CN101583624B - Compositions and methods for diagnosing and treating cancer - Google Patents

Abstract

An isolated antibody that specifically binds to an extracellular domain of human DLL4 and affects growth of a tumor comprising cancer stem cells is described. Also described is a method of treating cancer comprising administering a therapeutically effective amount of an anti-DLL4 antibody.

Description

Be used to diagnose and treat the composition and use thereof of cancer

Technical field

The present invention relates to oncology and novel compsns 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 solid tumor of anticancer stem cell labeling thing.

Background technology

Cancer is one of main causes of death in developed world, only in the U.S., just has every year the million people of surpassing to be diagnosed as the cancer patients, and has 500,000 people dead.In general, just there is being more than 1 people can suffer from the cancer of certain 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 carcinoma and prostate cancer wherein-account for half the (Jemal etc., 2003, Cancer J.Clin.53:5～26) above all new cases.

Breast cancer is a modal cancer among the women, according to estimates, has 12% women theirs the risk of suffering from this disease to be arranged in life.Although because the improvement of doing sth. in advance and treating of inspection, mortality ratio is reduced, breast cancer remains middle aged women's main causes of death, and the transitivity breast cancer still is the disease that can not cure.The patient that major part suffers from the transitivity breast cancer has only one or two tract to be affected when manifesting, but along with advancing of disease, is got involved in a plurality of positions.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 new 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 time manifest and is about 3 years.

At present breast cancer is diagnosed with by stages method and comprised that tumor nodule shifts (TNM) system; This system depends on (the American Joint Committee on Cancer:AJCC Cancer Staging Manual. Philadelphia that exists that existence and the far-end of tumour in tumor size, the lymphoglandula shift; Pa.:Lippincott-Raven Publishers; The 5th edition, 1997, the 171～180 pages; Harris, JR: " 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 property owing to have the tumour of similar histopathology outward appearance, therefore this method has critical limitations.At last, can use the mensuration of cell surface marker thing that some tumor type is divided into subclass.For example, an existence that factor is ERs (ER) of in the prognosis of breast cancer and treatment, being considered is because the positive breast cancer of ER-is replied making such as hormonotherapies such as tamoxifen or arimedexs than the negative tumour of ER-usually more easily.Although these analyses are useful, can only partly predict the clinical behavior of breast tumor, and in the breast cancer that current diagnostic tool can't detect and current therapy can't be treated, also have a large amount of phenotype variety.

Prostate cancer is the most common cancer among the male sex of developed world, in all New Development cases of cancers of the U.S., estimates to account for 33%, is the frequency time high cause of death (Jemal etc., 2003, CA CancerJ.Clin.53:5～26).Because the introducing of PSA (PSA) blood testing, the early 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 or metastatic disease (Muthuramalingam etc., 2004, Clin.Oncol.16:505～16) the most at last above 50%.

At present, radical prostatectomy and radiotherapy are that most local tumor of prostate provides curative therapy.Yet for late case, selectable regimen is very limited.For metastatic disease, the male sex hormone blocking-up of using luteinizing hormone-releasing hormone (LRH) (LHRH) excitomotor perhaps to be used in combination luteinizing hormone-releasing hormone (LRH) (LHRH) excitomotor and androgen antagonist separately is standard care.Although male sex hormone is farthest blocked, disease condition is development still almost, and major part develops into the androgen independence disease.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 carcinoma 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 carcinomas, 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 carcinoma is through circumferential growth and local the intrusion, then spread, stride through lymph diffusion, blood are capable that peritonaeum spreads and peripheral nerve spreads and invades in other position.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 position of the capable diffusion of blood comprises bone, kidney, suprarenal gland and brain.

The existing system by stages of colorectal carcinoma is passed having or not that degree and the tubercle of intestines wall get involved based on tumour.This system is by stages defined by 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 possibly pass colon or rectal wall; Duke C level disease comprises the intestines wall intrusion that has regional nodus lymphoideus transferring rate of any degree.Though excision is very effective for early stage colorectal carcinoma; In Duke A level patient, 95% curative ratio is provided; But this ratio drops to 75% in Duke B level patient, and the existence of positive lymph nodes indication recurrence possibility in 5 years is 60% in Duke C level disease.Chemotherapeutic postoperative course of treatment Duke C level patient's treatment can be reduced to 40%～50% with recurrence rate, this is these patients' a nursing standard at present.

Lung cancer is modal in the world cancer, is the third-largest cancer of the most often diagnosing 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 lung cancer that smoking causes accounts for all lung cancer is 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 lung cancer (SCLC) and nonsmall-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 type lung cancer root is to be close to the lamella that does not have cytoplasmic minicell in big central airway and histology composition.SCLC has more aggressive than NSCLC, and growth fast, shift early.NSCLC accounts for 80%～85% of all cases, and further is 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, therefore has only 6～12 months meta survival time after the diagnosis, and total 5 annual survival rates only have 5%～10%.Though operation provides the best chance of curing, have only few part patients with lung cancer to be fit to operation, major part depends on chemotherapy and radiotherapy.Although attempted controlling the opportunity and the dose intensity of these therapies, 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 multiple cancer show as the solid tumor of being made up of the heterology cell mass.For example, breast cancer is the mixtinite of cancer cells and normal cell (comprising a matter (matrix) cell, inflammatory cell and endotheliocyte).Several models of cancer are that the existence of this heterology provides different explanation.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 heterology stems from occurent sudden change in environmental factors and the cancer cells, thereby forms various carcinogenic cells crowd.This model is based on such viewpoint, that is, all colonies of tumour cell all have oncogenic potential (Pandis etc., 1998, Genes, Chromosomes&Cancer 12:122～129 to a certain degree; Kuukasjrvi etc., 1997, Cancer Res.57:1597～1604; Bonsing etc., 1993, Cancer 71:382～391; Bonsing etc., 2000, Genes Chromosomes&Cancer 82:173～183; Beerman H etc., 1991, Cytometry 12:147～54; Aubele M and Werner M, 1999, Analyt.Cell.Path.19:53; Shen L etc., 2000, CancerRes.60:3884).

A kind of substituting model of the solid tumor cell heterology that is observed stems from the influence of stem cell to tumor development.According to this model, cancer results from that the control healthy tissues is grown and the imbalance (Beachy etc., 2004, Nature 432:324) of the mechanism kept.In normal animal development process, the overwhelming majority or all cells of tissues 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 carry out the imparity cell fission to generate the filial generation that one or more multiplication potentialities and/or developmental potentiality descend; (3) thus can carry out symmetry cell fission self-regeneration or the oneself keeps.Being embodied as somatocyte regenerated best research example through differentiation of stem cells is the hematopoiesis system, wherein grows immature precursor (hemopoietic stem cell and progenitor cell) and responds molecular signal and form various hemocytes and lymphoidocyte type.Other cell (cell that comprises internal organ, latex dust system and skin) replenishes from what the microcommunity stem cell of each self-organization obtained 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 through symmetry and imparity fission process subsequently.In this stem cell model; Solid tumor contains obvious difference and limited (even possibly be rare) has the normally inferior group of cell of " stem cell " character, and reason is their extensive most of oncocytes that also forms other solid tumor stem cell (self-regeneration) effectively and form the shortage tumorigenesis potential of solid tumor of breeding.In fact, the sudden change in the long-lived population of stem cells can cause the formation of cancer stem cell, and formed cancer stem cell becomes tumor growth and the basis of keeping, and the existence of cancer stem cell causes the failure of current treat-ment.

The stem cell property of cancer at first obtains disclosing (Lapidot etc., 1994, Nature 17:645～8) in leukemia (acute myeloid leukemia (AML)).Recently, proved that pernicious human mammary tumour and colon tumor also have little and visibly different cancer stem cell crowd similarly, it can be formed tumour by enrichment in immunodeficient mouse.Find that in breast tumor, the tumorigenicity cell of ESA+, CD44+, CD24-/low, the enrichment of Lin-cell mass institute is 50 times (Al-Hajj etc., 2003, Proc.Nat ' l Acad.Sci.100:3983～8) of unassorted tumour cell.Similar is; Found that ESA+, CD44+ subgroup in the colorectum tumour only comprise the tumorigenicity cell; Can further enrichment colorectal carcinoma stem cell (CoCSC) (Dalerba etc. and in this subgroup (profile), add CD166; 2007, Proc.Nat ' l Acad.Sci.104:10158～63).Be expected to separate the feasible crucial biopathways that can study as the basis of the tumorigenicity in these cells of ability of tumorigenicity cancer cells, therefore be expected to develop better diagnostic assay method and remedy for the cancer patients.The purpose that this present invention just was directed against.

Summary of the invention

The invention provides a kind of antibody; Said antibodies specific combines human δ appearance part 4 (DLL4) epi-position; Said epi-position is combined to form by human DLL4N-end region (SEQ ID NO:27) and human DSL territory (SEQ ID NO:26), and wherein said antibody influences tumor growth.The present invention also provides a kind of pharmaceutical composition, and this pharmaceutical composition comprises disclosed antibody of the present invention and medicinal charge material (vehicle).The present invention also provides a kind of treatment method for cancer, and said method comprises the disclosed DLL4 antibody of the present invention of administering therapeutic significant quantity.

Other target of the present invention and advantage, a part will provide in the explanation subsequently, and a part is conspicuous according to said explanation, the perhaps acquistion through practice of the present invention.Target of the present invention and advantage will realize and reach by key element of specifically noting in the appended claims and combination.Should be understood that above general introduction and following detailed description all 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 and constitute the part of this specification sheets shows several embodiments of the present invention, its with said explanation in order to explain principle of the present invention.In this specification sheets and appended claims, " " of singulative, " a kind of " or " said " only if context offers some clarification in addition, otherwise comprise the referent of plural form.

Description of drawings

Fig. 1: the antibody 21M18 of anti-DLL4 combines with the proteic specificity of n cell surface DLL4.Will be with the antibody incubation of HEK 293 cells of the DLL4 of total length and GFP cotransfection and anti-DLL4 and through the FACS sorting.As between expressing through DLL4 antibodies and GFP linear relationship disclosed, the antibody 21M14 of anti-DLL4 and 21M18 show the specificity combination to the cell of expressing DLL4.

Fig. 2: the interaction of human DLL4 of DLL4 antibody blocking and Notch acceptor.A) in the presence of DLL4 or control antibodies, will express HEK 293 cells and Notch-Fc or the contrast Fc albumen incubation of DLL4.High fluorescent shows, combining of Notch and DLL4 in the presence of the antibody 21M12 of control antibodies (line 2) and anti-DLL4 (line 5), occur.Low fluorescence intensity shows, Notch and DLL4 do not interact under the situation that does not have Notch (line 1), and Notch and DLL4 interaction are damaged in the presence of the antibody 21M18 of anti-DLL4 (line 3) and 21M14 (line 4).B) will express HEK 293 cells and the mankind or the muroid DLL4Fc incubation of Notch 1.Combine and with facs analysis through fluorescently-labeled anti-Fc antibody test, high fluorescent shows combining between the cell of DLL4 and expression Notch 1.21M18 has blocked combining of human DLL4 (grey square) and Notch acceptor, but does not block combining of muroid DLL4 (black is circular) and Notch acceptor.

Fig. 3: the epitope mapping of the antibody of anti-DLL4.The fusion rotein of nested deletion of extracellular domain that A) will have a human DLL4 in ELISA measures with antibody 21M14 and the 21M18 incubation of anti-DLL4.In the presence of the fusion rotein that contains amino acid/11～154, fail to detect the combination (aa 1-96, the white post of band stain that are higher than background; Aa 1-154, the black post of band white point).Combine (aa 1-217, horizontal stripe post between the antibody that on the contrary, can detect anti-DLL4 and the amino acid/11 that contains DLL4～217 all fusion roteins of (comprising the DSL territory); Aa 1-251, slanted bar line post; Aa 1-283, the shade terminal; Aa 1-323, the gray columns of band white point).B) the Werstern trace shows human DLL4 (h-DLL4) C-terminal deletion albumen and muroid-human DLL4 chimeric fusion protein (anti-hFc; The top) expression.The DLL4 fusion rotein comprises one or more in the territory 1～6, and wherein territory 1 and 2 is-terminal amino acids 1～154; Territory 3 is the DSL territories from amino acid/11 55～217; Territory 4,5 and 6 each illustrated EGF territory among the C naturally.Have only the amino acid/11 of existence-217 (hDLL4 territory 1-3), antibody 21M18 just discerns h-DLL4 albumen.Opposite with human protein, the fusion rotein that contains muroid DLL4 (m-DLL4) amino acid/11-217 (territory 1-3) is not by 21M18 identification (1-3:h-DLL4 territory, m-DLL4 territory 4-6).And in the presence of muroid territory 3, the fusion rotein that comprises h-DLL4 amino acid/11-154 (territory 1-2) is by 21M18 identification (1-2:mDLL4 territory, h-DLL4 territory 3-6).C) shown the schematic diagram of the binding data of B.The top has shown the domain structure of DLL4, and the DLL4 fusion rotein is listed and schematically illustrated in the left side, and wherein human protein is represented with light gray, and the mouse proteinoid is represented with Dark grey.With "+" and "-" expression 21M18 and every kind of segmental combination of DLL4.D) elisa assay 21M18 and human residue combining by the corresponding substituted DLL4 protein fragments of muroid residue at select location.To in the amino acid position 68,69 and 71 (replacement Xie Ansuan, Xie Ansuan and proline(Pro)) or at amino acid/11 42 and 144 (replacement Methionin and L-Ala) substituted DLL4 protein fragments, the combination of 21M18 demonstration weakens.E) human residue the combining of elisa assay antibody 21M18 and 21M21 and select location in the DSL territory by the corresponding substituted DLL4 protein fragments of muroid residue.To (replacement Threonine and Serine) contains the human DLL4 protein fragments of aminoacid replacement in amino acid position 161 and 162, antibody 21M21 shows the combination that is weakened.21M21 does not influence the function (see figure 6) of DLL4 in signal transduction is measured, and this shows that not all and DSL district bonded antibody all influences the function of DLL4.

Fig. 4: the sequence alignment of variable region of heavy chain.A) shown parent's muroid 21M18 antibody sequence (m-21M18-Vh, top), human framework sequence (h-EST-framework, centre) and the humanization 21M18 weight chain variabl area sequence (21M18-H7, bottom) of expressing, conservative amino acid residues is marked with black shade.Three CDR of institute's mark show, remain with parent's muroid sequence in the humanization 21M18 antibody.In 21M18H7 and 21M18H9, the cysteine residues of Karbate position among the CDR2 (Kabat position) 52a has been changed into Serine and Xie Ansuan residue respectively and has not been lost the specificity combination to DLL4.Replacement in the framework region shown in the 4A is numbered as 1-6, the Karbate position 16,20,27,28,38,48 in the corresponding Vh chain.B) shown that parent's muroid 21M18 antibody sequence (m-21M18-Vh, top), the human kind are Vh sequence (h-kind system-Vh, centre) and humanization 21M18 weight chain variabl area sequence (21M18-H2, bottom), conservative amino acid residues is marked with black shade.Three CDR of institute's mark show, remain with parent's muroid sequence in the humanization 21M18 antibody.In 21M18H7 and 21M18H9, the cysteine residues of Karbate position 52a has been changed to Serine and Xie Ansuan residue respectively and has not been lost the specificity combination to DLL4 among the CDR2.Five muroid residues that kept in the variable framework region with all heavy chain variants are numbered 1～5 in their corresponding Karbate positions 20,28,38,48 and 69.

Fig. 5: the sequence alignment of variable region of light chain.Shown that parent's muroid 21M18 antibody sequence (m-21M18-Vk, top), the human kind are sequence (h-kind system-Vk, bottom) and humanization 21M18 light chain variable region sequence (21M18-L2, centre), conservative amino acid residues is marked with black shade.Three CDR of institute's mark show, remain with parent's muroid sequence among the humanized antibody 21M18.Two muroid residues that kept in the variable framework region are numbered as 1～2 in their corresponding Karbate positions 22 and 36.

Fig. 6: DLL4 antibody blocking Notch signal transduction.Under the situation of the antibody that has or do not exist anti-DLL4, cotransfection there be the HeLa cell and the DLL4-Fc albumen incubation of Hes1-Luc reporter gene and Renilla luciferase reporter gene carrier.Luciferase level reduces and to show that antibody 21M14 and 21M18 make the DLL4Notch approach can not activation.

Fig. 7: DLL4 antibody is regulated the expression of Notch target gene in colon tumor.A) the C8 colon tumor of antibody 21M18 or PBS (contrast) through anti-DLL4 being handled separates and passes through the expression of quantitative RT-PCR mensuration HES1 and ATOH-1.The relative genetic expression of comparing with the cell of control treatment (y axle) shows that the antibody treatment with anti-DLL4 has reduced the expression of HES1 and increased the expression of ATOH-1.B) shown HES1 in the mouse pedigree disappearance OMP-C11 colon tumor cell colony to the relative expression of ATOH1 than (y axle).HES1/ATOH1 expression ratio to cross the C11 colony (3T3+DLL4) that the 3T3 cell express DLL4 covers is higher than (3T3) that covers with the 3T3 cell or (contrast) colon cell that covers without cell.Through eliminating increasing of this HES1/ATOH1 expression ratio with 10 μ g/mL21M18 antibody (21M18) or 5 μ M-Secretase inhibitors DBZ (5 μ M GSI) incubation.

Fig. 8: DLL4 antibody slows down growth of tumor.With dissociated UM-C4 injection cell NOD/SCID mouse, and with the antibody 21M18 (n=5) of anti-DLL4 or PBS (n=10) treatment.Compare with the contrast (black) of injection PBS, the treatment of carrying out with antibody 21M18 (rhombus) slowed down growth of tumor since the 23rd day, observed this slowing down at the 48th day and can reach 54%.

Fig. 9: the quantity that has reduced the tumour cell of proliferation in vivo with the DLL4 Antybody therapy.To separating with the antibody 21M18 of anti-DLL4 or the C8 colon tumor of contrast Ab (antibody) treatment.The immunocytochemical assay that carries out with the antibody of anti-Ki67 shows, compares with contrast, and in the tumour of 21M18 treatment, the quantity of proliferative cell reduces.

Figure 10: the combination therapy of DLL4 antibody and Fluracil (5-FU) has slowed down growth of tumor.With dissociated UM-C4 injection cell NOD/SCID mouse, and antibody and/or the PBS with anti-DLL4 treats under the situation that has or do not exist 5-Fu.A) injection tumour cell after 46 days, with 5-FU (triangle, solid line) or 21M18 antibody (rhombus; Dotted lines) separately treatment compare and with the contrast (square of injection PBS; Solid line) compare, the combination therapy of antibody 21M18 and 5-FU (circle, dotted line) has slowed down tumor growth to a greater degree.The y axle is with mm ³Indicated gross tumor volume.B) the measurement of tumor figure that obtained by animal individual in the 46th day.Each point is represented an animal.Compare with contrast, the treatment of antibody 21M18 or 5-FU has slowed down tumour size (mm separately ³).And then, have additive effect with the combination therapy of antibody 21M18 and 5-FU, the tumour size is reduced to 1/5 of contrast size.

Figure 11: the combination therapy of the antibody of DLL4 antibody and anti-EGFR has slowed down growth of tumor.With dissociated UM-C4 injection cell NOD/SCDD mouse, and antibody and/or the PBS with anti-DLL4 treats under the situation of the antibody that has or do not exist anti-EGFR.Show the 46th day measurement of tumor figure by the animal individual acquisition.Each point is represented an animal.Compare with contrast, the treatment of the antibody of antibody 21M18 or anti-EGFR has slowed down tumour size (mm separately ³).And then the combination therapy of the antibody of antibody 21M18 and anti-EGFR has additive effect, and the tumour size is reduced to 1/5 of contrast size.

Figure 12: the mAb of anti-DLL4 (monoclonal antibody) 21M18 and irinotecan synergy and suppressed the growth of colon tumor.With dissociated C8 injection cell NOD/SCID mouse, and antibody and/or the control antibodies with anti-DLL4 treated under the situation that has or do not exist irinotecan.A) compare with the animal (black) of contrast treatment, reduced gross tumor volume (y axle mm separately with muroid 21M18 antibody (circle) or the independent treatment of irinotecan (triangle) ³).Yet the combination therapy of 21M18 and irinotecan (inverted triangle) has synergistic effect, can behind injection cell, reach 55 days and thoroughly eliminate tumor growth.B) than control antibodies (black) or control antibodies+irinotecan (triangle), humanization 21M18 (h21M18) has the similar efficient of combination therapy (triangle) with irinotecan (irtcn) with muroid 21M18 (m21M18) with the combination therapy (circle) of irinotecan (irtcn).

Figure 13: the 21M18 of anti-DLL4 and the combination therapy of irinotecan have prevented the regrowth of colon tumor.With dissociated C8 injection cell NOD/SCID mouse, and the uniting of antibody 21M18 of irinotecan or irinotecan and anti-DLL4 to treat (n=10, every group).A) with irinotecan independent treatment having slowed down colon tumor growth, but after the 56th day (* arrow) stopped treatment, quilts all except two examples were treated the tumour continued growth in the animal.B) opposite, the growth of colon tumor has been eliminated in the combination therapy of the antibody 21M18 of irinotecan and anti-DLL4, and this elimination is treated at all 10 to be run through therapeutic process in the animal and stopped to treat 5 weeks afterwards on the 56th day.Every line is represented the growth curve of an animal individual.

Figure 14: the 21M18 of anti-DLL4 and the combination therapy of irinotecan are more effective to the independent therapy for treating of rejection ratio of the growth of the colon tumor set up.With dissociated C8 injection cell NOD/SCID mouse, and antibody and/or the control antibodies with anti-DLL4 treated under the situation that has or do not exist irinotecan.Compare with the animal (black) of contrast treatment, separately with 21M18 antibody (rhombus) or irinotecan (triangle) the treatment reduction separately gross tumor volume (y axle, mm ³).Yet 21M18 adds the combination therapy (inverted triangle) of irinotecan and treats more effective separately to the rejection ratio 21M18 or the irinotecan of tumor growth.

Figure 15: show the minimizing of tumorigenicity cell quantity with the tumour of the Antybody therapy of anti-DLL4.In the experiment shown in Figure 14; After the associating (associating) of antibody 21M18 and irinotecan that adds antibody 21M18 or the anti-DLL4 of control antibodies, independent anti-DLL4 through control antibodies, irinotecan is treated; Tumour cell with the dosage that successively decreases comes the low mouse of injecting immune (n=10, every group).A) in the 81st day tumour become to live result of ratio.Gross tumor volume (mm with each treatment group ³) to the human tumor cell's that injected quantity: 900,300,100 and 50 mappings.Below the gross tumor volume figure of every kind of cell dosage; Record animal that having of every kind of tumour cell dosage can detect tumour with respect to 10 numbers of being injected animal; Through the tumour cell of contrast treatment on a left side (filled circles), through the tumour cell of the antibody 21M18 of anti-DLL4 treatment on a left side several second (hollow squares), through the tumour cell of irinotecan on the right side several second (solid triangle), the tumour cell of combination therapy is in right (open circles).B) calculated the 81st day stem cell frequency.Account for ratio (y axle) mapping through the tumour cell of treating with cancer stem cell: will compare through tumour cell (left side) that contrasts treatment and the tumour cell of treating through anti-DLL4 (several second an of left side), a tumour cell through irinotecan (right several second) and the tumour cell (right side) of combination therapy, fiducial interval is 95%.Compare with control group, the group of treating through anti-DLL4 has statistically significant difference (*); Organize (* *) separately with irinotecan with the independent group of contrast (*) and compare, combined group all has significant difference.

