mentioned FZD receptor antigen.Then the variable region antibody sequence that is identified is changed into again the form of the Ig expression vector that contains IgG 1 heavy chain and κ light chain, to be used at the Chinese hamster ovary celI express human antibody.

Embodiment 2

Discern the preparation of a plurality of FZD family members' antibody

For the human FZD acceptor of target more than one, a plurality of FZD receptor family of preparation specific recognition member's antibody.The soluble protein that comprises the ligand binding domain of any among the terminal Frizzled of N or Fri, FZD4, FZD5 and the FZD8 that is fused to human Fc in conjunction with and prevent to transmit the signal transduction (Fig. 2) of the Wnt part (comprising Wnt1, Wnt2, Wnn3, Wnt3a and Wnt7b) of all categories of signal by the mechanism that comprises the stabilisation that beta-catenin is white.Specifically, FZD Fc soluble recepter incubation under the situation that different Wnt parts (comprising Wnt1, Wnt2, Wnt3, Wnt3a and Wnt7b) exist of continuing to increase of HEK 293 cells that stable transfection had a 8xTCF luciferase reporter gene and its amount.FZD4Fc, FZD5Fc and FZD8Fc fusion have suppressed the ligand-mediated Wnt signal transduction (Fig. 2) by all 5 Wnt.Therefore, in some embodiments, generated the antibody of two above acceptors in specific recognition FZD4, FZD5 and the FZD8 acceptor.

In some embodiments, the method according to describing in detail among the embodiment 1 makes antibody with one or more FZD receptor antigen immune mouses.Test the antibody of prepared anti-each FZD acceptor and the cross reactivity of other FZD acceptors then.Identify and test specific recognition FZD2 and FZD6 according to the method for hereinafter describing in detail then; FZD7 and FZD10; FZD4 and FZD5; FZD4 and FZD8; FZD5 and FZD8; And the antibody of FZD4, FZD5 and FZD8 prevents the ability of growth of tumour cell.

In some embodiments, use phage display library to identify a plurality of FZD family members' of identification antibody.The bacteriophage of using the zone of height homology in the terminal extracellular domain of N of human FZD acceptor to come to show in the examination library antigen binding domain of two above FZD acceptors of specific recognition.For example, the homologous region of human FZD acceptor is expressed as FZD-Fc albumen, and recombinant protein is coated on the suitable surface with 10 μ g/mL.Then by the human phage library of two-wheeled enrichment elutriation (pan) (referring to for example Griffiths etc., EMBO J.12:715～34).Afterwards, reclaim the gene of coding for antigens from bacteriophage, and, make up complete human antibody molecule, to be used for expression in appropriate host cell system by this antigen binding domain is connected with constant region in conjunction with the territory.In some embodiments, according to the method for hereinafter describing in detail, identify and test identification FZD2 and FZD6; FZD7 and FZD10; FZD4 and FZD5; FZD4 and FZD8; FZD5 and FZD8; And the antibody of FZD4, FZD5 and FZD8 prevents the ability of growth of tumour cell.

In some embodiments, developed by transduce the antibody of antagonism Wnt signal transduction pathway by standard Wnt signal transduction pathway interference signal.For example, identified the antibody of the two or more FZD among specific recognition FZD4, FZD5 and the FZD8 by the phage display method.In some embodiments, developed by activating the antibody that antagonism non-standard Wnt signal transduction pathway comes antagonism Wnt signal transduction pathway.For example, identified the antibody of specific recognition FZD6 and FZD2 by the phage display method.

Embodiment 3

External test in order to the antibody of estimating anti-FZD acceptor

Present embodiment has been described representational external test, and this external test is used to test antibody on cell proliferation, pathway activation and the Cytotoxic activity of the anti-FZD acceptor that is generated.

