CN101583624A - 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 the method for cancer

Technical field

The present invention relates to oncology and novel compositions and the method that is used to diagnose and treat cancer is provided.The invention provides the antibody that is used to diagnose and treat 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 500,000 people's death are arranged.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 (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 checking of doing sth. in advance and treating, 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 and method by stages comprises that tumor nodule shifts (TNM) system, this system depends on (the American Joint Committee on Cancer:AJCC Cancer Staging Manual.Philadelphia that exists that the existence of tumour in tumor size, the lymphoglandula and far-end shift, Pa.:Lippincott-Raven Publishers, the 5th edition, 1997, the 171～180 pages; Harris, J R: " Staging of breast carcinoma " in Harris, J.R., Hellman, S., Henderson, I.C., Kinne D.W. (volume): Breast Diseases.Philadelphia, Lippincott, 1991).These parameters are in order to provide prognosis and to select suitable therapy.Can also estimate the form outward appearance of tumour, but can show tangible clinical change owing to have the tumour of similar histopathology outward appearance, therefore this method has critical limitations.At last, can use the mensuration of cell surface marker thing that some tumor type is divided into subclass.For example, an existence that factor is estrogen receptor (ER) of in the prognosis of breast cancer and treatment, being considered, negative tumour is easier replys making such as hormonotherapies such as tamoxifen or arimedexs because the positive breast cancer of ER-is usually than ER-.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 diversity.

Prostate cancer is the most common cancer among the male sex of developed world, estimates to account for 33% in all New Development cases of cancers of the U.S., is the frequency time high cause of death (Jemal etc., 2003, CA CancerJ.Clin.53:5～26).Because the introducing of prostate specific antigen (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 provide curative therapy for most local tumor of prostate.Yet for late case, selectable treatment plan is very limited.For metastatic disease, the male sex hormone blocking-up of using luteinizing hormone-releasing hormone (LRH) (LHRH) excitomotor separately or being used in combination luteinizing hormone-releasing hormone (LRH) (LHRH) excitomotor and androgen antagonist 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 by circumferential growth and local the intrusion, then spread, stride by lymph diffusion, blood are capable that peritonaeum spreads and peripheral nerve spreads and invades in other 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 the degree of intestines wall and tubercle 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 may 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, provide 95% curative ratio, but this ratio drops to 75% in Duke B level patient, and to be shown in 5 years the recurrence possibility in advance be 60% in the existence of positive lymph nodes in Duke C level disease.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 most normal the third-largest cancer of 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 class 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 is further divided into 3 main hypotypes based on histology: gland cancer, squamous cell carcinoma (epidermoid carcinoma) and maxicell undifferentiated carcinoma.

Lung cancer manifests later usually in its course of disease, 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 the small 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 mixture of cancer cells and normal cell (comprising a matter (matrix) cell, inflammatory cell and endotheliocyte).Several models of cancer provide different explanations for the existence of this heterology.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 group.This model is based on such viewpoint, that is, all colonies of tumour cell all have oncogenic potential (Pandis etc., 1998, Genes, Chromosomes to a certain degree; Cancer 12:122～129; Kuukasjrvi etc., 1997, Cancer Res.57:1597～1604; Bonsing etc., 1993, Cancer 71:382～391; Bonsing etc., 2000, Genes Chromosomes ﹠amp; Cancer 82:173～183; Beerman H etc., 1991, Cytometry 12:147～54; Aubele M and 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 cell in a organized way all come from normal precursor, promptly so-called stem cell (Morrison etc., 1997, Cell 88:287～98; Morrison etc., 1997, Curr.Opin.Immunol.9:216～21; Morrison etc., 1995, Annu.Rev.Cell.Dev.Biol.11:35～71).Stem cell is such cell: (1) has multiplication capacity widely; (2) can carry out the asymmetry 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 by 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) is from replenishing that the microcommunity stem cell of each self-organization obtains continuing, and research recently thinks that there is stem cell equally in most other adult tissues (comprising brain).The tumour that stems from " solid tumor stem cell " " cancer stem cell " of solid tumor (or come from) experiences unordered growth by symmetry and asymmetry fission process subsequently.In this stem cell model, solid tumor contains obvious difference and limited (even may be rare) has the normally cell subgroup of " stem cell " character, and reason is their extensive propagation and forms other solid tumor stem cell (self-regeneration) effectively and most of oncocyte of the shortage tumorigenesis potential of formation solid tumor.In fact, the sudden change in the long-lived population of stem cells can cause the formation of cancer stem cell, the basis that formed cancer stem cell becomes tumor growth and keeps, and the existence of cancer stem cell causes the failure of current methods of treatment.

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 group 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 tumorigenicity basis in these cells of ability of tumorigenicity cancer cells, therefore be expected to develop better diagnostic assay method and therapeutics for the cancer patients.This present invention just at purpose.

Summary of the invention

The invention provides a kind of antibody, described antibodies specific is in conjunction with human δ sample part 4 (DLL4) epi-position, described 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 antibody disclosed in this invention and medicinal charge material (vehicle).The present invention also provides a kind of treatment method for cancer, and described method comprises the DLL4 antibody disclosed in this 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 described explanation, the perhaps acquistion by 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 described explanation in order to explain principle of the present invention.In this specification and the appended claims, " " of singulative, " a kind of " or " described " unless context offers some clarification in addition, otherwise comprise the 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 by the FACS sorting.As between expressing by 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 of DLL4 and Notch-Fc or contrast Fc albumen incubation.High fluorescent shows, occurs combining of Notch and DLL4 in the presence of the antibody 21M12 of control antibodies (line 2) and anti-DLL4 (line 5).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.By fluorescently-labeled anti-Fc antibody test in conjunction with and with facs analysis, 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 circle) and Notch acceptor.

Fig. 3: the epitope mapping of the antibody of anti-DLL4.A) fusion rotein of nested deletion of extracellular domain that 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).On the contrary, combine (aa 1-217, horizontal stripe post between the antibody that 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 the DLL4 protein fragments that is replaced by corresponding muroid residue at the human residue of select location combining.To (replacing Xie Ansuan, Xie Ansuan and proline(Pro)) in the amino acid position 68,69 and 71 or at amino acid/11 42 and 144 DLL4 protein fragments that (replacing Methionin and L-Ala) replaces, the combination that 21M18 shows weakens.E) the DLL4 protein fragments that replaced by corresponding muroid residue of the human residue of elisa assay antibody 21M18 and 21M21 and select location in the DSL territory combining.To in amino acid position 161 and 162 (replace Threonine and Serine) contain the human DLL4 protein fragments of aminoacid replacement, 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 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 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 the Karbate position 20,28,38,48 and 69 of their correspondences.

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.The luciferase level reduction shows that antibody 21M14 and 21M18 can not activate DLL4 Notch approach.

Fig. 7: DLL4 antibody is regulated the expression of Notch target gene in colon tumor.A) the C8 colon tumor that antibody 21M18 or PBS (contrast) through anti-DLL4 are handled separates and measures by quantitative RT-PCR the expression of 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).Be higher than (3T3) that covers with the 3T3 cell or (contrast) colon cell that covers without cell with the HES1/ATOH1 expression ratio of crossing the C11 colony (3T3+DLL4) that the 3T3 cell express DLL4 covers.By 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 square) 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 that compared with the control, 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.Compared with the control, 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.Compared with the control, 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 square) 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 reach 55 days and thoroughly eliminate tumor growth behind injection cell.B) than control antibodies (black square) or control antibodies+irinotecan (triangle), humanization 21M18 (h21M18) has the similar efficient of combination therapy (triangle) to irinotecan (irtcn) to muroid 21M18 (m21M18) to 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, all quilts were treated the tumour continued growth in the animal except two examples.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 square) 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) that adds the antibody 21M18 of the antibody 21M18 of control antibodies, independent anti-DLL4 or anti-DLL4 and irinotecan 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) survive the result of ratio in the 81st day tumour.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 of the tumour cell through treating with cancer stem cell: the tumour cell (left side) through contrasting treatment is compared with the tumour cell for the treatment of 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, and fiducial interval is 95%.Compare with control group, the group for the treatment of through anti-DLL4 has statistically significant difference (*); Organize (* *) separately with irinotecan with the independent group of contrast (*) and compare, unite group and all have 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 delayed.

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 square, 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 be by at least one the antigen recognition site identification in this immunoglobulin molecules variable region and the immunoglobulin molecules of specificity combination such as the targets such as combination of albumen, polypeptide, peptide, carbohydrate, polynucleotide, lipid or aforementioned substances in order to refer to such immunoglobulin molecules.In some embodiments, antibody of the present invention comprises antagonist antibodies, and this antagonist antibodies combines and disturb the proteic downstream signal transduction of for example part combination, receptor dimerizationization, the proteic expression of cancer stem cell marker and/or cancer stem cell marker with cancer stem cell marker protein-specific.In some embodiments, disclosed antibody comprises agonist antibody, and this agonist antibody combines with cancer stem cell marker protein-specific and promotes part for example in conjunction with, receptor dimerizationization and/or the signal transduction that undertaken by cancer stem cell marker albumen.In some embodiments, disclosed antibody does not disturb or promotes the proteic biological activity of cancer stem cell marker, still discerns by 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) mutant, multi-specificity antibody (as 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 modified immunoglobulin molecules that comprises antigen recognition site of the antigen deciding section of antibody, as long as described antibody demonstrates required biologic activity.Antibody can belong to any one classification or its subclass (isotype) (for example IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) in 5 kinds of primary categories (being IgA, IgD, IgE, IgG and IgM) of immunoglobulin (Ig), is referred to as α, δ, ε, γ and μ respectively according to their feature of CH.That different classes of immunoglobulin (Ig) has is different, known subunit structure and 3-d modelling.Antibody can be expose or with such as other molecule couplings such as toxin, radio isotope.

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

" Fv antibody " is meant the minimum antibody fragment that contains 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 described two chains unite with similar dimeric structure by 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 half of Fv) can be used to identification and conjugated antigen, but avidity is lower usually.

" monoclonal antibody " used herein but be meant high specific identification and in conjunction with the homologous antibody group 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.Complete sum full length monoclonal antibodies and antibody fragment (for example Fab, Fab ', F (ab ') 2 and Fv), strand (scFv) mutant, the fusion rotein that comprises antibody moiety and other any modified immunoglobulin molecules that comprises antigen recognition site contained in term " monoclonal antibody ".In addition, " monoclonal antibody " refers to that also the described antibody that makes by any kind of mode, described 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 area (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.By the extra residue in the variable framework region and/or by the replacement of metathetical non-human residue, humanized antibody can also further be modified, to improve and optimization antibodies specific, avidity and/or ability.Usually, humanized antibody comprises the almost whole of at least one (be generally two or three or four) variable region, described variable region comprises all or nearly all CDR district corresponding with the non-human immunoglobulin (Ig), and all or nearly all FR district are the FR districts of human immunoglobulin consensus sequence.Humanized antibody also comprises at least a portion of constant region for immunoglobulin or territory (Fc), and what comprise usually is at least a portion of human immunoglobulin.At United States Patent (USP) 5,225, description arranged in 539 in order to the example of the method that generates humanized antibody.

Term used herein " human antibodies " is meant the antibody that produced by the mankind or by 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 make two kinds of different epi-positions or two kinds of different antigens to be discerned and combination from the heavy chain light chain of antibody to fitting together with different antigenic determinants zone 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.

Term " epi-position " or " antigenic determinant " are used interchangeably in this article, and refer to antigenic can identification and specificity bonded part by specific antibodies.When antigen was polypeptide, epi-position both can be formed by adjacent amino acid, also can be by forming through proteic three grades of folding and juxtaposed non-adjacent amino acid.The epi-position that is formed by adjacent amino acid is still kept 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.