Figure 16: the 21M18 of anti-DLL4 and the combination therapy of irinotecan have delayed the recurrence of tumour.With the mouse of dissociated C8 injection cell immunocompromised, and to about 150mm ³The tumour of setting up carry out 32 days combination therapy by a definite date: the antibody 21M18 of irinotecan (45mg/kg, all administrations 2 times)+anti-DLL4 or irinotecan (45mg/kg, all administrations 2 times)+control antibodies stop irinotecan afterwards.Continue treatment with control antibodies or 21M18.Compare with contrast (circle), the tumor recurrence in gross tumor volume (y axle) in the animal (triangle) of 21M18 treatment is postponed.

Figure 17: the 21M18 of anti-DLL4 and the combination therapy of irinotecan have delayed the recurrence of tumour.Shown the animal individual in the experiment shown in Figure 16.Shown that irinotecan stops total gross tumor volume of every back 47 days animal (y axle).

Figure 18: uniting of the 21M18 of anti-DLL4 and anti-VEGF slowed down growth of tumor.Implant the C17 tumour cell, and after 2 days with associating (circle, the dotted lines) begin treatment of control antibodies (black, solid line), 21M18 (triangle, dotted line), anti-VEGF (rhombus, solid line) or two kinds of antibody.Every kind of antibody is pressed 10mg/kg, administration biweekly, every group of 10 animals.21M18 and anti-VEGF have all subtracted slow growth of tumor, and the independent every kind of antibody of Combined Ration is more effective.

Embodiment

Term " antibody " promptly can combine the immunoglobulin molecules such as the targets such as combination of albumen, polypeptide, peptide, glucide, polynucleotide, lipid or aforementioned substances through at least one antigen recognition site identification and the specificity in this immunoglobulin molecules variable region in order to refer to such immunoglobulin molecules.In some embodiments; Antibody of the present invention comprises antagonist antibodies, and this antagonist antibodies combines with cancer stem cell affinity tag protein-specific and disturbs the proteic downstream signal of for example part combination, receptor dimerizationization, the proteic expression of cancer stem cell affinity tag and/or cancer stem cell affinity tag to transduce.In some embodiments, disclosed antibody comprises agonist antibody, this agonist antibody combine with cancer stem cell affinity tag protein-specific and promote that part for example combines, receptor dimerizationization and/or the signal transduction that carries out through cancer stem cell affinity tag albumen.In some embodiments, disclosed antibody does not disturb or promotes the proteic biological activity of cancer stem cell affinity tag, still discerns through for example antibody internalization and/or by immune system and suppresses tumor growth.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) two mutants, multi-specificity antibody (like the bi-specific antibody that is generated by at least two complete antibodies), chimeric antibody, humanized antibody, human antibodies, comprises fusion rotein and other any immunoglobulin molecules through modifying that comprises antigen recognition site of the antigen determining part of antibody, as long as said antibody demonstrates required BA.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 Tegeline, is referred to as α, δ, ε, γ and μ respectively according to their characteristic of CH.That different classes of Tegeline has is different, known subunit structure and 3-d modelling.Antibody can be expose or with such as other molecule couplings such as toxin, ri.

Be meant the part of complete antibody at this used term " antibody fragment ", also refer to the antigen determining variable region of complete antibody.The instance 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 complete antigen identification and antigen binding site; It can be double-stranded; Wherein a heavy chain and a variable region of light chain form non-covalent dimer; Also can be strand (scFv), thereby wherein a heavy chain and a variable region of light chain make said two chains unite with similar dimeric structure through flexible peptide linker is covalently bound.In this configuration, the complementary determining region of each variable region (CDR) thus interact and to limit the dimeric antigen-binding specificity of this Fv.As selection, single variable region (perhaps Fv's is half the) can be used to identification and conjugated antigen, but avidity is lower usually.

" monoclonal antibody " used herein but be meant high specific identification and combine the homologous antibody crowd of former determinant of monoclonal antibody or epi-position.This is opposite with polyclonal antibody, and polyclonal antibody generally includes the different antibodies of anti-different antigenic determinants.Term " monoclonal antibody " is contained complete sum full length monoclonal antibodies and antibody fragment (for example Fab, Fab ', F (ab ') 2 and Fv), strand (scFv) two mutants, is comprised fusion rotein and other any immunoglobulin molecules through modifying that comprises antigen recognition site of antibody moiety.In addition, " monoclonal antibody " refers to that also the said antibody that makes through any kind of mode, said mode include but not limited to that hybridoma, phage are selected, recombinant expressed and transgenic animal.

Term used herein " humanized antibody " is meant as the specific immunoglobulin chain, gomphosis immunoglobulin or its segmental various forms of non-humans (for example muroid) antibody that contain minimum non-human sequence.Usually; Humanized antibody is such human immunoglobulin, is wherein replaced by the residue from non-human species's (for example mouse, rat, rabbit, hamster) the CDR with required specificity, avidity and ability from the residue of the complementarity-determining region (CDR) in the antigen determining district (or hypervariable region) of the variable region of one or more antibody chains.In some cases, the residue of the variable chains framework region (FR) of human immunoglobulin is replaced from the corresponding residue in the antibody of the required specificity of having of non-human species, avidity and ability.Through the extra residue in the variable framework region and/or by the replacement of metathetical non-human residue, humanized antibody is all right further to be modified, to improve and optimization antibodies specific, avidity and/or ability.Usually; Humanized antibody comprises the almost whole of at least one (being generally two or three or four) variable region; Said variable region comprises all or nearly all CDR district corresponding with the non-human Tegeline, 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 USP 5,225, description arranged in 539 in order to the instance of the method that generates humanized antibody.

Term used herein " human antibodies " is meant the antibody that produced by the mankind or through having of utilizing that any technology known in the art the makes antibody with the corresponding aminoacid sequence of antibody that is produced by the mankind.This definition of human antibodies comprises the fragment of antibody complete or total length, 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 be from the heavy chain light chain of antibody to fitting together with different antigenic determinants zone, make two kinds of different epi-positions or two kinds of different antigens to be discerned and combine by the formed tetramer.

Term " chimeric antibody " is meant such antibody, and wherein the aminoacid sequence of immunoglobulin molecules is derived from two or more species.Usually; The two variable region of light chain and heavy chain is corresponding to the variable region with required specificity, avidity and ability of the antibody of species that come from Mammals (for example mouse, rat, rabbit etc.); And the sequence homology in constant region and the antibody that comes from another kind of species (be generally human), to avoid in these another kind species, causing immunne response.

The interchangeable in this article use of term " epi-position " or " antigenic determinant ", and refer to antigenic can identification and specificity bonded part by antibodies specific.When antigen was polypeptide, epi-position both can be formed by adjacent amino acid, also can by through proteic three grades folding and and the non-adjacent amino acid of putting form.The epi-position that is formed by adjacent amino acid still is able to keep when protein denaturation usually, and can be lost usually when the protein denaturation 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 space conformation.

Suppress the specificity bonded of reference antibody and common antigen through the Tegeline of wherein being tested and measure to confirm the competition between the antibody.Known have polytype competitive binding assay method; The for example direct or indirect radioimmunoassay of solid phase (RIA), the direct or indirect enzyme process immunoassay of solid phase (EIA), sandwich competition assay (seeing Stahli etc., Methods in Enzymology 9:242-253 (1983)); The direct vitamin H of solid phase-avidin EIA (seeing Kirkland etc., J.Immunol.137:3614-3619 (1986)); The direct marker determination of solid phase, the direct mark sandwich assay of solid phase (seeing Harlow and Lane, " Antibodies, A Laboratory Manual, " Cold Spring HarborPress (1988)); Utilize the direct mark RIA of solid phase (seeing Morel etc., Molec.Immunol.25 (1): 7-15 (1988)) of I-125 mark; The direct vitamin H of solid phase-avidin EIA (Cheung etc., Virology 176:546-552 (1990)); With direct mark RIA (Moldenhauer etc., Scand.J.Immunol.32:77-82 (1990)).Usually, said mensuration relates to using and is incorporated into the purified antigen of solid surface or contains this antigenic cell, unlabelled test Tegeline or through the reference Tegeline of mark.Through confirming in the presence of said test Tegeline, to be incorporated into the mark of solid surface or the amount of cell is measured competitive inhibition.The excessive existence of common said test Tegeline.The antibody of identifying through competition assay (competitive antibody) thus comprise antibody that institute's bonded epi-position is identical with reference antibody and antibody that the epi-position of institute's bonded epi-position and reference antibodies fully hinders near generation space.Usually, when competitive antibody is excessive when existing, it will suppress reference antibody and combine at least 50% or 75% with the specificity of common antigen.

Antibody " selective binding " or " specificity combination " be meant antibody more continually, more promptly, more enduringly, avidity more doughtily or some above combination and with epi-position reaction or associating (with the reaction of the substituting material that comprises incoherent albumen or unite and compare)." selective binding " perhaps " specificity combination " for example is meant antibody with at least about 0.1mM, but more commonly at least about the K of 1 μ M _DWith protein binding." selective binding " perhaps " specificity combination " is meant that sometimes antibody is sometimes with the K at least about 0.1 μ M _DPerhaps better K _DWith protein binding, other the time be meant with K at least about 0.01 μ M _DPerhaps better K _DWith protein binding.Because the specificity that the sequence identity between the homologous protein in the different plant species, specificity combine to comprise the proteic antibody of cancer stem cell affinity tag in the more than a kind of species of identification combines.

Term used herein " non-specific binding " and " background combination " 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 (; Antibody usually and protein binding, rather than combine with specific structure such as epi-position).

Term " separated " or " purified " are meant that material does not contain the composition that in native state, accompanies usually with this material basically or in fact.Purity and uniformity adopt usually to be confirmed such as analysis chemical technologies such as polyacrylamide gel electrophoresis or performance liquid chromatography.The present invention disclosed in preparation the albumen (for example antibody) or the nucleic acid of existing main kind passed through sufficient purifying.Particularly, separated nucleic acid separates with ORFs, natural both sides and the coding albumen different with the albumen of this coded by said gene that is positioned at this gene of this ORFs.Separated antibody separates with other NIg, and separates with other Tegeline with different antigens binding specificity.Said 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.

" cancer " is meant or describes the mammiferous physiological status that cell mass wherein has the dysregulated cellular growth characteristic to term used herein with " cancer ".The example of cancer includes but not limited to cancer, lymphoma, blastoma, sarcoma and white blood disease.The example more specifically of such cancer comprises squamous cell carcinoma; Small cell lung cancer; Nonsmall-cell lung cancer; The gland cancer of lung; The squamous cell carcinoma of lung; Peritoneal cancer; Hepatocellular carcinoma (hepatocellular cancer); Gastrointestinal cancer; Carcinoma of the pancreas; Glioblastoma; Cervical cancer; Ovarian cancer; Liver cancer; Bladder cancer; Hepatoma; Breast cancer; Colorectal carcinoma; Colorectal carcinoma; Carcinoma of endometrium or uterus carcinoma; Salivary-gland carcinoma; Kidney; Liver cancer; Prostate cancer; Carcinoma vulvae; Thyroid carcinoma; Liver cancer (hepatic carcinoma) and various types of Head and Neck cancer.

Term " proliferative disorders " 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 tissue block that is caused by cell transition growth or propagation, its be optimum (non-carcinous) or pernicious (carcinous), comprise precancerous lesion.

" transfer " used herein is meant cancer diffusion or transfer to other zone of health and the process of similar cancerous lesion is arranged in this new position development from position originally." transitivity " or " transfer " cell is to lose to contact with the adhesion of flanking cell and shift the cell of invading adjacent body structure from initial disease sites through blood flow or lymph.

Term " cancer stem cell ", " tumor stem cell " or " solid tumor stem cell " can exchange use in this article each other, and refer to the such cell mass from solid tumor: (1) has multiplication capacity widely; (2) can carry out the imparity cell fission, thereby generate the filial generation of the differentiation of one or more multiplication potentialities or developmental potentiality decline; (3) the symmetry cell fission 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 that comparing these cancer stem cells with the tumour cell that great majority can not form tumour has the ability that behind the mouse of being transplanted to immunocompromised continuously, forms the tumour of palpation.The self-regeneration and the differentiation of the no sequential mode of cancer stem cell experience, thus have can be because of the tumour of the time dependent abnormal cells type of undergoing mutation in formation.Like United States Patent(USP) No. 6,004,528 propositions, solid tumor stem cell is different from " cancer responsibility ".In this patent, " cancer responsibility " is defined as the progenitor cell type of slow growth, himself rare sudden change, and carry out the symmetry cell fission rather than carry out in cellular environment, occurring tumorigenicity changing caused imparity cell fission.This " cancer responsibility " hypothesis thereby proposition, as the result of unusual environment, a large amount of appearance are the tumour cell of sudden change, fast breeding highly, thereby causes normal relatively stem cell accumulation to be undergone mutation then, and this causes these cells to become tumour cell.United States Patent(USP) 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 ".United States Patent(USP) No. 6,004,528 described " the cancer responsibilities that sudden change lacks " can be counted as precancerous lesion, and solid tumor stem cell to be itself contain the cancer cells that begins to the whole process of later stage cancer, to cause the sudden change of tumorigenicity from the cancer last stage.That is, solid tumor stem cell (" cancer stem cell ") should be included in and United States Patent(USP) No. 6,004, in the cell of the visibly different height sudden change of 528 described " cancer responsibilities ".The second, the transgenation that causes cancer is that solid tumor stem cell institute is intrinsic and affected by environment basically.The solid tumor stem cell model prediction; Isolating solid tumor stem cell can form other tumour (can explain transfer thus) after transplanting; And " cancer responsibility " model prediction can not form new tumour through " cancer responsibility " cell of transplanting, and this is because their unusual environment has tumorigenicity.In fact, dissociated, the isolating human entity knurl of phenotype stem cell transplantation makes the present invention obviously be different from " cancer responsibility " model to the ability that mouse (enter into normal tumor environment very different environment) still can form new tumour.The 3rd, solid tumor stem cell carries out symmetry cell fission and imparity cell fission probably simultaneously, makes that symmetrical cell fission 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 rate of propagation is not the characteristic that truly has.

Term " cancer cells ", " tumour cell " and grammatical equivalents are meant the total crowd of the cell that is derived from tumour or precancerous lesion, and it comprises non-tumorigenic cell (great majority in the tumor cell group) and tumorigenicity stem cell (cancer stem cell).

" tumorigenicity " used herein is meant the functional character of solid tumor stem cell, comprise self-regeneration property (forming other tumorigenicity cancer stem cell) and generate all other tumour cells (differentiation takes place and therefore form the non-tumorigenic tumour cell) thus make solid tumor stem cell form the proliferative of tumour.

Term used herein " stem cell cancer affinity tag ", " cancer stem cell affinity tag ", " tumor stem cell affinity tag " or " solid tumor stem cell affinity tag " are meant one or more gene, or expressed albumen, polypeptide or the peptide of said one or more gene, the expression level of said one or more gene itself or the expression level during with the combination of other gene be associated with the existence of tumorigenicity cancer cells (comparing with the non-tumorigenic cell).This relevant increase that can relate to said genetic expression or minimizing (for example, the increase of the level of the peptide of the mRNA of said genes encoding or coding or reduce).

Term used herein " biopsy samples " or " biopsy " are meant tissue sample or the fluid sample of taking from study subject, and this sample is in order to confirm whether it contains cancerous tissue.In some embodiments, obtaining biopsy or fluid is to suffer from cancer because suspect study subject, whether has cancer so check said biopsy or fluid.

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

" medicinal " be meant by or can or list in USP or other receives universally recognized pharmacopeia and is used for animal (comprising the mankind) by federation management mechanism or state government's approval.

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

" pharmaceutical excipient, carrier or adjuvant " is meant such vehicle, carrier or adjuvant, promptly can be applied to study subject with the disclosed at least a antibody of the present invention, not destroy its pharmacological activity and be nontoxic when using by the dosage of the said compound that is enough to the delivering therapeutic amount.

" medicinal charge material " is meant thinner, adjuvant, vehicle or the carrier of using with the disclosed at least a antibody of the present invention.

" prodrug " is meant the verivate 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 significant quantity " is meant the amount of effective " treatment " study subject of antibody, polypeptide, polynucleotide, organic molecule or other medicines or mammiferous disease or illness.In the situation of cancer, the treatment significant quantity of medicine can reduce the quantity of cancer cells; Reduce the tumour size; Inhibition or prevention cancer cell infiltration comprise that to peripheral organs the for example metastasis of cancer is in soft tissue and 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 cells and/or kills existing cancer cells, it can be called cell inhibition and/or cytotoxicity.

" diagnosis is provided " used herein or " diagnostic message " are meant to can be used for confirming whether the patient suffers from disease or symptom and/or with disease or symptom is classified as the phenotype classification or in any information that has meaning aspect the prognosis of disease or symptom or possible treatment (can be that common treatment also can the be a particular treatment) reaction, and the existence that for example comprises cancer stem cell whether.Similarly; Diagnosis is meant the diagnostic message that any kind is provided, include but not limited to tumour that whether study subject possibly have symptom (for example tumour), a study subject whether comprise cancer stem cell, the information relevant with tumour character or classification for example excessive risk tumour or low risk tumour, the information relevant with prognosis and/or with can be used for selecting suitable information of treating.Treatment select to comprise particular chemical therapeutical agent or other form of therapy 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; Confirm through diagnostic method of the present invention) existence to study subject health in the future (for example; The morbidity or the death of expection; Suffer from the possibility of cancer and the risk of transfer) the relevant information of influence, for example comprise in the tumour of study subject whether having cancer stem cell.

All refer to 1 like " treatment " or " treatment " or " with treatment " or " alleviations " or terms such as " with alleviations ") cure, slow down, alleviate the symptom of the pathology symptom diagnosed or illness and/or the treatment measure and 2 of the progress of the pathology symptom that prevents to be diagnosed or illness) prevent and/or slow down target pathology symptom or illness development preventive measures or prevent measure.Therefore, need the patient of treatment to comprise the patient who has illness; The patient that tendency has illness; The patient that need prevent with illness.If study subject demonstrates one or more following effects then this patient successfully obtains " treatment " according to the method for the invention: the minimizing of cancer cells quantity or completely dissolve; Reducing of tumor size; Suppress or the cancer cells infiltration of organ towards periphery no longer occurs, comprise the for example diffusion of cancer 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 M & M; Improve quality of life; Or some combined effect.

Term used herein " polynucleotide " or " nucleic acid " are meant that a plurality of nucleotide units (ribonucleotide or deoxyribonucleotide or relevant structural variant) through the polymkeric substance 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.The sequence of any known base analogue that comprising of DNA and RNA contained in this term.Said base analogue includes but not limited to 4-acetylcytosine, 8-hydroxy-n 6-methyladenosine, aziridinyl cytosine(Cyt), false iso-cytosine, 5-(carboxyl hydroxymethyl) uridylic, 5 FU 5 fluorouracil, 5-bromouracil, 5-ethyloic aminomethyl-2-sulfo-uridylic, 5-ethyloic aminomethyl uridylic, dihydrouracil, inosine, N6-isopentenyl gland purine, 1-methyladenine, 1-methyl pseudouracil, 1-methyl guanine, 1-methylinosine, 2; 2-dimethylguanine, 2-methyladenine, 2-methyl guanine, 3-methylcystein, 5-methylcytosine, N6-methyladenine, 7-methyl guanine, 5-methyl aminomethyl uridylic, 5-methoxyl group aminomethyl-2-sulfo-uridylic, β-D-mannose group Q nucleosides (mannosylqueosine), 5 '-methoxycarbonyl 6-Methyl Uracil, 5-methoxyuracil, 2-methylthio group-N6-isopentenyl gland purine, uridylic-5-ethoxyacetic acid methyl ester, uridylic-5-ethoxyacetic acid, oxygen fourth nucleosides (oxybutoxosine), pseudouracil, Q nucleosides (queosine), 2-sulfo-cytosine(Cyt), 5-methyl-2-sulfo-uridylic, 2-sulfo-uridylic, 4-sulfo-uridylic, methyl uracil, N-uridylic-5 ethoxyacetic acid methyl ester, uridylic-5-ethoxyacetic acid, pseudouracil, Q nucleosides (queosine), 2-sulfo-cytosine(Cyt) and 2,6-diaminopurine.

Phrase " strict hybridization conditions " is meant such condition: under the described conditions, usually in the compounding mixture of nucleic acid, probe will with its target sequence hybridization, and not with other sequence hybridization.Stringent condition have sequence dependent and because of environment different different.Long sequence specific hybrid under higher temperature.Detailed guide about nucleic acid hybridization is found in Tijssen; Techniques inBiochemistry and Molecular Biology-Hybridization with Nucleic Probes, " Overview of principles of hybridization and the strategy of nucleic acidassays " (1993).Usually, selected stringent condition be low 5 ℃～10 ℃ approximately of the hot melt points (Tm) of bit sequencing row under the fixed ionic strength pH.Tm be (under fixed ionic strength, pH and the nucleic acid concentration) when the probe that is complementary to target compound 50% with the temperature of target sequence balance hybridization (because the excessive existence of target sequence, so at Tm, 50% of probe is occupied when balance).Can also realize stringent condition such as destabilizing agents such as methane amides through adding.For selective cross or specific hybrid, positive signal is at least 2 times of background, be preferably 10 times of background hybridization.Exemplary stringent hybridization condition is following: 50% methane amide, 5 * SSC and 1%SDS, at 42 ℃ of incubations, perhaps, 5 * SSC, 1% SDS, at 65 ℃ of incubations, washing and in 65 ℃ of SDS, washing in 0.2 * SSC 1%.

Term " gene " is meant and comprises nucleic acid (for example DNA) sequence that generates the required encoding sequence of polypeptide, precursor or RNA (for example rRNA, tRNA).Polypeptide can be encoded by complete encoding sequence, perhaps by any part coding of this encoding sequence, gets final product as long as remain with this complete encoding sequence or segmental required activity or functional property (for example, enzymic activity, part combination, signal transduction, immunogenicity etc.).Thereby also containing the coding region of structure gene, this term make this gene be equivalent to the sequence of full length mRNA length with contiguous this coding region and the above location for example of distance 5 ' end and 3 ' about 1kb of any end of end.Be positioned 5 of coding region ' hold and the appear at sequence on the mRNA and be meant 5 ' end non-translated sequence.The sequence that is positioned coding region 3 ' end or downstream and appears on the mRNA is meant 3 ' end non-translated sequence.The cDNA and the genome form of gene contained in term " gene ".The genome form of gene or gene clone comprise the coding region of being interrupted by non-coding sequence (being called " intron " or " inserting the district " or " insertion sequence ").Intron is the fragment of transcribing the gene in the nRNA (hnRNA); Intron can contain such as controlling elements such as enhansers.The intron quilt is from the nuclear transcript or primary transcribe is removed or " cutting off "; Therefore intron does not appear in messenger RNA(mRNA) (mRNA) transcript.MRNA plays aminoacid sequence or the effect of confirming in the newborn polypeptide in proper order in translation process.Except containing intron, 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 as " flank " sequence or " flank " district (these flanking sequences be positioned at 5 of the non-translated sequence that appears on the mRNA transcript ' or 3 ').5 ' flanking region can contain control or influence genetic transcription such as regulating and controlling sequences such as promotor and enhansers.3 ' flanking region can comprise the sequence that instructs Transcription Termination, transcribes back cutting and polyadenylation.