Proliferation assay

Use Taqman to analyze the expressed FZD acceptor of quantitative different carcinoma clone.Cultivate in the minitype plate at 96 hole tissues, by 10 ⁴The density of individual cells/well is carried out bed board and is allowed it launch 24 hours being accredited as the clone of expressing the FZD acceptor.In containing the fresh DMEM of 2%FCS, again cell was cultivated 12 hours subsequently, will be resisted this moment FZD antibody and control antibodies under the situation that has 10 μ mol/LBrdU, to be added in the culture medium.Behind the BrdU mark, remove culture medium and with cell in ethanol, fixing 30 minutes under the room temperature, and with anti-BrdU monoclonal antibody (BMG 6H8 clone, the Fab fragment) reaction of peroxidase coupling 90 minutes.In containing the solution of tetramethyl benzidine, make substrate colour developing, after 15 minutes with the 1mol/L H of 25 μ l ₂SO ₄Stop.Use the filter of 450nm to read plate instrument (UV Microplate Reader with automatic ELISA; Bio-RadLaboratories, Richmond CA) measures color reaction.All tests are all carried out 3 times and are repeated.Determine relatively that with control antibodies anti-FZD antibody suppresses the ability of cell proliferation.

Pathway activation is measured

In some embodiments, in the ability of the activation of the external antibody blocking Wnt signal transduction pathway of having determined anti-FZD acceptor.For example, with cultivate in the DMEM that is supplemented with antibiotic and 10%FCS the HEK293 cell with: 1) in order to activate the Wnt7B and the FZD10 expression vector of Wnt signal transduction pathway; 2) in order to the TCF that contains 3 or 8 copies in firefly luciferase reporter gene upstream of specifications of surveys Wnt signal transduction level TCF/Luc wild type or saltant reporter gene carrier (Gazit etc., 1999, Oncogene 18:5959～66) in conjunction with the territory; With 3) be used for sea pansy (Renilla) luciferase reporter gene (Promega as the internal contrast of transfection efficiency; Madison WI) carries out cotransfection.In cell culture medium, add anti-FZD10 and control antibodies then.After the transfection 48 hours, use two luciferase assay kit (Promega; Madison WI) measures luciferase level, wherein the firefly luciferase activity is standardized as the renilla luciferase activity.Repeat 3 independently tests with 3 times.Determine that thus FZD10 antibody suppresses the ability of Wnt pathway activation.

In some embodiments, in the external ability of having determined the antibody interferes with Wnt part combination of anti-human FZD10 acceptor.For example, with Wnt responsiveness luciferase reporter gene TOPFLASH (Upstate Group LLC; Article No. is 21-170) and Wnt3A expression vector transfection HEK293 cell to activate the endogenous Wnt signal transduction in the transfected cell.To contain the solubility FZD5Fc that is connected to the extracellular domain (the 1st～233 amino acid) of IgG 1Fc in the frame of FZD5 and add in the culture medium of transfectional cell, to combine (Fig. 3 with Wnt3A separately; The HT culture medium, Wnt3A, right side bar) or under the situation of each antibody that has the anti-FZD5 that is generated, combine (Fig. 1 C with Wnt3A; 44M1-32).Measure according to uciferase activity, under the situation that does not have FZD5 antibody, FZD5Fc has eliminated the Wnt signal transduction in the transfectional cell fully, but disturbs the adding of the FZD5 antibody that FZD5Fc combines with the part of Wnt3A to recover Wnt signal transduction (Fig. 3).

In some embodiments, external determined specificity in conjunction with the antibody of human FZD acceptor more than two (for example FZD2 and FZD6) by for example ability of the standard Wnt signal transduction of antagonism FZD mediation as the activator of non-standard Wnt signal transduction.For example, use Wnt responsiveness luciferase reporter gene TOPFLASH transfection HEK 293 cells.After the transfection 48 hours, with the antibody of the human FZD2 of specific recognition and FZD6 or isotype contrast with adding in the culture medium such as Wnt parts such as Wnt-3a.Then by measuring the activation that uciferase activity is determined existence or do not had standard Wnt signal transduction under the situation of antibody.

