CN1902488A - Screening assays and methods of tumor treatment - Google Patents

Abstract

The invention relates generally to the screening of candidate molecules for the treatment of tumor metastasis, and treatment methods using such molecules. Thus, the invention includes a method of screening comprising the steps of: (1) administering a plurality of test substances to a non-human syngeneic immunocompetent animal model bearing at least one soft tissue or bone metastasis, in the presence or absence of a primary tumor; (2) determining the effects of the test substances on the soft tissue or bone metastasis and growth of the primary tumor, if present; and (3) identifying a test substance that inhibits the growth of a soft tissue or bone metastasis, without adverse effect on the status of the primary tumor, if present.

Description

The screening experiment of oncotherapy and method

Background of invention

Invention field

The present invention relates in general to and is used for the treatment of tumour, comprises the screening of the candidate molecules of tumor metastasis, and uses the methods for the treatment of of described molecule.

The explanation of correlation technique

Tumour and cancer

The growth of higher organism body is characterised in that fissional exquisite pattern modulated on time and the space. The destruction of fissional normal physiological almost always is harmful to. A kind of this type of destruction is cancer, and this is a kind of disease that results from a series of genetic events.

By two kinds of heritable characteristics definition cancer cells, i.e. uncontrollable growth and uncontrollably invade normal structure. In the cell that causes localization growth (localized growth) or tumour, cancer cell can divide and not be subjected to normal growth restriction. In addition, some cancer cells also obtain from their initial site migration and the ability of other health tissues of intrusion patient. The combination of these two characteristics makes the cancer cell special hazard. Cancer develops by multistep process at human body, and this illustrates that multiple variation must occur normal cell to be changed into the cell with malignant phenotype. Class in the related gene comprises cellular oncogene (cellular oncogene), is activated or when being expressed inadequately, it is not subjected to normal cell controlling mechanism and promotes unrestraint cell proliferation when it suddenlys change.

The not controlled abnormal cell colony of growth that separates will cause tumour or neoformation (neoplasm). As long as neoformation remains on independent position, it just is considered to optimum, and expection can be cured fully by this material of exenterate. If tumour or neoformation are pernicious, that is to say, if its cell has the ability of invasion tissue on every side, just it is considered as cancer. Real pernicious when starting from cell and pass basalis (basal lamina) and begin to invade the connective tissue (connective tissue) of below. Pernicious generation cell obtains to dissociate from main tumour agglomerate (tumor mass), enters blood flow or lymphatic vessel, and form secondary tumor (secondary tumor) or in vivo other site occur in the situation of the ability that shifts. Metastases must be wider, more is difficult to eradicate and treatment.

As determined by epidemiological study and clinical research, most of cancers take stage slowly from slight benign development as malignant neoplasm. Malignant cancer begins with the optimum localization cell colony with the misgrowth characteristic that is called dysplasia (dysplasia) usually, described abnormal cell obtains to cause neoplastic unusual growth characteristics, it is characterized by local growth and the expansion (swelling) of cell colony. If do not treat, the neoplasia of original position (neoplasia) can develop malignant tumor and form. Morbidity from dysplastic initial sign to full-blown malignant cancer can be passed through several years or decades. In kinds cancer, observe this characteristic process.

Transforming growth factor-beta (TGF-β)

TGF-β be participate in regulation and control multiple bioprocess in such as fruit bat (drosophila) and people's different organisms growth factor (cell factor) greatly the member of superfamily (Grande,Proc.Soc.Exp.Biol.Med.， 214(1): 27-40 (1997)). Described process comprises that (embryogenesis), tissue repair and immune system (modulation) occur for cell proliferation and differentiation, extracellular matrix metabolism (extracellular matrix metabolism), the generation of bone form (bone morphogenesis), adhesion, Apoptosis (apoptosis), cell migration (cell migration), embryo. Every kind of cell in the actual upper body (for example, the cell of the cell of epithelium, endothelium, epithelial cell, hemopoietic cell, neuronal cell and phoirocyte) produces and has the acceptor of TGF-β.

In anaphylactic type TGF-'beta ' family a plurality of isotypes are arranged, be called TGF-β 1, TGF-β 2, TGF-β 3, TGF-β 4 and TGF-β 5, wherein the mammal isotype is TGF-β 1, TGF-β 2 and TGF-β 3. Every kind of isotype is expressed by different gene codes and with tissue-specific and the upper modulated mode of growth. For example, TGF-β 1 mRNA wide expression in epithelial cell, endothelial cell, hemopoietic cell and phoirocyte, and TGF-β 2 mRNA mainly express in epithelial cell and neuronal cell, and TGF-β mRNA mainly expresses in mesenchymal cell (mesenchymal cell). Described mammal isotype high conservative between kind, this has shown the important biological function of each isotype. No matter their similitude, these isotypes at them for different aspect the affinity (binding affinity) of TGF-beta receptor.

The phenotype that knocks out generation by three kinds of mammal TGF-β isotype TGF-β 1, TGF-β 2 and TGF-β 3 is very distinct and not overlapping. Mouse without TGF-β 1 suffers from autoimmunity-sample (autoimmune-like) inflammatory disease, TGF-β 2 knock-out mices show perinatal mortality (perinatal mortality) and serious developmental defect, and the mouse of TGF-β 3 defectives has cleft palate (cleft palate) and defectiveness in lung development. This illustrates that these parts have isotype-specific activity, and it can not be compensated by other family member.

The member of TGF-'beta ' family starts their cytosis by being incorporated into three kinds of high-affinity receptors (endoglin is another kind of TGF-beta receptor, and it enriches at endothelial cell) that are called I type, II type and III type. Described III receptor (being also referred to as the beta glycan) is the abundantest type such as existence, and it passs the I type and II type frizzled receptor plays a role by then they being in conjunction with all three kinds of TGF-β isotypes. The TGF-beta antagonists can be played in the extracellular soluble foreign lands of III receptor. (Vilchis Landeros etc.,Biochem.J.，355: 215-222 (2001)). Born of the same parents' internal area of I type and II receptor contains serine/threonine protein kitase, and it begins the intracellular signal transmission by the signal transducer phosphorylation with several being called " SMAD " (this term is derived from Sma and the MAD gene homolog of identifying in Caenorhabditis elegans (Caenorhabditis elegans) and Drosophila melanogaster (Drosophila melanogaster)). Although TGF-β can be in conjunction with the III receptor, then it is TGF-β and passs I type and II receptor, and TGF-β 1 and TGF-β 3 also can be directly in conjunction with the II receptors. Aglucon is incorporated into after the II receptor, the II receptor raise (recruit), in conjunction with and with I receptor phosphorylation, the thus protein kinase activity of costimulatory receptor. In this common mode, the transduction of TGF-β enabling signal.

TGF-β 1 quantitatively is main isotype, but one or more in three kinds of isotypes of every kind of tissue expression basically, together with their homoreceptor. In growth course and in the adult animal, the expression pattern of three kinds of isotypes (expression pattern) spatially with the time on all different, this illustrates that they play nonredundant effect. In order to support this viewpoint, the knock-out mice of three kinds of isotypes has non-overlapped phenotypic spectrum. All three kinds of TGF-β obviously are important in growth because knock out in these genes any one cause some the embryo's or perinatal death. Can be by the expression pattern of TGF-β (in not disturbed animal and the response to exciting), in the phenotype by the mouse of TGF-β function (by the application of genetic manipulation or TGF-beta antagonists) in damaged condition, and by showing that TGF-β infers adjection in the adult animal for the in vitro study of the effect of (specialized) cell type of specialization of difference. Therefore, TGF-β is at regulating cell propagation, differentiation and apoptosis, and function of immune system plays a crucial role in blood vessel generation and the tissue repair. Therefore, the numerous disease process is relevant with unusual TGF-β function. The forfeiture of TGF-β function is relevant with the pathogenesis of cancer, atherosclerotic (atherosclerosis) and autoimmune disease, and excessive TGF-β produces the immunosupress (parasite-induced immunosuppression) of inducing with fibroplasia disease (fibroproliferative disorders), parasite and transfer is relevant (referring to for example, Roberts and Sporn, The Transforming Growth Factor-β, in Sporn and Roberts (eds.)Handbook of Experimental Pharmacology：Peptide Growth Factors and Their Receptors, Springer Verlag, Berlin (1990), at pages 419-472; Flanders and Roberts, Transforming Growth Factor-β, in Oppenheim and Feldmann,Cytokine Reference, Academic Press, London (2000); Dunker and Krieglstein,Eur.J.Biochem.， 267: 6982-6988 (2000); Branton and Kopp,Microbes Infect.，1：1349-1365 (1999); With Chen and Wahl,Microbes Infect.，1：1367-1380(1999))。

The increase of TGF-β and minimizing and numerous disease interrelate, and described disease comprises atherosclerotic and the fibrotic conditions (fibrotic disease) of kidney, liver and lung. TGF-β, its acceptor, and/or the gene mutation in the intracellular signal molecule relevant with TGF-β also is important in cancer and hereditary hemorrhagic telangiectasia (hereditary hemorrhagic telangiectasia) specifically in (pathogenic) process of causing a disease.

TGF-β isotype occurs to play a part in (tumorigenesis) process complicated in the tumour of various tumours. In many cases, tumour cell becomes has resistance to TGF-β, this usually is because the intragenic sudden change of the following material of coding causes: (a) acceptor, (b) are directly involved in signal and transmit the molecule of (SMADS) or (c) downstream egg white matter, and it plays extremely important effect (such as CDK-mortifier, Rb albumen etc.) in the control of cell cycle. And some researchs have reported that the secretion that strengthens about TGF-β causes the propagation of adjacent tissue to be suppressed in tumour cell. The enhancing of TGF-β secretion also may promote blood vessel that (stimulating the generation of VEGF) occurs. Two kinds of effects have all stimulated tumor growth.

TGF-β is multiple-effect (pleiotropic) cell factor, its directly (by affecting Growth of Cells and differentiation) and indirectly (by regulate immune system, extracellular matrix upgrades (tumover) and blood vessel generation) affect tumor growth. Previous data tumor cells showed can change them for the replying of TGF-β, and are short metastatic (pro-metastatic) in the late tumor by changing into for the growth inhibition in the infantile tumour. TGF-signal beta approach has been considered to the promoter of tumor inhibitor approach and tumor development and intrusion. Referring to, for example, Derynck etc.,Nat.Genet.，29(2)：117-129(2001)。

In normal cell, TGF-β is by suppressing cell proliferation and/or can be used as tumor inhibitor by promotion Cell Differentiation or apoptosis. In tumorigenic process, many cells have been lost the growth inhibition of their TGF-β-mediation. After the growth inhibiting resistance of development to TGF-β, the TGF-β that the tumour cell in the tumour and stroma cell usually increase them produces. It is relevant to the transfer of organ at a distance with the invasiveness that increases and tumour cell that the TGF-β of this increase produces, at least in part because the interaction of the change of stimulation, cell mobility, immunosupress and tumour cell that the blood vessel of TGF-β-mediation occurs and extracellular matrix. Therefore, tumour cell causes the larger invasiveness of swollen neoplastic increase and those tumour cells for the excessive generation of the resistance of TGF-β and the TGF-beta ligands of following. In fact, TGF-β plays a very important role in healthy and disease with related (associated) acceptor.

TGF-β is the establishment thing of epithelial cell proliferation, and the TGF-beta system in many tissues, have the tumor inhibitor activity (referring to for example, Gold,Crit.Rev.Oncol.，10：303-360 (1999); Massague etc.,Cell，103：295-309 (2000); With Akhurst and Balmain,J.Pathol.， 187: 82-90 (1999)). Observe the minimizing of TGF-beta receptor or downstream signal composition or lose in many people's tumor types, described tumor type comprises gastroenteric tumor, tumor of breast and tumor of prostate. Use the research of xenograft (xenograft) of the tumor cell line of the mouse model of genetic modification or genetic manipulation to confirm the causal relation of the tumour of the TGF-β function that reduces and increase between occuring. Yet the effect of TGF-β in tumour occurs is complicated, and such as the increase that many people's tumour demonstration in late period TGF-β express, it increases relevant with prognosis mala with transfer. Seem that TGF-β occurs to play tumor inhibitor in early days in tumour, but late, they can play oncogene and promote aggressive to shift disease progression. Promote the mechanism that shifts to be believed to comprise the blood vessel generation of the invasive ability of tumor cell of enhancing, enhancing and the inhibition of immunological surveillance (immune surveillance) system. TGF-β 1 is the isotype that the most generally raises late period at human cancer, although related in some cases TGF-β 2 and TGF-β 3.

TGF-β expressed in many late periods to be increased in the human cancer and with the intrusion that strengthens and/or shift relevant. TGF-β 1 and TGF-β 3 are the isotypes that are usually directed to. Frequently, the blood plasma level of TGF-β also increases in suffering from the cancer patient of grave illness, and this explanation tumour just is secreted into the TGF-β of significant quantity in the circulation. The tumour that the TGF-β that demonstration raises expresses comprises cancer, melanoma, chondrosarcoma and the osteosarcoma of breast, colon, stomach, liver, pancreas, prostate, lung, kidney, bladder and nasopharynx.