Employed term " reorganization " is represented said cell, nucleic acid, albumen or carrier through importing heterologous nucleic acids or albumen or changing natural acid or albumen and being modified when relating to cell, nucleic acid, albumen or carrier, and perhaps said cell derives from the cell through modification like this.Therefore, for example, the gene that reconstitution cell can be expressed the cell that is not shown in natural (non-reorganization) form maybe can be expressed the natural gene of expression or unconventionality expression (for example be expressed as the fragment that non-natural exists or shear variant)." recombinant nucleic acid " of term used herein is meant such nucleic acid, its initial usually through manipulation nucleic acid (for example using polysaccharase and restriction endonuclease) in external formation, and for not being shown in natural form usually.Adopt this mode can realize not homotactic being operatively connected.Therefore, the separated nucleic acid of linear forms or all think to be used for the recombinant chou of purposes of the present invention through connecting the dna molecular that usually do not connect together at the expression vector of external formation.Should be understood that in case make recombinant nucleic acid and import host cell or organism, it duplicates non-reorganization ground (promptly utilizing the cells in vivo mechanism rather than the external manipulation of host cell); Yet such nucleic acid though duplicate, still is considered to be used for the recombinant chou 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 technology expression of above-described recombinant nucleic acid (promptly through).

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 also comprises for organism to be natural but to be changed the gene of (for example, that suddenlyd change, that add multiple copied, be connected to the non-natural regulating and controlling sequence, or the like) with certain mode.Heterologous gene obviously be different from the endogenous gene part be the heterologous gene sequence be typically connected to not find with karyomit(e) in gene order natural relevant or with the undiscovered chromosomal each several part related DNA sequence of nature (for example, at common gene of not expressing the locus expression of this gene).

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

" connection " is meant the process that between two double stranded nucleic acid fragments, forms diester linkage.Except as otherwise noted, otherwise connect the condition completion of the dna fragmentation to be connected of the about equimolar amount can use known damping fluid and 10 T4DNA of unit ligase enzyme (" ligase enzyme ")/0.5 μ g.The connection of nucleic acid can be used for two albumen are linked together in framework to generate single albumen or fusion rotein.

Term used herein " genetic expression " be meant through gene " transcribing " (for example; Enzymatic action through RNA polymerase) genetic information coded in the gene is converted into RNA (for example mRNA, rRNA, tRNA or snRNA), and " translation " through mRNA is converted into proteic process for protein coding gene.Can a lot of stage regulatory gene in this process express." rise " or " activation " is meant the regulation and control that improve gene expression product (for example, RNA or albumen) growing amount, and " downward modulation " or " inhibition " is meant the regulation and control that reduce growing amount.The molecule (for example, transcription factor) of participating in raising or reducing usually is hereinafter referred to as " activator " and " repressor ".

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

Term " amino acid " is meant the acid of naturally occurring amino acid and synthetic amino acid and has amino acid analogue and the amino acid analog thing with function like the naturally occurring amino acids.Naturally occurring amino acid is the amino acid of being modified by genetic code amino acids coding and those postmenstruations, for example oxyproline, Gla and O-Serine O-phosphate.Amino acid analogue is meant the compound with basic chemical structure identical with naturally occurring amino acid; 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(Met) methyl sulfonium.Said analogue can have through the R group of modifying (for example nor-leucine) or through the peptide backbone modified but still keep the basic chemical structure identical with naturally occurring amino acid.But the amino acid analog thing is meant to have the structure that is different from amino acid whose general chemistry structure have the 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 aminoacid sequence.For specific nucleotide sequence, conservative modify those nucleotide sequences that variant is meant the identical or essentially identical aminoacid 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 code, most of albumen are all by the identical nucleic acid encoding of a large amount of functions.For example, codon GCA, GCC, GCG and GCU amino acids coding all are L-Ala.Therefore, in each position that is defined as L-Ala by codon, this codon can be changed into another corresponding said 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 is also explained the silent variant of nucleic acid here.Those skilled in the art should know; In some cases, also can to each codon in the nucleic acid (except generally be methionine(Met) unique password AUG with generally be the TGG of unique password of tryptophane) modify with the identical molecule of acquisition function.Therefore, the silent variant of nucleic acid encoding lies in the said sequence about expression product, but does not lie in the said sequence about the actual probes sequence.For aminoacid sequence; Those skilled in the art should know; In the sequence of coding, change, increase or disappearance single amino acids or small percentage amino acid whose, be " the conservative variant of modifying " to independent replacement, disappearance or the increase of nucleic acid, peptide, polypeptide or protein sequence, the result who comprises change causes amino acid by the situation of aminoacid replacement like the chemofacies.Providing intimate amino acid whose conservative substituted form is known in this area.Except that comprising so conservative modification variant, do not get rid of homologue and allelotrope between polymorphum variant of the present invention, kind.Usually, conservative replacement comprises: 1) L-Ala (A), glycocoll (G); 2) aspartic acid (D), L-glutamic acid (E); 3) N (N), glutaminase (Q); 4) l-arginine (R), Methionin (K); 5) Isoleucine (I), bright amino acid (L), methionine(Met) (M), Xie Ansuan (V); 6) phenylalanine(Phe) (F), tyrosine (Y), tryptophane (W); 7) Serine (S), Threonine (T); With 8) halfcystine (C), methionine(Met) (M) (seeing Creighton for example, Proteins (1984)).

Term used herein " band epi-position label " is meant and comprises cancer stem cell affinity tag albumen or its territory sequence or the chimeric polyeptides partly that merges 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 proteic activity of cancer stem cell affinity tag.Suitable epi-position label has at least 6 amino-acid residues usually, is generally about 8～about 50 amino-acid residues, is about 10～about 20 residues sometimes.Epi-position label commonly used comprises Fc, HA, His and FLAG label.

The invention provides the compsn and the method that are used to study, diagnose, characterize and treat cancer.Particularly, the antibody that the invention provides solid tumor resisting stem cell labeling thing suppresses tumor growth and the method for cancer of treating human patients with these antibody of use.In some embodiments; Antibody of the present invention comprises antagonist antibodies, but said antagonist antibodies specificity combines cancer stem cell affinity tag albumen and disturbs for example part combination, receptor dimerizationization, the proteic expression of cancer stem cell affinity tag and/or the proteic signal transduction of cancer stem cell affinity tag.In some embodiments; Disclosed antibody comprises agonist antibody, and this agonist antibody can combine and promote for example part combination, receptor dimerizationization and/or the signal transduction that carries out through cancer stem cell affinity tag albumen with cancer stem cell affinity tag protein-specific.In some embodiments, disclosed antibody does not disturb or promotes the proteic biological activity of cancer stem cell affinity tag, still discerns through for example antibody internalization and/or by immune system and suppresses tumor growth.In some embodiments, but said antibody specific recognition is done thin affinity tag albumen more than a kind of solid tumor.

The invention provides and a kind of separated antibody of human DLL4 epitope specificity bonded; Said human DLL4 epi-position is combined to form by human DLL4N-stub area (SEQ ID NO:27) and human DSL (SEQ ID NO:26), and wherein said antibody influences growth of tumor.In some embodiments, said antibody is monoclonal antibody.In some embodiments, said antibody is chimeric antibody.In some embodiments, said antibody is humanized antibody.In some embodiments, said antibody is human antibodies.The present invention further provides the pharmaceutical composition that comprises disclosed antibody of the present invention and pharmaceutical carrier.

The present invention further provides a kind of treatment method for cancer, and said method comprises disclosed antibody of the present invention of administering therapeutic significant quantity or pharmaceutical composition.In some embodiments, with said antibody and the coupling of cytotoxicity part.In some embodiments, said method further comprises and uses at least a additional therapeutic agent that is suitable for carrying out conjoint therapy.In some embodiments, said tumour cell is selected from breast tumor, colorectum tumour, lung tumor, tumor of prostate, pancreas tumor and tumor of head and neck.

Be similar to it and come source tissue, solid tumor is made up of the allos cell mass.Most of these cells lack tumorigenicity; This show the development of solid tumor and keep also depend on groupuscule stem cell (promptly; The tumorigenicity cancer cells); These stem cells have multiplication capacity and form other tumor stem cell (self-regeneration) and most differentiation degree tumour cell (that is non-tumorigenic cancer cells) higher, that lack tumorigenesis potential effectively simultaneously.The notion of cancer stem cell is introduced after finding hemopoietic stem cell (HSC) soon first, and in acute myeloid leukaemia (AML), obtains experiment confirm (Park etc., 1971, JNatl.Cancer Inst.46:411～22; 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 the unique expression pattern of their cell surface receptor with to they self-regenerations and breed the evaluation of character and separated in culture and heterograft animal model.Finding, in order to form tumour, need be more than 50 times of unassorted tumour cell (Al-Hajj etc., 2003, Proc.Nat ' l.Acad.Sci.100:3983～8) with ESA+CD44+CD24-/low pedigree colony enrichment.The ability of from a large amount of non-tumorigenic tumour cells, isolating the tumorigenicity cancer stem cell makes can use microarray analysis to identify the cancer stem cell affinity tag, compare the gene that in cancer stem cell, has differential expression with non-tumorigenic tumour cell or normal breast epithelium.Cancer is diagnosed and treated to utilization of the present invention about the understanding of the cancer stem cell affinity tag that these are identified.

Cancer stem cell affinity tag of the present invention relates to human DLL4, a kind of Notch receptors ligand.The Notch signal transduction pathway is that the embryo sexual norm forms, the back embryonal connective tissue is kept one of several crucial adjusting factors of learning with stem cell biological.More particularly, the Notch signal transduction relates to the lateral inhibition process between the flanking cell destiny, and the decision of pair cell destiny plays a significant role in asymmetric fission process.Notch signal transduction out of control is relevant with a large amount of human cancers, and it can change the growth destiny of tumour cell and make them maintain not differentiation and vegetative state (Brennan and Brown, 2003, Breast Cancer Res.5:69).Therefore, carcinogenesis can be carried out (Beachy etc., 2004, Nature 432:324) through usurping the normal development that control undertaken by population of stem cells and the homeostatic mechanism of tissue repair.

The Notch acceptor is identified in the fruit bat two mutants first.The monoploid disappearance of fruit bat Notch produces damaged at the wing edge, afunction then produces embryo property lethal " neurogenic " phenotype, and wherein the destiny of epidermic cell is changed into nervous tissue (Moohr, 1919, Genet.4:252; Poulson, 1937, PNAS 23:133; Poulson, 1940, J.Exp.Zool.83:271).The Notch acceptor is the transmembrane receptor that single is striden film, and it contains a large amount of placed in-line Urogastrons (EGF) appearance Tumor-necrosis factor glycoproteins and the Notch/LIN-12 Tumor-necrosis factor glycoproteins (Wharton etc., 1985, the Cell 43:567 that are rich in halfcystine in large-scale extracellular domain; Kidd etc., 1986, Mol.Cell Biol.6:3094; At Artavanis etc., 1999, among the Science 284:770 summary is arranged).Four Mammals Notch albumen (NOTCH1, NOTCH2, NOTCH3 and NOTCH4) have been identified; Sudden change in these acceptors always causes heteroplasia and human pathological change (hereinafter will the detail) (Gridley that comprises several cancers; 1997, Mol.Cell Neurosci.9:103; Joutel and Tournier-Lasserve, 1998, Semin.Cell Dev.Biol.9:619-25).

δ, spination, delay-2 (Delta, Serrated, Lag-2) (DSL) but the single of family is striden the transmembrane ligand body activation Notch acceptor of film.The characteristic of known Mammals Notch part δ appearance 1 (Dll 1), δ appearance 3 (Dll 3), δ appearance 4 (Dll 4), Jagged (sawtooth appearance) 1 and Jagged 2 is the series connection EGF-appearance Tumor-necrosis factor glycoproteins in DSL territory and the extracellular domain.The extracellular domain of Notch acceptor and the extracellular domain interaction of its part on adjacent cell usually cause two kinds of proteolyze cuttings of Notch: by the extracellular cutting of ADAM proteolytic enzyme mediation with by the cutting in the membrane-spanning domain of gamma secretase mediation.A kind of cutting in back generates Notch born of the same parents' internal areas (NICD).NICD get in the nuclear subsequently and in nuclear activation as the CBF1 of main downstream effect thing; There is not hair suppressor gene (Suppressor of Hairless) [Su (H)]; The transcription factor that postpones-2 (CSL) family; Thereby strengthen (Artavanis etc., 1999, the Science 284:770 of transcribing of crinosity and the nuclear alkalescence helix-loop-helix transcription factor that divides enhanser [E (spl)] family; Brennan and Brown, 2003, Breast Cancer Res.5:69; Iso etc., 2003, Arterioscler.Thromb.Vase.Biol.23:543).Also possibly there are approach (Martinez etc. in the optional born of the same parents that relate to the cytoplasmic protein Deltex that in fruit bat, identifies in the Mammals; 2002; Curr.Opin.Genet.Dev.12:524-33); And this Deltex dependent pathway can be brought into play and suppress effect (Brennan etc., 1999, the Curr.Biol.9:707-710 that the Wnt target gene is expressed; Lawrence etc., 2001, Curr.Biol.11:375-85).

Hemopoietic stem cell (HSC) is to understand maximum stem cells in the body, the Notch signal transduction participated in simultaneously normally keeping of hemopoietic stem cell with white blood disease transform (Kopper and Hajdu, 2004, Pathol.Oncol.Res.10:69-73).HSC is the rare cell crowd 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 higher progenitor cell of degree of differentiation to build the ability of whole hematopoiesis system again.External and in secular reconstruction test; The composing type activation of Notch1 signal transduction can be set up the immortal cell line (Varnum-Finney etc. that produce lymphocyte and medullary cell simultaneously in HSC and the progenitor cell; 2000, Nat.Med.6:1278-81), and the existence of Jagged 1 can strengthen human bone marrow cell crowd's's (being enriched with HSC) transplanting (Karanu etc.; 2000, J.Exp.Med.192:1365-72).Recently, proved in vivo to have the Notch signal transduction among the HSC, and shown that the Notch signal transduction relates to the inhibition to the HSC differentiation.In addition, the HSC self-regeneration of Wnt mediation as if need the Notch signal transduction (Duncan etc., 2005, Nat.Immunol.6:314).

The Notch signal pathway is performance central role in the keeping of NSC also, and with NSC normally keep and the cancer of the brain all has relation (Kopper and Hajdu, 2004, Pathol.Oncol.Res.10:69-73; Purow etc., 2005, Cancer Res.65:2353-63; Hallahan etc., 2004, Cancer Res.64:7794-800).NSC produces neurocyte all in the mammalian nervous system and spongiocyte in growth course, and in the adult brain, has identified NSC recently (Gage, 2000, Science 287:1433-8).Notch1; Notch target gene Hes1,3 and 5; And the mouse of Notch signal transduction presenilin 1 (presenilinl) regulon disappearance (PS1) shows as the decline of embryo property NSC quantity.In addition, in the brain of PS1 heterozygote mouse, the adult NSC reduces (Nakamura etc., 2000, J.Neurosci.20:283-93; Hitoshi etc., 2002, Genes Dev.16:846-58).The minimizing of NSC possibly be since they be divided into too early neurone (Hatakeyama etc., 2004, Dev.131:5539-50), this shows Notch signal transduction regulation and control cell differentiation of nerve cord and self-regeneration.

Multiple human cancer relates to unusual Notch signal transduction.In a subclass of T cell acute lymphoblastic leukemia, identified human NOTCH1 gene first, the NOTCH1 gene of being identified is the translocated locus (Ellisen etc., 1991, Cell 66:649-61) that causes the Notch pathway activation.The composing type activation of the interior Notch1 signal transduction of T cell produces t cell lymphoma equally in the mouse model, and this shows its initiation (Robey etc., 1996, Cell 87:483-92; Pear etc., 1996, J.Exp.Med.183:2283-91; Yan etc., 2001, Blood 98:3793-9; Bellavia etc., 2000, EMBO is J.19:3337-48).Have been found that recently and usually have NOTCH1 point mutation, insertion and disappearance (Pear and the Aster that produces unusual NOTCH1 signal transduction in children and the adult T cell acute lymphoblastic leukemia/lymphoma; 2004, Curr.Opin.Hematol.11:416-33).

Mouse mammary tumour virus hints first with the activated Notch protein fragments that is produced the frequent insertion of Notch in the breast tumor 1 and Notch 4 locus and has Notch signal transduction (Gallahan and Callahan in the breast cancer; 1987, J.Virol 61:66-74; Brennan and Brown, 2003, Breast Cancer Res.5:69; Politi etc., 2004, Semin.Cancer Biol.14:341-7).Verified to the further research of transgenic mice in normal breast Notch effect in latex dust branch (ductal branching) between the growth period; And the composing type activated form of Notch 4 suppresses epidermal differentiation and causes tumour that (Jhappan etc. take place in the mammary epithelial cell; 1992, Genes&Dev.6:345-5; Gallahan etc., 1996, Cancer Res.56:1775-85; Smith etc., 1995, CellGrowth Differ.6:563-77; Soriano etc., 2000, Int.J.Cancer 86:652-9; Uyttendaele etc., 1998, Dev.Biol 196:204-17; Politi etc., 2004, Semin.CancerBiol.14:341-7).At present be limited to expression and they and dependency (Weijzen etc., 2002, Nat.Med.8:979-86 clinical effectiveness between of Notch acceptor in breast cancer about the evidence of the effect of Notch in human breast cancer; Parr etc., 2004, Int.J.MoI.Med.14:779-86).In addition, in cervical cancer (Zagouras etc., 1995; PNAS 92:6414-8), renal cell carcinoma (Rae etc., 2000, Int.J.Cancer 88:726-32), SCCHN (Leethanakul etc.; 2000, Oncogene19:3220-4), carcinoma of endometrium (Suzuki etc., 2000; Int.J.Oncol.17:1131-9) and neuroblastoma (van Limpt etc.; 2000, observed crossing of Notch approach in Med.Pediatr.Oncol.35:554-8) and expressed, this prompting Notch is at the developing latent effect of multiple knurl.What is interesting is Notch signal transduction possibly in the not differentiation attitude of the Ape two mutants oncocyte of keeping colon, play a role (van Es and Clevers, 2005, Trends Mol.Med.11:496-502).

Comprise propagation, migration, unstriated muscle differentiation, blood vessel takes place and the many aspects such as vascular development of arterial-venous differentiation in, relate to equally the Notch approach (Iso etc., 2003, Arterioscler.Thromb.Vase.Biol.23:543).For example, grow and during vitelline vessel formed, the heterozygous deletion of isozygoty null mutation and the DLL4 of Notch-1/4 and Jagged-1 caused serious and variable defective at artery.In addition, the inferior effect of Dll1 defective and Notch-2 mice embryonic occurs hemorrhage, and this possibly be because bad growth (Gale etc., 2004, PNAS, the 101:15949-54 of blood vessel structure; Krebs etc., 2000, Genes Dev.14:1343-52; Xue etc., 1999, Hum.Mel Genet.8:723-30; Hrabe de Angelis etc., 1997, Nature 386:717-21; McCright etc., 2001, Dev.128:491-502).In the mankind; The sudden change of JAGGED1 is relevant with Alagille syndrome, and Alagille syndrome is a kind of dysplasia that comprises vascular defect, and the sudden change of NOTCH3 is the reason of heredity vascular dementia (CADASIL); In the heredity vascular dementia; Blood vessel homeostasis defectiveness (Joutel etc., 1996, Nature 383:707-10).

The evaluation of the DLL4 that in cancer stem cell (comparing with normal newborn epithelium), expresses is shown that target Notch approach not only can eliminate most of non-tumorigenic cancer cells, can also eliminate and to cause solid tumor to form and the tumorigenicity cell of recurrence.In addition, because blood vessel occurs in the outstanding role of tumour in forming and keeping, the antibody target Notch approach through anti-DLL4 can also effectively suppress blood vessel and take place, makes cancer to lack nutrition and help to eliminate cancer.

Thus, the invention provides a kind of cancer stem cell affinity tag, the expression of said cancer stem cell affinity tag can be used for analyzing, with diagnosis or the monitoring disease relevant with cancer.In some embodiments, the expression of cancer stem cell affinity tag is for example expressed to confirm by the polynucleotide (for example mRNA) of the said cancer stem cell affinity tag of coding.Can be through any one detects and quantitative said polynucleotide in the known numerous methods of those skilled in the art.In some embodiments, employing is for example carried out the mRNA that in situ hybridization detects coding cancer stem cell affinity tag from the tissue slice of patient's biopsy samples.In some embodiments, RNA is separated from tissue and detect through 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 affinity tag to detect the expression of cancer stem cell affinity tag polynucleotide.

In some embodiments, can confirm the expression of cancer stem cell affinity tag through detecting corresponding polypeptide.Can detect and quantitative said polypeptide through in the known numerous means of those skilled in the art any one.In some embodiments, for example the operational analysis biochemical method for example electrophoresis, capillary electrophoresis, performance liquid chromatography (HPLC) or thin-layer chromatography (TLC) detect cancer stem cell affinity tag polypeptide.Can also check order to institute's isolated polypeptide according to standard technique.In some embodiments, utilize immunofluorescence or immunohistochemistry on the tissue slice for example, the antibody that produces with anticancer stem cell labeling albumen detects said albumen.As selection, can 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 with the proteic expression of fluorescently-labeled antibody test cancer stem cell affinity tag.In another approach, can utilize the antibody of mark in typical imaging system, the cell of expressing the cancer stem cell mark to be carried out detecting in the body.For example, can use the isotope-labeled antibody of paramagnetism to carry out nuclear magnetic resonance (MRI).

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

The detection of available then cancer stem cell marker representation provides prognosis and selects therapy.Prognosis can be expressed based on any known risk of cancer stem cell affinity tag indication.And the detection of cancer stem cell affinity tag can comprise the treatment of for example being carried out with the anti-proteic antibody of cancer stem cell affinity tag to be detected in order to select suitable therapy.In some embodiments, said antibodies specific combines such as proteic extracellular domains of cancer stem cell affinity tag such as Notch receptors ligand DLL4.