The complement-dependent cytotoxin is measured

In some embodiments, use to express the cancerous cell line of FZD acceptor or from patient's sample, be separated to as xenograft the cancer stem cell in the immunocompromised host mouse (as institute's detailed description hereinafter) that goes down to posterity, measurement is by the antibody-mediated CDC (CDC) of anti-FZD acceptor.By 10 ⁶Individual cell/ml is suspended in cell in the RPMI1640 culture medium that is supplemented with antibiotic and 5%FBS of 200 μ l.With the mixing of suspension cell and 200 μ l serum or hot inactivated serum or anti-FZD acceptor or control antibodies, establish 3 repetitions then.With cell mixture in 37 ℃ at 5%CO ₂Middle incubation 1～4 hour.Collect handled cell then, and it is resuspended in the annexin V that is diluted in 100 μ l FITC-marks in the culture medium and room temperature incubation 10 minutes.Add the 100 μ l propidium iodide solution (25 μ g/ml) be diluted among the HBSS, and incubation 5 minutes at room temperature.Collecting cell is suspended in it in culture medium, and uses flow cytometry.The flow cytometry of FITC staining cell provides total cell count, and the propidium iodide of dead cell absorbs (as the percentage of total cell number) and is used to measure cell death (comparing with control antibodies with hot inactivated serum) in the presence of serum and anti-FZD antibody.Determine the ability of the antibody-mediated CDC of anti-FZD thus.

Antibody dependent cellular born of the same parents poison is measured

In some embodiments, use to express the cancerous cell line of FZD acceptor or from patient's sample, be separated to as the go down to posterity cancer stem cell (as institute's detailed description hereinafter) of immunocompromised host mouse of xenograft, measurement is by the antibody-mediated antibody dependent cellular born of the same parents poison (ADCC) of anti-FZD acceptor.By 10 ⁶Individual cell/ml is suspended in being supplemented with not the containing in phenol red RPMI 1640 culture mediums of antibiotic and 5%FBS of 200 μ l with cell.From the heparinize peripheral blood, isolate peripheral blood lymphocytes (PBMC) by Ficoll-Paque density gradient centrifugation, to be used as the effector cell.Then under the situation that the antibody of at least a FZD acceptor or control antibodies exist, by 25: 1, the E/T ratio of 10: 1 and 5: 1 was blended in 96 orifice plates with target cell (T) and PBMC effector cell (E).Contrast incubation target cell and incubation effector cell separately separately under the situation that is included in the antibody existence.With cell mixture in 37 ℃ at 5%CO ₂Middle incubation 1～6 hour.Use colorimetric method (CytoTox96Non-radioactive Cytotoxicity Assay then; Promega; Madison WI) measures the lactic dehydrogenase discharged (LDH, the stable kytoplasm enzyme that discharges during a kind of lysis).Reading the plate instrument with standard 96 orifice plates is collected in the absorption data at 490nm place and carries out background correction.Calculate the percentage of specific cytotoxic according to following formula: % cytotoxicity=100 * (the spontaneous LDH of the spontaneous LDH release-target of test LDH release-effector discharges)/(the spontaneous LDH of the maximum LDH release-target of target discharges).Determine the ability of the antibody-mediated antibody dependent cellular born of the same parents poison of anti-FZD acceptor thus.

Embodiment 4

Use the antibody of anti-FZD acceptor to prevent tumor growth in the body

Present embodiment has been described the application in the tumor growth of antibody in preventing xenograft models of anti-FZD acceptor.In some embodiments, preparation from patient's sample (for example solid tumor biopsy samples or pleural effusion) as go down to posterity tumour cell in the mouse of xenograft, in the experimental animal that goes down to posterity again.Under aseptic condition, take out tumor tissues, be cut into small pieces, shred fully, and obtain single-cell suspension liquid by enzymic digestion and Mechanical Crushing with sterile razor blade.Specifically, pleural effusion cell or gained tumor mass are mixed in culture medium with ultrapure clostridiopetidase A III (200～250 unit clostridiopetidase As/mL) and 37 ℃ of incubations 3～4 hours, during inhaled up and down by the 10mL pipettor in per 15～20 minutes and to beat.By the cell that 45 μ M nylon net filters are digested,, and use the HBSS washed twice with the RPMI/20%FBS washing.Then the tumour cell that dissociates is subcutaneously injected in the mammary fat pad of NOD/SCID mouse to cause tumor growth.