The specificity bonded that suppresses reference antibody and common antigen by the immunoglobulin (Ig) wherein tested measures to determine 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, described mensuration relates to using and is incorporated into the purified antigen of solid surface or contains this antigenic cell, unlabelled test immunoglobulin (Ig) or through the reference immunoglobulin (Ig) of mark.By determining in the presence of described test immunoglobulin (Ig), to be incorporated into the mark of solid surface or the amount of cell is measured competitive inhibition.The excessive existence of common described test immunoglobulin (Ig).The antibody of identifying through competition assay (competitive antibody) thus comprise antibody that institute's bonded epi-position is identical with reference antibody and the epi-position of institute's bonded epi-position and reference antibodies fully near the antibody that steric restriction takes place.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 in conjunction with " be meant antibody more continually, more promptly, more enduringly, avidity more strongly 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 " or " specificity in conjunction with " 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 " or " 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 sequence identity between the homologous protein in the different plant species, specificity is in conjunction with the specificity combination that can comprise the proteic antibody of cancer stem cell marker in the more than a kind of species of identification.

Term used herein " non-specific binding " and " background in conjunction with " when using in the interaction that is relating to antibody and albumen or peptide, be meant the existence that does not rely on ad hoc structure interaction (, 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 accompanies usually with this material basically or in fact in native state.Purity and uniformity adopt usually to be determined such as analysis chemical technologies such as polyacrylamide gel electrophoresis or high performance liquid chromatography.Disclosed in this invention in preparation the albumen (for example antibody) or the nucleic acid of existing main kind passed through sufficient purifying.Particularly, separated nucleic acid separates with open reading frame, 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 open reading frame.Separated antibody separates with other NIg, and separates with other immunoglobulin (Ig) with different antigen-binding specificities.Described term refers to that also nucleic acid or albumen have at least 80% purity in some embodiments, has at least 85% purity in some embodiments, has at least 90% purity in some embodiments, have at least 95% purity in some embodiments, and have at least 99% purity in some embodiments.

" cancer " is meant or describes the mammiferous physiological status that cell mass wherein has the dysregulated cellular growth feature to term used herein with " cancer ".The example of cancer includes but not limited to cancer, lymphoma, blastoma, sarcoma and leukemia.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 incidence 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 that cancer is from the diffusion of position originally or transfer to other zone of health and the process of similar cancerous lesion is arranged in this new position development." transitivity " or " transfer " cell is to lose the cell that contacts and invade from initial disease sites transfer by blood flow or lymph adjacent body structure with the adhesion of flanking cell.

Term " cancer stem cell ", " tumor stem cell " or " solid tumor stem cell " can exchange use in this article mutually, and refer to the such cell mass from solid tumor: (1) has multiplication capacity widely; (2) can carry out the asymmetry cell 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 forms the tumour of palpation behind the mouse of being transplanted to immunocompromised continuously.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.As U.S. Patent No. 6,004,528 propositions, solid tumor stem cell is different from " cancer responsibility ".In this patent, " cancer responsibility " is defined as the slowly progenitor cell type of growth, himself rare sudden change, and carry out the symmetry cell fission rather than carry out in cellular environment, occurring tumorigenicity changing caused asymmetry 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.U.S. Patent No. 6,004,528 propose, and this model can be in order to promote the diagnosis of cancer.The solid tumor stem cell model is fundamentally different than " cancer responsibility " model, the result show " cancer responsibility " model the application that can not provide.The first, solid tumor stem cell is not " sudden change lacks ".U.S. Patent No. 6,004,528 described " the cancer responsibilities that sudden change lacks " can be counted as precancerous lesion, and solid tumor stem cell to be itself contain the cancer cells that begins to cause the sudden change of tumorigenicity from the cancer last stage to the whole process of later stage cancer.That is, solid tumor stem cell (" cancer stem cell ") should be included in and U.S. Patent No. 6,004, in the cell of the visibly different height sudden change of 528 described " cancer responsibilities ".The second, 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, " cancer responsibility " cell through transplanting can not form new tumour, 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 (entering into and the normal very different environment of tumor environment) still can form new tumour.The 3rd, solid tumor stem cell carries out symmetry cell fission and asymmetry 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 group 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 (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 marker ", " cancer stem cell marker ", " tumor stem cell marker " or " solid tumor stem cell marker " are meant one or more gene or expressed albumen, polypeptide or the peptide of described one or more gene, the expression level of described 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 described genetic expression or minimizing (for example, the increase of the level of the peptide of the mRNA of described 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 determine 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 described 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 described study subject is the recipient of particular procedure.When mentioning human subject in this article, term " study subject " and " patient " can exchange use usually mutually.

" medicinal " be meant by or can or list in American Pharmacopeia or other is subjected to 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 at least a antibody disclosed in this invention, not destroy its pharmacological activity and be nontoxic when using by the dosage of the described compound that is enough to the delivering therapeutic amount.

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

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

Term " treatment 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 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 determining 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 may 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 for the treatment of.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, determine by 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 as " 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's the method according to this invention successfully obtains " treatment ": 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 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) by 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.Described base analogue includes but not limited to the 4-acetylcytosine, 8-hydroxy-n 6-methyladenosine, the aziridinyl cytosine(Cyt), false iso-cytosine, 5-(carboxyl hydroxymethyl) uridylic, 5 FU 5 fluorouracil, 5-bromouracil, 5-carboxymethyl aminomethyl-2-sulfo-uridylic, 5-carboxymethyl aminomethyl uridylic, dihydrouracil, inosine, the N6-isopentenyl gland purine, the 1-methyladenine, 1-methyl pseudouracil, the 1-methyl guanine, the 1-methylinosine, 2, the 2-dimethylguanine, the 2-methyladenine, the 2-methyl guanine, the 3-methylcystein, 5-methylcytosine, the N6-methyladenine, the 7-methyl guanine, 5-methyl aminomethyl uridylic, 5-methoxyl group aminomethyl-2-sulfo-uridylic, β-D-mannose group Q nucleosides (mannosylqueosine), 5 '-the methoxycarbonyl 6-Methyl Uracil, the 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 hangs down 5 ℃～10 ℃ approximately by the hot melt point (Tm) of the row of bit sequencing under 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 by 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 as follows: 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 encode by any part of this encoding sequence, as long as remaining with this complete encoding sequence or segmental required activity or functional property (for example, enzymic activity, part combination, signal transduction, immunogenicity etc.) gets final product.Thereby also containing coding region and contiguous this coding region of structure gene, this term also make this gene be equivalent to the sequence of full length mRNA length in the above location of giving an 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.Intron is by from 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 determining 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 described cell, nucleic acid, albumen or carrier by 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 described cell derives from the cell through modification like this.Therefore, for example, the gene that reconstitution cell can be expressed the cell that does not see 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 by manipulation nucleic acid (for example using polysaccharase and restriction endonuclease) in external formation, and for not seeing natural form usually.Adopt this mode can realize not homotactic being operatively connected.Therefore, the separated nucleic acid of linear forms or all think to be used for the recombinant chou of purposes of the present invention by connecting the dna molecular that does not usually connect together at the expression vector of external formation.Should be understood that in 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 by).

Term used herein " heterologous gene " is meant the gene that is not in its natural surroundings.For example, heterologous gene comprise from a certain species but be directed to the gene of another kind of species.Heterologous gene 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) in some way.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 mutually with " carrier " sometimes and uses.Carrier comes from plasmid, phage or plant virus or animal virus usually.

" connection " is meant the process that forms diester linkage between two double stranded nucleic acid fragments.Except as otherwise noted, otherwise the condition that connects the dna fragmentation to be connected of the about equimolar amount can use known damping fluid and 10 T4 of unit dna ligase (" ligase enzyme ")/0.5 μ g finish.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 by gene " transcribing " (for example, enzymatic action by RNA polymerase) genetic information coded in the gene is converted into RNA (for example mRNA, rRNA, tRNA or snRNA), and " translation " by 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) that participates 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 mutually, 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 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-phosphoserine.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.Described analogue can have modified R group (for example nor-leucine) or modified peptide backbone but still keep the basic chemical structure identical with naturally occurring amino acid.But the amino acid analog thing is meant to have the structure that is different from amino acid whose general chemistry structure have 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 described codon and not change encoded polypeptide.Such nucleic acid variation is " silent variant ", is a kind of conservative modification variation.Each nucleotide sequence of coded polypeptide is also explained the silent variant of nucleic acid herein.Those skilled in the art should know, in some cases, also can to each codon in the nucleic acid (except generally be methionine(Met) unique password AUG with generally be the TGG of unique password of tryptophane) modified to obtain the identical molecule of function.Therefore, the silent variant of nucleic acid encoding lies in the described sequence about expression product, but does not lie in the described sequence about the actual probes sequence.For aminoacid sequence, those skilled in the art should know, in encoded sequence, 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.The form that provides intimate amino acid whose conservative replacement is known in this area.Except that comprising so conservative modification variant, do not get rid of homologue and allelotrope between polymorphism variant of the present invention, kind.Usually, conservative replacement comprises: 1) L-Ala (A), glycine (G); 2) aspartic acid (D), L-glutamic acid (E); 3) N (N), glutaminase (Q); 4) arginine (R), Methionin (K); 5) Isoleucine (I), bright amino acid (L), methionine(Met) (M), Xie Ansuan (V); 6) phenylalanine (F), tyrosine (Y), tryptophane (W); 7) Serine (S), Threonine (T); With 8) halfcystine (C), methionine(Met) (M) (seeing Creighton for example, Proteins (1984)).

Term used herein " band epi-position label " is meant and comprises cancer stem cell marker 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 marker.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 composition and the method that are used to study, diagnose, characterize and treat cancer.Particularly, the invention provides the antibody of solid tumor resisting stem cell labeling thing and the method for cancer that these antibody of use suppress tumor growth and treatment human patients.In some embodiments, antibody of the present invention comprises antagonist antibodies, but described antagonist antibodies specificity is in conjunction with cancer stem cell marker albumen and disturb for example part combination, receptor dimerizationization, the proteic expression of cancer stem cell marker and/or the proteic signal transduction of cancer stem cell marker.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 is undertaken by cancer stem cell marker albumen with cancer stem cell marker protein-specific.In some embodiments, disclosed antibody does not disturb or promotes the proteic biological activity of cancer stem cell marker, still discerns by for example antibody internalization and/or by immune system and suppresses tumor growth.In some embodiments, but described antibody specific recognition is done thin marker albumen more than a kind of solid tumor.

The invention provides and a kind of separated antibody of human DLL4 epitope specificity bonded, described 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, described antibody is monoclonal antibody.In some embodiments, described antibody is chimeric antibody.In some embodiments, described antibody is humanized antibody.In some embodiments, described antibody is human antibodies.The present invention further provides the pharmaceutical composition that comprises antibody disclosed in this invention and pharmaceutical carrier.

The present invention further provides a kind of treatment method for cancer, described method comprises the antibody disclosed in this invention or the pharmaceutical composition of administering therapeutic significant quantity.In some embodiments, with described antibody and the coupling of cytotoxicity part.In some embodiments, described method further comprises and uses at least a additional therapeutic agent that is suitable for carrying out conjoint therapy.In some embodiments, described tumour cell is selected from breast tumor, colorectum tumour, lung tumor, tumor of prostate, pancreatic neoplasm 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 obtains experiment confirm (Park etc., 1971, JNatl.Cancer Inst.46:411～22 in acute myeloid leukaemia (AML); Lapidot etc., 1994, Nature 367:645～8; Bonnet and Dick, 1997, Nat.Med.3:730～7; Hope etc., 2004, Nat.Immunol.5:738～43).From the stem cell of solid tumor recently based on they the cell surface receptor uniqueness expression pattern with the evaluation of their self-regeneration and propagation character in culture and heterograft animal model is separated.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 isolating the tumorigenicity cancer stem cell from a large amount of non-tumorigenic tumour cells makes can use microarray analysis to identify the cancer stem cell marker, compare the gene that has differential expression in cancer stem cell 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 marker that these are identified.