The immunohistochemical staining of TGF-β 1 and the PD of human breast cancer (Gorsch etc.,Canc. Res.，52：6949-6952 (1992)) link together, and with node positive (node positivity) and transfer interrelated (Walker and Dearing,Eur.J.Canc.，28：641-644 (1992)). Outer TGF-β 1 albumen of the born of the same parents of secretion increase at the progress edge of primary human breast carcinoma (advancing edge) with in lymph node (lymph node) shifts (Dalal etc.,Am.J.Pathol.，143：381-389 (1993)). TGF-β 1 increases in the blood plasma of the patients with mastocarcinoma of 81% new diagnosis, and this level by node-feminine gender (node-negative) patient rather than the surgical excision in node-positive patient with standardization, this explanation primary tumor and the TGF-β 1 that shifts significant quantity are secreted into (Kong etc. in the circulationAnn.Surg.，222：155-162 (1995)). The also discovery in having the patients with mastocarcinoma of positive lymph nodes of the blood plasma level of the increase of TGF-β 3 (Li etc.,Intl.J.Canc.，79：455-459 (1998)), and the lymph node relevance in the invasive tumor relevant with prognosis mala with the combination that positive TGF-β 3 expresses (Ghellal etc.,Anticancer Res.， 20：4413-4418(2000))。

For colorectal cancer patients, in the primary tumor of excision the strong dyeing of TGF-β 1 and disease progression to the metastatic disease significant correlation (Friedman etc.,Canc.Epidemiol.Biomarkers Prev.， 4：549-554 (1995)). In addition, when relatively the time, in the cancer cell of invading regional nodes, finding that the level of TGF-β 1 dyeing increases with primary tumor, and the TGF-β 1 that raises 75% be examined relate in the case transfer process (Picon etc.,Canc.Epidemiol.Biomarkers Prev.，7：497-504 (1998)). Plasma TG F-β 1 and TGF-β 2 levels increase in suffering from the patient of colorectal cancer, and the more serious level of disease just higher (Tsushima etc.,Gastroenterol.，110：375-382 (1996); With Bellone etc.,Eur.J.Canc.，37：224-233 (2001)). Similarly, plasma TG F-β 1 level of rising sees among the suffer from hepatocellular carcinoma patient of (hepatocellular carcinoma), and after tumor resection this level standard, the source that this explanation tumour is TGF-β 1 (Shirai etc.,Jpn.Canc.Res.， 83：676-679 (1992)). Positive staining and the serous coat of TGF-β 1 in stomach organization invaded (serosal invasion) and lymphatic metastasis is closely related (Maehara etc.,J.Clin.Oncol.，17：607-614 (1999)), and the serum levels rising of TGF-β 1 relevant with lymphatic metastasis and prognosis mala (Saito etc.,Anticancer Res.，20：4489-4493 (2000)). In addition, the expression that TGF-β 1,2 increases in 50% cancer of pancreas case and increases with 3 mRNA related with the survival rate of minimizing (Friess etc.,Gastroenterol.，105：1846-1856(1993)).

Tumour rank higher in TGF-β 1 dyeing that increases and the patients with prostate cancer relevant with transfer (Wikstrom etc.,Prostate，37：19-29 (1998)). For patient's survival rate, the TGF-β of increase by 1 dyeing be negative predictive factors (Stravodimos etc.,Anticancer Res.，20：3823-3828 (2000)). Higher level in those that the dyeing of TGF-β 1 ratio does not shift in the primary tumor that has shifted (Eastham etc.,Lab.Invest.，73：628-635 (1995)). In addition, plasma TG F-β 1 level shift suffering from clinically significantly (Adler etc.,J.Urol.，161：182-187 (1999)), or suffer from starting stage (primary stage) III/IV disease (Ivanovic etc.,Nat.Med.，1：282-284 (1995)) significantly raises among the patient.

Find that in the primary tumor of the patients with lung cancer with lymphatic metastasis those that do not shift with primary tumo(u)r are compared, extractible TGF-β 1 albumen increase (Hasegawa etc.,Canc.，91：964-971 (2001)). The blood plasma level of suffering from dissemination (disseminated) but not finding TGF-β 1 in the melanoma patient of regional area (loco-regional) disease raise and the blood plasma level rising amplitude of TGF-β 2 less (Krasagakis etc.,Br.J.Canc.，77：1492-1494 (1998)). In osteosarcoma, that the TGF-β 1 of rising or the immunohistochemical staining of TGF-β 3 and the follow-up lung of height ratio more shift is relevant (Yang etc.,J.Exp. Med.，184：133-142 (1998)). Plasma TG F-β 1 level also have chondrosarcoma (Gridley etc.,Canc.Detect.Prev.，22：20-29 (1998)) and clear-cell carcinoma (renal cell carcinomas) (Wunderlich etc.,Urol.Intl.，60：205-207 (1998); With Junker etc.,Cytokine，8：794-798 (1996)) significantly raise among the patient, this illustrates the TGF-β of the tumour secreting high levels of these types. Serum TG F-β 1 level is suffering from diffusivity but be not to increase among the patient of shallow phenotype (superficial) carcinoma of urinary bladder, although in suffering from the patient of metastatic disease, find further to increase (Eder etc.,J.Urol.， 156：953-957 (1996)). In the patient who suffers from relevant (Epstein-Barr virus-associated) nasopharyngeal carcinoma of Epstein-Barr virus, especially in the patient of the tumour of suffering from recurrence serum TG F-β 1 also be increase (Xu etc.,Intl.J.Canc.，84：396-399(1999))。

Find with TGF-β 1 albumen in the free serum culture thing of rat breast cancer cell system, carry out preliminary treatment causes lung transfer number after described injection cell is entered homogenic rat remarkable increase (Welch etc.,Proc.Natl.Acad.Sci.USA，87：7678-7682 (1990)). Discovery TGF-β 1 gene transfection primary human benign prostatic tumour cell stimulation transfer after coordination (orthotopic) is implanted the SCID mouse (Stearns etc.,Canc.Res.，5：711-720 (1999)). Utilize the rat prostate cancer cell (Steiner and Barrack,Mol.Endocrinol.，6：15-25 (1992)) and Chinese hamster ovary (Chinese hamster ovary) cell (Ueki etc.,Jpn.J.Canc.Res.，84：589-593 (1993)) obtains similar result.

Found with anti-TGF-beta 1,2 and 3 neutralizing antibody (neutralizing antibody) process athymic mouse be suppressed at the lung of human breast cancer cell line MDA-MB-231 intraperitoneal inoculation after shift formation (Arteaga etc.,J.Clin.Invest.，92：2569-2576 (1993)). When the B16F1 melanoma cells is injected into the tail vein of homogenic mouse, same antibody cause the transfer number that forms reduce by three times (Wojtowicz-Praga etc.,J.Immunother.Emphasis Tumor Immunol.，19：169-175 (1996)). In other report, find anti--TGF-β 1 monoclonal antibody reduce the generation shifted after will the athymic mouse of the subcutaneous implantation of human cancer cell line (Hoefer and Anderer,Canc.Immunol. Immunother.，41：302-308 (1995)). In all three these researchs, TGF-β is for relevant with the transfer efficiency of increase by the killer cell of the natural killer cell of host animal, monocyte or Lymphokine (the lymphokine-activated killer cell) inhibitory action of cell mediated immune surveillance. In addition, process the transfer characteristic that pernicious mouse fibrosarcoma (fibrosarcoma) cell has reduced these cells significantly with TGF-beta 1 specific ASON, this explanation is important (Spearman etc. by the TGF-β that tumour cell oneself produces in promoting transferGene，149：25-29(1994))。

In three different experimental systems, by disturbing breast tumor cell system to cause the remarkable minimizing (McEarchem etc. of the transfer efficiency of these cells for the responsiveness (responsiveness) of TGF-β with the transfection of dominant negative (dominant negative) II type TGF-beta receptorInt.J.Canc.，91：76-82 (2001); Oft etc.,Curr.Biol.，8：1243-1252 (1998); With Yin etc.,J.Clin.Invest.， 103：197-206 (1999)). In the situation of human breast cancer cell line MDA-MB-231, bone shift reduce significantly and also when using the xenograft models of athymic mouse existence prolong (Yin etc., same above). These presentation of results, in breast cancer, the TGF-β that directly works for tumour cell can increase transfer efficiency at least. Mechanism comprises the generation of the parathyroid hormone-related peptide of the invasiveness of enhancing and increase.

Yet TGF-β is not same short the transfer, as report with TGF-β carry out preliminary treatment with inhibition Chinese hamster chondrosarcoma cells (Fujisawa etc.,J.Exp.Med.，187：203-213 (2000)) formation shifted of lung, the metastatic that has reduced rat cancer cell of oral cavity system with the transfection of TGF-β 3 spread (Davies etc.,J.Oral.Pathol.Med.，29：232-240 (2000)), the overexpression of II type TGF-beta receptor reduced the thyroid cell that K-ras-transforms metastatic potential (Turco etc.,Intl.J.Canc.，80：85-91 (1999)). This explanation TGF-β promotes the ability that shifts to change with tumor type.

Because it is important like this that TGF-β plays a part in keeping normal cell homeostasis (homeostasis) in many tracts, the conceptual issues of the key of the pathology of use TGF-beta antagonists treatment TGF-β-driving is may produce undesirable side effect for normal structure, described side effect includes but not limited to abnormal cell proliferation and the tumour that causes owing to losing of the tumor inhibitor function of TGF-β in many epithelial cells forms and increases, and the problem that causes owing to immune system disorder (dysregulation) (for example, many focuses inflammation (multifocal inflammation), autoimmunity performance (autoimmune manifestation) and myelodysplastisches (myeloid hyperplasia)). These pathology has been predicted in research based on the mouse with tentative TGF-β functional defect (experimentally compromised TGF-β function).

The survival period that allow to prolong without the mouse generation non-metastatic colon cancer (Engle etc. without TGF-β 1 on the Rag2 genetic background, Canc.Res., 59:3379-3386 (1999)), this is consistent with endogenous TGF-β 1 plays tumor inhibitor in colonic epithelium concept. Only have the TGF-β 1 allelic TGF-β 1+ of a function/-mouse show glandular stomach (glandular stomach) paraplasm (Boivin etc.,Lab.Invest.，74：513-518 (1996)), and for the tumorigenic neurological susceptibility that carcinogenic substance (carcinogen) in liver and the lung is induced increase (Tang etc.,Nat.Med.，4：802-807 (1998)). Similarly, the overexpression of the target by dominant negative TGF-beta receptor disturb TGF-β responsiveness cause paraplasm and for the tumorigenic neurological susceptibility that carcinogenic substance in skin and the mammary gland is induced increase (Amendt etc.,Oncogene， 17：25-34 (1998); With Bottinger etc.,Canc.Res.，57：5564-5570 (1997)), and the increase of SMT generation (Gorska etc.,Proc.Am.Assoc.Canc.Res.，42：422(2001))。

Wean in the near future, die from quick exhaustion syndrome (rapid wasting syndrome) without TGF-β mouse, its with cause lymphocyte and macrophage to infiltrate through in a large number many organs, the relevant (Shull etc. of especially cardiorespiratory many focuses inflammatory reactionNature，359：693-699 (1992); With Kulkami etc.,Proc.Natl.Acad.Sci.USA，90：770-774 (1993)). Described syndrome has many signs of autoimmune disease, it comprise the enhancing of circulating antibody, immune complex deposit and major histocompatibility complex antigen (MHCI and MHCII) for NA expression (Dang etc.,J.Immunol.， 155：3205-3212 (1995)). In the repressed MCH-deficiency of inflammation background, have marrow paraplasm (Letterio etc.,J.Clin.Invest.，98：2109-2119 (1996)). These studies show that TGF-β 1 keeps the important function of normal homeostasis in immune a plurality of compartments (compartment). Consistent therewith, the TGF-β responsiveness that the transgene expression of dominant negative TGF-beta receptor in CD4+ and CD8+T-cell causes is low to subtract that to cause the T-Cell Differentiation be effect T-cell, this also cause autoimmunity-sample syndrome (Gorelik and Flavell,Immun.，12：171-181 (2000)), and the dominant negative acceptor in early days the expression in the T-cell cause CD8+T cell lymphocytic hyperplasia disease (lymphoproliferative disorder), its cause lymphoid organ a large amount of expansions (Lucas etc.,J.Exp.Med.，191：1187-1196 (2000))。

Before used TGF-beta antagonists (antibody, the SR2F of following discussion, antisense TGF-β DNA and dominant negative TGF-beta receptor) in many animal model systems, to treat the disease that TGF-β drives, specifically fibrillatable (fibrosis). Yet these are generally relative short-term experiment, and it often relates to the local delivery of antagonist, but also do not estimate long term exposure in the consequence of TGF-beta antagonists, especially occur about tumour and the consequence of function of immune system aspect.

The overexpression of TGF-β has related to the pathology of numerous disease, especially fibrotic conditions and TCA. Use the original research of TGF-beta antagonists to use anti-TGF-beta antibodies or naturally occurring TGF-β in conjunction with albumen. For example, anti-TGF-beta antibodies and proteoglycans decorin (proteoglycan decorin), its be TGF-β in conjunction with albumen, successfully in rat model, used with protection avoid experimental kidney fibrosis (Border etc.,Nature，360：361-364 (1992); With Border etc., Nature, 346:371-374 (1990)).