In some embodiments, before being expelled to experimental animal, at first elect the tumour cell branch that is dissociated as oncogenicity cell and non-tumorigenic cell according to the cell surface marker thing.Specifically, with Hepes buffered saline solution (HBSS) washing that contains 2% hot deactivation cow's serum (HICS) by the above-mentioned tumour cell that dissociates 2 times, and with 10 ⁶Individual cell/100 μ l suspend again.Add antibody,, use the HBSS/2%HICS washed twice then in incubation cell on ice 20 minutes.Antibody comprise anti-ESA (Biomeda, Foster City, CA), anti-CD44, anti-CD24 and kind be that the anti-CD2 of label, anti-CD3, anti-CD10, anti-CD16, anti-CD18, anti-CD31, anti-CD 64 and anti-CD140b (are referred to as Lin; PharMingen, San Jose, CA).Antibody directly is coupled on the fluorescent dye the cell of expressing these labels is carried out the positive or negative selection.By selecting anti-H2Kd+ cell to get rid of mouse cell, and by using viability dyestuff (viability dye) 7AAD to get rid of dead cell.(Becton Dickinson, Franklin Lakes NJ) go up the enforcement flow cytometry at FACSVantage.Utilize lateral angle scatter diagram and forward angle light scatter figure to get rid of cell mass.Then ESA+, CD44+, CD24-/low, the Lin-oncogenicity cell that is separated is subcutaneously injected in the NOD/SCID mouse to cause tumor growth.

In some embodiments, analyzed the ability that anti-FZD6 and anti-FZD5 antibody slow down the growth of UM-C4 colon tumor cell.The UM-C4 cell (10,000 cell/animals) that dissociates is subcutaneously injected into the flank district of the NOD/SCID mouse in 6～8 ages in week.Behind the tumor cell injection two days,, animal is carried out intraperitoneal injection (i.p.) with the anti-FZD6 of 10mg/kg or the antibody of anti-FZD5 acceptor by weekly twice.Monitor growth of tumor weekly until detecting growth, afterwards, in the time in 8 weeks altogether, weekly a tumor growth is carried out twice measurement.Compare with the contrast of injection PBS, can significantly slow down growth of tumor (Fig. 4) with anti-FZD6 antibody 23M2 and anti-FZD5 antibody 44M13 treatment animal.

Embodiment 5

Use the antibody of anti-FZD acceptor that tumour is carried out interior therapeutic

Application in the cancer of the antibody that present embodiment has been described anti-FZD acceptor in the treatment xenograft models.In some embodiments, preparation from patient's sample (for example solid tumor biopsy samples or pleural effusion) as go down to posterity tumour cell in the mouse of xenograft, in the experimental animal that goes down to posterity again.Take out tumor tissues, be cut into small pieces, shred fully, and obtain single-cell suspension liquid by enzymic digestion and Mechanical Crushing with sterile razor blade.Then the tumour cell that dissociates is subcutaneously injected into the NOD/SCID mouse mammary fat pad (being used for tumor of breast), be expelled to flank (being used for non-tumor of breast) to cause tumor growth.As selection, separate ESA+, CD44+, CD24-/low, Lin-oncogenicity tumour cell and inject by method described in detail above.

Behind the injection tumour cell, the tumor growth of monitoring animal.In case the average-size of tumour reaches about 150mm～200mm, promptly begin to carry out antibody treatment.In 6 weeks altogether,,, make the antibody or the control antibodies of every animals received 100 μ gFZD acceptors by intraperitoneal injection by 2～5 times weekly.In these 6 all processes, estimate twice of tumor size weekly.The antibody of determining the FZD acceptor thus prevent further tumor growth or reduce the ability of tumor size (with control antibodies relatively).