Within the scope of the invention, suitable antibody is the reagent of one or more effects below for example having: disturb the expression of cancer stem cell affinity tag; Disturb the activation of cancer stem cell signal transduction pathway through for example spatially suppressing interaction between cancer stem cell affinity tag and its part, acceptor or the co-receptor; The activation cancer stem cell signal transduction pathway through the combination of for example bringing into play part effect or promotion endogenic ligand; Or combine the cancer stem cell affinity tag 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 affinity tag protein function outward.In some embodiments, the intrinsic activation (for example kinase activity) that the outer combination of the born of the same parents of the antibody of anticancer stem cell labeling thing can be through for example suppressing the cancer stem cell affinity tag and/or through spatially suppress for example cancer stem cell affinity tag and its part, and its acceptor, and co-receptor or and extracellular matrix between interaction suppress the proteic signal transduction of cancer stem cell affinity tag.In some embodiments, the outer combination of the born of the same parents of the antibody of anticancer stem cell labeling thing can be transported the cell surface expression of reducing the cancer stem cell affinity tag through the cell surface of for example proteic internalization of cancer stem cell affinity tag or minimizing cancer stem cell affinity tag.In some embodiments, the outer combination of the born of the same parents of the antibody of anticancer stem cell labeling thing can combine to promote the proteic signal transduction of cancer stem cell affinity tag through effect or the increase part of for example bringing into play as the bait part.

In some embodiments; The antibody of anticancer stem cell labeling thing and cancer stem cell affinity tag protein binding also have following one or more effects: suppress the propagation of tumour cell, the necrocytosis of triggering tumour cell, promotion tumour cell are divided into the less cell type of tumorigenicity or prevent the transfer of tumour cell.In some embodiments, the antibody of anticancer stem cell labeling thing triggers necrocytosis through institute's link coupled toxin, chemotherapeutics, ri or other similar reagents.For example, with the antibody of anticancer stem cell labeling thing with in the tumour cell of expressing this cancer stem cell affinity tag through the albumen internalization be activated toxin conjugated.

In some embodiments, the antibody of anticancer stem cell labeling thing is expressed the necrocytosis of the proteic cell of said cancer stem cell affinity tag through cytotoxicity (ADCC) mediation that antibody relies on.ADCC participates in the cracking of cell through the effector cell of the Fc part of identification antibody.For example many lymphocytes, monocyte, tissue macrophages, granulocyte and eosinophilic granulocyte have the Fc acceptor and can the mediated cell cracking (Dillman, 1994, J.Clin.Oncol.12:1497).

In some embodiments, the antibody of anticancer stem cell labeling thing triggers the necrocytosis of expressing the proteic cell of cancer stem cell affinity tag through the cytotoxicity (CDC) of complement activation dependence.CDC participates in the combining and the activation of complement proteins cascade subsequently of Fc part of SC and antibody, thereby causes cytolemma destruction, and finally causes necrocytosis.The BA 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 effectively antibody classification with complement, secondly be IgG1, IgG3 and IgG2, but IgG4 lacks (Dillman, 1994, J.Clin.Oncol.12:1497 very much in the complement activation cascade; Jefferis etc., 1998, Immunol.Rev.163:59～76).According to the present invention, can prepare those classification antibody with required BA.

Any antibodies specific that can measure anticancer stem cell is through complement activation and/or ADCC mediation target cell cracked ability.The interested cell of vitro culture and mark; With antibody with SC or can be added in the cell culture by immune complex activatory immunocyte.Through for example detecting the lysis of target cell from the lysing cell 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 vitro test the antibody of complement activation or mediation ADCC be used for the treatment of this particular patient.

In some embodiments, the antibody of anticancer stem cell labeling thing can trigger necrocytosis, thereby suppresses vasculogenesis.Vasculogenesis is such process, that is, through vasculogenesis, by the new blood vessel of the vascularization of preexist, and vasculogenesis is the required primary process of normal growth in for example fetal development, wound healing and ovulation response process etc.Greater than 1mm ²～2mm ²Solid tumor growth also need vasculogenesis to supplement the nutrients and oxygen, if there is not vasculogenesis, tumour cell will be dead.In some embodiments, the antibody target of anticancer stem cell labeling thing is expressed the vascular cell of this cancer stem cell affinity tag, comprises the component of the extracellular matrix that for example endotheliocyte, smooth muscle cell or blood vessel assembling are required.In some embodiments, the antibody of anticancer stem cell labeling thing suppresses the growth factor signal transduction that vascular cell is raised, assembles, kept or survives required.

The antibody of anticancer stem cell labeling thing can be used for diagnosis as herein described and treat-ment.In some embodiments, antibody of the present invention can be in order to detect like the proteic expression of cancer stem cell affinity tag in the biological samples such as patient tissue biopsy samples, hydrothorax or blood sample.Can the biopsy sample be processed section and for example use immunofluorescence or immunohistochemistry detects albumen.In addition, can separate from the individual cells of sample and through facs analysis and detect the protein expression on fixed cell or viable cell.In some embodiments, can on protein arrays, use antibody to come the expression of the cancer stem cell affinity tag on test example such as the tumour cell, in the cell lysate or in other protein sample.In some embodiments, measure based on cells in vitro or body in the animal model etc., antibody of the present invention is used to suppress the growth of tumour cell through antibody is contacted with tumour cell.In some embodiments, the antibody of the anticancer stem cell labeling thing through the administering therapeutic significant quantity and the cancer that said antibody is used to treat the patient.

Can prepare antibody of the present invention through any ordinary method as known in the art.For example; Can come immune animal (for example rabbit, rat, mouse, donkey etc.) thereby the preparation polyclonal antibody through MSI or peritoneal injection related antigen (peptide fragment of purifying, total length recombinant protein, fusion rotein etc.), said antigen can be chosen and be diluted in coupling such as keyhole limpet hemocyanin (KLH) in the Sterile Saline, serum albumin 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.Let collected coagulation of blood, decant goes out serum, makes its clarification and measures antibody titers through centrifugal.Can comprise that affinity chromatography, ion-exchange chromatography, gel electrophoresis, dialysis etc. are purified into polyclonal antibody from serum or ascites according to the standard method of this area.

Can adopt for example Kohler and Milstein (1975), the described hybridoma method of Nature 256:495 prepares monoclonal antibody.Adopt the hybridoma method, but generate the antigenic antibody of specificity binding immunoassay property to cause lymphocyte according to as stated mouse, hamster or other suitable host animal being carried out immunity.Can also be at external immune lymphocyte.After the immunity, isolated lymphocytes, and for example use polyoxyethylene glycol that itself and suitable myeloma cell line are merged, thus form the hybridoma of selecting among the lymphocyte that can never merge subsequently and the myeloma cell.Measure (radioimmunoassay (RIA) for example through immunoprecipitation, immunoblotting or external combination; Enzyme-linked immunosorbent assay (ELISA)) comes definite hybridoma that can produce the selected antigenic monoclonal antibody of specific anti; Use standard method (Goding subsequently; Monoclonal Antibodies:Principles and Practice; Academic Press, 1986) external breeding or in culture as the said hybridoma of breeding in the animal ascites tumour body.Then from as above to monoclonal antibody purification the described substratum of polyclonal antibody or the ascites fluid.

As selection, can also use like USP 4,816,567 described recombinant DNA methods prepare monoclonal antibody.Use can specific amplification the Oligonucleolide primers of gene of heavy chain and light chain of coding monoclonal antibody; Isolate the polynucleotide of the said monoclonal antibody of coding through for example RT-PCR from mature B cell or hybridoma, and use conventional procedure to confirm their sequence.Then the polynucleotide of institute's separated coding heavy chain and light chain are cloned in the suitable expression; In the time of in this expression vector transfection being arrived the host cell that self does not generate Tegeline such as intestinal bacteria (E.coli) cell, ape COS cell, Chinese hamster ovary (CHO) cell or myeloma cell etc., 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 required species, isolate described 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 polynucleotide of coding monoclonal antibody with multitude of different ways, to produce substituting antibody.In some embodiments, can the light chain of for example mouse monoclonal antibody and the constant region of heavy chain 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, brachymemma or remove said constant region to produce the required antibody fragment of monoclonal antibody.Can use the site-directed mutagenesis of variable region or specificity that monoclonal antibody is optimized in high-density mutagenesis, avidity etc.

In embodiments more of the present invention, the monoclonal antibody of said anticancer stem cell labeling thing is a humanized antibody.Humanized antibody is the antibody that in the variable region, contains from the minimum sequence of non-human (for example muroid) antibody.Such antibody reduces antigenicity when in treatment, being used for human subject used and HAMA (human anti-mouse antibodies) replys.In practice, humanized antibody normally has the minimum human antibodies that does not even have the non-human sequence.Human antibodies is by the antibody of mankind's generation or has the antibody corresponding to the aminoacid sequence of the antibody that is produced by the mankind.

Can use various techniques known in the art to prepare humanized antibody.Can be by (Jones etc., 1986, Nature, 321:522～525; Riechmann etc., 1988, Nature, 332:323～327; Verhoeyen etc.; 1988; Science, 239:1534～1536) method, the CDR that replaces human antibodies with non-human antibody's (for example mouse, rat, rabbit, hamster etc.) with required specificity, avidity and ability CDR comes antagonist to carry out humanization.Can come further to modify humanized antibody through the replacement of variable framework region of the mankind and/or the intra-residue extra residue of institute's metathetical non-human, to improve and optimization antibodies specific, avidity and/or ability.

To the human heavy chain used in preparation humanized antibody process and/or the selection of variable region of light chain, be important for reducing antigenicity.According to " best fit " method, use the whole library of known human variable region amino acid sequence to screen the sequence of the variable region of rodents antibody.Therefore in some embodiments, will be with the amino acid sequence homology of the rodents antibody that therefrom takes out CDR the highest human amino acid sequence is as human framework region (FR) (Sims etc., 1993, J.Immunol, the 151:2296 of humanized antibody; Chothia etc., 1987, J.MoI.Biol, 196:901).Another kind method is used the specific FR of the consensus sequence of everyone antibody-like be derived from specific light chain or heavy chain subgroup, and this method can be used to several different humanized antibodies (Carter etc., 1992, PNAS, 89; 4285; Presta etc., 1993, J.Immunol, 151:2623).In some embodiments, use combined method to choose and be used for the human variable FR that humanized antibody is produced.

Further being understood that needs humanized antibody (for example rodents) to keep antigenic high-affinity and other favourable biological property.In order to realize this goal, can prepare humanized antibody through the method for analyzing from the parental array of the humanized rodents antibody of need and various alternative humanization sequence.Those skilled in the art can obtain and be familiar with the three-dimensional model of Tegeline.Computer program can be used to explain and show the possible three-dimensional conformation structure of selected alternative antibody sequence.Use this model can analyze residue possibly act in the function of the humanized antibody of need, promptly to the analysis of the residue that influences alternative its antigenic ability of antibodies.In this way, can and make up the humanized antibody that the FR residue is used to accept from the parental antibody selection, thereby realize required antibody characteristic.Usually, for antigen combines, reservation is from the residue among the CDR in the antigen determining district (or hypervariable region) of parental antibody (the rodents antibody that for example has required antigen-binding) in humanized antibody.In some embodiments, in humanized antibody, keep at least one extra residue in variable FR from parental antibody.In some embodiments, in humanized antibody, can keep nearly 6 extra residues in variable FR from parental antibody.

To be denoted as Hx and Lx from the amino acid of the variable region of the ripe heavy chain of Tegeline and light chain; Wherein x is that expression is according to Karbate figure (scheme of Kabat) (Sequences ofProteins of Immunological Interest; U.S.Department of Health and HumanServices; The numbering of amino acid position 1987,1991).The Karbate has listed the multiple amino acids sequence for the antibody of each subclass, and the Karbate has also listed the modal amino acid that forms consensus sequence for each residue position in this subclass.The Karbate uses the appointment method to specify a residue number for each amino acid in the listed sequence, and the method for this appointment residue numbering has become the standard in this area.Through compare the antibody studied and a consensus sequence among the Karbate with reference to conserved amino acid, can Karbate figure be expanded to other antibody that is not comprised in its tabulation.Use Karbate's number system can easily confirm equivalent locational amino acid in the different antibodies.For example, the amino acid position occupied on the L50 position of human antibodies is equivalent to the amino acid L50 position of mouse antibodies.And then, through using Karbate's number system, can compare any two kinds of antibody sequences separately, for example to confirm identity per-cent with the amino acid comparison that has same card Bart numbering in each amino acid in the antibody sequence and another sequence.After the comparison; If (for example with the test antibody zone; The whole ripe variable region of heavy chain or light chain) compares with the same area of reference antibody; Then test antibody zone with sequence identity per-cent between the reference antibody regions is: the positional number that is all occupied by same amino acid in test antibody zone and the reference antibody regions divided by two zones through comparing the sum (disregarding breach) of position, convert per-cent into thereby multiply by 100 again.

Embodiment 1 has hereinafter described the preparation of the humanized antibody of exemplary anti-DLL4; But said antibody specificity combines the human DLL4 (21M18H9L2 of the disclosed cancer stem cell affinity tag of the present invention; ATCC deposit number PTA-8427 and 21M18H7L2; ATCC deposit number PTA-8425 was in preservation on May 10 in 2007; (American type culture collection (American Type Culture Collection) is 10801University Blvd. (ATCC), Manassas, VA 20110-2209USA)).In some embodiments, said humanized antibody comprises the non-human antigen determining district from muroid monoclonal antibody 21M18.Particularly, in some embodiments, remain with one or more following heavy chain CDR:CDR1 (SEQ ID NO:1), CDR2 (SEQ ID NO:2 in the humanized 21M18 antibody from the rodents parental antibody; SEQID NO:3; Or SEQ ID NO:4,52a changes in the Karbate position for it) and CDR3 (SEQ IDNO:5).In some embodiments, remain with one or more following light chain CDR:CDR1 (SEQ ID NO:9), CDR2 (SEQID NO:10) and CDR3 (SEQ ID NO:11) in the humanized 21M18 antibody from the rodents parental antibody.In some embodiments, humanized antibody further comprises at least one FR replacement in human heavy chain or variable region of light chain.

In some embodiments; The invention provides the humanized antibody that specificity combines human DLL4 epi-position; Said epi-position is combined to form by human DLL4N-stub area (SEQ ID NO:27) and human DSL (SEQ ID NO:26), and wherein said antibody can influence tumor growth.In some embodiments, said humanized antibody is complete IgG antibody.In some embodiments, said humanized antibody is complete IgG ₂Antibody.In some embodiments, said humanized antibody is an antibody fragment.In some embodiments, said humanized antibody is the Fab fragment.

In some embodiments, humanized antibody of the present invention comprises weight chain variable (V _H) district, said weight chain variable (V _H) district comprises non-human antigen determining district and human variable framework region.In some embodiments, said non-human antigen determining district comprises the complementarity-determining region (CDR) in rodents source.In some embodiment, said non-human antigen determining district comprises the CDR from mouse antibodies.In some embodiment, said rodents CDR derives from monoclonal antibody 21M18, and wherein 21M18 contains the variable region of heavy chain of called after SEQ ID NO:6.In some embodiments, wherein humanized antibody comprises V _HThe district, said V _HThe district comprises (a) CDR1 (SEQ ID NO:1), CDR2 (SEQID NO:2; SEQ ID NO:3; Or SEQ ID NO:4) and the perhaps aminoacid sequence of (b) SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8 of CDR3 (SEQ ID NO:5).

In some embodiments, the human heavy chain variable framework region comprises the human sequence who is expressed.In some embodiments, at least one residue is substituted in the variable framework region of the said mankind.In some embodiments, at least one residue is positioned at the position that is selected from the group of being made up of position 16,20,27,28,38 and 48 (based on Karbate's number system) in the human heavy chain variable framework region.In some embodiments, the position 16,20,27,28,38 and 48 based on Karbate's number system is substituted.In some embodiments, the residue that at least one residue is occupied the corresponding position in the human variable framework region in the antibody that contains non-human antigen determining district replaces.

In some embodiments, said human heavy chain variable framework region contains IGH (V) 1-18.In some embodiments, at least one residue is substituted in the variable framework region of the said mankind.In some embodiments, at least one residue is positioned at the position that is selected from the group of being made up of 20H, 28H, 38H, 48H and 69H (based on Karbate's number system) in the human heavy chain variable framework region.In some embodiments, position 20H, 28H, 38H, 48H and the 69H based on Karbate's number system is substituted.In some embodiments, the residue that at least one residue is occupied the corresponding position in the human variable framework region in the antibody that contains non-human antigen determining district replaces.

In some embodiments, humanized antibody of the present invention comprises light chain variable (V _L) district, said light chain variable (V _L) district comprises non-human antigen determining district and human variable framework region.In some embodiments, said non-human antigen determining district comprises the CDR in rodents source.In some embodiments, said non-human antigen determining district comprises the CDR from mouse antibodies.In some embodiments, said CDR derives from monoclonal antibody 21M18, and wherein 21M18 contains the V of called after SEQ ID NO:12 _LThe district.In some embodiments, V _LThe district comprises the perhaps aminoacid sequence of (b) SEQ IDNO:12 of (a) CDR1 (SEQ IDNO:9), CDR2 (SEQ ID NO:10) and CDR3 (SEQ LD NO:11).

In some embodiments, said human light chain variable framework region contains IGK (V) 4-1.In some embodiments, at least one residue in the said human light chain variable framework region is substituted.In some embodiments, at least one residue in the variable framework region of the said mankind is positioned at the position that is selected from the group of being made up of position 22L and 36L (based on Karbate's number system).In some embodiments, position 22L and the 36L based on Karbate's number system is substituted.In some embodiments, at least one residue in the variable framework region of the said mankind is occupied the residue replacement of corresponding position in the antibody that contains non-human antigen determining district.

In some embodiments; Antibody of the present invention be for the antibody of the specificity bonded antibody 21M18 of human DLL4 competition, wherein antibody 21M18 comprises: (a) have called after SEQID NO:6, SEQ ID NO:7 or SEQ ID NO:8 the variable region heavy chain and (b) have a light chain of the variable region of called after SEQ ID NO:12.In some embodiments, said antibody is humanized antibody or human antibodies.

In some embodiments; Said humanized antibody specificity combines human DLL4 epi-position; Said epi-position is combined to form by human DLL4N-stub area (SEQ ID NO:27) and human DSL (SEQ IDNO:26); Wherein said antibody comprises variable region of heavy chain and variable region of light chain; Said variable region of heavy chain has the sequence identity with SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8 at least 90%, and said variable region of light chain has the sequence identity with SEQ ID NO:12 at least 90%.In some embodiments, said variable region of heavy chain has the sequence identity with SEQ ID NO:6, SEQID NO:7 or SEQ ID NO:8 at least 95%, and said variable region of light chain has the sequence identity with SEQ ID NO:12 at least 95%.In some embodiments, said variable region of heavy chain has the sequence identity with SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8 at least 99%, and said variable region of light chain has the sequence identity with SEQ ID NO:12 at least 99%.

In some embodiments; The invention provides isolating polynucleotide molecule; Said polynucleotide molecule coding specificity combines the humanized antibody of human DLL4 epi-position; Said epi-position is combined to form by human DLL4N-stub area (SEQ ID NO:27) and human DSL (SEQ ID NO:26), and wherein said antibody comprises V _HThe district, said V _HThe district comprises coding CDR1 (SEQ ID NO:1), CDR2 (SEQ ID NO:2; SEQ ID NO:3; Or SEQ ID NO:4) and the human variable framework region of the non-human antigen determining district of CDR3 (SEQID NO:5) and coding IGH (V) 1-18.In some embodiments; The invention provides isolating polynucleotide molecule; Said polynucleotide molecule coding specificity combines the humanized antibody of human DLL4 epi-position; Said epi-position is combined to form by human DLL4N-stub area (SEQ ID NO:27) and human DSL (SEQ ID NO:26), and wherein said polynucleotide molecule is selected from by (a) and the group (b) formed: the polynucleotide molecule that (a) is the aminoacid sequence of coding SEQ IDNO:6, SEQ ID NO:7 or SEQ ID NO:8; (b) be under stringent hybridization condition with the polynucleotide molecule of complementary strand (complement) hybridization of (a) polynucleotide molecule.In some embodiments; The invention provides isolating polynucleotide molecule; Said polynucleotide molecule coding specificity combines the humanized antibody of human DLL4 epi-position; Said epi-position is combined to form by human DLL4N-stub area (SEQ ID NO:27) and human DSL (SEQ IDNO:26), and wherein said polynucleotide molecule is selected from by following (a) and the group (b) formed: (a) SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:15; (b) under stringent hybridization condition with the polynucleotide molecule of the complementary strand hybridization of (a) polynucleotide molecule.

In some embodiments; The invention provides isolating polynucleotide molecule; Said polynucleotide molecule coding specificity combines the humanized antibody of human DLL4 epi-position; Said epi-position is combined to form by human DLL4N-stub area (SEQ ID NO:27) and human DSL (SEQ ID NO:26), and wherein said antibody comprises V _LThe district, said V _LThe district comprises the non-human antigen determining district of encode CDR1 (SEQ ID NO:9), CDR2 (SEQ ID NO:10) and CDR3 (SEQ ID NO:11) and the human variable framework region of coding IGK (V) 4-1.In some embodiments; The invention provides isolating polynucleotide molecule; Said polynucleotide molecule coding specificity combines the humanized antibody of human DLL4 epi-position; Said epi-position is combined to form by human DLL4N-stub area (SEQ ID NO:27) and human DSL (SEQ ID NO:26), and wherein said polynucleotide molecule is selected from by following (a) and the group (b) formed: (a) polynucleotide molecule of the aminoacid sequence of coding SEQ ID NO:12; (b) under stringent hybridization condition with the polynucleotide molecule of complementary strand (complement) hybridization of (a) polynucleotide molecule.In some embodiments; The invention provides isolating polynucleotide molecule; Said polynucleotide molecule coding specificity combines the humanized antibody of human DLL4 epi-position; Said epi-position is combined to form by human DLL4N-stub area (SEQ ID NO:27) and human DSL (SEQ ID NO:26), and wherein said polynucleotide molecule is selected from by following (a) and the group (b) formed: (a) SEQ ID NO:16; (b) under stringent hybridization condition with the polynucleotide molecule of the complementary strand hybridization of (a) polynucleotide molecule.

In some embodiments, the invention provides the expression vector that comprises isolating polynucleotide molecule of the present invention.In some embodiments, the invention provides the host cell that comprises the expression vector that contains isolating polynucleotide molecule of the present invention.

In some embodiments, the invention provides treatment patient's method for cancer, said method comprises the disclosed humanized antibody of the present invention to this patient's administering therapeutic significant quantity.In some embodiments, said cancer comprises breast cancer, colorectal carcinoma, lung cancer, carcinoma of the pancreas, prostate cancer or Head and Neck cancer.

In some embodiments; The invention provides and comprise the test kit that container reaches wherein contained compsn; Wherein said compsn comprises the disclosed humanized antibody of the present invention, and further comprises the package insert (package insert) that the indication said composition can be used to treat cancer.