Synthetic TGF-β is as biology potential type compound, and it must be activated before they can be incorporated into the frizzled receptor compound. The former district of precursor (pro-region) of the cracking by the former peptide of TGF-β (pro-peptide) gives incubation period (latency) with the non-covalent combination of mature T GF-β. The former district of described precursor is also referred to as the peptide relevant with incubation period (LAP), and TGF-β 1 LAP of purifying can be used as all three kinds of TGF-β isotypes antagonist (Bottinger etc.,Proc.Natl.Acad.Sci.USA，93：5877(1996))。

Usually, antibody with have relative low compatibility based on protein-bonded antagonist. The outer ligand binding domain of the born of the same parents of II type TGF-beta receptor have TGF-β 1 and TGF-β 3 the high-affinity binding site (O ' Connor-McCourt etc.,Ann.N.Y.Acad.Sci.，766：300-302 (1995)). When ligand binding domain was blended in the human immunoglobulin(HIg) Fc district that causes this ligand binding domain dimerization outside with extracellular soluble, compatibility further increased. With the Fc district add soluble cytokine receptor also increase they Half-life in vivo (Capon etc.,Nature，337：525-531 (1989)). Soluble T GF-beta receptor-Fc fusion (SR2F) produces in a lot of laboratories, and successfully used to seal or reduced pulmonary fibrosis and the rat balloon catheter of being induced by bleomycin (bleomycin) in radiation induced enteropathy (enteropathy), the hamster in N-nitrosodimethylamine (dimethylnitrosamine) or the liver fibrogenesis of being induced by the connection of ductus choledochus in the rat (common bile duct), the fibrillatable, mouse in EGN (glomerulonephritis) model and degraded (adventitial) fibrillatable of adventitia in (rat balloon catheter denudation) model and (intimal) damage (Ueno etc. of inner membranceHum.GeneTher.，11：33-42 (2000); George etc.,Proc.Natl.Acad.Sci.USA，96：12719-12724 (1999); Isaka etc.,Kidney Intl.， 55：465-475 (1999); Zheng etc.,Gastroenterol.，110：1286-1296 (2000); Wang etc.,Thorax，54：805-812 (1999); With Smith etc.,Circ.Res.，84：1212-1222 (1999)). In most of the cases, the albumen of SR2F antagonist as the injection purifying is given, although it gives with gene therapy method by cDNA being imported muscle that (Ueno etc. see above in two kinds of situations; With Isaka etc., see above). Neither one is mentioned disadvantageous side effect among the above-mentioned author, but all are the research of relative short-term.

With the synthetic TGF-β of the form of biologically hiding, described form must before TGF-β can be incorporated into acceptor, be activated and cause biological response (Munger etc.,Kidney Intl.，51：1376-1382 (1997)). Because difference and the difficulty of the quantitatively active and TGF-β that hides experimentally, relative with situation about the mechanism of TGF-β vivo activation seldom is known. Immunofluorescence technique (immunofluorescence technique) active and the TGF-β that hides is distinguished in use in frozen tissue section, recently show, (BarceUos-Hoff and Ewan can be in the epithelium of normal structure occur based on cell (cell-by-cell) one by one in hiding for the mammary gland activation of TGF-β very partlyBreast Canc.Res.，2：92-99 (2000)). By contrast, attack in the face of pathology, the activation of the much extensive TGF-β that hides may be arranged. For example, the irradiation mammary gland cause the TGF-β in the matter (adipose stroma) between epithelium, week-Epithelial and stromal (peri-epithelial stroma) and fat extensive activation (Barcellos-Hoff etc.,J.Clin.Invest.，93：892-899 (1994)). Similarly, the obvious TGF-β that hides of secretion of most of normal cells in the culture, and from the emiocytosis of the tumour in the late period more TGF-β of the activity of a large amount more. Significantly, in the research of the fibrosarcoma cell system that oncogene transforms, more a high proportion of activity form TGF-β identifies high metastatic fibrosarcoma by secretion, but the clone of all conversions all total TGF-β of secreting high levels (Schwarz etc.,Growth Factors，3：115-127(1990))。

U.S. Patent Application Publication No. 2002/0176758 and the United States Patent(USP) Nos. 5,571,714 published on November 28th, 2002; 5,772,998; 5,783,185 and 6,090,383 disclose the monoclonal antibody of anti-TGF-beta and the different purposes of these antibody.

U.S. Patent Application Publication No. 2002/0176758 and United States Patent(USP) Nos. 5,571,714 that on November 28th, 2002 published; 5,772,998; 5,783,185 and 6,090383 disclose for the monoclonal antibody of TGF-β and the multiple use of described antibody.

The U.S. Patent Application Publication No. 2003/0125251 of publishing on July 3rd, 2003 discloses TGF-beta antagonists " pathologic " TGF-β that selectively neutralizes. Particularly, it is provided for suppressing by soluble T GF-beta antagonists (SR2F) method and composition of transfer. This antagonist is comprised of the extracellular soluble foreign lands of the II type TGF-beta receptor in the Fc district that is blended in human IgG. Particularly, this application relates to the normal physiological effect that utilizes SR2F to prevent from shifting and do not affect TGF-β. Therefore, SR2F distinguishes " physiological " TGF-β and " pathologic " TGF-β by this way, so that the impact of only having " pathologic " TGF-β to be subject to using SR2F. It also discloses the transgenic nonhuman animal that comprises soluble T GF-beta antagonist, and preferably, wherein said soluble T GF-beta antagonist prevents the transfer of tumour in described transgenic animals.

The U.S. Patent Application Publication No. 2003/0028905 that on February 6th, 2003 published relates to gene at the cells of normal cell and tumour, and is specifically related to the saltant in conjunction with the TGF-β II acceptor of all TGF-β isotypes. It also relates to diagnosis and methods for the treatment of, it is for the diagnosis and treatment of the disease relevant with the TGF-β II receptor of sudden change, tumour for example, and relate to transgenic nonhuman animal, it is characterized in that it contains the insertion of TGF-β 1 code cDNA at first extron of TGF-β 2 encoding genes.

Emphasized the structural requirement in conjunction with albumen (LTBP4) to the TGF-beta that hides when not existing of lung and colon Elastic fiber, the shortage of the outer TGF-beta of born of the same parents relates to the LTBP4 in the TGF-beta signal. Because TGF-beta suppresses epithelial cell proliferation, especially in colon, can infer that it lacks the carcinogenic triggering reason (trigger) that is likely that colon cancer develops in mouse from the ECM of colon. In fact, some research interrelates the defective in the TGF-beta signal and colorectal cancer. For example, the mouse that in TGF-beta-signal-transducin Smad 3, has a null mutation occur with the tumour similar such as the tumour of in the 3C7 mouse, growing (Zhu etc.,Cell，94：703-714 (1998)). In addition, sudden change or the sudden change in the TGF-beta3II receptor among TGF-signal-transducin Smad 2 and the Smad 4 are very general in human colorectal cancer, this explanation TGF-beta3 and downstream target thereof have tumor inhibitor function (Markowitz etc.Science，268：1336-1338 (1995); Riggins etc.,Cancer Res.，57：2578-2580 (1997); Zhou etc.,Proc.Natl.Acad.Sci.USA，95：2412-2416(1998))。

WO 2003/015505 discloses the protein-bonded bifunctional animal model of demonstration TGF-beta. Described animal model does not produce the resting form TGF of functional resting form LTBP or generation suboptimal level in conjunction with albumen LTBP. This list of references also discloses method and kit, is used for cancer diagnosis, pulmonary emphysema (pulmonary emphysema) or cardiomyopathy (cardiomyopathy) and analyzes cancer and/or whether pulmonary emphysema and/or cardiomyopathy cause by the differential expression of LTBP or by the defective of LTBP-4 gene. This patent application has reported that LTBP-4 is important and prevents that oncogene from transforming, cancer cell is invaded and shift diffusion (metastatic spread) for the integrality of ECM.

United States Patent(USP) Nos. 6,455,757 and 6,175,057 has showed the expression or 2 of Alzheimer disease (non-human transgenic animal's model of Alzheimer ' s disease (AD) and CAA, wherein said transgenic animals are characterised in that 1) biologically active transforming growth factor-beta 1 (TGF-β 1)) expression of biologically active TGF-β 1 and the expression of people amyloid (amyloid) β precursor protein (APP) gene outcome.

Along with the progress in the test-and-treat of primary tumor, the death rate among the cancer patient is associated with the existence of secondary tumors (transfer) further. Shift in case the patient has bone, just think that cancer is incurable.

The transfer of tumour cell from original site to the secondary position relates to many steps. For understanding various compounds for former the effect with secondary tumor, zooscopy is basic. Unfortunately, a lot of tumour cells do not shift in animal model, especially do not transfer to bone.

Therefore, have the needs of the screening technique that uses animal model system, described animal model system allows growth inhibition and the front-transfer activity of difference TGF-β.

Develop in addition screening experiment is suitable for treating the molecule of secondary tumors with evaluation needs.

In addition, have exploitation treatment cancer, especially late period metastatic cancer the needs of new method, described method identification also shows primary and the different responsiveness of metastatic tumo(u)r for TGF-β and TGF-β mortifier or the antagonist dissimilar and stage. Specifically needing identification diagnosis is possible for reply patient good late period, metastatic cancer colony with the treatment of TGF-β mortifier or antagonist.

Also need to develop the methods for the treatment of of bone transfer and osteoclasia and/or bone loss, no matter whether above-mentioned illness is relevant with primary tumor.

Summary of the invention

Therefore, the present invention as requested. In one aspect, the present invention relates to screening technique, it comprises following steps: (1) uses multiple substances to the animal model of inhuman symimmunity activity being with or without in the situation of primary tumor, and this animal model suffers from least a soft tissue or bone shifts; (2) determination test material impact that soft tissue or bone are shifted, and if have primary tumor, described substances measured to the primary tumor affects on the growth; (3) qualification test material, it suppresses the growth of soft tissue or bone transfer, and if there is primary tumor, described substances does not have harmful effect for the situation of primary tumor.

In yet another aspect, the present invention relates to determine to be diagnosed as the method that the mammalian subject possibility of suffering from cancer is benefited from the treatment of using the TGF-beta antagonist, it comprises:

(a) cancer cell that obtains from the patient of test is for the sensitiveness of the growth inhibition effect of TGF-beta;

(b) obtain the gene expression atlas of the cancer cell that obtains from the patient, and its gene expression atlas with the cancer cell that obtains from animal model is compared, described animal model is for replying with the treatment of TGF-beta antagonist; With

(c) if the cancer cell that obtains from the patient is insensitive for the growth inhibition effect of TGF-beta, and its have to from the similar gene expression atlas of the gene expression atlas of the cancer cell of the described animal model gained that responds to described treatment, then the patient is accredited as and may benefits from the treatment that utilizes the TGF-beta antagonist.

If described cancer is breast cancer, comprise primary and metastatic breast cancer, so aforementioned prognostic method also comprises the step of the Her2 state of measuring the patient, and wherein Her2+ patient is common, although not always, may not be for not replying with the treatment of TGF-beta antagonist separately or replying very poor.

The method for the treatment of cancer also within the scope of the invention in being accredited as the patient that may benefit from the treatment of using TGF-beta antagonist and above-mentioned antagonist.

In yet another aspect, the present invention relates to treat the osteoclasia relevant with the tumor metastasis in the mammalian subject or the method for bone loss, it comprises the TGF-beta antagonist of the patient being used effective dose.

In yet another aspect, the present invention relates to treat the method for the mammalian subject that is diagnosed with cancer, it comprises the combination of described patient being used TGF-beta antagonist and chemotherapeutant or the cytotoxic agent of effective dose, and the monitoring patient is for the reaction of described combination, the effective dose sum that the effective dose of wherein said combination is lower than described TGF-beta antagonist and described chemotherapeutant or cytotoxic agent when separating administration as independent reagent. If described cancer is breast cancer, comprise metastatic breast cancer, described chemotherapeutant can, for example, be taxane such as taxol (paclitaxel) (Taxol ) or taxol (taxol) derivative (for example, Docetaxel (doxetaxel) (taxotere (Taxotere) )).

As substitute or the additives of chemotherapeutant or cytotoxic agent, can use the TGF-beta antagonist to the patient who is diagnosed as metastatic cancer and be exposed to radiotherapy. Particularly, the present invention also relates to treat the method for the mammalian subject that is diagnosed as cancer, it comprises TGF-beta antagonist and the radiocurable combination of described patient being used effective dose, and the effective dose of wherein said combination is lower than described TGF-beta antagonist and described radiocurable effective dose sum when separating administration as independent reagent. The preferred breast cancer of described cancer or metastatic breast cancer or colorectal cancer, and described method can also comprise described patient is used anti-angiogenic agent.

In yet another aspect, the present invention relates to treat the method for the mammalian subject that is diagnosed as cancer, it comprises uses the TGF-beta antagonist of effective dose and the combination of anti-angiogenic agent to described patient, and monitors described patient for the reaction of described combination. In a preferred embodiment, described anti-angiogenic agent is the antibody of specific bond VEGF, and/or the described TGF-beta antagonist antibody that is specific bond TGF-beta. In another preferred embodiment, described method also comprises chemotherapeutant or the cytotoxic agent of described patient being used effective dose. In yet another aspect, the method is a kind of such method, and the effective dose of wherein said combination is lower than the effective dose sum of described TGF-beta antagonist and described anti-angiogenic agent when separating administration as independent reagent.

In yet another aspect, the invention provides treatment and be diagnosed as the method for suffering from cancer and not replying or reply very poor mammalian subject through pre-mensuration for the TGF-beta antagonists, it comprises the combination of described patient being used TGF-beta antagonists and chemotherapeutant or the cytotoxic agent of effective dose, or TGF-beta antagonists and radiocurable combination, and the monitoring patient is for the reaction of described combination. In a preferred embodiment, described cancer is breast cancer. In another preferred embodiment, described chemotherapeutant is taxane.

In yet another aspect, the present invention relates to kit, the container that it comprises the antibody that contains the specific bond VEGF contains the container of the antibody of specific bond TGF-beta, and with the specification of two kinds of antibody of effective dose coupling with treatment cancer in mammalian subject.

The accompanying drawing summary

Figure 1A and 1B show that anti--TGF-β antibody is for the impact of primary tumor growth (Figure 1A) and plasma VEGF (Figure 1B) level in the 4T1 mouse breast cancer model.

Fig. 2 shows with respect to contrast, behind anti--TGF-β Antybody therapy, and the histological score of Secondary cases lung neoplasm in the 4T1 mouse breast cancer model.

Fig. 3 shows with respect to contrast, behind anti--TGF-β Antybody therapy, and the tissue weight of Secondary cases lung neoplasm in the 4T1 mouse breast cancer model.

Fig. 4 shows with respect to contrast (more shallow bar shaped), with behind anti--TGF-β Antybody therapy (darker bar shaped), the computed tomography of Secondary cases lung neoplasm (computed tomography) (CT) is worth in the 4T1 mouse breast cancer model.