Cancer stem cell marker 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) by 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 mutant first.The monoploid disappearance of fruit bat Notch produces damaged at the wing edge, afunction then produces embryo 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, it contains a large amount of placed in-line Urogastrons (EGF) sample tumor-necrosis factor glycoproteins and is rich in halfcystine in large-scale extracellular domain Notch/LIN-12 tumor-necrosis factor glycoproteins (Wharton etc., 1985, Cell 43:567; 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 comprises human pathological change (hereinafter will describe in detail the) (Gridley of several cancers, 1997, Mol.Cell Neurosci.9:103; Joutel and Tournier-Lasserve, 1998, Semin.Cell Dev.Biol.9:619-25).

(Lag-2) single of (DSL) family transmembrane ligand body of striding film can activate the Notch acceptor for Delta, Serrated for δ, spination, delay-2.The feature of known Mammals Notch part δ sample 1 (Dll1), δ sample 3 (Dll3), δ sample 4 (Dll4), Jagged (sawtooth sample) 1 and Jagged 2 is the series connection EGF-sample 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 proteolysis 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 enters in the nuclear subsequently and activate the CBF1 of the main downstream effect thing of conduct in nuclear, there is not hair suppressor gene (Suppressor of Hairless) [Su (H)], the transcription factor that postpones-2 (CSL) family, thereby strengthen (the Artavanis etc. that transcribe of crinosity and the nuclear alkalescence helix-loop-helix transcription factor that divides enhanser [E (spl)] family, 1999, Science 284:770; Brennan and Brown, 2003, Breast Cancer Res.5:69; Iso etc., 2003, Arterioscler.Thromb.Vase.Biol.23:543).Also may there be 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 the effect (Brennan etc. that the Wnt target gene is expressed, 1999, Curr.Biol.9:707-710; 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 leukemia transform (Kopper and Hajdu, 2004, Pathol.Oncol.Res.10:69-73).HSC is the rare cell group of the aperture tabernacle (stomal niche) that is arranged in adult marrow.These cells are characterised in that unique gene expression pattern and continue to produce the higher progenitor cell of degree of differentiation to build the ability of whole hematopoiesis system again.External and in secular reconstruction test, the activation of the composing type 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 group'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 neural stem cell also, and with neural stem cell 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).Neural stem cell produces neurocyte all in the mammalian nervous system and spongiocyte in growth course, and has identified neural stem cell recently in the adult brain (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 neural stem cell quantity.In addition, in the brain of PS1 heterozygote mouse, the adult neural stem cell reduces (Nakamura etc., 2000, J.Neurosci.20:283-93; Hitoshi etc., 2002, Genes Dev.16:846-58).The minimizing of neural stem cell may 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.Identified human NOTCH1 gene first in a subclass of T cell acute lymphoblastic leukemia, the NOTCH1 gene of being identified is the translocated locus (Ellisen etc., 1991, Cell 66:649-61) that causes the Notch pathway activation.The same t cell lymphoma that produces of the composing type of Notch1 signal transduction activation in the T cell in the mouse model, 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 the frequent insertion of Notch in the breast tumor 1 and Notch 4 locus and the activated Notch protein fragments that produced 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 ﹠amp; 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 about the evidence of the effect of Notch in human breast cancer be limited to the Notch acceptor in breast cancer expression and dependency (Weijzen etc., 2002, the Nat.Med.8:979-86 between they and the clinical effectiveness; Parr etc., 2004, Int.J.MoI.Med.14:779-86).In addition, at cervical cancer (Zagouras etc., 1995, PNAS 92:6414-8), renal cell carcinoma (Rae etc., 2000, Int.J.Cancer 88:726-32), squamous cell carcinoma of the head and neck (Leethanakul etc., 2000, Oncogene19:3220-4), carcinoma of endometrium (Suzuki etc., 2000, Int.J.Oncol.17:1131-9) and neuroblastoma (van Limpt etc., 2000, Med.Pediatr.Oncol.35:554-8) observed crossing of Notch approach in and expressed, this prompting Notch is at the developing latent effect of multiple knurl.What is interesting is Notch signal transduction may in the not differentiation attitude of the Ape mutant 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 may 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, 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 expresses in cancer stem cell (comparing with normal newborn epithelium) 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 by 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 marker, the expression of described cancer stem cell marker can be used for analyzing, with diagnosis or the monitoring disease relevant with cancer.In some embodiments, the expression of cancer stem cell marker is for example expressed to determine by the polynucleotide (for example mRNA) of the described cancer stem cell marker of coding.Can be by any one detects and quantitative described 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 marker from the tissue slice of patient's biopsy samples.In some embodiments, RNA is separated from tissue and detect by for example Northern trace, quantitative RT-PCR or microarray etc.For example, can from tissue sample, extract total RNA, and can utilize RT-PCR, use the specific hybrid and the primer of amplification cancer stem cell marker to detect the expression of cancer stem cell marker polynucleotide.

In some embodiments, can determine the expression of cancer stem cell marker by detecting corresponding polypeptide.Can detect and quantitative described polypeptide by 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, high performance liquid chromatography (HPLC) or thin-layer chromatography (TLC) detect cancer stem cell marker 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 described 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 marker.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 some embodiments of the present invention, diagnostic assay comprises and for example uses whether immunohistochemistry, in situ hybridization or RT-PCR determine the expression of cancer stem cell marker in tumour cell.In other embodiment, diagnostic assay comprises the expression level that uses quantitative RT-PCR for example to determine the cancer stem cell marker.In some embodiments, diagnostic assay for example also comprise with control tissue for example normal epithelial organize the expression level that relatively comes to determine the cancer stem cell marker.

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 marker indication.And the detection of cancer stem cell marker can comprise the treatment of for example being carried out with the anti-detected proteic antibody of cancer stem cell marker in order to select suitable therapy.In some embodiments, described antibodies specific combination is such as proteic extracellular domains of cancer stem cell marker such as Notch receptors ligand DLL4.

Within the scope of the invention, suitable antibody is to have for example reagent of following one or more effects: disturb the expression of cancer stem cell marker; Disturb the activation of cancer stem cell signal transduction pathway by for example spatially suppressing interaction between cancer stem cell marker and its part, acceptor or the co-receptor; By for example bringing into play the part effect or promoting the combination of endogenic ligand to activate the cancer stem cell signal transduction pathway; Or in conjunction with the cancer stem cell marker 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 marker protein function outward.In some embodiments, the born of the same parents of the antibody of anticancer stem cell labeling thing outer in conjunction with intrinsic activation (for example kinase activity) that can be by for example suppressing the cancer stem cell marker and/or by spatially suppress for example cancer stem cell marker and its part, and its acceptor, and co-receptor or and extracellular matrix between interaction suppress the proteic signal transduction of cancer stem cell marker.In some embodiments, the born of the same parents of the antibody of anticancer stem cell labeling thing are outer in conjunction with transporting the cell surface expression of reducing the cancer stem cell marker by the cell surface of for example proteic internalization of cancer stem cell marker or minimizing cancer stem cell marker.In some embodiments, the outer combination of the born of the same parents of the antibody of anticancer stem cell labeling thing can be by for example bringing into play effect or the increase part combination promotion the proteic signal transduction of cancer stem cell marker as the bait part.

In some embodiments, the antibody of anticancer stem cell labeling thing and cancer stem cell marker 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 by institute's link coupled toxin, chemotherapeutics, radio isotope 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 marker by 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 described cancer stem cell marker by cytotoxicity (ADCC) mediation that antibody relies on.ADCC participates in the cracking of cell by 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 marker by the cytotoxicity (CDC) of complement activation dependence.CDC participates in the combining and the activation of complement proteins cascade subsequently of Fc part of serum complement and antibody, thereby causes cytolemma destruction, and finally causes necrocytosis.The biologic activity of antibody is known to a great extent by the constant region of antibody molecule or Fc district decision (Uananue and Benacerraf, Textbook of Immunology, the 2nd edition, Williams and Wilkins, the 218th page (1984)).As belong to identical subclass but come from the antibody of different plant species, belonging to a different category with the antibody of subclass is different in this respect.As if in human antibodies, IgM combines the most effective antibody classification with complement, secondly be IgG1, IgG3 and IgG2, but IgG4 lacks (Dillman, 1994, J.Clin.Oncol.12:1497 very much in the complement activation cascade; Jefferis etc., 1998, Immunol.Rev.163:59～76).According to the present invention, can prepare those classification antibody with required biologic activity.

Any specific antibodies that can measure anticancer stem cell is by complement activation and/or ADCC mediation target cell cracked ability.The interested cell of vitro culture and mark; With antibody with serum complement or can be added in the cell culture by immune complex activatory immunocyte.By 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, by 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 marker, 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 methods of treatment.In some embodiments, antibody of the present invention can be in order to detect as the proteic expression of cancer stem cell marker in the biological samples such as patient tissue biopsy samples, hydrothorax or blood sample.The biopsy sample can be made section and for example use immunofluorescence or immunohistochemistry detects albumen.In addition, can separate from the individual cells of sample and by facs analysis and detect 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 marker 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 by antibody is contacted with tumour cell.In some embodiments, the antibody of the anticancer stem cell labeling thing by the administering therapeutic significant quantity and described antibody is used for the treatment of patient's cancer.

Can prepare antibody of the present invention by 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 by multiple subcutaneous injections or peritoneal injection related antigen (peptide fragment of purifying, total length recombinant protein, fusion rotein etc.), described 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.Allow collected coagulation of blood, decant goes out serum, makes its clarification and measures antibody titers by centrifugal.Can comprise that affinity chromatography, ion-exchange chromatography, gel electrophoresis, dialysis etc. are purified into polyclonal antibody from serum or ascites according to 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 to cause lymphocyte according to as mentioned above 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.By immunoprecipitation, immunoblotting or external in conjunction with measuring (radioimmunoassay (RIA) for example; Enzyme-linked immunosorbent assay (ELISA)) determines to produce the hybridoma that specificity resists selected antigenic monoclonal antibody, use standard method (Goding subsequently, Monoclonal Antibodies:Principles and Practice, Academic Press, 1986) external breeding or in culture as the described hybridoma of breeding in the animal ascites tumour body.Then from as above at monoclonal antibody purification described substratum of polyclonal antibody or the ascites fluid.

As selection, can also use as United States Patent (USP) 4,816,567 described recombinant DNA methods prepare monoclonal antibody.Use can specific amplification the Oligonucleolide primers of gene of the heavy chain of coding monoclonal antibody and light chain, isolate the polynucleotide of the described monoclonal antibody of coding by for example RT-PCR from mature B cell or hybridoma, and use conventional procedure to determine 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 immunoglobulin (Ig) 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, the light chain of for example mouse monoclonal antibody and the constant region of heavy chain can be substituted by 1) constant region of human antibodies for example, to produce chimeric antibody or 2) the NIg polypeptide, merge antibody to produce.In some embodiments, brachymemma or remove described 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 some embodiments of the present invention, the monoclonal antibody of described anticancer stem cell labeling thing is a humanized antibody.Humanized antibody is the antibody that contains in the variable region from the minimum sequence of non-human (for example muroid) antibody.Such antibody reduces antigenicity when being used for human subject used in treatment 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 the antibody that is produced by the mankind or has 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 the CDR of non-human antibody (for example mouse, rat, rabbit, hamster etc.) with required specificity, avidity and ability comes antagonist to carry out humanization.Can further modify humanized antibody by 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 by 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 immunoglobulin (Ig).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 may 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 the antigen combination, reservation is from the residue among the CDR of the antigen determining area (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 immunoglobulin (Ig) 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.Compare the antibody studied and a consensus sequence among the Karbate by the reference conserved amino acid, Karbate figure can be expanded to other antibody that is not comprised in its tabulation.Use Karbate's number system can easily determine 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, by using Karbate's number system, can compare any two kinds of antibody sequences separately, for example to determine identity per-cent with having the amino acid comparison of 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, be converted to per-cent thereby multiply by 100 again.