In addition, can use various techniques known in the art to come the directly complete human antibodies of preparation.Can produce through the human bone-marrow-derived lymphocyte of the immortalization of external immunity or from can generate to the antibody of target antigen through immune body, 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 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 in the transgenic mice that contains the human immunoglobulin gene seat, make human antibodies, said human immunoglobulin gene seat can be through immune all forming members that generate human antibodies under the situation that does not have endogenous immunoglobulin to generate.This method is at USP 5,545, description arranged in 807,5,545,806,5,569,825,5,625,126,5,633,425 and 5,661,016.

The bi-specific antibody of specific recognition cancer stem cell affinity tag is also contained in the present invention.Bi-specific antibody is can specific recognition and combine the antibody of at least two kinds of different epi-positions.Said different epi-position (for example can be in same a part; Same cancer stem cell affinity tag polypeptide) perhaps is on the different molecules in; Make for example said antibody can specific recognition and combine the cancer stem cell affinity tag and for example 1) such as the effector molecule on TXi Baoshouti (for example CD3) or the Fc acceptor white corpuscles such as (for example CD64, CD32 or CD16), perhaps 2) cytotoxic agent that will be explained below.Bi-specific antibody can be complete antibody or antibody fragment.

Exemplary bi-specific antibody can combine 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 TXi Baoshouti molecule (for example; CD2, CD3, CD28 or B7) or the white corpuscles such as Fc acceptor of IgG on the combination of triggering molecule bonded arm, with the cytophylaxis mechanism of the cell that is conceived to express specific antigen.Can also use bi-specific antibody cytotoxic agent to be directed at the cell of expressing specific antigen.These antibody have antigen brachium conjunctivum and the arm that can combine cytotoxic agent or radionuclide chelators such as EOTUBE, DPTA, DOTA or TETA.The technology of preparation bi-specific 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 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 penetrance.Known have a multiple technology that is used to generate antibody fragment.Traditionally, these fragments obtain (for example, Morimoto etc., 1993, Journal ofBiochemical andBiophysical Methods 24:107～117 through the proteolytic digestion of complete antibody; Brennan etc., 1985, Science, 229:81).In some embodiments, antibody fragment produces through reorganization.Fab, Fv and scFv antibody fragment can be expressed justacrine in intestinal bacteria or other host cell, thereby can generate these fragments in large quantities.Can also from antibody phage discussed above library, isolate said antibody fragment.Antibody fragment can also be a USP 5,641 for example, the linear antibody described in 870, and can be monospecific antibody or bi-specific antibody.Those skilled in the art know that other technology that generates antibody fragment.

According to the present invention, there are various technology to be applicable to that generation has specific single-chain antibody (referring to United States Patent(USP) No. 4,946,778) to polypeptide of the present invention.In addition; Also there is several different methods to be applicable to and makes up Fab expression library (Huse etc.; Science 246:1275～1281 (1989)), fast and effeciently to identify Notch receptors ligand DLL4 or derivatives thereof, fragment or homologue had required specific monoclonal antibody Fab fragment.Can generate through art technology and contain the antibody fragment to the idiotype of polypeptide of the present invention, said antibody fragment includes but not limited to: (a) F (ab ') 2 fragments that generate of the gastric pepsin digestion through antibody molecule; (b) the Fab fragment that produces through reduction F (ab ') 2 segmental disulphide bridgeses; (c) through using papoid and reductive agent to handle the Fab fragment that antibody molecule produces; (d) Fv fragment.

Better is that particularly in the situation of antibody fragment, antagonist is modified to prolong its serum half-life.This can realize through for example following method: will remedy the receptors bind epi-position through the sudden change of appropriate area in the antibody fragment and be incorporated in the antibody fragment; Perhaps said 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 through DNA or peptide.

Allos coupling antibody also within the scope of the invention.Allos coupling antibody is made up of two covalently bound antibody.Proposed for example to utilize such antibody that immunocyte is targeted on harmful cell (United States Patent(USP) No. 4,676,980).Also be susceptible to, can have used known method in the synthetic proteins chemistry, comprised the said antibody of the external preparation of the method that relates to linking agent.For example, can use disulfide exchange reaction or make up immunotoxin through forming thioether bond.The example that is used for the suitable reagent of this purpose comprises imino-thiolate (iminothiolate) and methyl-4-sulfydryl butyryl imines ester (methyl-4-mercaptobutyrimidate).

For purposes of the present invention, should be understood that the variable region that can comprise any kind that the polypeptide that makes said antibody and human DLL4 is associated through the antibody of modifying.In this respect, said variable region can comprise or come from and can be induced and strengthen the Mammals of the humoral response and any kind of the Tegeline that produces anti-required taa.Like this, can for example derive from the mankind, muroid, non-human primate (for example cynomolgus monkey, macaque etc.) or wolf through the variable region of modified antibodies.In some embodiments, the variable region of the Tegeline of warp modification and constant region all are human variable region and constant regions.In other embodiments, the variable region of consistency antibody (generally come from non-human source) can transform or the specificity customization with the bonding properties of improving this molecule or the immunogenicity that reduces this molecule.In this respect, can make the useful variable region humanization of the present invention through the aminoacid sequence that comprises introducing or this variable region is changed.

The displacement of part at least through one or more CDR can change the variable region in heavy chain and the light chain simultaneously, and if desired, can carry out said change through displacement of part frame district and sequence variation.Although CDR can come from the classification of the antibody that therefrom obtains framework region or or even the identical antibody of subclass, should see that also CDR can come from different classes of antibody, preferably come from the antibody of different plant species.The whole CDR that maybe not need be used for from the donor variable region 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.Know United States Patent(USP) No. 5,585, after the explanation that provides in 089,5,693,761 and 5,693,762, those skilled in the art have the ability through implementing conventional experiment or obtaining the functional antibodies that immunogenicity reduces through the trial and error method of testing fully.

Though changed the variable region; But those skilled in the art should recognize; Antibody through modification of the present invention will comprise such antibody or its immunoreactivity fragment; Wherein, When the antibody that has approximate identical immunogenicity, comprise natural or unaltered constant region is compared, thereby at least a portion in the one or more constant zones in the said antibody has changed by deletion or through alternate manner required biochemical characteristic is provided, like the serum half-life of enhanced tumor-localizing or shortening.In some embodiments, the constant region of the antibody of warp modification comprises human constant region.The modification that the constant region compatible with the present invention carried out is included in has one or more amino acid whose interpolations, disappearance or replacement in one or more territories.That is, the antibody through modification 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 embodiments more of the present invention, can expect in the constant region of modifying excalation or lack one or more territories fully.In some embodiments, comprise territory disappearance construct or the variant (Δ CH2 construct) of wherein having removed whole C H2 territory through the antibody of modifying.The constant zone of being saved in some embodiments, 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 replace.

Except their configuration, known in the art is that constant region mediates several effector functions.For example, the C1 component of complement and antibody combines the complement activation system.The conditioning and the cracking of complement activation pair cell pathogenic agent are important.Complement activation also stimulates Inflammatory response, and can participate in the autoimmunization hypersensitivity.In addition, antibody combines with cell through 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.The combining of Fc acceptor on antibody and the cell surface can be triggered many important and various biological answer-replies; Comprise that the antibody sandwich particulate is engulfed and destruction, the removing of immunocomplex, the cracking (the cell-mediated cytotoxicity that is called the antibody dependence, or ADCC) that the killer cell antagonist encapsulates target cell, the release of inflammatory mediators, the placenta that Tegeline 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 do not limit the scope of the invention, it is believed that the antibody of the constant region that comprises warp modification as described herein can provide altered effector function, this has influenced the biological characteristics of institute's administration of antibodies conversely again.For example, the disappearance in constant zone or inactivation (through point mutation or alternate manner) can reduce the antibody of warp modification and the combining of Fc acceptor in the circulation, strengthen the location of tumour thus.In other situation, be that constant region is modified and regulated complement and combine and reduce serum half-life and the cytotoxic non-specific associating of link coupled thus with the present invention coincide.In addition, other that can use constant region modified and eliminated disulfide linkage or oligosaccharides part, thereby makes and can improve antigen-specific or antibody is flexible, therefore can add strong fix.Similarly, can use biological chemistry well known to those skilled in the art or molecular engineering technology to come easily to realize the modification of constant region being carried out according to the present invention.

Should be noted in the discussion above that the hinge area that can directly be fused to the antibody of corresponding warp modification through the antibody of modifying by transformation and with the CH3 territory.In other construct, possibly hope provides the peptide introns at hinge area with between the CH2 that modifies and/or CH3 territory.For example, can express the consistency construct, wherein the CH2 territory lacks, and remaining CH3 territory (modification or unmodified) is connected on the hinge area through 5～20 amino acid introns.Can increase that such introns for example keep free with the controlling 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 situation, can confirm the unwanted immunne response that the amino acid introns have immunogenicity and can bring out anti-said construct.Therefore, any introns that are added to said construct are non-immunogenicity relatively, if perhaps can keep the required biochemical property through the antibody of modifying, even can save introns fully.

Except lacking whole constant zone, should be understood that, can antibody of the present invention be provided through the replacement of excalation or perhaps a small amount of or even single amino acids.For example, the sudden change of the monamino acid in institute's favored area in CH2 territory possibly be enough to significantly reduce the location that Fc combines and strengthen thus tumour.Similarly, possibly hope to lack simply that part of the may command in one or more constant zones effector function (for example complement CLQ combines) to be regulated.The selected characteristics (serum half-life) that this excalation of constant region can be improved antibody keeps the integrity with other required function of this constant zone association simultaneously.And, mention one or more amino acid whose sudden change that can be through can improving gained construct characteristic or replace the constant region of modifying disclosed antibody indirectly like preceding text.In this respect, might be able to destroy the activity (for example Fc combines) that provides by conservative binding site, basic simultaneously configuration and the immunogenicity characteristic that keeps through the antibody of modifying.Some embodiment can comprise the one or more amino acid of interpolation to constant region, to strengthen the function of desired characteristic such as effector, the absorption of more cytotoxin or glucide is provided perhaps.In such embodiment, possibly hope to insert or duplicate specific sequence from selected constant zone.

The present invention also comprises basic and chimeric antibody, humanized antibody and the human antibodies of this paper proposition or their antibody fragment homologous variant and equivalent.These variants or equivalent can contain the for example conservative sudden change that replaces, and promptly one or more amino acid are by similar aminoacid replacement.For example; Conservative replacement is meant that an amino acid is by same big type of another interior aminoacid replacement; For example an acidic amino acid is replaced by another acidic amino acid, and a basic aminoacids is replaced by another basic aminoacids, and perhaps a neutral amino acids is replaced by another neutral amino acids.The substituted purpose of conserved amino acid is known in the art.

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 (the 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 said immune conjugate comprises for example methotrexate, Zorubicin, Dx, melphalan, ametycin, TV, daunorubicin or other intercalator.Available enzyme activity toxin and fragment thereof comprise diphtheria A chain; The non-binding active fragments of diphtheria toxin; The exotoxin A chain; Ricin A chain; Abrin A chain; The plain A chain of enlightening not; α-Zhou Qujunsu (α-sarcin); Tung oil tree albumen (Aleurites fordii protein); The China pink toxalbumin; Dyers' grapes (Phytolacaamericana) albumen (PAPI; PAPII and PAP-S); Momordica charantia inhibitor (momordica charantiainhibitor); Curcin; Crotin; Saponaria officinalis suppressor factor (sapaonaria officinalisinhibitor); White tree toxalbumin; Silk splits albumen (mitogellin); Restrictocin; Phenomycin; Enomycin; And trichothecene (tricothecene).In some embodiments, can use multiple known sequestrant or guide in the label any with said antibody and ri coupling, ri for example has ⁹⁰Y, ¹²⁵I, ¹³¹I, ¹²³I, ¹¹¹In, ¹⁰⁵Rh, ¹⁵³Sm, ⁶⁷Cu, ⁶⁷Ga, ¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁸⁶Re with ¹⁸⁸Re.In other embodiments, the disclosed compsn of the present invention can comprise with medicine, prodrug or like lymphokine link coupled antibody such as Interferon, rabbit.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), imino-THTP (IT; Iminothiolane), the difunctionality verivate of imines ester (like diimine for dimethyl adipate HCL), active ester (like disuccinimidyl suberate), aldehyde (like LUTARALDEHYDE), double azido compound (like two (to the azido benzoyl base) hexanediamine), two-tetroxide derivative (like two-(pyrazine formyl radical)-quadrols), vulcabond are (like methylene phenyl 2; The 6-vulcabond) and dual-active property fluorine cpd (as 1; 5-two fluoro-2, the 4-dinitrobenzene).Can also use the conjugate of antibody and one or more small molecules toxin, said small molecules toxin for example has an active verivate of toxin for calicheamycin, maytansinol (maytansinoid), single-ended born of the same parents' verticillium toxin (trichothene) and CC1065 and these toxin.In some embodiments, can be with compound through antibody and other immunocompetence part (for example antibody or its fragment) modified, wherein resulting molecule can combine oncocyte and simultaneously like effect cells such as T cells.

No matter obtain how useful amount, can use antibody of the present invention with in numerous coupling forms (that is immune conjugate) or the not coupling form any one.As selection, antibody of the present invention can use to utilize the natural immunology defense of study subject with non-coupling form or " exposing " form, comprises that the cytotoxicity (CDC) that complement relies on eliminates malignant cell with the cytotoxicity (ADCC) of antibody dependence.Select to use coupling or not the link coupled modified antibodies will depend on type and residing stage, the use of assisting therapy (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 through any means known in the art.Operable immunoassay includes but not limited to use such as the BIAcore analytical method; The facs analysis method; Immunofluorescence technique; Immunocytochemical method; The Western blotting; Radioimmunoassay; ELISA; " sandwich " immunoassay; Immunoprecipitation assay; The precipitin reaction method; The GDP reaction method; The immunodiffusion(ID) assay method; Agglutination assay; The complement fixation(CF) assay method; The immunoradiometric assay(IRMA) assay method; The competitiveness and the noncompetitive of technology such as fluorescence immunoassay and albumin A immunoassay are measured system.Such assay method be in this area routine and known method (see for example volume such as Ausubel, 1994, Current Protocols in Molecular Biology; The 1st volume, John Wiley&Sons, Inc.; New York all introduces this paper in this mode through reference with it).

In some embodiments, use ELISA to confirm the immunologic opsonin of the antibody of anticancer stem cell labeling thing.The ELISA assay method comprises preparation antigen, with the plate hole of antigen coated 96 hole microtiter plates; But in plate hole, add the antibody of the anticancer stem cell labeling thing that is coupled to detection compound such as enzymatic substrate (for example horseradish peroxidase or SEAP), incubation for some time is also detected antigenic existence.In some embodiments, but the antibody of anticancer stem cell labeling thing not with the detection compound coupling, but in plate hole, add and the second coupling antibody that can discern the antibody of this anticancer stem cell labeling thing substitutingly.In some embodiments, need not antigen coated plate hole, but but can and after in the plate hole that encapsulates, adding antigen, add and detection compound link coupled SA with the antibody sandwich plate hole of anticancer stem cell labeling thing.It will be appreciated by those skilled in the art that and to change parameter and strengthen signal to be detected and other ELISA known in the art changes and (for example sees volumes such as Ausubel, 1994; Current Protocols in Molecular Biology; The 1st volume, John Wiley&Sons, Inc.; NewYork is in 11.2.1).

Can confirm the dissociation rate of antibody and the antigenic binding affinity of cancer stem cell affinity tag and antibody-AI through the competitive binding assay method.An example of competitive binding assay method is a radioimmunoassay, and this method is included under the ever-increasing situation of amount of unlabelled antigen, will be through the antigen of mark (for example ³H or ¹²⁵I) or its fragment or variant and interested antibody incubation, detect then and be attached to antigenic antibody through mark.Can confirm the avidity and combination dissociation rate of the antibody of anticancer stem cell labeling thing through the scatchard mapping analysis by data.In some embodiments, use the BIAcore dynamic analysis to confirm the combination and the speed of dissociating of the antibody of anticancer stem cell labeling thing.The BIAcore dynamic analysis comprises analyzing and is fixed with combining and dissociating of the antigenic chip of cancer stem cell affinity tag on antibody and the surface.

In some embodiments, separated polynucleotide are contained in the present invention, and this polynucleotide encoding contains antibody or its segmental polypeptide of anti-human DLL4.Therefore, term " polynucleotide of coded polypeptide " is contained the polynucleotide of the encoding sequence that only comprises this polypeptide and is comprised the extra encoding sequence and/or the polynucleotide of non-coding sequence.Polynucleotide 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 polynucleotide of fragment, analogue and verivate of encoding.The variant of polynucleotide can be the variant that the non-natural of the naturally occurring allele variant of these polynucleotide or these polynucleotide exists.In some embodiments, polynucleotide can have such encoding sequence, and this encoding sequence is the naturally occurring allele variant of the encoding 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 Nucleotide still do not have the function of the coded polypeptide of material change that has of polynucleotide sequence.

In some embodiments; Polynucleotide comprise the encoding sequence of mature polypeptide, and this encoding sequence is blended in same reading frame and helps the for example polynucleotide of expression and secrete polypeptide (for example as the leader sequence of control polypeptide from the secretion sequence of transit cell fortune) in host cell.Polypeptide with leader sequence is a kind of precursor protein, and the leader sequence that is had can be by the host cell excision to form the polypeptide of mature form.Polynucleotide such precursor protein of can also encoding, this precursor protein is that maturation protein adds 5 extra ' amino-acid residue.Maturation protein with precursor sequence is a kind of precursor protein, and is this proteic inactive form.After the excision precursor sequence, what stay promptly is to have active maturation protein.

In some embodiments, polynucleotide comprise the encoding sequence of mature polypeptide, and this encoding sequence is blended in same reading frame can be used for the for example flag sequence of the coded polypeptide of purifying.For example; In the situation of host bacterium; This flag sequence can be the mature polypeptide 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 nucleotide sequence that this nucleic acid molecule had contains the antibody of anti-human DLL4 with coding or the polynucleotide of its segmental polypeptide have at least 80% identity, at least 85% identity, at least 90% the same sex, at least 95% identity; In some embodiments, has at least 96%, 97%, 98% or 99% identity.

Have with " identity " of contrast nucleotide sequence polynucleotide and be meant for for example at least 95% nucleotide sequence; Except polynucleotide sequence can comprise at the most the Nucleotide of 5 point mutation/100 contrast nucleotide sequence, the nucleotide sequence of polynucleotide was identical with control sequence.In other words; In order to obtain to have and the identity of contrast nucleotide sequence polynucleotide at least 95% nucleotide sequence; Have 5% Nucleotide to lack in the control sequence at the most or replaced by other Nucleotide, the Nucleotide 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 N-terminal position or the C-terminal position of contrast nucleotide sequence; The perhaps optional position between these terminal positions; Perhaps individually be dispersed among the Nucleotide of control sequence, perhaps exist in the control sequence with one or more adjacent groups.

As a kind of practical situation, (Wisconsin Sequence Analysis Package is used for the 8th version of Unix can to use known computer program such as Bestfit program; GeneticsComputer Group; University Research Park, 575Science Drive, Madison; WI 53711); Whether conventional definite any specific nucleic acid molecule has at least 80% identity, at least 85% identity, at least 90% identity with control sequence, in some embodiments, whether has at least 95%, 96%, 97%, 98% or 99% identity.Bestfit uses local homology's algorithm of Smith and Waterman (Advances in Applied Mathematics 2:482489 (1981)) to find the best homology fragment between the two sequences.When using Bestfit or other sequence alignment program confirms that whether a specific sequence for example has 95% identity with control sequence of the present invention arbitrarily, can list at the contrast nucleotides sequence of total length and calculate identity per-cent and allow total 5% the homology breach at the most of Nucleotide that accounts for control sequence thereby parameter can be set.

The polynucleotide variant can contain in coding region and/or non-coding region and changes.In some embodiments, the polynucleotide variant contains character or the active variation that produces reticent replacement, interpolation or disappearance but do not change coded polypeptide.In some embodiments, utilize the degeneracy of genetic codon to replace the generation nucleotide variants by silence.Can for example optimize the codon expression (human mRNA's codon being changed into the codon of host bacterium such as intestinal bacteria preference) that is used for specific host and produce the polynucleotide variant according to various reasons.

Polypeptide of the present invention can be antibody or its segmental recombinant polypeptide, natural polypeptides or the synthetic polypeptide that comprises anti-human DLL4.What should know in this area is can change aminoacid sequences more of the present invention and proteic structure of not obvious influence or function.Therefore, the present invention also comprises the polypeptide variants that demonstrates primary activity or comprises the polypeptide variants that resists the proteic antibody of human DLL4 or its segmental zone.Such two mutants comprises that disappearance, insertion, inversion, repetition and typical case replace (typesubstitution).

Polypeptide and analogue can be by further modification to contain extra chemical part (proteic non-common part).These derivative moieties can improve proteic solubleness, biological half time or absorptivity.Said part can also reduce or eliminate any required spinoff 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.

Isolated polypeptide as herein described can be through any appropriate means preparation known in the art.Said method comprises from the albumen direct synthesis technique to the dna sequence dna that makes up the separated peptide sequence of coding and with these sequences and is expressed in suitable expressing through transforming the host.In some embodiments, utilize recombinant technology to come the constructed dna sequence through the dna sequence dna of separation or the interested wild-type protein of composite coding.Optional is to come the said sequence of mutagenesis so that the functional analogue of this sequence to be provided through site-directed mutagenesis.See for example Zoeller etc., Proc.Nat ' l.Acad.Sci.USA 81:5662～5066 (1984) and United States Patent(USP) No. 4,588,585.

In some embodiments, use oligonucleotide synthesizer to make up the dna sequence dna of the interested polypeptide of coding through chemosynthesis.Can design such oligonucleotide based on required amino acid sequence of polypeptide and those codons of selecting to produce the host cell preference of interested recombinant polypeptide with rice.Can the application standard method come the isolating polynucleotide sequence of the interested isolated polypeptide of composite coding.For example, can use complete aminoacid sequence to make up the gene (back-translated gene) of retroversion.In addition, can synthesize the DNA oligomer that contains the nucleotide sequence that is useful on the isolating specific polypeptide of coding.Several short oligonucleotide that for example, can synthesize the each several part of the required polypeptide that is used to encode connect then.Each bar oligonucleotide contains usually and is useful on 5 of complementary assembling ' or 3 ' protruding terminus.

In case assembling (through synthetic, site-directed mutagenesis or other method); The polynucleotide sequence of interested isolating specific polypeptide of encoding will be inserted in the expression vector, and operably will be connected to be applicable to the expression control sequenc that said albumen is expressed in required host.Can confirm the exactness of assembling through nucleotide sequencing, estriction map, the expression of biologically active polypeptides in suitable host.As known in the field, in order in the host, to obtain the high expression level of rotaring redyeing gene, this gene must operably be connected to has transcribing and the accurate translation control sequence of function in selected expressive host.