Fig. 5 A and 5B show normal cancellous bone (trabecular bone) (Fig. 5 A) in the 4T1 mouse breast cancer model and are shifted MicroCT (the X ray microbedding the is analysed photography) image of (Fig. 5 B) by the bone that the diffusion of primary tumor causes.

Fig. 6 has described in the mouse model of trastuzumab (trastuzumab) (Trastuzumab (HERCEPTIN) )-sensitiveness Her2+ breast cancer (clone F2-1282) anti--TGF-β Antybody therapy for the primary tumor affects on the growth.

Fig. 7 has described in the mouse model of trastuzumab-sensitiveness Her2+ breast cancer (clone F2-1282) anti--TGF-β Antybody therapy for the impact for level of plasma VEGF.

Fig. 8 has described in the mouse model of trastuzumab-sensitiveness Her2+ breast cancer (clone Fo5) anti--TGF-β Antybody therapy for the primary tumor affects on the growth.

Fig. 9 has described in the mouse model of trastuzumab-sensitiveness Her2+ breast cancer (clone Fo5) anti--TGF-β Antybody therapy for the impact of level of plasma VEGF.

Figure 10 and 11 has illustrated that treatment with anti--TGF-β antibody has increased the survival rate (being respectively F10 and BL6) in two melanoma mouse models.

Figure 12 and 13 is images (being respectively MicroCT and optical image) of Secondary cases lung neoplasm in the melanoma mouse model.

Figure 14 and 15 is images (being respectively MicroCT and optical image) of Secondary cases lung neoplasm in the melanoma mouse model.

Figure 16 shows with the anti--treatment of TGF-β antibody and has reduced the number of Secondary cases lung neoplasm in the melanoma mouse model.

Figure 17 shows with the anti--treatment of TGF-β antibody and has reduced the incidence of disease of lung neoplasm in the melanoma mouse model.

Figure 18 A and 18B show the contrast with respect to IgG, use the treatment of anti--TGF-β antibody for the volume (Figure 18 A) of PyMT tumour and the impact of weight (Figure 18 B). In Figure 18 B, right-hand bar is the IgG contrast for the bar on anti--TGF-β antibody left side.

Figure 19 A and 19B have described mouse monoclonal antibody 2G7 (being respectively SEQ ID Nos.1 and 2); The V of humanized huxTGFB (version)_LAnd V_HTerritory (V5H.V5L) (being respectively SEQ ID Nos.3 and 4), and people V_LAnd V_HTotal framework (hum κ 1, light chain kappa subgroup I; HumIII, heavy chain subgroup III) variable region of light chain (V of (being respectively SEQ ID Nos.5 and 6)_L) (Figure 19 A) and variable region of heavy chain (V_H) the amino acid sequence comparison in (Figure 19 B) territory. Between the humanized huxTGFB of asterisk sign and the mouse monoclonal antibody 2G7 or humanized huxTGFB and people V_LAnd V_HDifference between the total framework region. Complementarity-determining region (CDR) is underlined, and actual ethnic group be the CDR of sequence below total framework region with as a comparison (SEQ ID NOS:7-10).

The dna sequence dna (SEQ ID NOS:11-17) in the different CDR districts of Figure 20 code displaying (SEQ ID NOS:18-24).

Figure 21 shows the amino acid sequence (SEQ ID NO:25) of 709.1andH.IgG1; The amino acid sequence of H2NI.V5L (SEQ ID NO:26), the amino acid sequence of V11H.V11L (SEQ ID NO:27), the amino acid sequence of V5H.V5L (SEQ ID NO:28), the amino acid sequence of chimL.chimH (SEQ ID NO:29), and the amino acid sequence of V5H.g1L2 (SEQ ID NO:30).

Figure 22 shows the nucleotide sequence (SEQ ID NOS:31-42) of the burst of the sequence that does not have and have coding Figure 21.

Figure 23 shows for expressing the as described in Example 2 sequence of the plasmid pDR1 of light chain immunoglobulin (SEQ ID NO:44; 5391bp). PDR1 contain the sequence of the incoherent antibody of encoding and humanized anti-CD3 light chain of antibody (Shalaby etc.,J.Exp.Med.，175：217-225 (1992)), wherein the initial sum termination codon form that gives runic and underscore represents.

Figure 24 shows for expressing the as described in Example 2 sequence of the plasmid pDR2 of heavy chain immunoglobulin (SEQ ID NO:45; 6135bp). PDR2 contains sequence and the humanized anti-CD3 heavy chain of antibody (Shalaby etc. see above) of the incoherent antibody of encoding, and initial sum terminator codon wherein represents with the form of runic and underscore.

DESCRIPTION OF THE PREFERRED

I. definition

As using herein, " TGF-beta " refers to all isotypes of TGF-beta. The isotype (1-5) that 5 kinds of known TGF-beta are arranged at present, they all are that homology (60-80% homogeneity) and they all form the homodimer of about 25KD, and act on common TGF-beta cell receptor (I, II and III type). The heredity of TGF-beta and molecular biology be in the art known (referring to, for example, Roberts,Miner.Electrolyte and Metab.，24(2-3)：111-119(1998)；Wrana， Miner.Electrolyte and Metab.，24(2-3)：120-130(1998))。

As used in this article, " bioactive TGF-β 1 " refers to comprise any biologically active form of TGF-β 1 polypeptide, for example, wherein the TGF-β 1 that replaced by serine of the position 223 of TGF-β 1 propetide and 225 cysteine (referring to Samuel etc.,EMBO J.，11：1599-1605(1992)；Brunner， J. Biol.Chem.，264：13660 (1989)), or its biologically-active moiety, isotype, homologue, variant or analog.

Term " antagonist " refers to suppress molecule or the compound of " natural " or " nature " function of chemical compound. Antagonist can with these native compounds in homology or non-homology aspect conformation, electric charge or other characteristic. Therefore, by being identified antagonist by the identical or different acceptor of activator (agonist) identification. Antagonist can have the allosteric effect (allosteric effect) that hinders agonist activity. Perhaps, antagonist can hinder the function of activator. With activator contrast, the compound of antagonism does not cause physiological and/or biochemical change in the cell, so that cell same mode when existing as native compound was reacted to existing of antagonist.

As using herein, term " TGF-beta antagonists " refers to any reagent (for example, natural or synthetic reagent, biomolecule or organic compound, etc.), and this reagent can reduce amount or the activity of TGF-β in cell or in physiological system. Preferably, the TGF-beta antagonist works to reduce amount or the activity of TGF-β 1,2 or 3. For example, the TGF-beta antagonists can be in the level of transcribing, react, processing or transporting and suppresses the molecule that TGF-β expresses; It can affect the stability of TGF-β or the conversion (conversion) that precursor molecule arrives active mature form; It can affect the ability of TGF-β and one or more cell receptors (for example, I, II or III type) combination; Or it can disturb the transmission of TGF-signal beta, such as the cell response that suppresses normally TGF-β-mediation by the level at the TGF-beta receptor (for example, stop TGF-β and receptors bind or inhibition by the signal induction of bound TGF-β), by with TGF-signal beta approach in the interaction of the factor suppress specifically TGF-signal beta approach, or suppress TGF-signal beta approach so that the minimizing by the beta mediated cell response of TGF-to be provided usually by alternate manner.

The TGF-beta antagonists comprises one or more isotypes for TGF-β, such as the antibody of TGF-beta1, TGF-beta2 and/or TGF-beta3, comprise monoclonal and polyclonal antibody (Dasch etc., U.S. Patent No. 5 for one or more isotypes of TGF-β, 571,714; Also referring to, WO 97/13844 and WO 00/66631), chimeric antibody, humanized antibody and human antibodies; TGF-beta receptor such as dominant negative TGF-beta receptor and soluble form and the fragment of being combined with TGF-β, specifically TGF-β II receptor (TGFBIIR) or TGF-β III receptor (TGFBIIIR, or beta glycan), it comprises, for example, the extracellular domain of TGFBIIR or TGFBIIIR, most preferably the soluble T GF-beta receptor (rsTGFBIIR or rsTGFBIIIR) of restructuring, they all can import effectively by transgenosis, such as explanation herein; Antibody (Segarini etc., U.S. Patent No. 5,693,607 for the TGF-beta receptor; Lin etc., U.S. Patent No. 6,001,969, U.S. Patent No. 6,010,872, U.S. Patent No. 6,086,867, U.S. Patent No. 6,201,108; WO 98/48024; WO 95/10610; WO 93/09228; WO 92/00330); SR2F receptor antibody, antisense TGF-β DNA; The peptide relevant with incubation period (WO 91/08291); The TGF-β that hides for a long time (WO 94/09812); The proteoglycans of TGF-β inhibition, such as myosin (fetuin) (U.S. Patent No. 5,821,227), (WO 91/10727 for decorin (decorin) and other proteoglycans such as biglycan (biglycan), fibromodulin (fibromodulin), lumican and endoglin; Ruoslahti etc., U.S. Patent No. 5,654,270, U.S. Patent No. 5,705,609, U.S. Patent No. 5,726,149; Border, U.S. Patent No. 5,824,655; WO 91/04748; Letarte etc., U.S. Patent No. 5,830,847, U.S. Patent No. 6,015,693; WO 91/10727; WO 93/09800; With WO 94/10187); Somat (somatostatin) (WO 98/08529); Mannose-6-phosphate or mannose-1-phosphate salt (Ferguson, U.S. Patent No. 5,520,926); Prolactin (prolactin) (WO 97/40848); Insulin-like growth factor II (WO 98/17304); IP-10 (WO 97/00691); Tripeptides arg-gly-asp and peptide (Pfeffer, U.S. Patent No. 5,958,411 of containing this tripeptides; WO 93/10808); Extract (EP-A-813875 from the TGF-β of plant, fungi or bacterium-inhibition; JP 8119984; With Matsunaga etc., U.S. Patent No. 5,693,610); ASON, described oligonucleotides for example can suppress TGF-β genetic transcription or translation (Chung, U.S. Patent No. 5,683,988; Fakhrai etc., U.S. Patent No. 5,772,995; Dzau, U.S. Patent No. 5,821,234, U.S. Patent No. 5,869,462; With WO 94/25588); The albumen that relates to the TGF-signal beta, (EP-A-874 046 to comprise SMAD such as SMAD6 and SMAD7 and MADs; WO 97/31020; WO 97/38729; WO 98/03663; WO 98/07735; WO 98/07849; WO 98/45467; WO 98/53068; WO 98/55512; WO 98/56913; WO 98/53830; WO 99/50296; Falb, U.S. Patent No. 5,834,248; Falb etc., U.S. Patent No. 5,807,708; With Gimeno etc., U.S. Patent No. 5,948,639); Ski or Sno (Vogel,Science，286：665 (1999); With Stroschein etc.,Science， 286：771-774 (1999)); Any mutant, fragment or the derivative of the above-mentioned molecule of the ability of the activity of maintenance inhibition TGF-β; And organic molecule.

Preferably, the TGF-beta antagonists is TGF-beta1, TGF-beta2 or TGF-beta3 antagonist. More preferably, antagonist is the TGF-beta1 antagonist. In preferred embodiments, the TGF-beta antagonists is human monoclonal antibodies, and described antibody stops TGF-β to be combined described fragment such as F (ab) with its acceptor or its fragment₂" antibody " of other form of the ability that fragment, Fv fragment, list-chain antibody and maintenance are combined with TGF-β. In one embodiment, the TGF-beta antagonists is the people's antibody (WO 00/66631) that produces by phage display (phage display). In another preferred embodiment, the TGF-beta antagonists is people or humanized monoclonal antibody, and described antibody stops TGF-β and its acceptor (or its fragment such as F (ab)₂Other form or the fragment of the antibody of the ability that fragment, Fv fragment, list-chain antibody and maintenance are combined with TGF-β) combination. Preferred monoclonal antibody be such as embodiment 1 described mouse monoclonal antibody 2G7 and 4A11 herein, and its people or humanized form as listing among this paper embodiment 2, with from hybridoma (hybridoma) 1D11.16 (Dasch etc., U.S. Patent No. 5, the ATCC accession number HB 9849 that describes in 783,185) mouse monoclonal antibody that obtains. The people of more preferably above-mentioned Muridae antibody or humanized form, for example, those that describe among this paper embodiment 2. In order to screen the antibody of the epi-position combination on the TGF-beta of being combined by target antibody, can carry out conventional intersection enclosed experiment (cross-blocking assay) such asAntibodies，A Laboratory Manual，Those that describe among the Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988). Optional, or in addition, can carry out by means commonly known in the art epitope mapping (epitope mapping) (referring to, for example this paper Figure 19 A and 19B).

Be used for function mutation body, variant, derivative and analog that suitable TGF-beta antagonists of the present invention also will comprise above-mentioned TGF-beta antagonists, as long as keep them to suppress TGF-β amount or active ability. As using herein, " mutant, variant, derivative and analog " refers to have similar shape or structure and maintenance as the molecule of the ability of TGF-beta antagonist with parent compound. For example, can be with any crystallization in the TGF-beta antagonist disclosed herein, and can be designed with reasoningly based on the coordinate of avtive spot shape the analog of usefulness.

Optional, those of ordinary skill need not too much experiment, can modify activity, half-life, biological usability or other required characteristic of the increase of the functional group (functional group) of known antagonist and the molecule that screening is modified. When the TGF-beta antagonist is polypeptide, can produce simplification, activity, half-life that the modification of fragment and polypeptide is sent with increase, wait (for example, humanized antibody or function antibody fragment, as mentioned above). Level according to technical staff in the field of synthetic polypeptide and recombinant polypeptide generation need not too much experiment and just can obtain above-mentioned modification. Those skilled in the art also can be according to the crystal structure of TGF-beta antagonist as herein described and/or the new mortifier of knowledge design of avtive spot.