Embodiment 1 has hereinafter described the preparation of the humanized antibody of exemplary anti-DLL4, but described antibody specificity is in conjunction with human DLL4 (the 21M18 H9L2 of cancer stem cell marker disclosed in this invention, ATCC deposit number PTA-8427 and 21M18 H7L2, ATCC deposit number PTA-8425 was in preservation on May 10 in 2007; (American type culture collection (American Type Culture Collection) is 10801 University Blvd. (ATCC), Manassas, VA 20110-2209 USA)).In some embodiments, described humanized antibody comprises the non-human antigen determining area 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, it changes at Karbate position 52a) 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 of specificity in conjunction with human DLL4 epi-position, described 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, described humanized antibody is complete IgG antibody.In some embodiments, described humanized antibody is complete IgG ₂Antibody.In some embodiments, described humanized antibody is an antibody fragment.In some embodiments, described humanized antibody is the Fab fragment.

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

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 described 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 area replaces.

In some embodiments, described 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 described 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 area replaces.

In some embodiments, humanized antibody of the present invention comprises light chain variable (V _L) district, described light chain variable (V _L) district comprises non-human antigen determining area and human variable framework region.In some embodiments, described non-human antigen determining area bag comprises the CDR in rodents source.In some embodiments, described non-human antigen determining area comprises the CDR from mouse antibodies.In some embodiments, described 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 (a) CDR1 (SEQ IDNO:9), CDR2 (SEQ ID NO:10) and CDR3 (SEQ LD NO:11) or (b) aminoacid sequence of SEQ IDNO:12.

In some embodiments, described human light chain variable framework region contains IGK (V) 4-1.In some embodiments, at least one residue in the described human light chain variable framework region is substituted.In some embodiments, at least one residue in the variable framework region of the described 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 described mankind is occupied the residue replacement of corresponding position in the antibody that contains non-human antigen determining area.

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 the light chain of the variable region of called after SEQ ID NO:12.In some embodiments, described antibody is humanized antibody or human antibodies.

In some embodiments, described humanized antibody specificity is in conjunction with human DLL4 epi-position, described 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, described 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 described variable region of light chain has the sequence identity with SEQ ID NO:12 at least 90%.In some embodiments, described 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 described variable region of light chain has the sequence identity with SEQ ID NO:12 at least 95%.In some embodiments, described 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 described 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, described polynucleotide molecule coding specificity is in conjunction with the humanized antibody of human DLL4 epi-position, described 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, described 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 area of CDR3 (SEQID NO:5) and coding IGH (V) 1-18.In some embodiments, the invention provides isolating polynucleotide molecule, the assorted acid molecule coding of described multinuclear specificity is in conjunction with the humanized antibody of human DLL4 epi-position, described 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, described polynucleotide molecule coding specificity is in conjunction with the humanized antibody of human DLL4 epi-position, described 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, described polynucleotide molecule coding specificity is in conjunction with the humanized antibody of human DLL4 epi-position, described 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, described V _LThe district comprises the non-human antigen determining area of coding 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, described polynucleotide molecule coding specificity is in conjunction with the humanized antibody of human DLL4 epi-position, described 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, described polynucleotide molecule coding specificity is in conjunction with the humanized antibody of human DLL4 epi-position, described 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, described method comprises the humanized antibody disclosed in this invention to this patient's administering therapeutic significant quantity.In some embodiments, described cancer comprises breast cancer, colorectal carcinoma, lung cancer, carcinoma of the pancreas, prostate cancer or incidence cancer.

In some embodiments, the invention provides and comprise the test kit that container reaches wherein contained composition, wherein said composition comprises humanized antibody disclosed in this 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 directly to prepare complete human antibodies.Can produce through the human bone-marrow-derived lymphocyte of the immortalization of external immunity or from can generate at 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 United States Patent (USP) 5,750,373).And, can from the phage library of express human antibody wherein, select human antibodies (Vaughan etc., 1996, Nat.Biotech., 14:309～314; Sheets etc., 1998, Proc.Nat ' l.Acad.Sci., 95:6157～6162; Hoogenboom and Winter, 1991, J.Mol.Biol., 227:381; Marks etc., 1991, J.Mol.Biol., 222:581).Can also make human antibodies in the transgenic mice that contains the human immunoglobulin gene seat, described human immunoglobulin gene seat can generate all forming members of human antibodies through immunity under the situation that does not have endogenous immunoglobulin to generate.This method is at United States Patent (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 marker is also contained in the present invention.Bi-specific antibody is can specific recognition and in conjunction with the antibody of at least two kinds of different epi-positions.Described different epi-position (for example can be in same a part, same cancer stem cell marker polypeptide) in or be on the different molecules, make for example described antibody can specific recognition and in conjunction with cancer stem cell marker 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 be in conjunction with two kinds of different epi-positions, and wherein at least one epi-position comes from polypeptide of the present invention.As selection, can with the antigen arm of immunoglobulin molecules with can with such as the 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 can be in conjunction with the arm of 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 United States Patent (USP) 5,731,168).Also imagination has the antibody more than two valencys.For example, can prepare three-specific antibody (Tutt etc., J.Immunol.147:60 (1991)).

In some embodiments, provide antibody fragment for example to improve the tumour penetrance.Known have a multiple technology that is used to generate antibody fragment.Traditionally, these fragments obtain (for example, Morimoto etc., 1993, Journal of Biochemical andBiophysical Methods 24:107～117 by the proteolytic digestion of complete antibody; Brennan etc., 1985, Science, 229:81).In some embodiments, antibody fragment produces by reorganization.Fab, Fv and scFv antibody fragment can 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 described antibody fragment.Antibody fragment can also be a United States Patent (USP) 5,641 for example, the linear antibody described in 870, and can be monospecific antibody or 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 U.S. Patent 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 the antibody fragment contain the idiotype of polypeptide of the present invention by art technology, described antibody fragment includes but not limited to: (a) F (ab ') 2 fragments that generate of the gastric pepsin digestion by antibody molecule; (b) the Fab fragment that produces by reduction F (ab ') 2 segmental disulphide bridgeses; (c) by 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 by for example following method: will remedy the receptors bind epi-position by the sudden change of appropriate area in the antibody fragment and be incorporated in the antibody fragment, perhaps described epi-position is incorporated in the peptide tag, then peptide tag is fused to the centre (for example synthetic) of the arbitrary end or the antibody fragment of antibody fragment by DNA or peptide.

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

For purposes of the present invention, should be understood that modified antibody can comprise the variable region of any kind that the polypeptide that makes described antibody and human DLL4 is associated.In this respect, described variable region can comprise or come from and can be induced and strengthen humoral response and produce the Mammals of any kind of the immunoglobulin (Ig) of anti-required tumor associated antigen.Like this, the variable region of modified antibody can for example derive from the mankind, muroid, non-human primate (for example cynomolgus monkey, macaque etc.) or wolf.In some embodiments, the variable region of modified immunoglobulin (Ig) 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 by the aminoacid sequence that comprises introducing or this variable region is changed.

By one or more CDR can change variable region in heavy chain and the light chain simultaneously to small part displacement, and if desired, can carry out described change by 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.May not need the whole CDR that are used for from the donor variable region to replace all CDR so that the antigen binding capacity of a variable region is transferred to another variable region.More suitably be only to need to shift to keeping active necessary those residues of antigen binding site.Know U.S. Patent 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 fully by implementing conventional experiment or obtaining the functional antibodies that immunogenicity reduces by the trial and error method of testing.

Though changed the variable region, but those skilled in the art should recognize, modified antibody of the present invention will comprise such antibody or its immunoreactivity fragment, wherein, when 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 described antibody is deleted or provide required biochemical characteristic by the alternate manner change, as the serum half-life of enhanced tumor-localizing or shortening.In some embodiments, the constant region of modified antibody comprises human constant region.The modification that the constant region compatible with the present invention carried out is included in one or more amino acid whose interpolations, disappearance or replacement in one or more territories.That is, modified antibody disclosed herein can comprise one or more constant regions of three CH (CH1, CH2 or CH3) and/or change or the modification that constant region of light chain (CL) is carried out.In some embodiments of the present invention, can expect in the modified constant region excalation or lack one or more territories fully.In some embodiments, modified antibody comprises territory disappearance construct or the variant (Δ CH2 construct) of wherein having removed whole C H2 territory.In some embodiments, the constant zone of being saved will can be provided usually the short amino acid introns (for example 10 residues) of some molecular flexibility of being given by the disappearance constant region to replace.

Except their configuration, known in the art is that constant region mediates several effector functions.For example, the complement activation system that combines of the C1 component of complement and antibody.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 by the Fc district, and wherein the Fc acceptor site in the antibody Fc district is attached to the Fc acceptor (FcR) on the cell.Have in a large number, comprise that IgG (γ acceptor), IgE (η acceptor), IgA (α acceptor) and IgM (μ acceptor) have specific Fc acceptor different classes of antibody.Antibody can trigger many important and various biological answer-replies with the combining of Fc acceptor on the cell surface, comprise that the antibody sandwich particulate is engulfed and destruction, the removing of immunocomplex, killer cell antagonist bag are shifted and control by the placenta of the release of the cracking of target cell (the cell-mediated cytotoxicity that is called the antibody dependence, or ADCC), inflammatory mediators, immunoglobulin (Ig) generation.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 that comprises modified constant region 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 (by point mutation or alternate manner) can reduce combining of modified antibody and Fc acceptor in the circulation, strengthen the location of tumour thus.In other situation, with the present invention coincide be, constant region modify regulated complement in conjunction with and reduce serum half-life and the cytotoxic non-specific associating of link coupled thus.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 flexibility, therefore can add strong fix.Similarly, can use biological chemistry well known to those skilled in the art or molecular engineering technology easily to realize the modification of constant region being carried out according to the present invention.