The DNA that uses recombinant expression vector to increase and express coding cancer stem cell affinity tag polypeptide syzygy.Recombinant expression vector is reproducible DNA construct; It has the synthetic DNA fragment of coding cancer stem cell affinity tag polypeptide syzygy or bioequivalence analogue or the dna fragmentation in cDNA source, and said syzygy or bioequivalence analogue operably are connected to suitable the transcribing or the translational control element that comes from Mammals, mikrobe, virus or insect genes.Transcription unit generally comprises the assembly of following material: (1) has one or more gene elements of regulating and controlling effect in genetic expression; For example transcripting promoter or enhanser; (2) can be transcribed into mRNA and translate into proteic structure sequence or encoding sequence; (3) suitable transcribe and beginning and terminator sequence are opened in translation, the sequence of describing in detail like preceding text.These controlling elements can comprise the operon sequence that control is transcribed.Ability (generally providing) of duplicating in can the extra host of being introduced in and the selection gene of being convenient to discern transformant by replication orgin.When each DNA district is interrelated on function, they are operably connected.For example, when participating in polypeptide excretory precursor expression, the dna sequence dna of signal peptide (secreted leader sequence) operably is connected to the DNA of polypeptide at it; When the promotor control sequence was transcribed, promotor operably was connected to encoding sequence; Perhaps when the ribose binding site was positioned with the permission translation, the ribose binding site operably was connected to encoding sequence.Usually, operably connect to mean it is adjacent, and in the situation of secreted leader sequence, mean adjacent and be positioned at and read frame.Plan is used in the leader sequence that structural element in the yeast expression system comprises the proteic exocytosis that can make that host cell carries out being translated.As selection, under the situation of the recombinant protein of expressing no leading sequence or transit sequence, said structural element can comprise the terminal methionine residues of N-.This residue can be chosen wantonly from expressed recombinant protein excision so that final product to be provided subsequently.

Host's selection is depended in the selection of expression control sequenc and expression vector.Can adopt multiple 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 expression vector that is used for host bacterium comprises known bacterial plasmid (Tathagata comprises pCR 1, pBR322, pMB9 and verivate thereof from the plasmid of intestinal bacteria), wider host range plasmid (like M13) and thread single stranded DNA phage.

Be applicable to that expressing the proteic host cell of cancer stem cell affinity tag comprises prokaryotic organism, yeast, insect or the senior eukaryotic cell that receives suitable promotor control.Prokaryotic organism comprise gram negative organism or gram-positive organism, for example intestinal bacteria or genus bacillus (bacilli).Senior eukaryotic cell comprises the clone of having established in Mammals source as mentioned below.Also can adopt the cell free translation system.Said (the Cloning Vectors:A Laboratory Manual of clone and expression vector such as the Pouwels etc. that bacterium, fungi, yeast and mammalian cell host are suitable for; Elsevier; N.Y., 1985), introduce this paper through the mode of the reference disclosure that it is relevant at this.

Can also advantageously adopt various Mammalss or insect cell culture system to come express recombinant protein.Can be in mammalian cell express recombinant protein because such albumen generally can and have complete function by correctly folding, suitable modification.The example of suitable mammalian host cell line comprises (the Cell 23:175 like Gluzman; 1981) the COS-7 clone of described MK cells with can express other clone of suitable carrier, for example comprise L cell, C 127,3T3, Chinese hamster ovary (CHO), HeLa and bhk cell system.Mammalian expression vector can comprise and is connected to the non-transcribed element of treating expressing gene such as replication orgin, suitable promotor and enhanser and other 5 ' or 3 ' flank non-transcribed sequence and 5 ' or 3 ' non-translated sequence, like essential ribosome bind site, polyadenylation site, shear donor and acceptor site and transcription termination sequence.Be used for producing the summary (Bio/Technology 6:47 (1988)) that the baculovirus system of heterologous protein is seen Luckow and Summers at insect cell.

Can be according to any appropriate means purifying by the albumen that produces through the conversion host.Such standard method comprises chromatography (for example ion exchange chromatography, affinity chromatography and fractionation column chromatography), centrifugal, difference solubility method or is used for any other standard method of protein purification.Can be with combining affinity labels such as territory, influenza tunicle sequence (influenza coat sequence) and glutathione-S-transferase to be connected on the albumen to be easy to carrying out column purification through suitable affinity column such as six Histidines, SANMALT-S.Can also use technology such as proteolyze, nucleus magnetic resonance and x ray Laue method that isolating albumen is carried out physics characterizes.

The albumen thickening filtration device that for example, at first can use commercially available acquisition for example Amicon or Millipore Pellicon ultra filtration unit concentrates from the supernatant that recombinant protein is secreted into the system in the substratum.After enrichment step, can liquid concentrator be added in the suitable purifying matrix.As selection, can adopt anionite-exchange resin, for example gather matrix or base material that outstanding diethylamino ethyl (DEAE) group that attaches is arranged.Matrix can be acrylic amide, agarose, VISOSE, Mierocrystalline cellulose or in protein purification other type commonly used.As selection, can adopt cation-exchange step.Suitable cationite comprises the various insoluble matrixs that contain sulfo group propyl group or ethyloic.At last, can adopt one or more RPLC steps of use hydrophobicity RPLC (RP-HPLC) medium (for example having the outstanding methyl that attaches or the silica gel of other aliphatic group) to be further purified cancer stem cell albumen-Fc compsn.Also can adopt the various combinations of some or all aforementioned purification steps that the homology recombinant protein is provided.

Can be for example through the initial extraction from cell precipitation, carry out then one or morely concentrating, saltout, water-based IX or size exclusion chromatography step are come the recombinant protein that generates in the separation of bacterial culture.Can use performance liquid chromatography (HPLC) to carry out last purification step.Can come the used microorganism cells of broken express recombinant protein through any ordinary method, said method comprises circulating freezing resistance method, ultrasonic method, mechanical crushing method or uses the method for lysis agent.

The invention provides the method for the growth of the tumorigenicity cell that suppresses expression cancer stem cell affinity tag, this method is used the antibody of anti-cancer stem cell affinity tag as herein described.In some embodiments, the said inhibition method of growth of expressing the tumorigenicity cell of cancer stem cell affinity tag is included in and external said 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 affinity tag are cultivated in substratum, this substratum is added with the antibody of anti-expressed cancer stem cell affinity tag 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, hydrothorax or blood sample; And it is cultivated in substratum, this substratum is added with the antibody of anticancer stem cell labeling thing to suppress the growth of cell.

In some embodiments, the method for growth that suppress to express the tumorigenicity cell of cancer stem cell affinity tag comprises makes said cell contact with the antibody of anticancer stem cell labeling thing in vivo.In some embodiments, the tumorigenicity cell is contacted with the antibody of anticancer stem cell labeling thing.For example, the xenotransplantation body of expressing the cancer stem cell affinity tag is grown in the mouse (for example NOD/SCID mouse) of immunocompromised, this mouse is used the antibody of anticancer stem cell labeling thing to suppress growth of tumor.In some embodiments; From for example isolating the cancer stem cell of expressing the cancer stem cell affinity tag patient's samples such as biopsy sample, hydrothorax or blood sample; And it is expelled in the mouse of immunocompromised, the antibody of then this mouse being used anticancer stem cell labeling thing is to suppress the growth of tumour cell.In some embodiments, when the tumorigenicity cell being imported in the animal or the antibody of using anticancer stem cell labeling thing in the short period of time afterwards to prevent growth of tumor.In some embodiments, after the tumorigenicity cell has grown into certain size, the antibody of anticancer stem cell labeling thing is used as therapeutant.

The present invention also provides the pharmaceutical composition of the antibody that comprises target cancer stem cell affinity tag.Said pharmaceutical composition can be used to suppress the cancer of growth of tumour cell and treatment human patients.

Through being prepared, the combination of antibody purification of the present invention and medicinal charge material (for example carrier, vehicle) is used to the preparation (Remington, The Science and Practice ofPharmacy (the 20th edition) Mack Publishing, 2000) preserving and use.Suitable medicinal charge material includes but not limited to non-toxicity buffer reagent, like phosphoric acid salt, Citrate trianion and other organic acid; Such as salt such as sodium-chlor; Inhibitor comprises xitix and methionine(Met); Sanitas (stearyl dimethyl benzyl ammonium chloride for example; Hexamethonium chloride; Benzalkonium chloride; Benzethonium chloride; Phenol, butanols or phenylcarbinol; Alkyl paraben is like methyl paraben or propylben; Pyrocatechol; Resorcinol; Hexalin; The 3-amylalcohol; And meta-cresol); Low molecular weight polypeptide (for example less than about 10 amino-acid residues); Albumen is like serum albumin, gelatin or Tegeline; Hydrophilic polymer is like Vinylpyrrolidone polymer; Amino acid is like glycocoll, Stimulina, l-asparagine, Histidine, l-arginine or Methionin; Glucide is like monose, disaccharides, glucose, seminose or dextrin; Sequestrant is like EDTA; Sugar is like sucrose, maltose alcohol, trehalose or Sorbitol Powder; The salify gegenion is like sodium; Metal complex (for example Zn-protein complex); And nonionogenic tenside, like TWEEN or polyoxyethylene glycol (PEG).

Can use pharmaceutical composition of the present invention to be used for topical therapeutic or whole body therapeutic with the any-mode in numerous modes.Using can be topical application (mucosal administration for example, comprise vagina is carried with rectum carry), like the employing transdermal patch, and the using of ointment, lotion, ointment, gelifying agent, drops, suppository, sprays, liquor and pulvis; Pulmonary administration (for example through powder or aerocolloidal suction or be blown into, comprises and uses using of atomizer; In the tracheae, in the nose, epidermis and transdermal administration); Orally administered; Or parenteral uses, and comprises intravenously, intra-arterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; Or encephalic (for example sheath is interior or Intraventricular) is used.

Said treatment preparation can be a unit dosage form.Said preparation comprises and is used for oral, parenteral administration, rectal administration or the tablet of using through suction, pill, capsule, pulvis, granule, the solution in water or non-aqueous media or suspension agent or suppository.In such as solids compsns such as tablets, can main active component be mixed with pharmaceutical carrier.Conventional compressing tablet composition comprises W-Gum, lactose, sucrose, Sorbitol Powder, talcum, Triple Pressed Stearic Acid, Magnesium Stearate, dicalcium phosphate or natural gum; And other thinner (for example water), contain preparation compsns before the solid of homogeneous mixture of The compounds of this invention or its non-toxicity pharmaceutical salts with formation.Then preparation compsns before the said consubstantiality is further divided into the unit dosage form of the above-mentioned type.Can carry out dressing to the tablet of said novel compsns, pill etc., perhaps carry out being mixed of alternate manner and the dosage form with long-acting advantage is provided.For example, said tablet or pill can comprise the inner composition that is encapsulated by outer component.And said two kinds of components can be separated by enteric layer, the effect that this enteric layer plays anti-disintegration and makes internal composition perhaps delay to discharge through stomach intactly.Have various materials can be used for said enteric layer or dressing, such material comprises the mixture of numerous polymeric acids and polymeric acid, and such material for example is shellac, the pure and mild FM of hexadecyl.

Pharmaceutical prepn comprises and liposome compound antibody of the present invention (Epstein etc., 1985, Proc.Natl.Acad.Sci.USA 82:3688; Hwang etc., 1980, Proc.Natl.Acad.Sci.USA77:4030; With USP 4,485,045 and 4,544,545).USP 5,013 discloses the liposome of the cycling time with prolongation in 556.Some liposome can evaporate through the anti-phase that use comprises the lipid composition of phosphatidylcholine, SUV and PEG deutero-phosphatidylethanolamine (PEG-PE) and prepare.Liposome can be extruded the liposome that obtains to have required diameter through the strainer with definite aperture.

Said antibody can also be encapsulated in the microcapsule.Such microcapsule can prepare through for example condensation technique or interfacial polymerization; (for example for example be respectively at the colloid drug delivery system; Liposome, albumin microsphere spheroid, micro emulsion, nano particle and Nano capsule) or macro emulsion in hydroxy-methyl cellulose or gelatin-microcapsule with gather (TEB 3K) microcapsule; As at Remington, described in TheScience and Practice 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 semipermeability matrix of the solid hydrophobic property polymkeric substance that contains said antibody, and said matrix is moulding article form (for example film or microcapsule).The example of sustained-release matrix comprises polyester, hydrogel (for example gather (methylacrylic acid-2-hydroxyethyl ester) or gather (vinyl alcohol)), polylactide (USP 3; 773,919), the multipolymer of L-L-glutamic acid and 7-ethyl-L-glutamate, nondegradation ethane-acetic acid ethyenyl ester, degradation property lactic acid-ethanol copolymer (like LUPRON DEPOT TM (the Injectable microspheres body of forming by lactic acid-ethanol copolymer and TAP-144), sucrose acetate isobutyrate and gather-D-(-)-3-hydroxybutyric acid).

In some embodiments, treatment comprises the mixture of co-administered antibody of the present invention and chemotherapeutics or multiple different chemotherapeutics.The treatment of carrying out with antibody can be before using chemotherapeutics, simultaneously or carry out afterwards.The chemotherapeutics that the present invention expects comprises and can be purchased the chemical substance or the medicine of acquisition that for example Dx, 5 FU 5 fluorouracil, cytosine arabinoside (" Ara-C "), endoxan, plug are for group, busulfan, cytotoxin (Cytoxin), taxol, methotrexate, cis-platinum, melphalan, vinealeucoblastine(VLB) and carboplatin known in the art.Use altogether and use successively co-administered can comprising; Said using altogether can be single using of pharmaceutical prepn of planting; Also can be to use using of preparation separately; Said using successively can be with the using of random order, but normally in the regular hour, uses successively, so that all promoting agents can be brought into play their BA simultaneously.The preparation of said chemotherapeutics and dosage regimen can be used according to the specification sheets of manufacturers, are perhaps rule of thumb confirmed by skilled practitioner.Said chemotherapeutic preparation and dosage regimen be at ChemotherapyService Ed., M.C.Perry, and Williams and Wilkins, Baltimore, Md. also has description in (1992).

In some embodiments of the present invention, said treatment relates to the co-administered antibody of the present invention and second therapeutical agent.Here used " second therapeutical agent " includes but not limited to the antibody of chemotherapeutic, radiotherapy, cytokine and anti-other taa.

In other embodiment, treatment comprises antibody of the present invention and radiotherapeutic co-administered.The treatment of carrying out with antibody can be before implementing radiotherapy, simultaneously or carry out afterwards.Can confirm to use said radiotherapeutic any dosage regimen according to skilled practitioner.

In other embodiment; Treatment can comprise the co-administered of antibody of the present invention and other antibody that resists other taas, and said other antibody includes but not limited to and EGF acceptor (EGFR) (

), erbB2 acceptor (HER2) (

) and VEGF (VEGF) ( ) bonded antibody.And treatment can comprise using of one or more cytokines, and this treatment can follow exenterate or the treatment doctor of cancer cells to think necessary other any treatment.

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

The invention provides and comprise antibody as herein described and can be in order to implement the test kit of methods described herein.In some embodiments, test kit comprises at least a antibody purification of the anticancer stem cell labeling thing that is arranged in one or more containers.In some embodiments, said test 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 is used for interpretation of result and any necessary software of displaying.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.

The disclosed embodiment of the present invention can be through further being explained with reference to following examples, and said embodiment has described the preparation of the disclosed antibody of the present invention and the method for use of the disclosed antibody of the present invention in detail.Those skilled in the art can know clearly, can under the situation that does not deviate from the present disclosure scope, carry out many changes to material and method.Identical Reference numeral in whole accompanying drawing all as much as possible in order to represent same or analogous part.Used singulative " ", the plural form that " perhaps " and " being somebody's turn to do " comprises referent in this paper and the appended claims, but clear from context point out except.Therefore, when for example, mentioning " a kind of antibody ", be meant the plural form or one or more antibody and the equivalent thereof that comprise said antibody, this equivalent is well known by persons skilled in the art.In addition, except as otherwise noted, otherwise all numerals of the amount of used expression composition, reaction conditions, purity, polypeptide and polynucleotide length or the like are all modified with speech " approximately " or " pact " in this specification sheets.Therefore, the numerical parameter described in this specification sheets and claims is the approximation that can required character according to the present invention changes.

Embodiment

The preparation of embodiment 1 mono-clonal DLL4 antibody and humanization DLL4 antibody

Antigen prepd

The recombinant polypeptide fragment for preparing the extracellular domain of human DLL4 is used antigen as Antibody Preparation.Use the standard recombinant dna technology to separate the polynucleotide of the 1st～No. 522 amino acid (SEQ IDNO:25) of encoding D LL4.These polynucleotide with N-end in the framework 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 cultures program make the recombinant insect cell (O ' Reilley etc. that express corresponding DLL4 polypeptide; Baculovirus expression vectors:A Laboratory Manual, Oxford:Oxford University Press (1994)).

The cutting that use cutting forecasting software SignalP 3.0 comes the endogenous signal sequence of simulating human DLL4, still actual body inscribe cutpoint possibility is difference owing to the amino acid coupling of arbitrary direction.The prediction cutting of DLL4 is between the 1st～No. 26 amino acid, so the DLL4 antigen protein comprises about No. 27 amino acid～No. 522 amino acid.Use albumin A and Ni++ chelating affinity chromatography from the insect cell conditioned medium, to be purified into antigen protein.Subsequently about 1mg/ml is dialysed, is concentrated into to purified antigen protein with PBS (pH=7), and asepticly be filtered into immunity and use preparation.

Immunity

Use standard technique with purified DLL4 antigen protein (Antibody Solutions; Mountain View, CA) immune mouse (n=3).Behind initial immunity about 70 days, adopt blood that ELISA and facs analysis come the individual mouse of examination to antigenic identification (following detailed description).Select two the highest animals of antibody titers to carry out last antigen booster immunization, isolate splenocyte afterwards and be used for the hybridoma preparation.With hybridoma in 96 orifice plates by 1 cells/well bed board, through ELISA and facs analysis the supernatant of each plate hole is directed against antigen protein and carries out examination.Select the high hybridoma of several antibody titerss and use enlarged culturing in the bottle in static cultivation.Use albumin A or Protein G agarose chromatography to be purified into antibody from the hybridoma supernatant.Purified monoclonal anti body and function FACS detects once more and carries out the isotype analysis to select IgG and IgM antibody.

Facs analysis

The proteic monoclonal antibody of identification n cell surface DLL4 in order to select to be generated by the hybridoma clone has adopted the FACs analysis.With the full length cDNA clone of encoding D LL4 and the expression vector cotransfection HEK293 cell of transfection affinity tag GFP.After the transfection 24～48 hours, collect in the suspension cell and on ice with the antibody of anti-DLL4 or in order to the contrast IgG incubation of detection background antibodies.Washed cell has the two anti-detections one of the anti-mouse of fluorescent chromophore to resist with coupling.Then through the FACS sorting through the cell of mark, with the antibody of the anti-DLL4 that identifies the proteic cell surface expression of specific recognition n cell surface DLL4.Monoclonal antibody 21M14 and the DLL4 (Fig. 1) of 21M18 identification on transfectional cell.The hybridoma cell line that produces muroid monoclonal antibody 21M18 is deposited in American type culture collection (AmericanType Culture Collection) (ATCC) on September 28th, 2007, and deposit number is PTA-8670.

Next utilize flow cytometry to confirm interactional ability between antibody interferes with DLL4 and the Notch of anti-DLL4.Will be with antibody or the reference protein-Fc+ control antibodies incubation of the antibody of HEK 293 cells of the stable transduction of DLL4cDNA and the anti-DLL4 of Notch1-EGF10-15-Fc+, Notch1-EGFlO-15-Fc+ control antibodies, the anti-DLL4 of reference protein-Fc+.Through PE-link coupled goat anti Fc antibody and Flow cytometry combining of Fc fusion rotein and the cell of expressing DLL4.Confirmed the bonded inhibition ability of the antibody of anti-DLL4 through the reduction of fluorescence intensity like this to Notch and DLL4.Shown in Fig. 2 A, observed the Notch bonded with rodent antibody 21M14 and 21M18 and suppressed, 21M12 does not then have.In addition, rodent antibody 21M18 specificity combines human DLL4 (but not combining muroid DLL4 specifically), and blocks combine (Fig. 2 B) of human DLL4 (but not muroid DLL4) and the cell of expressing Notch1.These data show, in the heterograft experiment, and the human DLL4 that expresses on the 21M18 target tumor cell but not expressed muroid DLL4 on the vascular system.

Epitope mapping

In order to identify the antibody of the specific region that can discern the DLL4 extracellular domain, carried out epitope mapping.Use standard recombinant dna technology has prepared the Mammals expression plasmid carrier that comprises the CMV promotor at the polynucleotide upper reaches that coding is blended in the nested serial deletion fragment of the proteic DLL4 extracellular domain of Fc.The same standard recombinant dna technology of using has prepared coding and has been blended in proteic segmental other construct of chimeric DLL4 as human and muroid DLL4 of Fc.That has also designed another series comprises specific amino acids substituted DLL4-fc fusion rotein.In the HEK of transient transfection 293 cells, express these recombination fusion proteins, and after transfection 24 hours～48 hours therefrom the collection condition substratum to be used for ELISA.DLL4 fusion rotein fragment is trapped on the plate that is coated with anti-human Fc antibody.The antibody that makes anti-DLL4 subsequently with interacted by bonded DLL4 fragment, and through subsequently with the incubation of the anti-mouse antibodies of HRP link coupled with detect the HRP activity and measure and combine (Fig. 3 A).As shown in Figure 3, contained epi-position in the 1st～No. 217 amino acid of muroid monoclonal antibody 21M14 and 21M18 identification DLL4.The motif (Tax etc., 1994, Nature 368:150-4) that is present in " DSL (δ/spination/delay-2 (Delta/Serrate/lag-2)) " territory by name in several Notch parts is contained in this zone.In addition, checked the mAb of anti-DLL4 to combine (Fig. 3 B) with the DLL4 fusion rotein is segmental through the western engram analysis.This work shows, when the DSL territory in amino acid/11 55-217 existed, 21M18 with DLL4 in the amino acid/11-154 human specificity took place and combines.This has shown the importance of this N-end sequence to the DLL4 function, and this importance is incognizant before this.With graphic form this work has been carried out concluding (Fig. 3 C), represented the combination of 21M18 or not combination with "+" or "-" respectively.Through the combination that combines further to have characterized 21M18 of check 21M18 and a series of DLL4 protein fragments (DLL4 territory 1-6), said a series of DLL4 protein fragments contain with corresponding muroid amino acid and exchange the amino acid whose specific amino acids replacement of human DLL4.Through the ELISA examination combinations of these fusion roteins to 21M18.Evaluation shows that it is important (shown in Fig. 3 D) that several positions combine 21M18.To (substituting Xie Ansuan, Xie Ansuan and proline(Pro)) in the amino acid position 68,69 and 71 or amino acid position 142 and 144 (substituting Methionin and L-Ala) has substituted DLL4 protein fragments, 21M18 to show combination to weaken.On the contrary, the antibody 21M21 of a uniqueness combines contained epi-position (Fig. 3 E) in the DSL district, but as shown in Figure 6, this antibody does not influence the function of DLL4, and this shows with DSL bonded antibody not mean that it is functional antibodies.