At the antibody treatment terminal point, the results tumour is further to analyze.In some embodiments, a part of tumour by the immunofluorescence technique analysis to estimate antibody penetrating and tumor response to tumour.Each tumour aquatic foods in liquid nitrogen of handling mouse available from the antibody treatment mouse and the control antibodies of anti-FZD acceptor of a part freeze, and are embedded among the O.C.T., and are cut into the 10 μ m section that is positioned on the slide with cryostat.In some embodiments, the part of each tumour is cut into the 10 μ m section that is positioned on the slide with formalin fixed, FFPE and with ultramicrotome.To described section carry out the back fixing and with the antibody incubation of the specific recognition institute injection of antibodies of chromophore mark, to detect the antibody or the control antibodies of the anti-FZD acceptor that exists in the tumor biopsy sample.In addition, can use following antibody or method to estimate antibody treatment to for example angiogenesis, the effect of tumor growth and tumour form: for example detect for example anti-VE catenin of antibody (CD144) of the cell type that different tumours and tumour raise or anti-PECAM-1 (CD31) antibody to detect vascular endothelial cell, anti-smooth muscle α-Ji Dongdanbai antibody is to detect vascular smooth muscle cell, anti-Ki67 antibody is to detect proliferative cell, TUNEL measures to detect dying cell, the white antibody of anti-beta-catenin is with detection Wnt signal transduction, and anti-born of the same parents' internal area (ICD) Notch fragment antibody is to detect the Notch signal transduction.

In some embodiments, also estimated the effect of the antibody treatment of anti-FZD acceptor to tumour cell gene expression.From each tumour of a part, extract total RNA, and carry out quantitative RT-PCR with this total RNA available from FZD antibody treatment mouse and control antibodies processing mouse.The cancer stem cell label that had before identified (for example CD44) of analyzing the component (comprising that for example Wnt1 and beta-catenin are white) of FZD acceptor, Wnt signal transduction pathway and being added is with respect to the expression as the house-keeping gene GAPDH of internal contrast.The variation of tumour cell gene expression when determining the antibody treatment of FZD acceptor thus.

In addition, estimated the effect of the antibody treatment of anti-FZD acceptor to the existence of the cancer stem cell in the tumour.To be cut into small pieces from the tumor sample of FZD antibody treatment mouse and control antibodies processing mouse, shred fully with sterile razor blade, and obtain single-cell suspension liquid by enzymic digestion and Mechanical Crushing.Press above method described in detail then,, adopt facs analysis, analyze the existence of the oncogenicity cancer stem cell of the tumour cell that is dissociated according to ESA+, CD44+, CD24-/low, Lin-superficial cell marker representation.

Then can be according to the oncogenicity of institute's isolated cells after ESA+, CD44+, the anti-FZD antibody treatment of CD24-/low, Lin-expression evaluation.ESA+, the CD44+, CD24-/low, the Lin-cancer stem cell that separate from FZD antibody treatment mouse and control antibodies processing mouse are subcutaneously injected in the mammary fat pad of NOD/SCID mouse again.According to forming the required quantity of being injected cell of reliable tumour (consistent tumorformation), determine the oncogenicity of cancer stem cell then.

Embodiment 6

Use the Antybody therapy human cancer of anti-FZD acceptor

Present embodiment has been described the method for use in order to the antibodies for treating cancer of the anti-FZD acceptor of target tumor, and described tumour comprises cancer stem cell and/or the tumour cell that has wherein detected the FZD expression of receptor.At first, can determine the existence of cancer stem cell marker representation from the tumor biopsy sample.The biopsy samples taking-up tumour cell of under aseptic condition, suffering from the patient of cancer from diagnosis.In some embodiments, biopsy sample aquatic foods are frozen in liquid nitrogen, be embedded among the O.C.T. and utilize cryostat to be cut into to be positioned at 10 μ m section on the slide.In some embodiments, the biopsy sample with formalin fixed, FFPE, and is cut into the 10 μ m section that is positioned on the slide with ultramicrotome.With the antibody incubation of section, to detect protein expression with anti-FZD acceptor.