Term " material " is similar to " compound " implication and refer to can be used for treating or preventing any chemical individual (chemical entity), medicine, medicine etc. of the disease, illness, discomfort of body function or imbalance. Compound comprises known and potential treatment compound. The screening technique screening of the application of the invention can determine that compound is therapeutic agent. " known therapeutic agent compound " all as is known chemotherapeutants or cytotoxic agent, its (for example, by animal experiment or to what the people used experience being arranged) shown it is effective in described treatment. In other words, known treatment compound is not limited to for the treatment symptom compounds effective relevant with the pathology factor that relates to such as TGF-β.

Term used herein " substances " refers to any material, comprise, and be not limited to, polypeptide, protein, peptide and and organic molecule, test its purposes useful in screening experiment of the present invention or animal model. Described substances comprises antibody particularly, comprises mouse-anti body, chimeric antibody, humanized antibody and people's antibody.

Term used herein " primary tumor " refers to that order or the time by development is the tumour of first (first).

Term used herein " secondary tumors " refers to spread (transfer) to another organ of health or the tumour of another part from the organ or the position that occur first. Therefore, the mammary gland cancer that has been diffused into bone is not osteocarcinoma, and Secondary cases (transfer) breast cancer, because regardless of their position, cancer cell remains breast cancer cell.

Term used herein " transfer " refers to the diffusion of cancer from a part of health to another part. The process of described transfer relates to series of steps, described step comprise intrusion, in ooze (intravasation), transportation, suppress (arrest), exosmose (extravasation) and grow, it must be finished by cancer cell before shifting formation remote.

" for the side effect of situation (status) " of term primary tumor used herein refers to any the cause growth of primary tumor or the effect of primary tumor cell migration (diffusion).

" non-human animal " of the present invention comprises any non-human animal, comprise vertebrate such as rodent (rodent), inhuman primate (primate), sheep class (ovine), bovine (bovine), ruminant (ruminant), Lagomorpha (lagomorph), Swine (porcine), goat class (caprine), horse class (equine), dog class (canine), cat class (feline), birds (avian) etc. Preferred non-human animal is selected from lower group: Swine (for example, pig) and rodent such as mouse (for example, rat and mouse), most preferably rodent is such as mouse. Yet, be not intended that the invention be limited to any concrete non-human animal.

As using herein, term " mammal " refers to classify as mammiferous any animal, includes, but not limited to people, non-human primates, rodent etc., and it will be the recipient of concrete treatment, non-human animal model or the people of preferred this paper.

Term " cancer " and " carcinous " refer to or describe physiological conditions in the mammal that its feature is not modulated Growth of Cells usually. The example of cancer includes, but are not limited to cancer, lymthoma (lymphoma), blastoma (blastoma), sarcoma (sarcoma) and leukaemia (leukemia). The more specifically example of above-mentioned cancer comprises gland cancer (adenocarcinoma), lung carcinoma squamosum, peritonaeum (peritoneum) cancer, hepatocellular carcinoma, human primary gastrointestinal cancers, cancer of pancreas, glioblastoma (glioblastoma), cervix (cervical) cancer, ovary (ovarian) cancer, liver cancer, carcinoma of urinary bladder, liver cancer (hepatoma), melanoma, breast cancer, colon cancer, colorectal cancer, endometrium (endometrial) or uterus (uterine) cancer of squamous cell carcinoma (squamous cell cancer), cellule (small-cell) lung cancer, non-small cell lung cancer, lung, salivary gland (salivary gland) cancer, the cancer of kidney, liver cancer, prostate cancer, vulva (vulval) cancer, thyroid gland (thyroid) cancer, liver (hepatic) cancer and all kinds of head and neck.

As using herein, " treatment " refers to attempt to change the clinical intervention of the natural process of the individuality that is treated or cell, and can be for preventing (prophylaxis) or implementing in clinicopathologic process. The required effect for the treatment of comprises and prevents that disease from occuring or again occur, mitigation symptoms, and any direct or indirect pathological examination that eliminates a disease prevents from shifting, and reduces the speed of PD, improves or alleviate symptom, and that take a turn for the better or improved prognosis. Therefore, this term comprises and improvement and/or the reverse of the pathology factor such as the relevant symptom of TGF-β. Can use any measuring method described herein to assess non-human animal of the present invention " improvement in the physiological function ", and for any impact of animal dis motility rate; Use replying of animal that any experiment described herein relatively treats and untreated animal. When being used for screening technique of the present invention, cause to be defined as thus treating compound with the material of the pathology factor such as the relevant any parameter improvement of TGF-β.

" effective dose " or " effective dose " refers to effectively measure on the time of dosage and needs, to obtain required treatment or prevention result. " the treatment effective dose " of antibody can be according to factor change, the disease condition of described key element such as individuality, age, sex and body weight, and antibody causes required ability of replying in this individuality. The treatment effective dose also is a kind of amount, and wherein beneficial effect any toxicity of antibody or that illeffects is treated surpasses. " prevention effective dose " refers to effectively measure on the time of dosage and needs, to obtain required prevention result. Usually, because preventive dose is before disease or the early stage of disease is used for the curee, so the prevention effective dose will be less than the treatment effective dose.

The material that term used herein " cytotoxic agent " refers to suppress or stop the function of cell and/or causes cytoclasis. Mean that this term comprises radio isotope (radioactive isotope) (At for example²¹¹、I ¹³¹、 I ¹²⁵、Y ⁹⁰、Re ¹⁸⁶、Re ¹⁸⁸、Sm ¹⁵³、Bi ²¹²、P ³²Radio isotope with Lu), and toxin such as bacterium, fungi, plant or animal origin little-molecule toxin or enzyme activity toxin, comprise its fragment and/or variant.

" chemotherapeutant " is the compound that is used for the treatment of cancer. The example of chemotherapeutant comprises that alkylating agent (alkylating agent) is such as phosphinothioylidynetrisaziridine (thiotepa) and endoxan (cyclosphosphamide) (CYTOXAN^TM); Alkylsulfonate (alkyl sulfonate) such as busulfan (busulfan), Improsulfan (improsulfan) and piposulfan (piposulfan); Aziridine (aziridine) such as benzodopa, card ripple quinone (carboquone), meturedopa and uredopa; Aziridine (ethylenimine) and methylaminacrine (methylamelamine) comprise hemel (altretamine), tretamine (triethylenemelamine), APO (trietylenephosphoramide), TESPA (triethylenethiophosphoramide) and front three melamine (trimethylolomelamine); Acetogenin (acetogenin) (especially bullatacin and bullatacinone); Camptothecine (camptothecin) (comprising synthetic analog Hycamtin (topotecan)); Bryostatin (bryostatin); Callystatin; CC-1065 (comprising its Adozelesin (adozelesin), Carzelesin (carzelesin) and the synthetic analog of Bizelesin (bizelesin)); Cryptophycin (especially cryptophycin 1 and cryptophycin 8); Dolastatin (dolastatin); Duocarmycin (comprising synthetic analog KW-2189 and CBI-TMI); Eleutherobin; Pancratistatin; Sarcodictyin; Spongistatin; Mustargen (nitrogen mustard) such as Chlorambucil (chlorambucil), Chlornaphazine (chlomaphazine), cholophosphamide, Estramustine (estramustine), ifosfamide (ifsfamide), mustargen (mechlorethamine), nitrobin hydrochloride (mechlorethamine oxide hydrochloride), melphalan (melphalan), novoembichin (novembichin), phenesterin (phenesterine), prednimustine (prednimustine), Trofosfamide (trofosfamide), uracil mastard (uracil mustard); Nitrosureas such as BCNU (carmustine), chlorozotocin (chlorozotocin), Fotemustine (fotemustine), lomustine (lomustine), Nimustine (nimustine), Ranimustine (ranimustine); Antibiotic such as enediyne (enediyne) antibiotic (for example Calicheamicin (calicheamicin), especially Calicheamicin γ 1I and Calicheamicin θ I1, referring to, for example, Agnew,Chem Intl.Ed.Engl.，33：183-186 (1994); Anthracycline antibiotic (dynemicin) comprises anthracycline antibiotic A; Esperamicin; And neoearcinostain chromophore (neocarzinostatin chromophore) and relevant chromoprotein enediyne antibiotic chromophore), aclacinomycin (aclacinomysin), D actinomycin D (actinomycin), authramycin, azaserine (azaserine), bleomycin (bleomycin), act-C (cactinomycin), carabicin, carminomycin (carminomycin), cardinophyllin (carzinophilin), chromomycin (chromomycin), actinomycin D (dactinomycin), daunorubicin (daunorubicin), Detorubicin (detorubicin), 6-diazonium-5-oxygen-L-nor-leucine (6-diazo-5-oxo-L-nor leucine), Doxorubicin (doxorubicin) (comprises morpholino (morpholino)-Doxorubicin, cyano group morpholino (cyanomorpholino)-Doxorubicin, 2-pyrrolinyl-Doxorubicin and deoxidation Doxorubicin), epirubicin (epirubicin), esorubicin (esorubicin), idarubicin (idarubicin), marcellomycin (marcellomycin), mitomycin (mitomycin), Mycophenolic Acid (mycophenolic acid), nogalamycin (nogalamycin), olivomycin (olivomycin), Peplomycin (peplomycin), potfiromycin, puromycin (puromycin), triferricdoxorubicin (quelamycin), rodorubicin (rodorubicin), streptonigrin (streptonigrin), streptozotocin (streptozocin), tubercidin (tubercidin), ubenimex (ubenimex), Zinostatin (zinostatin), zorubicin (zorubicin); Antimetabolite (anti-metabolite) such as methotrexate (methotrexate) and 5 FU 5 fluorouracil (5-fluorouracil) are (5-FU); Folacin such as denopterin (denopterin), methotrexate, pteropterin (pteropterin), Trimetrexate (trimetrexate); Purine analogue such as fludarabine (fludarabine), 6-MP (6-mercaptopurine), ITG (thiamiprine), thioguanine (thioguanine); Pyrimidine analogue such as ancitabine (ancitabine), azacitidine (azacitidine), 6-azauridine (6-azauridine), Carmofur (carmofur), cytarabine (cytarabine), two BrdU (dideoxyuridine), doxifluridine (doxifluridine), enocitabine (enocitabine), floxuridine (floxuridine), 5-FU; Androgen (androgen) such as Calusterone (calusterone), dromostanolone (dromostanolone propionate), epithioandrostanol (epitiostanol), Mepitiostane (mepitiostane), Testolactone (testolactone); Anti--adrenaline (anti-adrenal) such as aminoglutethimide (aminoglutethimide), mitotane (mitotane), Trilostane (trilostane); Folic acid supplement (replenisher) is such as frolinic acid; Aceglatone (aceglatone); Aldophosphamide glucosides (aldophosphamide glycoside); Aminol evulinic acid (aminolevulinic acid); Amsacrine (amsacrine); Bestrabucil; Bisantrene (bisantrene); Edatrexate (edatraxate); Defosfamide (defofamine); Demecolcine (demecolcine); Diaziquone (diaziquone); Elfornithine; Elliptinium Acetate (elliptinium acetate); Epothilone; Ethoglucid (etoglucid); Gallium nitrate (gallium nitrate); Hydroxycarbamide (hydroxyurea); Lentinan (lentinan); Lonidamine (lonidamine); Maytansinoid (maytansinoid) is such as maytansine (maytansine) and ansamitocin (ansamitocin); Mitoguazone (mitoguazone); Mitoxantrone (mitoxantrone); Mopidamol (mopidamol); C-283 (nitracrine); Pentostatin (pentostatin); Phenamet (phenamet); THP (pirarubicin); Podophyllic acid (podophyllinic acid); 2-acethydrazide (2-ethylhydrazide); Procarbazine (procarbazine); PSK  (coriolan (krestin)); Razoxane (razoxane); Rhizomycin (rhizoxin); Sizofiran (sizofiran); Spirogermanium (spirogermanium); Tenuazonic acid (tenuazonic acid); Triethyleneiminobenzoquinone (triaziquone); 2,2 ', 2 "-RA3 (2,2 ', 2 "-trichlorotriethylamine); Trichothecenes (trichothecenes) (especially T-2 toxin, verracurin A, roridin (roridin) A and anguidin (anguidine); Urethane (urethan); Eldisine (vindesine); Dacarbazine (dacarbazine); Mannomustine (mannomustine); Dibromannitol (mitobronitol); Mitolactol (mitolactol); Pipobroman (pipobroman); Gacytosine; Cytarabine (arabinoside) (" Ara-C "); Endoxan; Phosphinothioylidynetrisaziridine; Taxanes, for example taxol (paclitaxel) (TAXOL , Bristol-Myers Squibb Oncology, Princeton, NJ) and Docetaxel (TAXOTERE , Rh  ne-Poulenc Rorer, Antony, France); Chlorambucil; Gemcitabine (gemcitabine); The 6-thioguanine; Mercaptopurine; Methotrexate; Platinum analogs such as cis-platinum (cisplatin) and carboplatin (carboplatin); Vincaleukoblastinum (vinblastine); Platinum (platinum); Etoposide (etoposide) (VP-16); Ifosfamide; Mitomycin C; Mitoxantrone; Vincristine (vincristine); Vinorelbine (vinorelbine); NVB (navelbine); Novantrone (novantrone); Teniposide (teniposide); Daunomycin (daunomycin); Aminopterin (aminopterin); XELODA  (capecitabine); Ibandronate (ibandronate); CPT-11; Topoisomerase enzyme inhibitor (topoisomerase inhibitor) RFS 2000; DFMO (difluoromethylornithine) (DMFO); Retinoic acid (retinoic acid); Capecitabine (capecitabine); With above-mentioned any pharmaceutically useful salt, acid or derivative. Comprise also in this definition that adjusting or inhibitory hormone are for the antihormone agent (anti-hormonal agent) of the effect of tumour, such as antiestrogen (anti-estrogen), it comprises, for example, TAM (tamoxifen), Raloxifene (raloxifene), aromatase enzyme inhibition 4 (5)-imidazoles, 4-hydroxy-tamoxifen (4-hydroxytamoxifen), Trioxifene (trioxifene), keoxifene, LY117018, Onapristone (onapristone) and Toremifene (toremifene) (fareston (FARESTON) ); And antiandrogen (anti-androgen) such as Flutamide (flutamide), Nilutamide (nilutamide), Bicalutamide (bicalutamide), leuprorelin acetate (leuprolide) and Goserelin (goserelin); With above-mentioned any pharmaceutically useful salt, acid or derivative.