Chimeric antibody

After identifying the monoclonal antibody of specific recognition DLL4, modify these antibody to overcome human anti-mouse antibodies (HAMA) immunne response when using rodents antibody as therapeutical agent.In some embodiments, from the variable region that hybridoma separates the heavy chain and the light chain of selected monoclonal antibody, and this variable region is connected to the IgG in the mammalian expression vector in the framework respectively through RT-PCR ₁Heavy chain and κ constant region of light chain.As selection, use such as human Ig expression vector such as TCAE 5.3 grades, this carrier contains the IgG on same plasmid ₁Heavy chain and κ constant region of light chain gene (Preston etc., 1998, Infection&Immunity 66:4137～42).To encode then chimeric heavy chain and light chain the expression vector cotransfection to Chinese hamster ovary (CHO) cell with the preparation chimeric antibody.The immunoreactivity and the avidity that compare chimeric antibody and parent's rodent antibody through ELISA and FACS.

Humanized antibody

In some embodiments, the humanized antibody that has prepared anti-DLL4.Use degenerate pcr from hybridoma cell strains, to separate variable domain and the order-checking of muroid monoclonal antibody 21M18; This process is basic like Larrick; J.M. etc. (1989, Biochem.Biophys.Res.Comm.160:1250) and Jones, S.T. and Bendig; M.M. (1991, Bio/Technology 9:88) are said.Choose subsequently and possibly be used for humanized human framework region with the structural similar human heavy chain and the conduct of light chain variable framework region of parent 21M18 antibody aminoacid sequence.In order to identify alternative human framework region, use BLAST that the human sequence who stores among the Genbank is searched for, the V that is relatively predicted by 21M18 _HAnd V _LMuroid variable domain encoded protein matter sequence and the human sequence antibody of encoding by expressed human cDNA.Utilize this method, (for example genbank AY393019 DC295533) does further to analyze in design heavy chain framework to select expressed human cDNA sequence.

Possible importance to the amino acid difference between alternative humanization framework heavy chain and the parent's muroid monoclonal antibody 21M18 heavy chain is assessed, and whether each locational difference is helped the correct folding judgement of making of antibody 21M18.The crystalline structure of having resolved through studying other antibody fragment (for example Trakhanov etc., Acta Crystallogr D Biol Crystallogr, 1999, the structure of the fab 2E8 described in the 55:122-28) instructs this analysis.Use comprises Jmol, fast the computer software of PDB and Pymol is simulated structure.That need to consider has, the possibility that the interaction between potential impact, heavy chain variable domain and the light chain variable territory of the accumulation of βZhe Die framework, degree that amino acid side chain is exposed to solvent and amino acid is influenced the CDR loop mapping at the amino acid on the given position.According to this analysis, chemosynthesis with five alternative V of IgG 2 constant region framework endomixis _HChain.Said alternative heavy chain comprises: i) contain according to the analysis that possibly influence of 21M18 combined function and selected substituted functional human class framework and ii) parent's rodent antibody 21M18CDR (SEQ ID NO:1,2 and 5) at synthetic framework region.

Similar is, has identified the amino acid difference between selected people's class framework IGK (V) 4-1 light chain and the parent's muroid monoclonal antibody 21M18 light chain, subsequently whether each locational difference is helped the correct folding judgement of making of antibody 21M18.According to this analysis, chemosynthesis five alternative V _LChain.First alternative light chain comprises: i) complete IGK (V) 4-1 people's class framework and ii) parent's rodent antibody 21M18CDR (SEQ ID NO:9,10 and 11).Other four alternative light chains comprise: the human framework region of IGK (V) 4-1 and the ii) parent's rodent antibody 21M18CDR (SEQ ID NO:9,10 and 11) that i) remain with the muroid residue of the 21M18 that quantity increases progressively in the framework region.

Test the functional of every kind of alternative variant humanization heavy chain and light chain through cotransfection in mammalian cell.With each and muroid 21M18 light chain cdna cotransfection in above-mentioned five kinds of alternative humanization 21M18 heavy chains in HEK 293 cells, and the DLL4 antigen-binding activity through ELISA test condition substratum.Select to show the strongest bonded 21M18 heavy chain variant.This variant-" 21M18H2 "-except containing muroid CDR, also 6 frame positions in the Vh framework contain replacement, and said 6 frame positions are Karbate position 16,20,27,28,38 and 48 (Fig. 4 A).Subsequently with each cotransfection in 21M18H2 humanization heavy chain and the five kinds of alternative humanization light chains in HEK 293 cells, and through ELISA once more the antigen of test condition substratum combine.Find independent light chain variant-" 21M18L2 "-demonstrate better combination than other alternative light chain, it remains with muroid residue (Fig. 5) in Karbate position 22 and 36.

Next, changed independent cysteine residues among the CDR2H2 (SEQ ID NO:2).Particularly, the cysteine residues with Karbate position 52a is modified to Serine (variant H7; SEQID NO:3) residue or Xie Ansuan (variant H9; SEQ ID NO:4) residue, thereby two heavy chain variants of synthetic H2.With these heavy chains and L2 cotransfection in HEK 293 cells, and test condition substratum once more.Two variants (21M18H7L2 and 21M18H9L2) show all that through ELISA specific antigens combines.Thereby 21M18 heavy chain CDR2 comprises SEQ ID NO:2,3, or 4, wherein the residue of Karbate position 52a comprises cysteine residues, serine residue or Xie Ansuan residue.

In some embodiments, the humanized antibody that has prepared anti-DLL4.Use degenerate pcr from hybridoma cell strains, to separate variable domain and the order-checking of muroid monoclonal antibody 21M18; This process is basic like Larrick; J.M. etc. (1989, Biochem.Biophys.Res.Comm.160:1250) and Jones, S.T. and Bendig; M.M. (1991, Bio/Technology 9:88) are said.Choose human heavy chain the most similar and light chain variable framework region subsequently as being used for humanized human framework region with parent 21M18 antibody aminoacid sequence.In order to identify the most similar said human framework region, use BLAST that the human genomic sequence of storing among the Genbank is searched for, the V that is relatively predicted by 21M18 _HAnd V _LMuroid variable domain encoded protein matter sequence and by the Ig variable domain of Human genome group coding.Utilize this method, selection IGH (V) I-18 is as the human heavy chain framework region and select IGK (V) 4-1 as human light chain framework region.

Amino acid difference between selected people's class framework IGH (V) I-18 heavy chain and the parent's muroid monoclonal antibody 21M18 heavy chain is identified subsequently whether each locational difference is helped the correct folding judgement of making of antibody 21M18.The crystalline structure of having resolved through studying other antibody fragment (for example Trakhanov etc., Acta Crystallogr D Biol Crystallogr, 1999, the structure of the fab 2E8 described in the 55:122-28) comes instructing this analysis.Use comprises Jmol, fast the computer software of PDB and Pymol is simulated structure.That need to consider has: the possibility that the interaction between potential impact, heavy chain variable domain and the light chain variable territory of the accumulation of βZhe Die framework, degree that amino acid side chain is exposed to solvent and amino acid is influenced the CDR loop mapping at the amino acid on the given position.According to this analysis, chemosynthesis with five alternative V of IgG 2 constant region framework endomixis _HChain.Article one, alternative heavy chain comprises: i) complete IGH (V) 1-18 people's class framework and ii) parent's rodent antibody 21M18CDR (SEQ ID NO:1,2 and 5).Other four alternative heavy chains comprise i) remain with the human framework region of IGH (V) 1-18 of the 21M18 muroid residue that quantity increases progressively and ii) parent's rodent antibody 21M18CDR (SEQ IDNO:1,2 and 5) in the framework region.

Similar is, has identified the amino acid difference between selected people's class framework IGK (V) 4-1 light chain and the parent's muroid monoclonal antibody 21M18 light chain, subsequently whether each locational difference is helped the correct folding judgement of making of antibody 21M18.According to this analysis, chemosynthesis five alternative V _LChain.Article one, alternative light chain comprises: i) complete IGK (V) 4-1 people's class framework and ii) parent's rodent antibody 21M18CDR (SEQ ID NO:9,10 and 11).Other four alternative light chains comprise: i) remain with the human framework region of IGK (V) 4-1 of the 21M18 muroid residue that quantity increases progressively and ii) parent's rodent antibody 21M18CDR (SEQ ID NO:9,10 and 11) in the framework region.

Test the functional of every kind of alternative variant humanization heavy chain and light chain through cotransfection in mammalian cell.With each and muroid 21M18 light chain cdna cotransfection in above-mentioned five kinds of alternative humanization 21M18 heavy chains in HEK 293 cells, and the DLL4 antigen-binding activity through ELISA test condition substratum subsequently.Select to show the strongest bonded 21M18 heavy chain variant.This variant-" 21M18H2 "-except containing muroid CDR, also contain the muroid residue at 5 frame positions, said 5 frame positions are Karbate position 20,28,38,48 and 69 (Fig. 4).Subsequently with each cotransfection in 21M18H2 humanization heavy chain and the five kinds of alternative humanization light chains in HEK 293 cells, and through ELISA once more the antigen of test condition substratum combine.Independent light chain variant-" 21M18L2 " of test discovery-shown better combination than other alternative light chain, it remains with muroid residue (Fig. 5) in Karbate position 22 and 36.

Next, changed independent cysteine residues among the CDR2H2 (SEQ ID NO:2).Particularly, the cysteine residues with Karbate position 52a is modified to Serine (variant H7; SEQID NO:3) residue or Xie Ansuan (variant H9; SEQ ID NO:4) residue, thereby two heavy chain variants of synthetic H2.With these heavy chains and L2 cotransfection in HEK 293 cells, and test condition substratum once more.ELISA shows all specificity conjugated antigens of two variants (21M18H7L2 and 21M18H9L2).Thereby 21M18 heavy chain CDR2 comprises SEQ ID NO:2,3, or 4, wherein the residue of Karbate position 52a comprises cysteine residues, serine residue or Xie Ansuan residue.

Further characterized humanized antibody 21M18 subsequently.Particularly, use Biacore to confirm binding affinity by the humanized antibody 21M18 of albumin A chromatography purification.Through measuring, for 21M18 variant H2L2, avidity is about 0.33nM.

Humanized antibody 21M18 is deposited in ATCC (preservation on May 10 in 2007, the ATCC deposit number of 21M18H9L2 is PTA-8427, and the ATCC deposit number of 21M18H7L2 is PTA-8425).

Human antibodies

In some embodiments, but use display technique of bacteriophage to separate the human antibodies of the extracellular domain of specific recognition DLL4.Utilization contains the synthetic antibody libraries of human antibodies variable domain, according to the antigenic specificity of above-mentioned DLL4, high-affinity identification carrying out examination.Flank restriction site through uniqueness comes specially to exchange CDR box in this library to optimize antibody.To be cloned into through the human variable region of optimizing subsequently and contain IgG ₁In the Ig expression vector of heavy chain and κ light chain, to be used at Mammals Chinese hamster ovary celI express human antibody.

Embodiment 2: the external test of estimating the antibody of anti-DLL4

Present embodiment has been described representational external test, and this external test is used to test anti-DLL4 and the antibody on cell proliferation, Notch pathway activation and the Cytotoxic activity that generate.

Proliferation assay

Use Taqman to analyze the expression of the DLL4 of quantitative different carcinoma clone.In 96 hole tissue culture minitype plates, with 10 ⁴The density of individual cells/well will be accredited as the clone bed board of expressing DLL4, and let it launch 24 hours.Subsequently in containing the fresh DMEM of 2%FCS again with cell cultures 12 hours, will resist this moment the antibody of DLL4 and control antibodies under the situation of existence 10 μ mol/LBrdU, to be added to substratum.Behind the BrdU mark, remove substratum, under the room temperature with cell in ethanol fixing 30 minutes, and with monoclonal antibody (BMG 6H8 clone, the Fab fragment) reaction of the anti-BrdU of px link coupled 90 minutes.Substrate is developed the color in containing the solution of TMB, after 15 minutes with the 1mol/L H of 25 μ l ₂SO ₄Stop.Read plate appearance (UV Microplate Reader with automatic ELISA; Bio-Rad Laboratories, Richmond, CA) the filter measurement color reaction of use 450nm.All experiments all repeat 3 times.Through coming relatively to confirm that with control antibodies the antibody of anti-DLL4 suppresses the ability of cell proliferation.

Pathway activation is measured

In some embodiments, in the external activatory ability of having confirmed the antibody blocking Notch signal transduction pathway of anti-DLL4.With the Hela cell of cultivating among the DMEM that is supplemented with microbiotic and 10%FCS with: 1) in order to be determined to the Notch signal transduction level (Jarriault etc. in the response of DLL4 part; 1995; Nature 377:355-8) the Hes1-Luc report carrier and 2 of Hes1 promotor is contained at the upper reaches at the Photinus pyralis LUC reporter gene) as the renilla luciferase reporter gene (Promega of transfection efficiency confidential reference items contrasts; Madison WI) carries out cotransfection.To add the culture plate that spends the night and encapsulate through 10 μ g/ml DLL4-Fc albumen through cells transfected then.Next will resist the antibody of DLL4 to add this cell culture medium.After the transfection 48 hours, use two luciferase assay test kit (Promega; Madison WI) measures luciferase level, wherein the Photinus pyralis LUC activity is standardized as the renilla luciferase activity.Confirmed the inhibition ability of antibody thus to DLL4 inductive Notch pathway activation.Rodent antibody 21M14 and 21M18 with anti-DLL4 observe the DLL4 activatory inhibition (Fig. 6) to the Notch pathway activation.On the contrary, though the antibody 21M21 of anti-DLL4 can combine the DS1 territory (Fig. 3 E) of DLL4,21M21 does not suppress Notch and combines (Fig. 6).

In some embodiments, measured the antibody regulation and control downstream gene activatory ability of anti-DLL4.From animal separation of C 8 colon tumor cells, and pass through the expression that RT-PCR measures Notch pathway gene HES1 and ATOH-1 through rodent antibody 21M18 treatment (hereinafter detailed description).(Valencia CA) separates total RNA according to the operation instruction of manufacturers from tumor tissues for RNeasy Fibrous Tissue kit, Qiagen with RNeasy fibrous tissue test kit.Recently quantitative with 260nm/280nm to the RNA sample.Confirm the integrity of RNA through a RNA aliquot sample of electrophoresis on the painted sex change sepharose of ethidium bromide (EtBr).Use the FluorChem photographic camera to make 28s rRNA on the gel to the ratio of 18s rRNA visual (presenting) by AphaEasa FC software.The RNA sample is eluted in the water of no RNase and is stored in-80 ℃.With contain 3 '-TAMRA FAM (6-Fluoresceincarboxylic acid) reporting dyes and 3 '-the non-expansion probe of two fluorescence (nonextendable probe) of TAMRA (6-carboxyl-tetramethyl-rhodamine) carries out real-time RT-PCR.The total RNA of 100 μ g is used for PCR in real time, and final volume is 25 μ L, wherein contain reversed transcriptive enzyme, I * Tagman damping fluid (Applied Biosystems, Foster City, CA) and primer/probe mixture.Be reflected at ABI 7900HT quick real-time PCR system (Applied Biosystems, Foster City carry out in CA): 48 ℃ 30 minutes, at 95 ℃ of 10 minutes and 40 circulations (95 ℃ 15 seconds and 60 ℃ 1 minute).Use SDS2.3 software (Applied Biosystems) analytical results.All primers and probe groups all available from Applied Biosystems (Foster City, CA).The expression level of target gene is represented to the expression level stdn of house-keeping gene GusB and with relative quantity.Compare with the tumour of contrast treatment, use the treatment of the rodent antibody 21M18 of anti-DLL4 to reduce the expression of HES1 and increased the expression (Fig. 7 A) of ATOH-1.

In some embodiments, use the culture condition of the external maintenance tumorigenicity of known ability cell to set up mouse pedigree disappearance OMP-C11 tumour cell colony.At 10 μ g/mL muroid 21M18 or 5 μ M inhibitors of gamma-secretase (GSI; Be DBZ) existence/do not exist down, with the 3T3 cell (3T3) that do not have human DLL4 or comprise that the 3T3 cell (DLL4) of crossing the human DLL4 that expresses on the cell surface covers these tumour cell colonies.Wherein also comprised unlapped contrast.Though 3T3-DLL4 cell induction HES1 also suppresses ATOH1 genetic expression (ratio with HES1: ATOH1 is represented), independent 21M18 or GSI have suppressed DLL4 inductive Notch target gene and have changed.

The CTA that complement relies on

In some embodiments; Cancerous cell line of expressing DLL4 or the cancer stem cell that from patient's sample, is separated to are gone down to posterity in the mouse of immunocompromised (like hereinafter institute detaileds description) as heterograft, use the cytotoxicity (CDC) of this cancerous cell line or cancer stem cell measurement by the antibody-mediated complement dependence of anti-DLL4.With cell with 10 ⁶Individual cell/ml is suspended in the RPMI that is supplemented with microbiotic and 5%FBS 1640 substratum of 200 μ l.To contain the antibody of anti-DLL4 with 200 μ l through the cell that suspends then or the serum or the hot inactivated serum of control antibodies mixes, establish 3 repetitions.With cell mixture in 37 ℃ at 5%CO ₂Middle incubation 1～4 hour.Collect treated cell then, and it is resuspended in the annexin V that is diluted in 100 μ l FITC-marks in the substratum and room temperature incubation 10 minutes.Add to be diluted in 100 μ l propidium iodide solution (25 μ g/ml) among the HBSS, and incubation 5 minutes at room temperature.Collecting cell suspends in substratum, and analyzes with flow cytometry.The flow cytometry of FITC staining cell provides total cell count, and the propidium iodide (as the per-cent that accounts for total cell count) that dead cell is absorbed is used to measure the necrocytosis in the presence of the antibody (comparing with control antibodies with hot inactivated serum) at serum and anti-DLL4.Confirm the Cytotoxic ability of the antibody-mediated complement dependence of anti-DLL4 thus.

The cell born of the same parents poison that antibody relies on is measured

In some embodiments; Cancerous cell line of expressing DLL4 or the cancer stem cell that from patient's sample, is separated to are gone down to posterity in the mouse of immunocompromised (like hereinafter institute detaileds description) as heterograft, use this cancerous cell line or cancer stem cell to measure cell born of the same parents malicious (ADCC) by the antibody-mediated antibody dependence of anti-DLL4.With cell with 10 ⁶Individual cell/ml is suspended in no phenol red RPMI 1640 substratum that are supplemented with microbiotic and 5%FBS of 200 μ l.From the peripheral blood of heparinization, isolate PMBC (PBMC) through Ficoll-Paque density gradient centrifugation, to be used as the effector cell.Then under the situation that the antibody of at least a DLL4 or control antibodies exist, with 25: 1, the E/T of 10: 1 and 5: 1 was than in 96 orifice plates, mixing with target cell (T) and PBMC effector cell (E).Contrast independent incubation target cell and independent incubation effector cell under the existence that is included in antibody.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 d/d serum lactic dehydrogenase (LDH, the stable kytoplasm enzyme that discharges during a kind of lysis).Reading the plate appearance with standard 96 orifice plates gathers absorbance data and carries out background correction at the 490nm place.Per-cent according to the computes specific cytotoxic: % cytotoxicity=100 * (the spontaneous LDH of the spontaneous LDH release-target of experiment LDH release-effector discharges)/(the spontaneous LDH of the maximum LDH release-target of target discharges).Confirm the malicious ability of cell born of the same parents of the antibody-mediated antibody dependence of anti-DLL4 thus.

Embodiment 3: use the antibody of anti-DLL4 to prevent growth in the tumour body

Present embodiment has been described the antibody of using anti-DLL4 and has been prevented the tumor growth in the xenograft models.In some embodiments, preparation is from the tumour cell that has gone down to posterity in mouse as heterograft of patient's sample (for example solid tumor biopsy samples or hydrothorax), in the laboratory animal of going down to posterity again.Under aseptic condition, take out tumor tissues, be cut into small pieces, shred fully, and obtain single cell suspension through enzymic digestion and Mechanical Crushing with sterile razor blade.Specifically, hydrothorax cell or gained tumor mass are mixed in substratum with ultrapure collagenase III (200～250 unit collagenases/mL) and 37 ℃ of incubations 1～4 hour were inhaled up and down through the 10mL pipettor and to be beaten in per 15～20 minutes.Through the cell of 40 μ M nylon net filters, with Hank ' s buffered salts solution (HBSS) (pH 7.4) washing that contains 2% hot deactivation calf serum (HICS) and 25mM HEPES through digestion.Then dissociated tumour cell is subcutaneously injected in the mammary fat pad of NOD/SCID mouse to cause tumor growth.

In some embodiments, before being expelled to laboratory animal, at first elect the dissociated tumour cell branch of institute as tumorigenicity cell and non-tumorigenic cell according to the cell surface marker thing.Specifically, press above-mentioned dissociated tumour cell 2 times with HEPES buffered salts solution (HBSS) washing that contains 2% hot deactivation calf serum (HICS), 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 (Miltenyi Biotec, Aubern, CA), anti-CD44, anti-CD24 and kind be that the anti-CD2 of affinity tag, anti-CD3, anti-CD10, anti-CD16, anti-CD18, anti-CD31, anti-CD 64 and anti-CD140b (are referred to as Lin; BD Biosciences, San Jose, CA).Antibody directly is coupled on the fluorescent chromophore the cell of expressing these affinity tags is carried out the positive or negative selection.Through selecting anti-H2Kd+ and muroid CD45+ cell to get rid of mouse cell, and through using viability dyestuff (viability dye) DAPI to get rid of dead cell.(BD Biosciences, San Jose carry out flow cytometry on CA) at FACS Aria.Utilize lateral scattering figure and direct scattering figure to get rid of cell mass.Then the isolating ESA+ of institute, CD44+, CD24-/low, Lin-tumorigenicity cell are subcutaneously injected in the NOD/SCID mouse to cause tumor growth.

In some embodiments, the antibody of having analyzed anti-DLL4 slows down the ability that the UM-C4 colon tumor cell is grown.Dissociated UM-C4 cell (10,000 cell/animals) is subcutaneously injected into the flank district, right side of the NOD/SCID mouse in 6～8 ages in week.Behind the tumor cell injection first day, animal is carried out peritoneal injection (i.p.), twice weekly of experimental session with rodent antibody 21M18 (n=5) or the PBS (n=10) of the anti-DLL4 of 10mg/kg.Monitor growth of tumor weekly,, weekly tumor growth is being carried out twice measurement in the time in totally 8 weeks by a definite date after this time point until detecting growth.Compare with the contrast of injection PBS, with antibody 21M18 treatment, tumor growth slows down 54% (Fig. 8).