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

Except lacking whole constant zone, should be understood that, can by excalation or a small amount of or or even the replacement of single amino acids antibody of the present invention is provided.For example, the sudden change of the monamino acid in institute's favored area in CH2 territory may be enough to significantly reduce Fc in conjunction with the location of also strengthening tumour thus.Similarly, may wish to lack simply that part of the may command in one or more constant zones effector function to be regulated (for example complement CLQ in conjunction with).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 by can improving gained construct characteristic or replace the constant region of modifying disclosed antibody as mentioned indirectly.In this respect, might be able to destroy the activity (for example Fc combination) that provides by conservative binding site, the configuration and the immunogenicity characteristic of the antibody that basic simultaneously maintenance is modified.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, perhaps provides the absorption of more cytotoxin or carbohydrate.In such embodiment, may wish 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 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 another aminoacid replacement in the same big class, 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 purpose that conserved amino acid replaces 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 (for example enzyme activity toxin of bacterium, fungi, plant or animal-origin or its fragment), radioactive isotope (that is radioactivity conjugate) etc.The chemotherapeutics that can be used for producing described immune conjugate comprises for example methotrexate, Zorubicin, Dx, melphalan, ametycin, Chlorambucil, 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 inhibitor (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 described antibody and radio isotope coupling, radio isotope for example has ⁹⁰Y, ¹²⁵I, ¹³¹I, ¹²³I, ¹¹¹In, ¹⁰⁵Rh, ¹⁵³Sm, ⁶⁷Cu, ⁶⁷Ga, ¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁸⁶Re and ¹⁸⁸Re.In other embodiments, composition disclosed in this invention can comprise with medicine, prodrug or as 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-tetramethylene sulfide (IT; iminothiolane); the difunctionality derivative of imines ester (as diimine for dimethyl adipate HCL); active ester (as disuccinimidyl suberate); aldehyde (as glutaraldehyde); double azido compound (as two (to the azido benzoyl base) hexanediamine); two-tetroxide derivative (as two-(pyrazine formyl radical)-quadrols); vulcabond is (as methylene phenyl 2; the 6-vulcabond) and the dual-active 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, described small molecules toxin for example has an active derivative 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 modified antibody and other immunocompetence part (for example antibody or its fragment) is compound, wherein resulting molecule can be simultaneously in conjunction with oncocyte with as 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 and the cytotoxicity (ADCC) of antibody dependence eliminate malignant cell.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 by 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 of technology such as fluorescence immunoassay and albumin A immunoassay and noncompetitive are measured system.Such assay method be routine in this area and known method (for example see volumes such as Ausubel, 1994, Current Protocols in Molecular Biology, the 1st volume, John Wiley ﹠amp; Sons, Inc., New York all is incorporated herein it in this mode by reference).

In some embodiments, use ELISA to determine 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 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 alkaline phosphatase) in plate hole, 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 alternatively and the second coupling antibody that can discern the antibody of this anticancer stem cell labeling thing.In some embodiments, need not antigen coated plate hole, but but add and detection compound link coupled second antibody after can adding antigen with the antibody sandwich plate hole of anticancer stem cell labeling thing and in the plate hole of Xiang Jingbao quilt.Those skilled in the art should know, can change parameter and strengthen detected signal and other ELISA known in the art change (for example see volumes such as Ausubel, 1994, Current Protocols in Molecular Biology, the 1st volume, John Wiley ﹠amp; Sons, Inc., New York is in 11.2.1).

Can determine the dissociation rate of antibody and the antigenic binding affinity of cancer stem cell marker and antibody-AI by the competitive binding assay method.An example of competitive binding assay method is a radioimmunoassay, and this method is included under the 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 the antigenic antibody that is attached to through mark then.Can by the scatchard mapping analysis determine by data anticancer stem cell labeling thing antibody avidity and in conjunction with dissociation rate.In some embodiments, use the BIAcore dynamic analysis to determine the combination and the speed of dissociating of the antibody of anticancer stem cell labeling thing.The BIAcore dynamic analysis comprises analyzing on antibody and the surface and is fixed with combining and dissociating of the antigenic chip of cancer stem cell marker.

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 derivative 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 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 excised to form the polypeptide of mature form by host cell.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 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, can use known computer program such as Bestfit program (Wisconsin Sequence Analysis Package, the 8th version that is used for Unix, GeneticsComputer Group, University Research Park, 575 Science Drive, Madison, WI 53711), whether conventional definite any specific nucleic acid molecule has at least 80% identity with control sequence, at least 85% identity, at least 90% identity, in some embodiments, whether have at least 95%, 96%, 97%, 98% or 99% identity.Bestfit use Smith and Waterman (Advances in Applied Mathematics 2:482489 (1981)) interior local homology algorithm find the best homology fragment between the two sequences.When using Bestfit or other sequence alignment program determines that whether a specific sequence for example has 95% identity with control sequence of the present invention arbitrarily, thereby 5% the homology breach at the most that parameter can list calculating identity per-cent at the contrast nucleotides sequence of total length and allow the Nucleotide sum that accounts for control sequence 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 mutant comprises that disappearance, insertion, inversion, repetition and typical case replace (typesubstitution).

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

Isolated polypeptide as herein described can be by any appropriate means preparation known in the art.Described 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 be expressed in suitable expressing through transforming the host.In some embodiments, utilize recombinant technology to come the constructed dna sequence by the dna sequence dna of separation or the interested wild-type protein of composite coding.Optionally, can come the described sequence of mutagenesis so that the functional analogue of this sequence to be provided by site-directed mutagenesis.See for example Zoeller etc., Proc.Nat ' l.Acad.Sci.USA 81:5662～5066 (1984) and U.S. Patent No. 4,588,585.

In some embodiments, use oligonucleotide synthesizer to make up the dna sequence dna of the interested polypeptide of coding by chemosynthesis.Those codons of can be based on required amino acid sequence of polypeptide and selecting to be used for to produce the host cell preference of interested recombinant polypeptide design such oligonucleotide.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.For example, several short oligonucleotide that 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 (by 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 described albumen is expressed in required host.Can confirm the exactness of assembling by nucleotide sequencing, estriction map, the expression of biologically active polypeptides in suitable host.As known in the art, in order to obtain the high expression level of rotaring redyeing gene in the host, 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 marker polypeptide syzygy.Recombinant expression vector is reproducible DNA construct, it has the synthetic DNA fragment of coding cancer stem cell marker polypeptide syzygy or bioequivalence analogue or the dna fragmentation in cDNA source, and described syzygy or bioequivalence analogue operably are connected to suitable the transcribing or the translational control element that comes from Mammals, microorganism, 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, sequence described in detail are as mentioned opened in translation.These controlling elements can comprise the operon sequence that control is transcribed.Ability (generally providing by replication orgin) of duplicating among the host and the selection gene of being convenient to discern transformant can additionally be provided.When each DNA district is interrelated on function, they are operably connected.For example, when participating in polypeptide excretory precursor expression, the dna sequence dna of signal peptide (secretion property 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 secretion property leader sequence, mean adjacent and be positioned at and read frame.Plan is used in structural element in the yeast expression system and comprises and can make host cell carry out the leader sequence of the proteic exocytosis translated.As selection, under the situation of the recombinant protein of expressing no leading sequence or transit sequence, described structural element can comprise the terminal methionine residues of N-.This residue can be chosen wantonly from expressed recombinant protein excision subsequently so that final product to be provided.

Host's selection is depended in the selection of expression control sequenc and expression vector.Can adopt 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 derivative thereof from the plasmid of intestinal bacteria), wider host range plasmid (as M13) and thread single stranded DNA phage.

Be applicable to that expressing the proteic host cell of cancer stem cell marker comprises prokaryotic organism, yeast, insect or the senior eukaryotic cell that is subjected to 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.Clone that bacterium, fungi, yeast and mammalian cell host are suitable for and expression vector are as (Cloning Vectors:A Laboratory Manual as described in Pouwels etc., Elsevier, N.Y., 1985), be incorporated herein by 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 as Gluzman, 1981) the COS-7 clone of described monkey-kidney cells and can express other clone of suitable carrier comprises for example 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 for the treatment of 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, as 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 will be connected on the albumen to be easy to carrying out column purification in conjunction with affinity labels such as territory, influenza tunicle sequence (influenza coat sequence) and glutathione-S-transferases such as six Histidines, maltose by suitable affinity column.Can also use technology such as proteolysis, nucleus magnetic resonance and x ray Laue method that isolating albumen is carried out physics characterizes.

For example, for example Amicon or Millipore Pellicon ultra filtration unit concentrate from the supernatant liquor that recombinant protein is secreted into the system in the substratum albumen thickening filtration device that at first can use commercially available acquisition.After enrichment step, concentrated solution can be added in the suitable purifying matrix.As selection, can adopt anionite-exchange resin, for example gathering has matrix or the base material that hangs attached diethylamino ethyl (DEAE) group.Matrix can be acrylamide, agarose, dextran, 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 carboxymethyl.At last, can adopt and use one or more RPLC steps of hydrophobicity RPLC (RP-HPLC) medium (for example having the outstanding attached methyl or the silica gel of other aliphatic group) to be further purified cancer stem cell albumen-Fc composition.Also can adopt the various combinations of some or all aforementioned purification steps that the homology recombinant protein is provided.

Can be for example by the initial extraction from cell precipitation, carry out then one or morely concentrating, saltout, water-based ion-exchange or size exclusion chromatography step are come the recombinant protein that generates in the separation of bacterial culture.Can carry out last purification step with high performance liquid chromatography (HPLC).Can come the used microorganism cells of broken express recombinant protein by any ordinary method, described 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 marker, this method is used the antibody of anti-cancer stem cell marker as herein described.In some embodiments, the described inhibition method of growth of expressing the tumorigenicity cell of cancer stem cell marker is included in and external described cell is contacted with the antibody of anticancer stem cell labeling thing.For example, the immortalized cell line or the cancerous cell line of expressing the cancer stem cell marker are cultivated in substratum, this substratum is added with the antibody of anti-expressed cancer stem cell marker 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 marker comprises described cell is contacted with the antibody of anticancer stem cell labeling thing.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 marker is grown in the mouse (for example NOD/SCID mouse) of immunocompromised, this mouse is applied 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 marker patient's samples such as biopsy sample, hydrothorax or blood sample, and it is expelled in the mouse of immunocompromised, then this mouse is used the antibody of anticancer stem cell labeling thing to suppress the growth of tumour cell.In some embodiments, when importing the tumorigenicity cell in the animal or in the short period of time afterwards, use the antibody of anticancer stem cell labeling thing 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 marker.Described pharmaceutical composition can be used to suppress the cancer of growth of tumour cell and treatment human patients.

By 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 that preserves and use, The Science and Practice ofPharmacy (the 20th edition) Mack Publishing, 2000).Suitable medicinal charge material includes but not limited to non-toxicity buffer reagent, as phosphoric acid salt, Citrate trianion and other organic acid; Such as salt such as sodium-chlor; Antioxidant 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 as methyl p-hydroxybenzoate or propylparaben; Pyrocatechol; Resorcinol; Hexalin; The 3-amylalcohol; And meta-cresol); Low molecular weight polypeptide (for example less than about 10 amino-acid residues); Albumen is as serum albumin, gelatin or immunoglobulin (Ig); Hydrophilic polymer is as polyvinylpyrrolidone; Amino acid is as glycine, glutamine, l-asparagine, Histidine, arginine or Methionin; Carbohydrate is as monose, disaccharides, glucose, seminose or dextrin; Sequestrant is as EDTA; Sugar is as sucrose, maltose alcohol, trehalose or Sorbitol Powder; The salify gegenion is as sodium; Metal complex (for example Zn-protein complex); And nonionogenic tenside, as 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 comprises that vagina is carried and rectum is carried), as the employing transdermal patch, and the using of ointment, lotion, ointment, gelifying agent, drops, suppository, sprays, liquor and pulvis; Pulmonary administration (for example by 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.

Described treatment preparation can be a unit dosage form.Described preparation comprises and is used for oral, parenteral administration, rectal administration or the tablet of using by suction, pill, capsule, pulvis, granule, the solution in water or non-aqueous media or suspension agent or suppository.In such as solids compositions such as tablets, main active component can be mixed with pharmaceutical carrier.Conventional compressing tablet composition comprises W-Gum, lactose, sucrose, Sorbitol Powder, talcum, stearic acid, Magnesium Stearate, dicalcium phosphate or natural gum, and other thinner (for example water), contain preparation compositions before the solid of homogeneous mixture of The compounds of this invention or its non-toxicity pharmaceutical salts with formation.Then preparation compositions before the described solid is further divided into the unit dosage form of the above-mentioned type.Can carry out dressing to the tablet of described novel compositions, pill etc., perhaps carry out being mixed of alternate manner and the dosage form with long-acting advantage is provided.For example, described tablet or pill can comprise the inner composition by outer set subpackage quilt.And described two kinds of components can be separated by enteric layer, this enteric layer play anti-disintegration and make internal composition can be in good condition by stomach or the effect that delays to discharge.Have various materials can be used for described 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 cellulose acetate of hexadecyl.