As using herein, " taxane (taxoid) " or " taxane (taxane) " refers to Diterpenes family and the derivative thereof of the complexity that exists in the bark of mountain mahogany tree (Pacific Yew tree) (mountain mahogany (Taxus brevifolia)) and the leaf. The member of taxane or taxane family comprises; but be not limited to; taxol (PTX (TAXOL) ) and derivative thereof; baccatin (baccatin) III; cephalomannine (cephalomannine); 10-deacetylation baccatin (10-deacetylbaccatin) III; 10-deacetylation taxol (10-deacetyltaxol); 7-epi-10-deacetylation taxol; 7-xylosyl-10-deacetylation taxol; 7-shows (epi)-taxol; baccatin V; 7-table (epi)-10-deacetylation-Baccatine III; Docetaxel (TAXOTERE ); 2-debenzoylation-2-(p-trifluoromethyl benzoyl) taxol (2-debenzoyl-2-(p-trifluromethylbenzoyl) taxol) and 20-acetoxyl group-4-deacetylation-5-epi-20; O-open loop taxol (20-acetoxy-4-deacetyl-5-epi-20, O-secotaxol).

Term " cell factor " is that it acts on another cell as intercellular mediators (mediator) by the general terms of the albumen of a cell colony release. The example of described cell factor is lymphokine (lymphokine), monokine (monokine) and traditional polypeptide hormone. Comprise growth hormone such as human growth hormone (HGH), N-methionyl human growth hormone (HGH) and BGH in the cell factor; Parathormone (parathyroid hormone); Thyroxine (thyroxine); Insulin (insulin); Proinsulin (proinsulin); Relaxain (relaxin); Relaxation precipitinogen (prorelaxin); Glycoprotein hormones such as follicle-stimulating hormone (FSH) (follicle-stimulating hormone) (FSH), thyrotropic hormone (thyroid-stimulating hormone) (TSH) and luteinising hormone (luteinizing hormone) (LH); LGF; Fibroblast (fibroblast) growth factor; Prolactin (prolactin); Galactagogin (placental lactogen); TNF (necrosis factor)-α and-β; Mullerian inhibitor (mullerian-inhibiting substance); Mouse gonadotropic hormone related peptide (gonadotropin-associated peptide); Inhibin (inhibin); Nandrolone Phenylpropionate (activin); Blood vessel endothelium (vascular endothelial) growth factor; Integrin (integrin); TPO (thrombopoietin) (TPO); Nerve growth factor such as NGF-β; Blood platelet-growth factor; Insulin-like growth factor-I and-II; Hematopoietin (erythropoietin) (EPO); Bone-inducing factor (osteoinductive factor); Interferons (interferons) such as interferon-' alpha ' ,-β, and-γ; Colony stimulating factor (CSF) is such as macrophage-CSF (M-CSF); GM-CSF (GM-CSF); And granulocyte-CSF (G-CSF); Interleukins class (interleukins) is (ILs) such as IL-1, IL-1 α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; TNF is such as TNF-α or TNF-β; Comprise LIF and element part (kit ligand) (KL) with other polypeptide factor. As using herein, the term cell factor comprises from natural origin or from the protein of recombinant cell culture thing and the biologically active coordinate of natural-sequence cell factor.

" growth inhibitor " refers to suppress cell when using in this article, especially express the cancer cell of TGF-beta at compound or composition external or in vivo growth. Therefore, growth inhibitor is the percentage of expressing the cell of TGF-beta in the interim remarkable minimizing of S. The example of growth inhibitor comprises the reagent (except the position of S the phase) of blocking-up cell-loop, as induces the reagent that G1 suppresses and the M-phase suppresses. Classical M-phase closure comprises that Changchun amine (vincas) (vincristine and vincaleukoblastinum), taxanes and topo II mortifier are such as Doxorubicin, epirubicin, daunorubicin, Etoposide and bleomycin. Those reagent that suppress G1 also spill into the S-phase to be suppressed, for example, and DNA alkylating agent such as TAM, metacortandracin (prednisone), Dacarbazine, mustargen, cis-platinum, methotrexate, 5 FU 5 fluorouracil and ara-C. Further information canThe Molecular Basis of Cancer，Mendelsohn and Israel, eds., the exercise questions such as Murakami are, and " cell cycle regulation; oncogenes; and antineoplastic drugs " finds in (WB Saunders:Philadelphia, 1995) the 1st chapter, specifically the 13rd page.

The example of " growth inhibition " antibody is to be combined with TGF-beta and to suppress those of growth of cancer cells of overexpression TGF-beta. Preferred growth inhibition anti--growth of TGF-beta antibody suppression SK-BR-3 breast tumor cell in cell culture be greater than 20%, and in the situation of the AC of about 0.5 to 30 μ g/ml, suppress described growth and be preferably greater than 50% (for example about 50% to about 100%), wherein measured afterwards growth inhibition in six days (referring to the U.S. Patent No. 5 of promulgating on October 14th, 1997 the SK-BR-3 cell being exposed to antibody, 677,171). In this patent with hereinafter described in more detail the experiment of SK-BR-3 cell-growth inhibition.

The antibody of " inducing cell death " causes living cells to become can not surviving. Described cell is expressed the TGF-beta acceptor usually, concrete described cell overexpression TGF-beta acceptor. Preferably, described cell is cancer cell, for example mammary gland, ovary, stomach, endometrium, salivary gland, lung, kidney, colon, thyroid gland, pancreas or bladder cell. External, described cell can be SK-BR-3, BT474, Calu 3, MDA-MB-453, MDA-MB-361 or SKOV3 cell. Can when not having complement and immune effector cell, measure the cell death that cell in vitro death is induced by the cell-mediated cell toxicant of complement-dependent (ADCC) or complement-dependent cell toxicant (CDC) with difference. Therefore, can use heat-inactivated serum (namely when lacking complement) and when lacking immune effector cell, carry out the experiment of cell death. Whether can inducing cell death in order to measure antibody, with respect to untreated cell, can determine as by iodate the third ingot (propidium iodide) (PI), trypan blue (trypan blue) (referring to Moore etc.,Cytotechnology， 17：1-11 (1995)) or the film integrality loss estimated of the picked-up of 7AAD. Preferred cell-death-inductivity antibody is those (below the seeing) of inducing the PI picked-up in PI picked-up experiment in the BT474 cell.

The antibody of " cell death inducing " can be induced apoptosis, as by in conjunction with annexin (annexin) V, dna fragmentation, cellular contraction, endoplasmic reticulum (endoplasmic reticulum) expansion, smudge cells and/or form membrane vesicle (membrane vesicle) (being called apoptotic bodies) and measure. The common overexpression TGF-beta of described cell acceptor. Preferred described cell is tumour cell, for example, and mammary gland, ovary, stomach, endometrium, salivary gland, lung, kidney, colon, thyroid gland, pancreas or bladder cell. External, described cell can be SK-BR-3, BT474, Calu 3 cells, MDA-MB-453, MDA-MB-361 or SKOV3 cell. Distinct methods can be used for estimating and apoptosis-related cell event. For example, can be by annexin in conjunction with measuring (PS) transposition of phosphatidylserine (phosphatidyl serine); The DNA fragmentation can divide ladder (laddering) evaluation by DNA; And can estimate by any increase of hypodiploid (hypodiploid) cell with the nuclear/Chromatin condensation of dna fragmentation. Preferably, the antibody of cell death inducing is such antibody, with respect to the annexin that uses the BT474 cell in conjunction with experiment in for the untreated cell, about 2 to 50 times of described antibody induction, preferably approximately 5 to 50 times, and most preferably about 10 to 50 times annexin in conjunction with (below seeing). Sometimes former-Apoptosis antibody will further seal TGF-beta in conjunction with (for example 2G7 antibody); Namely the antibody of this antibody and anti-TGF-beta is shared biological characteristics. In other situation, the not obvious sealing of described antibody TGF-beta's. In addition, described antibody can be a kind of when cell death inducing, the antibody (for example only inducing the percentage of these cells to reduce the antibody of about 0-10% with respect to contrast) of not inducing the interim cell percentage of S significantly to reduce.

In broad sense, (for example use term " antibody " and comprise monoclonal antibody, polyclonal antibody, multivalent antibody, polyspecific (multispecific) antibody, bispecific antibody), full length antibody and antibody fragment, as long as they show required biologically active. Naturally occurring antibody comprises four polypeptide chains, by interconnective two the identical weights of disulfide bond (H) chain light (L) chain identical with two. Each heavy chain is comprised of variable region of heavy chain (VH) and CH, and CH is comprised of three districts with its native form, CH1, CH2 and CH3. Each light chain is comprised of variable region of light chain (VL) and constant region of light chain. Constant region of light chain is comprised of a district CL. VH and VL district further can be divided into super changeability district again, be called complementarity-determining region (CDR), it frequently with more conservative district, is called framework region (FR) and interrupts. Each VH and VL are comprised of three CDR and four FR, arrange in the following order from the amino terminal to the carboxyl terminal: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

Rely on the amino acid sequence of the constant region of their heavy chains, antibody (immunoglobulin (Ig)) can be belonged to different classifications. The immunoglobulin (Ig) that five large classes are arranged: IgA, IgD, IgE, IgG and IgM, and also some in these can further be divided into subclass (isotype), for example, IgG-1, IgG-2, IgA-1, IgA-2 etc. CH corresponding to inhomogeneous immunoglobulin (Ig) is called as respectively α, δ, ε, γ and μ. The subunit structure of inhomogeneous immunoglobulin (Ig) and three-dimensional configuration be known and also, for example, Abbas etc.,Cellular and Mol.Immunology，Carrying out generality among the 4th ed. (2000) describes. Light chain from the antibody of any invertebrate species can be attributed in two kinds of diverse types a kind of, described two types based on the amino acid sequence of their constant regions and be called as kappa (κ) and lambda (λ). Preferably, antibody is immunoglobulin G herein, more preferably immunoglobulin G.

As used in this article, term " monoclonal antibody " refers to the antibody that obtains from the colony of the antibody of homology basically, that is, the independent antibody that comprises colony is identical, but the sudden change of the possible natural generation that can exist in a small amount. Monoclonal antibody is high degree of specificity, for single antigen. In addition, compare from the Anti-TNF-α body preparation that generally includes for the different antibodies of different determinants (determinant) (epi-position), each monoclonal antibody is for the single determinant on the antigen. Modifier " monoclonal " need to be not understood to produce antibody by any specific method. For example, monoclonal antibody used according to the invention can be by first by Kohler etc.,Nature，256：495 (1975) the hybridoma method preparations of describing, maybe can prepare by recombinant DNA method (referring to, for example, U.S. Patent No. 4,816,567). Also can use such as at Clackson etc.,Nature，352：624-628 (1991) or Marks etc.,J.Mol.Biol.， 222：The method of describing among the 581-597 (1991) is separated " monoclonal antibody " from phage antibody library.

" full length antibody " refers to such as the complete antibody that will exist with native form and is not fragment.

" antibody fragment " includes only the part of complete antibody, generally includes the antigen-binding site of described complete antibody and keeps thus the ability of conjugated antigen. The example of the antibody fragment of being contained by this definition comprises: (i) Fab fragment, and it has VL, CL, VH and CH1 territory,, contains the variable region of light chain and first constant region (CH1) of constant region and heavy chain that is; (ii) Fab ' fragment, it adds several residues by the carboxyl terminal in heavy chain CH1 territory and is different from the Fab fragment, comprising one or more cysteines from antibody hinge region; (iii) Fab '-SH fragment, it is Fab ' fragment, wherein the cysteine residues of constant region contains freely mercapto; (iv) Fv fragment, it has VL and the VH territory of the single armed of antibody; (v) F (ab ')₂Fragment, it is to comprise by the divalence fragment of disulfide bond in two Fab ' fragments of hinge area connection; (vi) single-chain antibody molecule (scFv for example; ScFv) (Bird etc.,Science，242：423-426 (1988); With Huston etc.,Proc.Natl.Acad.Sci.USA，85：5879-5883 (1988)); (vii) have " bivalent antibody (diabody) " of two antigen-binding sites, it comprise the variable region of heavy chain (VH) that is connected with variable region of light chain (VL) in the same polypeptide chain (referring to, for example, EP 404,097; WO 93/11161; With Hollinger etc.,Proc.Natl.Acad.Sci.USA，90：6444-6448(1993))。

Have the antibody in " native sequences " or " natural-sequence " antibody or its district or antibody or its zone that its zone refers to have the amino acid sequence identical with the counterpart that is derived from natural antibody. Therefore, the antibody that has a native sequences can have the amino acid sequence from the corresponding antibody of any mammiferous naturally occurring antibody. Above-mentioned antibody with native sequences can be derived from from antibody natural separation or that pass through restructuring or synthetic method generation.

Variant antibody or its district refer to, has bioactive antibody or its zone of about at least 80% amino acid sequence identity with the corresponding antibodies with native sequences or its zone. Described variant comprises, for example, full length antibody and antibody fragment or its light chain or heavy chain district are wherein at the N-in antibody or fragment or zone or C-is terminal or adding or lack one or more amino acid residues in antibody, fragment or district. Normally, variant will have about at least 80% amino acid sequence identity with the corresponding antibodies with native sequences or its zone, more preferably about at least 90% amino acid sequence identity, and even more preferably about at least 95% amino acid sequence identity.