The antibody of having measured anti-DLL4 subsequently influences the ability of proliferation in vivo.With rodent antibody 21M18 or control antibodies treatment animal, confirm the expression of cell proliferation affinity tag Ki67 from separation of C 8 colon tumors through the animal of treatment and through immunocytochemistry.Particularly, will be cut into the section of 4 μ m thickness through formalin fixed, paraffin-embedded tumour.To cut into slices and in YLENE, take off paraffin and hydration again in zero(ppm) water.Carry out the immune group chemical analysis according to standard method.In simple terms, in decoking chamber (Decoking chamber), will cut into slices immerse in the water-bath citrate buffer (pH 6) thus in obtained antigen in 20 minutes.Slide glass was cooled off about 45 minutes and with the PBS rinsing.Add one anti-before, will cut into slices with hydrogen peroxide (Sigma-Aldrich, St Louis, MO) room temperature incubation 10 minutes, thus the removal endogenous peroxydase.Be diluted in this dilution buffer liquid of person of outstanding talent (horse dilution buffer) to each section adding with 1: 50 and (contain 1%NHS; 1%BSA; 0.1%Tx-100; The PBS of 0.05%NaN3) rabbit in resists human Ki67, and (Vector Laboratories Inc., Burlingame is CA) and 4 ℃ of incubations 1 hour or spend the night.With slide glass rinsing 3 times in the lavation buffer solution (containing 10% gelatin, the PBS of 10% Tx-100), each 5 minutes.To slide glass add coupling and have the two anti-solution of the anti-rabbit of HRP (Immpress dilutes anti-rabbit antibody in advance, Vector Laboratories Inc., Burlingame, CA) and incubation 30 minutes.After the lavation buffer solution thorough washing, add the carrier nova red (Vector Nova Red, Vector Laboratories Inc., Burlingame, CA).With the water rinse slide glass, redye and with permanent mounting medium (Vectamount, Vector Laboratories Inc., Burlingame, CA) mounting with phenodin (hematoxilin).Compare with the tumour of contrast treatment, reduced the cell quantity (Fig. 9) of expressing K i67 with the treatment of the rodent antibody 21M18 of anti-DLL4.

Embodiment 4: in conjoint therapy, grow in the body of the antibody prevention of the anti-DLL4 of use and treatment tumour

The associating of DLL4 antibody and Fluracil

In some embodiments, antibody and the chemotherapeutic ability of uniting growth in the body that slows down the UM-C4 colon tumor cell of anti-DLL4 have been analyzed.Dissociated UM-C4 cell (10,000 cell/animals) is subcutaneously injected into the flank district, right side of the NOD/SCID mouse in 6～8 ages in week.Behind the tumor cell injection first day, with rodent antibody 21M18 or the PBS of the anti-DLL4 of 10mg/kg animal is carried out peritoneal injection (i.p.), experimental session is injected weekly twice; Perhaps in injection said antibody or PBS with metabolic antagonist chemotherapeutics Fluracil (5-FU) co-therapy, use weekly 1 time.Monitor growth of tumor weekly,, weekly tumor growth is being carried out twice measurement in the time in totally 8 weeks by a definite date after this time point until detecting growth.The rodent antibody 21M18 of anti-DLL4 has slowed down growth of tumor (Figure 10) with the combination therapy of 5-FU than any independent treatment to a greater degree.

The associating of DLL4 antibody and EGFR antibody or VEGF antibody

In some embodiments, with anti-EGF acceptor (EGFR) antibody combined in tested anti-DLL4 antibody influence the ability of in-vivo tumour surviving rate (tumor take frequency).Dissociated UM-C4 cell (10,000 cell/animals) is subcutaneously injected into the flank district, right side of the NOD/SCID mouse in 6～8 ages in week.Behind the tumor cell injection first day, with associating or the PBS of 10mg/kg to the antibody of the rodent antibody 21M18 of the anti-DLL4 of animal (n=10) peritoneal injection (i.p.), anti-EGFR, anti-DLL4 antibody and anti-egfr antibodies.All with animal anti-DLL4 Antybody therapy or anti-egfr antibody therapy in and 9 in 10 control animals merely hit and all detect tumour.On the contrary, have only 2 after treatment several weeks, to have detectable tumour (Figure 11) in the animal that 10 are carried out combination therapy with antibody and the anti-egfr antibodies of anti-DLL4.In addition, the combination therapy of the antibody of the rodent antibody 21M18 of anti-DLL4 and anti-EGFR is treated separately any and has all been reduced tumour incidence (Figure 11).

In some embodiments, with anti-EGF acceptor (EGFR) antibody combined in tested anti-DLL4 antibody influence the ability of in-vivo tumour surviving rate.With the C17 tumour cell implant mouse (n=10/ group) and after 2 days with the associating begin treatment of control antibodies, muroid 21M18, VEGF antibody or two kinds of antibody.Every kind of antibody administration dosage is 10mg/kg, and a week is given 2 times.21M 18 has all slowed down growth of tumor with VEGF antibody, and any independent antibody of Combined Ration is (Figure 18) more effectively.

DLL4 antibody and irinotecan associating

In some embodiments, tested the associating of antibody and the chemotherapeutic irinotecan of anti-DLL4.In some embodiments, dissociated OMP-C8 tumour cell (10,000 cell/animals) is subcutaneously injected into the flank district, right side of the NOD/SCID mouse in 6～8 ages in week.Behind the tumor cell injection first day, with rodent antibody 21M18 or the control antibodies of the anti-DLL4 of 10mg/kg animal is carried out peritoneal injection (i.p.), experimental session is injected weekly twice; Perhaps in injection of antibodies with chemotherapeutic irinotecan co-therapy, dosage is 7.5mg/kg, uses weekly 1 time.Monitor growth of tumor weekly until detecting growth, weekly tumor growth is carried out twice measurement after this time point.The rodent antibody 21M18 of anti-DLL4 slows down tumor growth (Figure 12 A) with the irinotecan combination therapy than any independent treatment to a greater degree.And; Though separately with the 7.5mg/kg irinotecan treat weekly stop after tumour continued growth or accelerating growth in most of animals, the irinotecan of the 21M18 of the anti-DLL4 of twice 10mg/kg and weekly 7.5mg/kg unites the growth (Figure 13) that has further stoped colon tumor in 5 all times that surpasses after treatment in the 56th day stops weekly.

In some embodiments, to check the humanized antibody H7L221M18 of anti-DLL4 with the mode of irinotecan associating.In some embodiments, dissociated C8 tumour cell (10,000 cell/animals) is subcutaneously injected into the flank district, right side of the NOD/SCID mouse in 6～8 ages in week.Behind the tumor cell injection first day, with humanization 21M18 antibody, rodent antibody 21M18 or the control antibodies of the anti-DLL4 of 10mg/kg animal is carried out peritoneal injection (i.p.), experimental session is injected weekly twice; Perhaps in injection of antibodies with chemotherapeutic irinotecan co-therapy, dosage is 7.5mg/kg, uses weekly 1 time.Monitor growth of tumor weekly until detecting growth, weekly tumor growth is carried out twice measurement after this time point.Neoplasm growth effect that the humanized antibody 21M18 of anti-DLL4 and the combination therapy of irinotecan show and similar (Figure 12 B) of muroid 21M18.

In some embodiments, the muroid 21M18 of anti-DLL4 and irinotecan are united be used to treat the colon tumor of having set up.Dissociated C8 cell (10,000 cell/animals) is subcutaneously injected into the flank district, right side of the NOD/SCID mouse in 6～8 ages in week.When institute's injected cells generates about 60mm ³Tumour the time, begin treatment.Rodent antibody 21M18 or contrast with the anti-DLL4 of 10mg/kg are carried out peritoneal injection (i.p.) to animal, and experimental session is injected weekly twice; Perhaps in injection of antibodies with chemotherapeutic irinotecan co-therapy, dosage is 7.5mg/kg, uses weekly 1 time.The rodent antibody 21M18 of anti-DLL4 treats separately with any one with the irinotecan combination therapy and compares the growth (Figure 14) that has slowed down the colon tumor of having set up to a greater degree.

In some embodiments, after conjoint therapy, delay the recurrence of tumour with Antybody therapy.Dissociated C8 cell (10,000 cell/animals) is subcutaneously injected into the flank district, right side of the NOD/SCID mouse in 6～8 ages in week.When institute's injected cells generates about 150mm ³Tumour the time, begin treatment.To rodent antibody 21M18 or control antibodies (10mg/kg, twice weekly) and the irinotecan (7.5mg/kg, 1 time weekly) of the co-administered anti-DLL4 of animal intraperitoneal (i.p.), 32 days by a definite date altogether.Interrupt combination therapy subsequently, in the experiment of remainder, carry out Antybody therapy with DLL4 antibody 21M18 or control antibodies.Compare with the animal of contrast treatment, the antibody 21M18 with anti-DLL4 behind the conjoint therapy treats the generation again (Figure 16) that has significantly delayed tumor growth.Shown that also irinotecan stops back 47 days each tumor size (Figure 17).

Irinotecan has strengthened the reduction of DLL4 antibody to the cancer stem cell frequency

In some embodiments, use restricted dilution analysis to confirm antibody 21M18 independent or the antibody 21M18 of anti-DLL4 and the ability that irinotecan is united reduction cancer stem cell frequency of anti-DLL4.Mouse is treated in associating with control antibodies or DLL4 rodent antibody 21M18, irinotecan or DLL4 rodent antibody 21M18 and irinotecan in a manner described, the treatment 38 days after from mouse separation of C 8 colon tumors.Handle isolating tumour (n=3/ experimental group) in the following manner.Take out tumour, shred with sterile razor blade.In order to obtain single cell suspension; Tumour suspension and digestion solution mixed being incorporated in 37 ℃ of incubations 1 hour, said digestion solution is at MEBM substratum (Cambrex, East Rutherford; NJ) contain collagenase/Unidasa in: Dispase II (1: 1: 8; 10 *) and 1: 100 the dilution deoxyribonuclease I (Worthington, Lakewood, NJ).Eccentric visual cell also (is dissolved in the 0.15M NH of zero(ppm) water at the ACK of 1mL substratum ₄Cl, 10mM KHCO ₃, 0.1mMNa ₂EDTA) resuspended in, in placing 2 minutes to remove erythrocyte on ice.Eccentric visual cell and with 1 * 10 ⁷The concentration of cell/ml is resuspended in the FACS damping fluid, subsequently on ice with biotinylated mouse antibodies (1: 200 diluent of α-mouse CD45-vitamin H and rat α-mouse H ₂1: 100 diluent of Kd-vitamin H, BioLegend, San Diego, CA) incubation is 30 minutes, and (Invitrogen, Carlsbad is CA) to remove mouse cell to add streptavidin (strepavadin) magnetic bead then.Collect human cell remaining in the suspension, counting and dilution are for further using required concentration.Serial dilutions with the human cell is recycled in the immunocompromised mouse subsequently.Particularly, injection 900,300,100 or 50 isolating human tumor cells (n=10, every group) in flank district, the right side of mouse.The gross tumor volume of testing and assessing weekly twice.

When research finished in the 81st day; Compare through the group of the tumour cell of contrast or the independent treatment of irinotecan with injection; The per-cent that in the group of the tumour cell of DLL4 antibody 21M18 treatment, has the mouse that can detect tumour in all injections reduces, and further reduces (Figure 15 A) at injection this per-cent in the group of the tumour cell of DLL421M18-irinotecan.Utilize these tumour occurrence frequencies, through L-Calc ^TMSoftware (downloading from http://www.stemcell.com/search/default.asp) has calculated the stem cell frequency with Poisson statistics.Concise and to the point, according to the Poisson's distribution statistics,, then in the injection cell of dose known amounts, there is 1 stem cell just if tumour does not appear in 37% animal.Treat from the tumour of control treatment 1: 93 of quantity that tumour makes cancer stem cell separately with irinotecan and be increased to 1: 82.On the contrary, the antibody of anti-DLL4 make in the tumour that is reduced to the DLL4 Antybody therapy at 1: 93 of cancer stem cell frequency from the tumour of contrast treatment 1: 238 and DLL421M18-irinotecan combination therapy tumour cell in 1: 573 (Figure 15 B).

Embodiment 5: use the antibody of anti-DLL4 that tumour is carried out interior therapeutic

Present embodiment has been described the application of the cancer in the humanized antibody 21M18 treatment xenograft models of anti-DLL4.In some embodiments, prepared the tumour cell that has gone down to posterity in mouse as heterograft, in the laboratory animal of going down to posterity again from patient's sample (solid tumor biopsy samples or hydrothorax).Take out tumor tissues, be cut into small pieces, shred fully, and obtain single cell suspension through enzymatic digestion and Mechanical Crushing with sterile razor blade.Then with dissociated tumour cell be subcutaneously injected into the NOD/SCID mouse mammary fat pad (for breast tumor), be expelled in the flank (for non-breast tumor) to cause tumor growth.As selection, the method for describing in detail by preceding text is separated ESA+, CD44+, CD24-/low, Lin-tumorigenicity tumour cell and is injected.

Behind the injection tumour cell, the tumor growth of monitoring animal.In case tumour reaches about 100mm ³Mean sizes, promptly begin Antybody therapy.In 6 weeks altogether,, make the humanized antibody 21M18 of the anti-DLL4 of every animals received 100 μ g or the peritoneal injection of control antibodies by 2～5 times weekly.In this 6 all process, estimate twice of tumor size weekly.Definite thus DLL4 humanized antibody is compared the ability that further prevents tumor growth or reduce tumor size with control antibodies.

At the Antybody therapy terminal point, the results tumour is further to analyze.In some embodiments, through immunofluorescence technique analysis part tumour to estimate antibody penetrating and tumor response to tumour.With antibody or the control antibodies treatment mouse of anti-DLL4, part aquatic foods liquid nitrogen of every kind of tumour that will obtain from the mouse of being treated freeze, and are embedded among the O.C.T., and are switching on the slide glass with 10 μ m on the cryostat.In some embodiments, with the part of every kind of tumour with formalin fixed, paraffin embedding and switching on the slide glass with 10 μ m on the ultramicrotome.To 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-DLL4 acceptor that exists in the tumor biopsy sample.The cell type (for example anti-VE catenin (CD144) or anti-PECAM-1 (CD31) antibody are to detect vascular endothelial cell, anti-unstriated muscle α-Ji Dongdanbai antibody to detect VSMC, anti-Ki67 antibody to detect proliferative cell, TUNEL mensuration to detect dying cell, anti-born of the same parents' internal area (ICD) Notch fragment antibody with detection Notch signal transduction) that in addition, can use different tumours of detection and tumour to raise is estimated Antybody therapy to the for example influence of vasculogenesis, tumor growth and tumour form.

In some embodiments, also estimated the influence of the humanized antibody treatment of anti-DLL4 to tumour cell genetic expression.From extracting total RNA the part available from the tumour of the mouse of the mouse of DLL4 Antybody therapy and control antibodies treatment respectively, and be used for quantitative RT-PCR.The cancer stem cell affinity tag that had before identified (for example CD44) of analyzing the component of DLL4, Notch acceptor, Notch signal transduction pathway and being added is with respect to the expression level as the house-keeping gene GAPDH of confidential reference items.Confirm that thus the DLL4 Antybody therapy is to the caused variation of tumour cell genetic expression.

In addition, estimated of the influence of the Antybody therapy of anti-DLL4 to the existence of the cancer stem cell in the tumour.To be cut into small pieces from the tumor sample of the mouse of the mouse of DLL4 Antybody therapy and control antibodies treatment, shred fully with sterile razor blade, and through enzymic digestion and Mechanical Crushing acquisition single cell suspension.The method of describing in detail by preceding text then according to ESA+, CD44+, CD24-/low, Lin-superficial cell marker representation, adopts facs analysis, analyzes dissociated tumour cell and whether has the tumorigenicity cancer stem cell.

Can estimate behind the Antybody therapy of anti-DLL4 the tumorigenicity of expressing institute's isolated cells according to ESA+, CD44+, CD24-/low, Lin-then.The ESA+, CD44+, CD24-/low, the Lin-cancer stem cell that separate the mouse of treating from the mouse and the control antibodies of DLL4 Antybody therapy are subcutaneously injected in the mammary fat pad of NOD/SCID mouse again.According to forming the required quantity of being injected cell of tumour all the time, confirm the tumorigenicity of cancer stem cell then.

Embodiment 6: the humanized antibody treatment human cancer that uses anti-DLL4

Present embodiment has been described the humanized antibody that uses anti-DLL4 and has been come target tumor and treat method for cancer, and said tumour comprises cancer stem cell and/or the tumour cell that has wherein detected Notch acceptor or the expression of Notch receptors ligand.At first, can confirm to exist the cancer stem cell marker representation from the tumor biopsy sample.The biopsy samples of under aseptic condition, suffering from the patient of cancer from diagnosis takes out tumour cell.In some embodiments, biopsy sample aquatic foods in liquid nitrogen are frozen, be embedded among the O.C.T. and switching on the slide glass with 10 μ m on the cryostat.In some embodiments, the biopsy sample with formalin fixed, paraffin embedding, and is being switched on the slide glass with 10 μ m on the ultramicrotome.With the antibody incubation of section, to detect protein expression with anti-DLL4.

Can also confirm 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 with sterile razor blade.Then with the antibody incubation of dissociated tumour cell and anti-ESA, anti-CD44, anti-CD24, anti-Lin and anti-DLL4, detecting cancer stem cell, and confirm ESA+ through the flow cytometry that preceding text are described in detail; CD44+; CD24-/low, Lin-, the existence of DLL4+ tumor stem cell.

Its tumour of humanized antibody treatment of using anti-DLL4 is after diagnosing for expressing the cancer patients of Notch acceptor or Notch receptors ligand.In some embodiments, the humanized antibody of the above-mentioned anti-DLL4 that makes of purifying is also with the suitable medicinal charge material preparation that is used to inject.In some embodiments, at least 10 weeks, at least once used DLL4 Antybody therapy patient in every month.In some embodiments, at least about 14 weeks, at least once use DLL4 Antybody therapy patient weekly.Each amount of application of antibody should be a pharmacy effective dose.In some embodiments, use the antibody of the anti-DLL4 of about 2mg/ml～about 100mg/ml.In some embodiments, use the antibody of the anti-DLL4 of about 5mg/ml～about 40mg/ml.Before can or using the chemotherapy regimen of one or more chemotherapeutics (for example oxaliplatin, Fluracil, LV or streptozocin) in the standard radiation treatment plan, simultaneously or use said antibody afterwards.For example according to tumor regression, new tumour reduce, tumour antigen express reduce, cancer stem cell quantity reduces or other means of assess disease prognosis, and the patient is monitored, and whether has produced antitumor reaction with definite such treatment.

Consider that from the explanation and the practice of invention disclosed herein other embodiment of the present invention is conspicuous to those skilled in the art.Said explanation and embodiment should be regarded as being merely the example purpose, and actual range of the present invention and essence are provided by appended claims.With all communiques, patent and patented claim that this paper quoted by reference mode all incorporate in the present disclosure.

Claims (28)

1. monoclonal antibody; Said antibodies specific is combined in the N-terminal fragment of the human δ appearance part 4 (DLL4) of the 217th amino acids place brachymemma; Wherein said antibody comprises variable region of heavy chain and variable region of light chain; The aminoacid sequence of said variable region of heavy chain is selected from the group of being made up of SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8, and the aminoacid sequence of said variable region of light chain is SEQ ID NO:12, and the aminoacid sequence of said human DLL4 is SEQ ID NO:25.

2. a specific specificity combines the antibody of human DLL4, and wherein said antibody comprises:

I. the variable region of heavy chain that contains cdr amino acid sequence C DR1, CDR2 and CDR3, wherein the CDR1 sequence is SEQ ID NO:1; The CDR2 sequence is SEQ ID NO:2, SEQID NO:3 or SEQ ID NO:4; And the CDR3 sequence be SEQ ID NO:5 and

Ii contains the variable region of light chain of cdr amino acid sequence C DR1, CDR2 and CDR3, and wherein the CDR1 sequence is SEQ ID NO:9; The CDR2 sequence is SEQ ID NO:10; And the CDR3 sequence is SEQ ID NO:11.

3. antibody as claimed in claim 2, wherein said variable region of heavy chain contain cdr amino acid sequence C DR1, CDR2 and CDR3, and wherein the CDR1 sequence is SEQ ID NO:1, and the CDR2 sequence is SEQ ID NO:3, and the CDR3 sequence is SEQ ID NO:5; And said variable region of light chain contains cdr amino acid sequence C DR1, CDR2 and CDR3, and wherein the CDR1 sequence is SEQ ID NO:9, and the CDR2 sequence is SEQ ID NO:10, and the CDR3 sequence is SEQ ID NO:11.

4. a specific specificity combines the antibody of human DLL4, and wherein said antibody comprises variable region of heavy chain and variable region of light chain, and the aminoacid sequence of said variable region of heavy chain is SEQ ID NO:7, and the aminoacid sequence of said variable region of light chain is SEQ ID NO:12.

5. antibody, the variable region of heavy chain of said antibody is identical with the plasmid-encoded antibody that by the ATCC deposit number is PTA-8427 or PTA-8425 with the variable region of light chain peptide sequence.

6. it is contained variable region of heavy chain cdr amino acid sequence and the variable region of light chain cdr amino acid sequence of 21M18 antibody that the hybridoma of PTA-8670 produces that monoclonal antibody, said monoclonal antibody contain by the ATCC deposit number.

7. like each described antibody in the claim 1 to 6, said antibody is humanized antibody.

8. like each described antibody in the claim 1 to 6, said antibody is human antibodies.

9. like each described antibody in the claim 1 to 6, said antibody is bi-specific antibody.

10. like each described antibody in the claim 1 to 6, said antibody is IgG1 antibody.

11. like each described antibody in the claim 1 to 6, said antibody is IgG2 antibody.

12. like each described antibody in the claim 1 to 6, said antibody debond muroid DLL4.

13. like each described antibody in the claim 1 to 6, said antibody has the K of the combination people DLL4 that is equal to or less than 0.1 μ M _D

14. pharmaceutical composition that comprises like each described antibody and pharmaceutical carrier in the claim 1 to 13.

15. be used for treating the application of the medicine of cancer in manufacturing like each described antibody in the claim 1 to 13.

16. application as claimed in claim 15, wherein said antibody reduces the frequency of cancer stem cell in the solid tumor.

17. application as claimed in claim 15, wherein said cancer are colorectal carcinoma, breast cancer, lung cancer or carcinoma of the pancreas.

18. application as claimed in claim 16, wherein said cancer are colorectal carcinoma, breast cancer, lung cancer or carcinoma of the pancreas.

19. the cell of each said antibody in expression such as the claim 1 to 13.

20. an ATCC deposit number is the hybridoma of PTA-8670.

21. polynucleotide, each described antibody in said polynucleotide encoding such as the claim 1 to 13.

22. polynucleotide, the sequence of wherein said polynucleotide are SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15 or SEQ ID NO:16.

23. carrier that comprises like the said polynucleotide of claim 21.

24. carrier that comprises like the said polynucleotide of claim 22.

25. like the application of each said antibody in the claim 15 to 18, wherein said cancer therapy comprises the co-administered said antibody and second therapeutical agent.

26. like the application of the said antibody of claim 25, wherein said second therapeutical agent is chemotherapeutics or SA.

27. like the application of the said antibody of claim 25, wherein said second therapeutical agent is the antibody of anti-VEGF.

28. like the application of the said antibody of claim 25, wherein said second therapeutical agent is a chemotherapeutics.

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