Pharmaceutical preparation 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 United States Patent (USP) 4,485,045 and 4,544,545).United States Patent (USP) 5,013 discloses the liposome of the cycling time with prolongation in 556.Some liposome can prepare by the anti-phase evaporation that use comprises the lipid composition of phosphatidylcholine, cholesterol and PEG deutero-phosphatidylethanolamine (PEG-PE).Liposome can be extruded the liposome that obtains to have required diameter by the strainer with definite aperture.

Described antibody can also be encapsulated in the microcapsule.Such microcapsule can prepare by 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 and poly-(methyl methacrylate) 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 polymkeric substance that contains described antibody, and described matrix is moulding product form (for example film or microcapsule).The example of sustained-release matrix comprises polyester, hydrogel (for example poly-(methacrylic acid-2-hydroxyethyl ester) or poly-(vinyl alcohol)), polylactide (United States Patent (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 (as LUPRON DEPOT TM (the Injectable microspheres body of forming by lactic acid-ethanol copolymer and leuprorelin acetate), sucrose acetate isobutyrate and poly--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 chemical substance or medicine known in the art and can be commercially available, and 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.Use altogether and use successively co-administered can comprising, described using altogether can be single using of pharmaceutical preparation of planting, also can be to use using of preparation separately, described using successively can be using with random order, but normally use successively in the regular hour, so that all promoting agents can be brought into play their biologic activity simultaneously.The preparation of described chemotherapeutics and dosage regimen can be used according to the specification sheets of manufacturers, are perhaps rule of thumb determined by skilled practitioner.Described 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, described 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 tumor associated antigen.

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 determine to use described radiotherapeutic any dosage regimen according to skilled practitioner.

In other embodiment, treatment can comprise other antibody co-administered of antibody of the present invention and anti-other tumor associated antigens, described other antibody include but not limited to EGF acceptor (EGFR) (

), erbB2 acceptor (HER2) (

) and vascular endothelial growth factor (VEGF) (

) bonded antibody.And treatment can comprise using of one or more cytokines, and this treatment can follow the exenterate of cancer cells or treatment doctor 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 process, the disease of disease to be treated, the purpose of using this antibody are therapeutic purpose or preventative purpose, previous therapy, patient's clinical medical history etc., and all these depends on treatment doctor's judgement.Antibody can be used once, also can use in continuing the serial therapy of a couple of days to several months, or use until realizing recovery from illness or reaching 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 doctor in charge can easily determine 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 treatment doctor can be according to residence time and the concentration repetition rate of estimating administration 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, described 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.

Embodiment disclosed in this invention can obtain further instruction by reference following examples, and described embodiment has described the preparation of antibody disclosed in this invention and the using method of antibody disclosed in this invention in detail.Those skilled in the art can know clearly, can carry out many changes to material and method under the situation that does not deviate from the present disclosure scope.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 context clear 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 described 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 of extracellular domain for preparing human DLL4 is as Antibody Preparation antigen.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 forecasting software SignalP 3.0 comes the endogenous signal sequence of simulating human DLL4 is cut in use, and the interior cut point possibility of still actual body is difference owing to the amino acid coupling of either 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 ⁺⁺The chelating affinity chromatography is purified into antigen protein from the insect cell conditioned medium.Subsequently about 1mg/ml is dialysed, is concentrated into to purified antigen protein with PBS (pH=7), and the aseptic immunity preparation that is filtered into.

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 ELISA and facs analysis to come the blood of the individual mouse of examination to antigenic identification (as detailed below).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, by ELISA and facs analysis the supernatant liquor of each plate hole is carried out examination at antigen protein.Select the high hybridoma of several antibody titerss and enlarged culturing in static cultivation usefulness bottle.Use albumin A or Protein G agarose chromatography to be purified into antibody from the hybridoma supernatant liquor.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 marker 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 by 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 determine interactional ability between the antibody interferes with DLL4 of anti-DLL4 and the Notch.Will be with antibody or the reference protein-Fc+ control antibodies incubation of the antibody of HEK 293 cells of the stable transduction of DLL4 cDNA and the anti-DLL4 of Notch1-EGF10-15-Fc+, Notch1-EGF1O-15-Fc+ control antibodies, the anti-DLL4 of reference protein-Fc+.By anti-Fc antibody of PE-link coupled goat and Flow cytometry combining of Fc fusion rotein and the cell of expressing DLL4.Determined the bonded inhibition ability of the antibody of anti-DLL4 by 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 is in conjunction with human DLL4 (but specifically in conjunction with muroid DLL4), 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 the standard recombinant dna technology, prepared the Mammals expression plasmid carrier that comprises the CMV promotor in the polynucleotide upstream 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.Also designed the DLL4-fc fusion rotein that specific amino acids replaces that comprises of another series.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 of anti-DLL4 and combined DLL4 fragment are interacted, and combine (Fig. 3 A) by measuring with detection HRP activity with the incubation of the anti-mouse antibodies of HRP link coupled subsequently.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 by 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 combination or the not combination of 21M18 respectively with "+" or "-".By the combination that has further characterized 21M18 that combines of check 21M18 and a series of DLL4 protein fragments (DLL4 territory 1-6), described 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.By the ELISA examination combinations of these fusion roteins to 21M18.Evaluation shows that combination is important (shown in Fig. 3 D) to 21M18 in several positions.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 the DLL4 protein fragments of replacement, 21M18 shows in conjunction with weakening.On the contrary, the antibody 21M21 of a uniqueness is in conjunction with epi-position contained in the DSL district (Fig. 3 E), 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, separate the variable region that the heavy chain and the light chain of antibody fall in institute's menu gram from hybridoma, and this variable region is connected to IgG in the mammalian expression vector respectively in the framework by 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 ﹠amp; 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 by 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 as 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 described.Choose subsequently and may be used for humanized human framework region to 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, relatively the V that is 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 by 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.Described alternative heavy chain comprises: i) contain according to the analysis that may influence of 21M18 combined function and the functional human class framework of selected replacement and ii) parent's rodent antibody 21M18 CDR (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 21M18 CDR (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 21M18 CDR (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 by 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 by ELISA test condition substratum.Select to show the strongest bonded 21M18 heavy chain variant.This variant-" 21M18 H2 "-except containing muroid CDR, also 6 frame positions in the Vh framework contain replacement, and described 6 frame positions are Karbate position 16,20,27,28,38 and 48 (Fig. 4 A).Subsequently with each cotransfection in 21M18 H2 humanization heavy chain and the five kinds of alternative humanization light chains in HEK 293 cells, and by the ELISA antigen combination of test condition substratum once more.Find an independent light chain variant-" 21M18 L2 "-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 CDR2 H2 (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 (2,1M1 8 H7L2 and 2,1M1 8 H9L2) all show the specific antigens combination by ELISA.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 as 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 described.Choose human heavy chain the most similar and light chain variable framework region subsequently as being used for humanized human framework region to parent 21M18 antibody aminoacid sequence.In order to identify the most similar described human framework region, use BLAST that the human genomic sequence of storing among the Genbank is searched for, relatively the V that is 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 by 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 21M18 CDR (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 21M18 CDR (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 21M18 CDR (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 21M18 CDR (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 by 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 by ELISA test condition substratum subsequently.Select to show the strongest bonded 21M18 heavy chain variant.This variant-" 21M18 H2 "-except containing muroid CDR, also contain the muroid residue at 5 frame positions, described 5 frame positions are Karbate position 20,28,38,48 and 69 (Fig. 4).Subsequently with each cotransfection in 21M18 H2 humanization heavy chain and the five kinds of alternative humanization light chains in HEK 293 cells, and by the ELISA antigen combination of test condition substratum once more.Independent light chain variant-" 21M18 L2 " 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 CDR2 H2 (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 (2,1M1 8 H7L2 and 2,1M1 8 H9L2).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 determines the binding affinity by the humanized antibody 21M18 of albumin A chromatography purification.After measured, 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 21M18 H7L2 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 by uniqueness specially exchanges 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 allow 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 cell being fixed 30 minutes in ethanol, and with monoclonal antibody (BMG 6H8 clone, the Fab fragment) reaction of the anti-BrdU of peroxidase link coupled 90 minutes.Substrate is developed the color in containing the solution of tetramethyl benzidine, after 15 minutes with the 1mol/L H of 25 μ l ₂SO ₄Stop.Read plate instrument (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.By coming relatively to determine 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 determined the antibody blocking Notch signal transduction pathway of anti-DLL4.Be supplemented with the Hela cell cultivated among the DMEM of 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 in the upstream 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.Then will through cells transfected add through 10 μ g/ml DLL4-Fc albumen spend the night the bag quilt culture plate.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.Determined the inhibition ability of antibody thus to DLL4 inductive Notch pathway activation.The DLL4 activatory of observing the Notch pathway activation with the rodent antibody 21M14 of anti-DLL4 and 21M18 suppresses (Fig. 6).On the contrary, though the antibody 21M21 of anti-DLL4 can be in conjunction with the DS1 territory (Fig. 3 E) of DLL4,21M21 does not suppress Notch in conjunction with (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 measure the expression of Notch pathway gene HES1 and ATOH-1 by RT-PCR through rodent antibody 21M18 treatment (described below).(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.Determine the integrity of RNA by 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 the quick real-time PCR system of ABI 7900 HT (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 Gus B 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 energy 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 cytotoxic assay that complement relies on

In some embodiments, the cancer stem cell of expressing the cancerous cell line of DLL4 or being separated to from patient's sample is gone down to posterity in the mouse of immunocompromised (as institute's detaileds description hereinafter) 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.Then will cell contain the antibody of anti-DLL4 with 200 μ l or the serum or the hot inactivated serum of control antibodies mixes, establish 3 repetitions through suspending.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 the 100 μ l propidium iodide solution (25 μ g/ml) be diluted among the HBSS, and incubation 5 minutes at room temperature.Collecting cell 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 absorbs is used to measure necrocytosis in the presence of the antibody (comparing with control antibodies with hot inactivated serum) at serum and anti-DLL4.Determine 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, the cancer stem cell of expressing the cancerous cell line of DLL4 or being separated to from patient's sample is gone down to posterity in the mouse of immunocompromised (as institute's detaileds description hereinafter) as heterograft, use the cell born of the same parents poison (ADCC) of this cancerous cell line or cancer stem cell measurement 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 peripheral blood lymphocytes (PBMC) by Ficoll-Paque density gradient centrifugation, to be used as the effector cell.Then under the situation that the antibody of at least a DLL4 or control antibodies exist, with 25: 1, the E/T of 10: 1 and 5: 1 was than mixing in 96 orifice plates 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 (CytoTox96 Non-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 instrument with standard 96 orifice plates gathers absorbance data and carries out background correction at the 490nm place.Calculate the per-cent of specific cytotoxic according to following formula: % 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).Determine the ability of the cell born of the same parents poison that the antibody-mediated antibody of anti-DLL4 relies on thus.

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

Present embodiment has been described the tumor growth that the antibody of using anti-DLL4 prevents that xenograft models is interior.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 by 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 by the 10mL pipettor and to be beaten in per 15～20 minutes.By 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 marker, 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 markers is carried out the positive or negative selection.By selecting anti-H2Kd+ and muroid CD45+ cell to get rid of mouse cell, and by 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, from separation of C 8 colon tumors through the animal of treatment and determine the expression of cell proliferation marker Ki67 by 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 dimethylbenzene, take off paraffin and rehydrated in distilled 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 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 (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: grow in the body of the antibody prevention of the anti-DLL4 of use and treatment tumour in conjoint therapy

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 described 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 to a greater degree than any independent treatment.