" percentage (%) amino acid sequence identity " is defined as the percentage of amino acid residue in the candidate sequence identical with amino acid residue in the sequence of selecting in this article, if necessary, in the described sequence of comparison and after importing breach, obtaining maximum percentage sequence homogeneity, and any conservative replacement is not regarded as the part of sequence homogeneity. Can obtain in order to measure the comparison of percentage amino acid sequence identity by the whole bag of tricks in the art, for example, use computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software that can openly obtain. Those skilled in the art can be identified for measuring the suitable parameter of comparison, comprise for the right any required algorithm of high specific that obtains to be compared the sequence total length. Yet for purpose herein, % amino acid sequence identity value is as described below by using sequence comparison computer program ALIGN-2 to obtain. By Genentech, Inc. the ALIGN-2 sequence comparison computer program of establishment is submitted U.S.Copyright Office to customer documentation, Washington D.C., 20559, be registered as U.S.Copyright Registration No.TXU510087 at this place, and pass through Genentech, Inc., the San Francisco, south, California can obtain publicly. Should work out described ALIGN-2 program with in UNIX operating system, use among the UNIX V4.0D of digit preference. Described sequence comparative parameter is by ALIGN-2 program setting and constant.

For purpose herein, given amino acid sequence A with (to) and (with) or for the % amino acid sequence identity of (against) given amino acid sequence B (it is optional to be expressed as given amino acid sequence A, its with and or have or contain certain % amino acid sequence identity for given amino acid sequence B) be calculated as follows:

100 multiply by mark X/Y

Wherein X is by series arrangement program ALIGN-2 number as the amino acid residue of identical match score in the arrangement of the A of this program and B, and wherein Y is the sum of amino acid residue among the B. Be appreciated that the % amino acid sequence identity of A and B will be not equal to the % amino acid sequence identity of B and A when being uneven in length during length in amino acid sequence B of amino acid sequence A.

" function " or " biologically active " antibody be can be in event structure, regulation and control, biochemical, biophysics one or more antibody in its natural activity of performance. For example, function antibody ability and the described combination that can have specific bond antigen can cause or change again cell event or molecular events such as signal transduction or enzymatic activity. Function antibody also can seal the ligand activation (ligand activation) of acceptor or as agonist antibody. One or more the ability that antibody is brought into play in its natural activity depends on several factors, comprises the assembling of correct folding and polypeptide chain. As using herein, the function antibody that produces by disclosed method has two H chains that identical L chain is identical with two usually, and they connect by a plurality of disulfide bond and be correctly folding.

Unless additionally point out, use statement " multivalent antibody " to comprise the antibody of three or more antigen-binding sites with expression in this specification. Described multivalent antibody is preferably designed for has three or more antigen-binding sites and be not natural-sequence IgM or IgA antibody usually.

Antibody specific ground herein comprises " chimeric " antibody, wherein the part of heavy chain and/or light chain be derived from concrete species or belong to the identical or homology of corresponding sequence in the antibody of antibody specific classification or subclass, and the remainder of described chain be derived from another species or belong to the identical or homology of corresponding sequence in the antibody of another antibody isotype or subclass, as long as they show required biologically active (U.S. Patent No. 4,816,567; With Morrison etc.,Proc.Natl.Acad.Sci.USA，81：6851-6855(1984))。

" humanized " antibody is chimeric antibody, and it contains the minmal sequence that is derived from non--human immunoglobulin(HIg). Largely, humanized antibody is human immunoglobulin(HIg) (receptor antibody), the residue that wherein comes the super variable region of autoreceptor is by from inhuman species (donor antibody), substitutes such as mouse, rat, rabbit or the residue of super variable region with inhuman primate of required specificity, compatibility and capacity (capacity). In some cases, the framework region of human immunoglobulin(HIg) (FR) residue is substituted by the inhuman residue of correspondence. In addition, humanized antibody can comprise receptor antibody or non-existent residue in donor antibody. Carrying out these modifies with the further antibody performance of improving. Usually, humanized antibody will comprise basically whole at least one, with common two, the variable region, wherein all or basically whole super variable loop (hypervariable loop) is corresponding to those non-human immunoglobulin, and all or basically whole FR is those human immunoglobulin(HIg) sequences. Humanized antibody also will comprise constant region for immunoglobulin (Fc), normally at least a portion of the constant region of human immunoglobulin(HIg) alternatively. About further details, referring to Jones etc.,Nature， 321：522-525 (1986); Riechmann etc.,Nature，332：323-329 (1988); And Presta,Curr. Op.Struct.Biol.，2：593-596(1992)。

" people's antibody " is a kind of such antibody, its amino acid sequence corresponding to produced by the people and/or used as disclosed herein amino acid sequence for the preparation of the antibody of any method preparation of people's antibody. The definition of this person's antibody has been got rid of particularly and has been contained non-human antigen-in conjunction with the humanized antibody of residue. Can use the whole bag of tricks known in the art to produce people's antibody. In one embodiment, people's antibody is selected from phage library, wherein this phage library express people's antibody (Vaughan etc.,Nature Biotechnology， 14：309-314 (1996); Sheets etc.,PNAS(USA)，95：6157-6162 (1998)); Hoogenboom and Winter,J.Mol.Biol.，227：381 (1991); Marks etc.,J.Mol.Biol.，222：581 (1991)). People's antibody also can prepare by human immunoglobulin gene's seat is imported transgenic animals, and for example, endogenous immunoglobulin gene is by the mouse of inactivation partially or completely. In case excite, to observe people's antibody and produce, the situation that this extremely is similar in all fields seen in the people comprises gene rearrangement, assembling and antibody library (antibody repertoire). The method exists, for example, and United States Patent(USP) Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and at Marks etc.,Bio/Technology，10：779-783 (1992); Lonberg etc.,Nature，368：856-859(1994)； Morrison， Nature，368：812-813 (1994); Fishwild etc.,Nature Biotechnology，14： 845-51(1996)；Neuberger， Nature Biotechnology，14：826 (1996); Lonberg and Huszar, Intern.Rev.Immunol.，13：Describe among the 65-93 (1995). Optional, people's antibody can prepare by producing the lymphocytic infinite multiplication of people B-(immortalization) for the antibody of target antigen (described bone-marrow-derived lymphocyte can from individuality reclaim or can be external by immunity). Referring to, for example, Cole etc.,Monoclonal Antibodies and Cancer Therapy，Alan R.Liss, p.77 (1985); Boerner etc.,J.Immunol.，147(1)：86-95 (1991); With US Pat No.5,750,373.

Term " variable " refers to such fact, and the some parts of variable region is widely different and be used for each specific antibody to combination and the specificity of its concrete antigen on sequence in the middle of the antibody. Yet changeability is not the variable region that is distributed in fifty-fifty antibody. Changeability concentrates on three sections that are called super variable region in light chain and the variable region of heavy chain. The part of the more high conservative of variable region is called framework region (FR). Each comprises four FR the variable region of natural heavy chain and light chain, and described FR takes the beta-folding configuration mostly, connects by three super variable regions, and it forms loop connecting, and the beta-foldable structure of forming section in some cases. By FR the super variable region in every chain is very closely combined and, with the super variable region from other chain, help to form antibody antigen-binding site (referring to Kabat etc.,Sequences of Proteins of Immunological Interest，5th Ed.Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Constant region is not directly involved in the combination of antibody and antigen, but shows different effector functions, such as the participation of the cytotoxicity (ADCC) of antibody antagonist-dependent cell-mediation.

" compatibility-maturation " antibody is the antibody that has one or more variation in its one or more CDR, and described variation causes described antibody to be compared with the corresponding parental generation antibody that does not have those variations, and the former improves for the compatibility of antigen. That the antibody of preferred compatibility-maturation will have a nanomole or even the target antigen compatibility of picomole. Produce the antibody of compatibility-maturation by methods known in the art. Marks etc.,Bio/Technology，10：779-783 (1992) has described the compatibility ripe (affinity maturation) of resetting by VH and VL territory. The random mutation of CDR and/or framework residue is by Barbas etc.,Proc.Nat.Acad.Sci，USA，91：3809-3813 (1994); Schier etc.,Gene，169：147-155 (1995); Yelton etc.,J.Immunol.，155：1994-2004 (1995); Jackson etc.,J.Immunol.， 154(7)：3310-3319 (1995); With Hawkins etc.,J.Mol.Biol.，226：889-896 (1992) describes.

" separate " or " recovery " antibody are a kind of identified and antibody of separating and/or reclaiming from the component of its natural surroundings. The pollution components of its natural surroundings is to disturb the diagnosis of antibody or the material of therapeutical uses, and can comprise the solute of enzyme, hormone and other oroteins or nonprotein. In preferred embodiments, described antibody will be purified to as be determined as 95 % by weight greater than polypeptide by the Lowry method by (1), and most preferably greater than 99 % by weight, (2) by utilizing rotary cup sequenator (spinning-cup sequenator) to be purified to the degree of at least 15 terminal or inner residues of enough acquisition amino acid sequence N-, or (3) are purified to homogeneous (homogeneity) by using Coomassie blue (Coomassie blue) or preferred silver to dye to carry out SDS-PAGE under reduction or non-reduced condition. Antibody that separate or that reclaim comprises the antibody of original position in the recombinant cell, because at least a component of the natural surroundings of antibody will not exist. Yet normally, antibody separation or that reclaim will be by at least one purification step preparation.

Term " antigen " fully understanding in the art, and comprises immunogenic material, namely, immunogene (immunogens), and induction of immunity nonreply, or the material of reactionless (anergy), that is, reactionless former (anergen). When antigen was polypeptide, it can be transmembrane molecule (for example acceptor) or part such as growth factor. Representational antigen comprises molecule such as feritin (renin); Growth hormone comprises human growth hormone (HGH) and BGH; Somatotropin releasing factor (growth-hormone releasing factor); Parathormone; Thyrotropic hormone; Lipoprotein (lipoprotein); AAT (antitrypsin); INSULIN A-chain; Insulin B-chain; Proinsulin; Follicle-stimulating hormone (FSH); Calcitonin (calcitonin); Luteinising hormone; Hyperglycemic factor (glucagon); Clotting factor is such as Factor IX C, factors IX and the von Willebrands factor; Anti--clotting factor is such as PROTEIN C; ANF (atrial natriuretic factor); Pulmonary surfactant; Activator of plasminogen (plasminogen activator) is such as urokinase (urokinase) or people's urine or tissue-type activator of plasminogen (t-PA); Bombesin (bombesin); Fibrin ferment (thrombin); Hemopoieticgrowth factor; TNFalpha and-beta; Enkephalinase (enkephalinase); RANTES (regulating the activation of the T-cell of normal expression and secretion); Human macrophage inflammatory protein (MIP-1-alpha); Seralbumin is such as human serum albumins; The Muellerian-inhibiting substances; Relaxain A-chain; Relaxain B-chain; Relaxation precipitinogen; Mouse gonadotropic hormone (gonadotropin)-related peptide; Microprotein is such as the beta-lactamase; DNase; IgE; Cytotoxic t-lymphocyte related antigen (CTLA) is such as CTLA-4; Inhibin (inhibin); Nandrolone Phenylpropionate (activin); VEGF (VEGF); The acceptor of hormone or growth factor; Albumin A or D; Rheumatoid factor (rheumatoid factor); Neurotrophic factor (neurotrophic factor) such as the neurotrophic factor that is derived from bone (BDNF), neurotrophic factor (neurotrophin)-3 ,-4 ,-5 or-6 (NT-3, NT-4, NT-5 or NT-6), or nerve growth factor such as NGF-β; The growth factor (PDGF) in blood platelet source; Fibroblast growth factor such as aFGF and bFGF; EGF (EGF); TGF (TGF) comprises TGF-β 1, TGF-β 2, TGF-β 3, TGF-β 4 or TGF-β 5 such as TGF-alpha and TGF-beta; Insulin-like growth factor-I and-II (IGF-I and IGF-II); Des (1-3)-IGF-I (brain IGF-I), insulin-like growth factor is in conjunction with albumen; CD albumen such as CD3, CD4, CD8, CD19 and CD20; Hematopoietin; Bone-inducing factor; Immunotoxin (immunotoxin); BMP (BMP); Interferon such as Alpha-IFN ,-beta and-gamma; Colony stimulating factor (CSF), for example, M-CSF, GM-CSF and G-CSF; Interleukins (IL), for example, IL-1 to IL-10; Superoxide dismutase; T-cell receptors; Surface membrane protein; Decay accelerating factor (decay-accelerating factor); Viral antigen as, for example, the part of AIDS coating (envelope); Transport protein; Homing receptor (homing receptor); Addressin (addressin); Modulin; Integrin such as CD11a, CD11b, CD11c, CD18, ICAM, VLA-4 and VCAM; Tumor-associated antigen such as HER2, HER3 or HER4 acceptor; With any fragment in the polypeptide listed above.

The specific antigen of the used antibody of the inventive method or described antibody for antigen preferably TGF-β 1, TGF-β 2, TGF-β 3, TGF-β 4, TGF-β 5, IFN-γ, FGF, EGF, and the acceptor of natural TGF-beta polypeptides, such as TGF β-RI and TGF β-RII. Other preferred antigen is the antigen that exists in the TGF-signal beta approach, such as, for example, Smad2, Smad3, Smad2/3, Smad4, Smad7, JNK, p38MAPK, erk MAPK, TAK1/MEKK1, Ras, RhoA, PP2A, MKK3/6, MKK4, p160Rock and S6K.