Can also determine the existence of cancer stem cell.The biopsy sample is cut into small pieces, shreds fully, and pair cell carries out enzymic digestion and Mechanical Crushing, to obtain single-cell suspension liquid with sterile razor blade.Then with the tumour cell and anti-ESA, anti-CD44, anti-CD24, anti-Lin and the anti-FZD antibody incubation that are dissociated, detecting cancer stem cell, and determine the existence of ESA+, CD44+, CD24-/low, Lin-, FZD+ tumor stem cell by flow cytometry described in detail above.

Express the cancer patient of FZD acceptor after diagnosing with its tumour of Antybody therapy of anti-FZD acceptor.In some embodiments, the humanization of the above-mentioned anti-FZD acceptor that makes of purifying or human monoclonal antibody and be used for injection with the preparation of the acceptable charge material of suitable pharmacy.In some embodiments, at least 10 weeks, press every month at least once, use FZD antibody that the patient is treated.In some embodiments, at least about 14 weeks,, use FZD antibody that the patient is treated by weekly at least once.Each amount of application of antibody should be a pharmacy effective dose.In some embodiments, use the antibody of the anti-FZD of about 2mg/ml～about 100mg/ml.In some embodiments, use the antibody of the anti-FZD of about 5mg/ml～about 40mg/ml.Before described antibody can or use the chemotherapy regimen of one or more chemotherapeutics (for example oxaliplatin, fluorouracil, folinic acid or chain assistant star) at the conventional planning therapeutic scheme, simultaneously or use afterwards.For example according to tumor regression, new tumour a situation arises reduces, tumour antigen express reduce, cancer stem cell quantity reduces or other means of assess disease prognosis, the patient is monitored, to determine whether such treatment has produced antitumor reaction.

Consider that from the explanation and the practice of invention disclosed herein other execution modes of the present invention are conspicuous to those skilled in the art.Described explanation and embodiment should be considered as only being the example purpose, and actual range of the present invention and spirit are provided by appended claims.