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 rodent antibody 21M18, the anti-EGFR of the anti-DLL4 of animal (n=10) peritoneal injection (i.p.), 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.The C17 tumour cell implanted 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.21M18 and VEGF antibody have all been slowed down growth of tumor, any independent antibody of Combined Ration more effective (Figure 18).

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 to a greater degree than any independent treatment.And, though separately with the 7.5mg/kg irinotecan treat weekly stop after tumour continued growth or accelerating growth in most of animals, weekly the irinotecan of the 21M18 of the anti-DLL4 of twice 10mg/kg and weekly 7.5mg/kg unite after treatment in the 56th day stops above the growth (Figure 13) that has further stoped colon tumor in the time in 5 weeks.

In some embodiments, to check the humanized antibody H7L2 21M18 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 for the treatment of 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 the cell of being injected 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 the cell of being injected 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, significantly delayed the generation again (Figure 16) of tumor growth behind the conjoint therapy with the antibody 21M18 treatment of anti-DLL4.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 determine 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, with tumour suspension and mixed 37 ℃ of incubations 1 hour that is incorporated in of digestion solution, described digestion solution is at MEBM substratum (Cambrex, East Rutherford, NJ) contain collagenase/Unidasa in: Dispase (1: 1: 8,10 *) and 1: 100 the dilution deoxyribonuclease I (Worthington, Lakewood, NJ).Eccentric cell also (is dissolved in the 0.15M NH of distilled 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 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, flank district, the right side of mouse injection 900,300,100 or 50 isolating human tumor cells (n=10, every group).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 has the mouse that can detect tumour in all injections in the group of the tumour cell of DLL4 antibody 21M18 treatment reduces, and further reduces (Figure 15 A) at injection this per-cent in the group of the tumour cell of DLL4 21M18-irinotecan.Utilize these tumour occurrence frequencies, pass 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 DLL4 21M18-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 by enzymatic digestion and Mechanical Crushing with sterile razor blade.Then dissociated tumour cell is 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, separate ESA+, CD44+, CD24-/low, Lin-tumorigenicity tumour cell and inject by method described in detail above.

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, by 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.In addition, 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 vascular smooth muscle cell, 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 can use different tumours of detection and tumour to raise is estimated Antybody therapy to 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 marker 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.Determine 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 obtain single cell suspension by enzymic digestion and Mechanical Crushing.Press above method described in detail then,, adopt facs analysis, analyze dissociated tumour cell and whether have the tumorigenicity cancer stem cell according to ESA+, CD44+, CD24-/low, Lin-superficial cell marker representation.

Can estimate the tumorigenicity of expressing institute's isolated cells behind the Antybody therapy of anti-DLL4 according to ESA+, CD44+, CD24-/low, Lin-then.ESA+, the CD44+, CD24-/low, the Lin-cancer stem cell that separate the mouse for the treatment of 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, determine 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 described 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 determine to exist the cancer stem cell marker representation from the tumor biopsy sample.The biopsy samples taking-up tumour cell of under aseptic condition, suffering from the patient of cancer from diagnosis.In some embodiments, biopsy sample aquatic foods 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, with biopsy sample formalin fixed, paraffin embedding, and switching 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 determine the existence of cancer stem cell.The biopsy sample is cut into small pieces, shreds fully, and pair cell carries out enzymic digestion and Mechanical Crushing, to obtain single cell suspension 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 determine ESA+ by flow cytometry described in detail above, CD44+, CD24-/low, Lin-, the existence of DLL4+ tumor stem cell.

Treat its tumour after diagnosing for expressing the cancer patients of Notch acceptor or Notch receptors ligand with the humanized antibody of anti-DLL4.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, folinic acid or streptozocin) in the standard radiation treatment plan, simultaneously or use described 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.Described explanation and embodiment should be considered as only being the example purpose, and actual range of the present invention and essence are provided by appended claims.All communiques, patent and patent application that this paper quoted are all incorporated in the present disclosure by reference.

Sequence table

SEQ ID NO:1 (heavy chain CDR1)

TAYYIH

SEQ ID NO:2 (heavy chain CDR2, H2)

YISCYNGATNYNQKFKG

SEQ ID NO:3 (heavy chain CDR2 H7)

YISSYNGATNYNQKFKG

SEQ ID NO:4 (heavy chain CDR2 H9)

YISVYNGATNYNQKFKG

SEQ ID NO:5 (heavy chain CDR3)

RDYDYDVGMDY

SEQ ID NO:6 (variable region of heavy chain, H2)

QVQLVQSGAEVKKPGASVKISCKASGYSFTAYYIHWKQAPGQGLEWIGYISCYNGATNYNQ

KFKGRVTFTTDTSTSTAYMELRSLRSDDTAVYYCARDYDYDVGMDYWGQGTLVTVSS

SEQ ID NO:7 (variable region of heavy chain, H7)

QVQLVQSGAEVKKPGASVKISCKASGYSFTAYYIHWVKQAPGQGLEWIGYISSYNGATNYN

QKFKGRVTFTTDTSTSTAYMELRSLRSDDTAVYYCARDYDYDVGMDYWGQGTLVTVSS

SEQ ID NO:8 (variable region of heavy chain, H9)

QVQLVQSGAEVKKPGASVKISCKASGYSFTAYYIHWVKQAPGQGLEWIGYISVYNGATNYN

QKFKGRVTFTTDTSTSTAYMELRSLRSDDTAVYYCARDYDYDVGMDYWGQGTLVTVSS

SEQ ID NO:9 (light chain CDR1)

RASESVDNYGISFMK

SEQ ID NO:10 (light chain CDR2)

AASNQGS

SEQ ID NO:11 (light chain CDR3)

QQS?KEVPWTFGG

SEQ ID NO:12 (variable region of light chain)

DIVMTQSPDSLAVSLGERATISCRASESVDNYGISFMKWFQQKPGQPPKLLIYAASNQGSGVP

DRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSKEVPWTFGGGTKVEIK

SEQ ID NO:13 (nucleotide sequence of heavy chain, H2, variable region)

ATGGACTGGACCTGGAGCATCCTGTTCCTGGTGGCTGCTGCTACAGGAGCTCACTCCCAG

GTTCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCTAGCGTGAAGATCAG

CTGCAAGGCTAGCGGATACTCCTTTACAGCTTACTACATCCACTGGGTGAAGCAGGCCCC

TGGACAAGGGCTGGAGTGGATCGGATATATCAGCTGTTACAACGGAGCTACAAACTACA

ACCAGAAGTTCAAGGGCAGGGTCACCTTCACAACAGACACAAGCACAAGCACAGCCTAC

ATGGAGCTGAGGAGCCTGAGAAGCGACGACACAGCCGTGTACTACTGTGCTAGGGACTA

CGACTACGACGTGGGGATGGACTACTGGGGCCAAGGAACCCTGGTCACCGTCAGCTCA

SEQ ID NO:14 (nucleotide sequence of heavy chain, H7, variable region)

ATGGACTGGACCTGGAGCATCCTGTTCCTGGTGGCTGCTGCTACAGGAGCTCACTCCCAG

GTTCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCTAGCGTGAAGATCAG

CTGCAAGGCTAGCGGATACTCCTTTACAGCTTACTACATCCACTGGGTGAAGCAGGCCCC

TGGACAAGGGCTGGAGTGGATCGGATATATCAGCTCCTACAACGGAGCTACAAACTACA

ACCAGAAGTTCAAGGGCAGGGTCACCTTCACAACAGACACAAGCACAAGCACAGCCTAC

ATGGAGCTGAGGAGCCTGAGAAGCGACGACACAGCCGTGTACTACTGTGCTAGGGACTA

CGACTACGACGTGGGGATGGACTACTGGGGCCAAGGAACCCTGGTCACCGTCAGCTCA

SEQ ID NO:15 (nucleotide sequence of heavy chain, H9)

ATGGACTGGACCTGGAGCATCCTGTTCCTGGTGGCTGCTGCTACAGGAGCTCACTCCCAG

GTTCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCTAGCGTGAAGATCAG

CTGCAAGGCTAGCGGATACTCCTTTACAGCTTACTACATCCACTGGGTGAAGCAGGCCCC

TGGACAAGGGCTGGAGTGGATCGGATATATCAGCGTCTACAACGGAGCTACAAACTACA

ACCAGAAGTTCAAGGGCAGGGTCACCTTCACAACAGACACAAGCACAAGCACAGCCTAC

ATGGAGCTGAGGAGCCTGAGAAGCGACGACACAGCCGTGTACTACTGTGCTAGGGACTA

CGACTACGACGTGGGGATGGACTACTGGGGCCAAGGAACCCTGGTCACCGTCAGCTCA

SEQ ID NO:16 (nucleotide sequence of light chain)

ATGGTGCTCCAGACCCAGGTCTTCATTTCCCTGCTGCTCTGGATCAGCGGAGCCTACGGG

GACATCGTGATGACCCAGTCCCCTGACTCCCTGGCTGTGTCCCTGGGCGAGAGGGCCACC

ATCTCCTGCAGAGCCAGCGAATCCGTCGATAATTATGGCATTTCCTTTATGAAGTGGTTCC

AGCAGAAACCAGGACAGCCTCCTAAGCTGCTCATTTACGCTGCATCCAACCAAGGGTCCG

GGGTCCCTGACAGGTTCTCCGGCAGCGGGTCCGGAACAGATTTCACTCTCACCATCAGCA

GCCTGCAGGCTGAAGATGTGGCTGTCTATTACTGTCAGCAAAGCAAGGAGGTGCCTTGGA

CATTCGGAGGAGGGACCAAGGTGGAAATCAAACGTACGGTGGCTGCCCCCTCCGTCTTC

ATCTTCCCCCCCAGCGATGAGCAGCTGAAAGCGGCACTGCCAGCGTGGTGTGCCTGCTGA

ATAACTTCTATCCCCGGGAGGCCAAAGTGCAGTGGAAGGTGGATAACGCCCTCCAAAGC

GGCAACTCCCAGGAGAGCGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAG

CAGCACCCTGACCCTGAGCAAAGCCGACTACGAGAAACACAAAGTCTACGCCTGCGAAG

TCACCCATCAGGGCCTGAGCAGCCCCGTCACAAAGAGCTTCAACAGGGGCGAGTGTTGA

SEQ ID NO:17 (nucleotide sequence of heavy chain CDR1)

ACAGCTTACTACATCCAC

SEQ ID NO:18 (nucleotide sequence of heavy chain CDR2, H2)

ATATCAGCTGTTACAACGGAGCTACAAACTACAACCAGAAGTTCAAGGGC

SEQ ID NO:19 (nucleotide sequence of heavy chain CDR2, H7)

ATATCAGCTCCTACAACGGAGCTACAAACTACAACCAGAAGTTCAAGGGC

SEQ ID NO:20 (nucleotide sequence of heavy chain CDR2, H9)

ATATCAGCGTCTACAACGGAGCTACAAACTACAACCAGAAGTTCAAGGGC

SEQ ID NO:21 (nucleotide sequence of heavy chain CDR3)

AGGGACTACGACTACGACGTGGGGATGGACTAC

SEQ ID NO:22 (nucleotide sequence of light chain CDR1)

AGAGCCAGCGAATCCGTCGATAATTATGGCATTTCCTTTATGAAG

SEQ ID NO:23 (nucleotide sequence of light chain CDR2)

GCTGCATCCAACCAAGGGTCC

SEQ ID NO:24 (nucleotide sequence of light chain CDR3)

CAGCAAAGCAAGGAGGTGCCTTGGACATTCGGAGGA

SEQ ID NO:25 (human DLL4 antigen)