Sequence table

SEQ?ID?NO：1

The terminal extracellular domain of FZD10N-

MQRPGPRLWLVLQVMGSCAAISSMDMERPGDGKCQPIEIPMCKDIGYNMTRMPNLMGHENQREAAIQLH

EFAPLVEYGCHGHLRFFLCSLYAPMCTEQVSTPIPACRVMCEQARLKCSPIMEQFNFKWPDSLDCRKLPNK

NDPNYLCMEAPNNGSDEPTRGSGLFPPLFRPQRPHSAQEHPLKDGGPGRGGCDNPGKFHVEKSASCAPLC

TPGVDVYWSREDKRFA

SEQ?ID?NO：2

The terminal extracellular domain of FZD7N-

MRDPGAAAPLSSLGLCALVLALLGALSAGAGAQPYHGEKGISVPDHGFCQPISIPLCTDIAYNQTILPNLLGH

TNQEDAGLEVHQFYPLVKVQCSPELRFFLCSMYAPVCTVLDQAIPPCRSLCERARQGCEALMNKFGFQWPE

RLRCENFPVHGAGEICVGQNTSDGSGGPGGGPTAYPTAPYLPDLPFTALPPGASDGRGRPAFPFSCPRQLKV

PPYLGYRFLGERDCGAPCEPGRANGLMYFKEEERRFARL

SEQ?ID?NO：3

The terminal extracellular domain of FZD5N-

MARPDPSAPPSLLLLLLAQLVGRAAAASKAPVCQEITVPMCRGIGYNLTHMPNQFNHDTQDEAGLEVHQF

WPLVEIQCSPDLRFFLCSMYTPICLPDYHKPLPPCRSVCERAKAGCSPLMRQYGFAWPFRMSCDRLPVLGR

DAEVLCMDYNRSEATTAPPRPFPAKPTLPGPPGAPASGGECPAGGPFVCKCREPFVPILKESHPLYNKVRTG

QVPNCAVPCYQPSFSADERT

SEQ?ID?NO：4

The terminal extracellular domain of FZD6N-

MEMFTFLLTCIFLPLLRGHSLFTCEPITVPRCMKMAYNMTFFPNLMGHYDQSIAAVEMEHFLPLANLECSPN

IETFLCKAFVPTCIEQIHVVPPCRKLCEKVYSDCKKLIDTFGIRWPEELECDRLQYCDETVPVTFDPHTEFLGP

QKKTEQVQRDIGFWCPRHLKTSGGQGYKFLGIDQCAPPCPNMYFKSDELEFAKSFIGTVSI

SEQ?ID?NO：5

The terminal extracellular domain of FZD4N-

MLAMAWRGAGPSVPGAPGGVGLSLGLLLQLLLLLGPARGFGDEEERRCDPIRISMCQNLGYNVTKMPNLV

GHELQTDAELQLTTFTPLIQYGCSSQLQFFLCSVYVPMCTEKINIPIGPCGGMCLSVKRRCEPVLKEFGFAWP

ESLNCSKFPPQNDHNHMCMEGPGDEEVPLPHKTPIQPGEECHSVGTNSDQYIWVKRSLNCVLKCGYDAGL

YSRSAKEFTDI

SEQ?ID?NO：6

The FZD8Fri territory

MEWGYLLEVTSLLAALALLQRSSGAAAASAKELACQEITVPLCKGIGYNYTYMPNQFNHDTQDEAGLEVH

QFWPLVEIQCSPDLKFFLCSMYTPICLEDYKKPLPPCRSVCERAKAGCAPLMRQYGFAWPDRMRCDRLPEQ

GNPDTLCMDYNRTDLTT

Claims (18)

1. isolated antibody, described antibody specificity be in conjunction with the extracellular domain of two or more human FZD acceptors, and suppress the growth of tumour cell.

2. antibody as claimed in claim 1, wherein said antibody specificity is in conjunction with the extracellular domain of human FZD2 and FZD6.

3. antibody as claimed in claim 1, wherein said antibody specificity is in conjunction with the extracellular domain of human FZD7 and FZD10.

4. antibody as claimed in claim 1, wherein said antibody specificity is in conjunction with the extracellular domain of human FZD4 and FZD5.

5. antibody as claimed in claim 1, wherein said antibody specificity is in conjunction with the extracellular domain of human FZD4 and FZD8.

6. antibody as claimed in claim 1, wherein said antibody specificity is in conjunction with the extracellular domain of human FZD5 and FZD8.

7. isolated antibody, described antibody specificity is in conjunction with the extracellular domain of human FZD acceptor more than three kinds.

8. antibody as claimed in claim 7, wherein said antibody specificity is in conjunction with the extracellular domain of human FZD4, FZD5 and FZD8.

9. as each described antibody of claim 1～8, wherein said antibody is monoclonal antibody.

10. antibody as claimed in claim 9, wherein said antibody is chimeric antibody.

11. antibody as claimed in claim 9, wherein said antibody is humanized antibody.

12. antibody as claimed in claim 9, wherein said antibody is human antibodies.

13. a pharmaceutical composition, this pharmaceutical composition comprise each described antibody of claim 1～12 and the acceptable charge material of pharmacy.

14. a hybridoma, described hybridoma produces each described antibody of claim 1～12.

15. comprising, a treatment method for cancer, described method use each described antibody of claim 1～12 or the described pharmaceutical composition of claim 13 that suppresses the growth of tumour cell effective dose.

16. method as claimed in claim 15, wherein said antibody and the coupling mutually of cytotoxicity part.

17. further comprising, method as claimed in claim 15, described method use at least a additional therapeutic agent that realizes conjoint therapy that is applicable to.

18. method as claimed in claim 15, wherein said tumour cell are selected from tumor of breast, colorectum tumour, lung neoplasm, pancreatic neoplasm, tumor of prostate and head and neck neoplasm.

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