MAAASRSASGWALLLLVALWQQRAAGSGVFQLQLQEFINERGVLASGRPCEPGCRTFFRVC

LKHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLPFNFTWGTFSLIIEAWHAP

GDDLRPEALPPDALISKIAIQGSLAVGQNWLLDEQTSTLTRLRYSYRVICSDNYYGDNCSRLC

KKRNDHFGHYVCQPDGNLSCLPGWTGEYCQQPICLSGCHEQNGYCSKPAECLCRPGWQGRL

CNECIPHNGCRHGTCSTPWQCTCDEGWGGLFCDQDLNYCTHHSPCKNGATCSNSGQRSYTC

TCRPGYTCVDCELELSECDSNPCRNGGSCKDQEDGYHCLCPPGYYGLHCEHSTLSCADSPCF

NGGSCRERNQGANYACECPPNFTGSNCEKKVDRCTSNPCANGGQCLNRGPSRMCRCRPGFT

GTYCELHVSDCARNPCAHGGTCHDLENGLMCTCPAGFSGPRCEVRTSIDACASSPCFNRATC

YTDLSTDTFVCNCPYGFVGSRCEFPVG

SEQ ID NO:26 (human DLL4 DSL zone)

WLLDEQTSTLTRLRYSYRVICSDNYYGDNCSRLCKKRNDHFGHYVCQPDGNLSCLPGWTGE

YC

SEQ ID NO:27 (human DLL4 N-stub area)

MAAASRSASGWALLLLVALWQQRAAGSGVFQLQLQEFINERGVLASGRPCEPGCRTFFRVC

LKHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLPFNFTWPGTFSLIIEAWHA

PGDDLRPEALPPDALISKIAIQGSLAVGQN

The PCT/RO/134 table

Applicant or attorney docket FCI09US0603C

International application no PCTUS2007/020889

Explanation about microbial preservation

(detailed rules and regulations 13 two)

PCT/RO/134 table (in July, 1998, reprint in January, 2004)

Claims (63)

1. separated antibody, this antibodies specific ground is in conjunction with human DLL4 epi-position, and described human DLL4 epi-position contains the amino acid in the human DLL4N-end region (SEQ ID NO:27), and wherein said antibody influence contains the growth of tumor of cancer stem cell.

2. separated antibody as claimed in claim 1, wherein said human DLL4 epi-position is combined to form by described human DLL4N-end region (SEQ ID NO:27) and human DLL4DSL (SEQ ID NO:26).

3. separated antibody as claimed in claim 1, wherein said human DLL4 epi-position further comprise the amino acid among the human DLL4DSL (SEQ ID NO:26).

4. separated antibody as claimed in claim 2, wherein said human DLL4 epi-position contains the amino acid 66～73 (QAVVSPGP) of described human DLL4N-end region.

5. separated antibody as claimed in claim 4, wherein said human DLL4 epi-position contains amino acid V68, V69 and the P71 of described human DLL4N-end region.

6. separated antibody as claimed in claim 2, wherein said human DLL4 epi-position contains the amino acid/11 39～146 (LISKIAIQ) of described human DLL4N-end region.

7. separated antibody as claimed in claim 6, wherein said human DLL4 epi-position contains the amino acid K142 and the A144 of described human DLL4N-end region.

8. separated antibody as claimed in claim 2, wherein said human DLL4 epi-position contain the amino acid 66～73 (QAVVSPGP) and the amino acid/11 39～146 (LISKIAIQ) of described human DLL4N-end region.

9. separated antibody as claimed in claim 8, wherein said human DLL4 epi-position contains amino acid V68, V69, P71, K142 and the K144 of described human DLL4N-end region.

10. the described separated antibody of arbitrary as described above claim, wherein said influence is reducing of gross tumor volume.

11. as each described antibody in the claim 1～9, wherein said antibody is monoclonal antibody.

12. as each described antibody in the claim 1～9, wherein said antibody is chimeric antibody.

13. as each described antibody in the claim 1～9, wherein said antibody is humanized antibody.

14. as each described antibody in the claim 1～9, wherein said antibody is human antibodies.

15. humanized antibody as claimed in claim 13, described humanized antibody are IgG antibody.

16. humanized antibody as claimed in claim 15, wherein said IgG is IgG ₂

17. humanized antibody as claimed in claim 13, described humanized antibody is an antibody fragment.

18. humanized antibody as claimed in claim 17, wherein said antibody fragment are the Fab fragments.

19. as each described separated antibody in the claim 1～9, described antibody comprises:

I. contain cdr amino acid sequence C DR1 (SEQ ID NO:1); CDR2 (SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4); And the variable region of heavy chain of CDR3 (SEQ ID NO:5); With

Ii. contain cdr amino acid sequence C DR1 (SEQ ID NO:9); CDR2 (SEQ ID NO:10); And the variable region of light chain of CDR3 (SEQ ID NO:11).

20. separated antibody as claimed in claim 19, described antibody is produced by hybridoma, and described hybridoma is preserved in ATCC on September 28th, 2007, and the ATCC deposit number is PTA-8670.

21. an antibody, this antibody and the antibody competition that produced by following hybridoma are to the specificity combination of human DLL4, and described hybridoma is preserved in ATCC on September 28th, 2007, and the ATCC deposit number is PTA-8670.

22. humanized antibody, this antibodies specific ground is in conjunction with human DLL4 epi-position, described human DLL4 epi-position contains the amino acid in the human DLL4N-end region (SEQ ID NO:27), wherein said antibody comprises: (i) comprise the variable region of heavy chain of non-human antigen determining area and human heavy chain variable framework region and (ii) comprise non-human antigen determining area and the variable region of light chain of human light chain variable framework region.

23. humanized antibody as claimed in claim 22, wherein said human DLL4 epi-position is combined to form by described human DLL4N-end region (SEQ ID NO:27) and human DLL4DSL (SEQ ID NO:26).

24. humanized antibody as claimed in claim 22, wherein said human DLL4 epi-position further comprise the amino acid among the human DLL4DSL (SEQ ID NO:26).

25. humanized antibody as claimed in claim 23 has at least 1 residue to be substituted in the wherein said human heavy chain variable framework region.

26. humanized antibody as claimed in claim 25, wherein said at least one replacement are the replacements that occupies the residue of corresponding position in the antibody that contains described non-human antigen determining area.

27. humanized antibody as claimed in claim 25 has 5 or 6 residues to be substituted in the wherein said human heavy chain variable framework region.

28. humanized antibody as claimed in claim 25, described at least one replacement in the variable framework region of the wherein said mankind is positioned at the position that is selected from the group of being made up of 16H, 20H, 27H, 28H, 38H and 48H according to Karbate's number system.

29. humanized antibody as claimed in claim 25, described at least one replacement in the variable framework region of the wherein said mankind is positioned at the position that is selected from the group of being made up of 20H, 28H, 38H, 48H and 69H according to Karbate's number system.

30. humanized antibody as claimed in claim 27, wherein position 16H, 20H, 27H, 28H, 38H and the 48H according to Karbate's number system is substituted.

31. humanized antibody as claimed in claim 27, wherein position 20H, 28H, 38H, 48H and the 69H according to Karbate's number system is substituted.

32. as each described humanized antibody in the claim 22～31, wherein said heavy chain non-human antigen determining area comprises cdr amino acid sequence C DR1 (SEQ ID NO:1); CDR2 (SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4); And CDR3 (SEQ ID NO:5).

33., have at least one residue to be substituted in the wherein said human light chain variable framework region as each described humanized antibody in the claim 22～31.

34. humanized antibody as claimed in claim 33, wherein said at least one replacement are the replacements that occupies the residue of corresponding position in the antibody that contains described non-human antigen determining area.

35. humanized antibody as claimed in claim 33 has two residues to be substituted in the wherein said human light chain variable framework region.

36. humanized antibody as claimed in claim 33, described at least one residue in the variable framework region of the wherein said mankind is positioned at the position that is selected from the group of being made up of 22L and 36L according to Karbate's number system.

37. humanized antibody as claimed in claim 35, wherein position 22L and the 36L according to Karbate's number system is substituted.

38. as each described humanized antibody in the claim 33～37, wherein said light chain non-human antigen determining area comprises CDR1 (SEQ ID NO:9); CDR2 (SEQ ID NO:10); And CDR3 (SEQ ID NO:11).

39. humanized antibody as claimed in claim 22, wherein said variable region of heavy chain comprise SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8.

40. humanized antibody as claimed in claim 22, wherein said variable region of light chain comprise SEQ ID NO:12.

41. humanized antibody as claimed in claim 22, wherein said variable region of heavy chain comprise SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8 and described variable region of light chain and comprise SEQ ID NO:12.

42. a separated antibody, this antibodies specific ground is in conjunction with human DLL4 epi-position, and described human DLL4 epi-position contains the amino acid in the human DLL4N-end region (SEQ ID NO:27), and wherein said antibody inhibition DLL4 combines with the Notch acceptor.

43. an antibody, described antibody with combine by the specificity of following plasmid-encoded antibody competition human DLL4, described plasmid is preserved in ATCC, the ATCC deposit number is PTA-8427 or PTA-8425.

44. a humanized antibody, this humanized antibody is the plasmid-encoded of PTA-8427 or PTA-8425 by the ATCC deposit number.

45. a pharmaceutical composition, described pharmaceutical composition comprises:

I. each described antibody in the aforementioned claim; With

Ii. pharmaceutical carrier.

46. a separated polynucleotide carrier, each described antibody in the described polynucleotide carrier coding claim 1～44.

47. a host cell, described host cell comprise one or more polynucleotide carriers of appointing a described antibody in the coding claim 1～44.

48. as each described antibody in the claim 1～44, described antibody is used for the treatment of cancer.

49. each described antibody is used for the treatment of application in the medicine of cancer in manufacturing in the claim 1～44.

50. it is co-administered in the patient that application as claimed in claim 49, wherein said application comprise the described antibody and second therapeutical agent of treatment significant quantity.

51. application as claimed in claim 50, the wherein said antibody and second therapeutical agent give simultaneously or give successively.

52. application as claimed in claim 50, wherein said second therapeutical agent is a chemotherapeutic.

53. application as claimed in claim 52, wherein said chemotherapeutic are Fluracil or irinotecan.

54. application as claimed in claim 50, wherein said second therapeutical agent are the second treatment antibody.

55. application as claimed in claim 54, the wherein said second treatment antibody is the antibody of anti-EGF acceptor or the antibody of anti-VEGF.

56. application as claimed in claim 50, wherein said second therapeutical agent is a radiotherapy.

57. the method for a diagnosing cancer, described method comprise patient's sample is contacted with each described antibody in the claim 1～44, and come diagnosing cancer according to the location that DLL4 in described patient's sample expresses.

58. a method of making each described antibody in the claim 1～44, described method comprises:

I. with one or more polynucleotide expression vector transformed host cells of encoding said antibody;

Ii. make the described host cell of cultivation under the condition of described antibody expression; With

Iii. the described antibody of purifying from substratum.

59. one kind express each described antibody in the claim 1～44 through cell transformed be.

60. the hybridoma of a secretory antibody 21M18, described hybridoma is deposited in ATCC on September 28th, 2007, and the ATCC deposit number is PTA-8670.

61. the plasmid of encode 21M18 H9L2 (ATCC deposit number PTA-8427) or 21M18H7L2 (ATCC deposit number PTA-8425).

62. a method for preparing antibody 21M18H9L2 or antibody 21M18 H7L2, the ATCC deposit number is arranged is that the cell of the plasmid of PTA-8427 or PTA-8425 carries out to described method by cultivating transfection.

63. test kit, described test kit comprises container and the composition that is contained in the described container, and comprise that further the described composition of indication can be used to treat the package insert of cancer, wherein said composition contains each described antibody in the claim 1～44.

Images