Among the present invention, use interchangeably term " ErbB2 ", " ErbB2 acceptor ", " c-erb-B2 ", " HER2 " and " Her2 ", and, except as otherwise noted, refer to natural-sequence ErbB2 human polypeptides, or its functional deriv. " Her2 ", " erbB2 " and " c-erb-B2 " refer to corresponding people's gene. Term in this context " natural-sequence " or " natural " refer to have the polypeptide of naturally occurring peptide sequence, and no matter the pattern of its preparation. Like this natural-sequences polypeptide can maybe can be by restructuring or synthetic method from natural separation, or produce by any combination of these or similar approach.

Humanized resisting-ErbB2 antibody comprises huMAb4D5-1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 and huMAb4D5-8 (trastuzumab (trastuzumab) (HERCEPTIN )), as clearly incorporating this paper United States Patent (USP) 5 as a reference into, described in 821,337 the table 3; Humanized 520C9 (WO93/21319) and humanized 2C4 antibody.

Use interchangeably term " to express the cancer (tumour) of Her2 " and " Her2+ cancer (tumour) ", they refer to wrap celliferous cancer (tumour), and the surface of described cell has Her2 albumen. " expressing the cancer of Her2 " is such cancer, produces the Her2 of enough levels on the surface of its cell, so that anti--Her2 antibody can be combined thereon, and have result for the treatment of for cancer. The cancer of Her2-or Her2-feminine gender (tumour) is the celliferous tumour of bag, and described cell does not have the Her2 albumen that is positioned at the surface.

In known animal model or people's clinical testing, when with untreated or compare with placebo treatment, or when comparing to replying with the initial treatment of trastuzumab but with the tumour that treatment continues to increase, " trastuzumab-resistance tumor " do not show the statistically evident improvement of replying for the treatment of for trastuzumab (HERCEPTIN ). By contrast, in known animal model or people's clinical testing, when with treatment not or when comparing with placebo treatment, " trastuzumab-reply " or " trastuzumab-sensitivity " tumour show the statistically evident improvement of replying for the treatment of for trastuzumab.

Unless additionally point out, term " monoclonal antibody 2G7 " refers to such antibody, and it has the antigen of the mouse 2G7 antibody of following embodiment-in conjunction with residue, or is derived from the antigen of described mouse 2G7 antibody-in conjunction with residue. For example, monoclonal antibody 2G7 can be mouse monoclonal antibody 2G7 or its variant, and such as humanized antibody 2G7, it has the amino acid residue of the conjugated antigen of mouse monoclonal antibody 2G7.

The following examples 2 have been described the generation of representational humanized resisting in conjunction with TGF-beta-TGF-beta antibody. Humanized antibody herein comprises the inhuman super variable region residue of incorporating people's variable region of heavy chain (variable heavy domain) into, and further be included in the framework region (FR) that is selected from lower group of position and replace: 48,49,67,69,71,73 and 78, described location application is in Kabat etc., the variable region coded system of listing in seeing above. In one embodiment, humanized antibody is in the position 48,49,67,69,71,73, and the two or more positions in 78 comprise FR and replace; And in other embodiments, in above-mentioned position three or four or multiposition more. In preferred embodiments, antibody is in the position 49,67 and 71, position 48,49 and 71, or position 49,69 and 71, or position 49,69,71 and 73, or position 49,71 and 73, or 49,71 and 78 comprise FR and replace in the position. Preferably have still less rather than more framework substitution so that immunogenicity minimizes, also be very important Consideration but render a service. In fact the amino acid that replaces is preferred conservative so that do not change those of immunogenicity or effectiveness. In the position 48, change and preferably become isoleucine by valine, in the position 49, change and preferably to become glycine by alanine, in the position 67, change and preferably become alanine by phenylalanine, in the position 69, change and preferably to become alanine by phenylalanine, in the position 71, change and preferably become alanine by arginine, in the position 73, change and preferably to become lysine by asparagine, and in the position 78, change and preferably become alanine by leucine.

Interested representational humanized antibody comprises the residue GYAFTNYLIE (SEQ ID NO:21) that determines the variable region of heavy chain complementarity herein; VNNPGSGGSNYNEKFKG (SEQ ID NO:22) or VINPGSGGSNYNEKFKG (SEQ ID NO:43); And/or SGGFYFDY (SEQ ID NO:23), comprise alternatively the amino acid modified of those CDR residues, for example wherein said modification keeps or improves the compatibility of antibody basically. For example, the target antibody variant can have about one extremely about seven or approximately five amino acid replacement in above-mentioned variable region of heavy chain CDR sequence. Described antibody variants can prepare by the compatibility maturation, and is for example, as described below. Preferably, described residue is two or more in following: GYAFTNYLIE (SEQ ID NO:21); VNNPGSGGSNYNEKFKG (SEQ ID NO:22) or VINPGSGGSNYNEKFKG (SEQ ID NO:43); And/or SGGFYFDY (SEQ ID NO:23), most preferably be whole three kinds. Most preferred humanized antibody comprises the weight chain variable region amino acid sequence among the SEQ ID NO:4 or has GYAFTNYLIE (SEQ ID NO:21); The sequence of VINPGSGGSNYNEKFKG (SEQ ID NO:43) and SGGFYFDY (SEQ ID NO:23).

Humanized antibody can comprise residue RASQSVLYSSNQKNYLA (SEQ ID NO:18) or the RASQGISSYLA (SEQ ID NO:7) that determines the variable region of light chain complementarity; WASTRES (SEQ ID NO:19) or YASSLQS (SEQ ID NO:8); And/or HQYLSSDT (SEQ ID NO:20), for example replenishing as the variable region of heavy chain CDR residue described in the last period. Described humanized antibody comprises the amino acid modified of above-mentioned CDR residue alternatively, and for example wherein said modification keeps or improve the compatibility of antibody basically. For example, the target antibody variant can have about one extremely about seven or approximately five amino acid replacement in above-mentioned variable region of light chain CDR sequence. Described antibody variants can prepare by the compatibility maturation, and is for example, as described below. Preferably, described residue is two or more in following: RASQSVLYSSNQKNYLA (SEQ ID NO:18); WASTRES (SEQ ID NO:19); And/or HQYLSSDT (SEQ ID NO:20), most preferably be whole three kinds. Most preferred humanized antibody comprises the light chain variable region amino acid sequence among the SEQ ID NO:3.

The application also relates to the affine sexually matured antibody in conjunction with TGF-beta. Parental generation antibody can be people's antibody or humanized antibody, for example, contains respectively the light chain of SEQ ID Nos.3 and 4 and/or the antibody of weight chain variable sequence (being modification (version) 5). Described affine sexually matured antibody preferably (for example improves about twice or about four times with the affinity that surpasses mouse 2G7 or variant 5, to about 100 times or about 1000 times, for example, the affinity as using TGF-beta-extracellular domain (ECD) ELISA to determine) be combined with TGF-beta.

In external or animal model or people's clinical trial of generally acknowledging during the responsiveness of the tumour of test patient, when with not the treatment or with placebo treatment, or wherein the patient to replying with the initial treatment of TGF-beta antagonist, but described replying is instantaneous and the tumour situation that continues to increase with treatment when comparing, the patient " predetermined to not replying with the treatment of TGF-beta antagonist, or reply very poor " not show statistically evident improvement to replying with the TGF-beta antagonist for treating.

" anti--angiogenic agent " refers to the compound except the TGF-beta antagonist, and it is blocked to a certain extent, or disturbs the generation of blood vessel. Described resisting-angiogenesis factor can for example be little molecule or antibody, and it is combined with the growth factor or the growth factor receptors that relate to the generation of promotion blood vessel. Example is the antagonist of VEGF (VEGF), such as the antibody of specific bond VEGF, such as bevacizumab (bevacizumab) (AVASTIN ).

II. implement mode of the present invention

As previously mentioned, TGF-β plays a part complicated in carcinogenesis. The TGF-beta pathway the epithelial cell carcinogenesis in early days as tumor inhibitor. Along with the heredity of precancerous cell and cancer cell and the variation of outer heredity (epigenetic) background, the TGF-β responsiveness of cell descends, and until evening of tumor development pre--TGF-β that the transfer phase is just observed increase expresses/activates, and in the diffusional transfer cancer, the TGF-beta pathway short-carcinogenic (pro-oncogenic) effect becomes very outstanding. Further details is referring to Roberts and Wakefield,Proc.Natl.Acad.Sci.USA，100(15)：8621-8623 (2003). More known tumours such as various cancers, are escaped the inhibition that Growth of Cells is subjected to TGF-β as the result of the inactivation type in TGF-beta receptor sudden change. TGF-β (with other members of TGF-beta pathway) can the direct fact as tumor promotor be proved by the fact that many tumours do not have the TGF-beta receptor of inactivation; Therefore, the formation of described tumour and diffusion can not be explained by the cell growth inhibition that causes escaping TGF-β owing to the sudden change of inactivation type.

In many tumor models system, with the TGF-β preliminary treatment of purifying or cause the increase of metastatic potential with TGF-β 1 cDNA transfection. On the contrary, the blocking-up tumour cell to the responsiveness of TGF-β or in and TGF-β produce and reduced transfer efficiency in the body. This illustrates that effectively TGF-β can promote to shift. The possible mechanism that has obtained evidence comprises: (i) inhibition of immunosurveillance; (ii) promotion of diffusivity and activity; (iii) promotion of blood vessel generation. Yet, do not need to understand described mechanism and can use the present invention. And do not mean that any concrete mechanism that is confined to of the present invention in fact.

The present invention is based on by in several animal models, testing anti--TGF-β antibody and obtain experimental data, comprise by use from the clone of spontaneous tumor and by use those antibody from the primary cell generation of the tumour preparation of oncogene driving. (heterogeneity) is similar with the heterogeneity of observing in people's tumour, and animal model shows to using the TGF-beta antagonists, such as various the replying of anti--TGF-β Antybody therapy. The information that produces in these animal models allows the difference between the activity of the various TGF-β-induce for tumour cell, and for identifying particular type, stage or the form that is used for cancer, such as Secondary cases (metastatic) tumour, breast cancer is with respect to the cancer of other types, and the material of the preferential treatment of different subclass of breast cancer etc. has important implication. As a result, the experimental data that consists of basis of the present invention provides the information of the individualized type (personalizing) for the treatment of of cancer that important being used for make human patients. Because metastatic cancer is the main cause with death of solid tumor (solid tumor), one aspect of the present invention concentrates on identifies effective material in the treatment of secondary tumors.

Therefore, in one embodiment, the present invention is the screening technique that cancer is had the material of therapeutic activity, it comprises following steps: (1) uses multiple substances to the animal model of inhuman symimmunity activity being with or without in the situation of primary tumor, and this animal model suffers from least a soft tissue or bone shifts; (2) measure described substances to the impact of soft tissue or bone transfer, and if have primary tumor, described substances also measured to the primary tumor affects on the growth; (3) qualification test material, it suppresses the growth that soft tissue or bone shift, and if have primary tumor, described substances does not have harmful effect for the situation of primary tumor.

In the variation of the method, the using and treat cancer of described substances, especially the other standards therapy of metastatic cancer, as, for example radiotherapy combination.

In one embodiment, the substances of described animal being used comprises known chemotherapeutant or cytotoxic agent such as taxane. Aspect the method preferred, described animal is applied two kinds of substances, wherein a kind of is the TGF-beta antagonist, and another kind is chemotherapeutant or cytotoxic agent, and described two kinds of substances have been measured for the coupling effect of soft tissue or bone transfer and primary tumor growth, if there is primary tumor. In a more preferred embodiment, the TGF-beta antagonist is the antibody of specific bond TGF-beta, and chemotherapeutant or cytotoxic agent are taxanes.

The animal that is used for screening experiment in this body can be the animal of any kind except the people, but the preferred example of described animal comprises rodent, such as Mouse and rat, rabbit, miniature pig (miniature pig) and pig, more preferably mouse.

Being used for animal models more of the present invention shows for cancer such as breast cancer or melanomatous special pathology late period, that shift, therefore and can be used for identifying material, for example chemotherapeutant and/or cytotoxic agent, the treatment that it is provided at above-mentioned invasive, TCA comprises the benefit in the treatment that soft tissue and bone shift.

In order to produce the animal model of tumor metastasis, but being entered animal, tumor cell injection must cause having the primary of the repetition time pattern that primary and secondary tumors occur and the formation of secondary tumors; Described system must be isogenic; And secondary tumors must be really to shift, and namely must be formed by the cell of primary tumor. In addition, should in vitro culture be used for the tumour cell of injection, and may obtain rational transfection efficiency.

The transgenic animals that are carried at the transformed gene under the control of viral promotors provide the animal of the primary tumor with spontaneous generation. Yet described animal usually dies from serious primary tumor rather than forms the dissemination tumour cell of secondary tumors, and thus, is not the best model of research metastatic cancer. Yet they can be as being used for being injected into another animal so that the tumour cell source of the animal model that exploitation is fit to.

Therefore, the transplantable 4T1 mouse breast cancer that is derived from BALB/c is the model that is used for the research metastatic cancer of determining. Referring to, for example Aslakson and Miller,Cancer Res.，52：1399-1405 (1992); Pulaski and Ostrand-Rosenberg,Cancer Res.，58：1486-1493 (1998); With Pulasky etc.,Cancer Res.，60：2710-2715 (2000). With the 4T1 tumor cell inoculation behind the mammary fat pad (fat pad) of acceptor mouse, lung, liver and other soft tissues little by little and are spontaneously transferred in primary tumor growth, and transfer to bone. Specifically aggressive gland cancer is similar with human breast carcinoma, and the cell of transfer is being bred away from the position that has primary tumor, and continues propagation behind the exenterate primary tumor. Therefore, the 4T1 model be suitable for when primary tumor exists and the exenterate primary tumor after study tumor metastasis.

In order to study the different tests material for the impact of the metastatic breast cancer of expressing Her-2/neu, but the Her-2/neu of overexpression human breast cancer cell is inoculated into the mammary fat pad of acceptor mouse, and treats with substances. Optional, can be with described tumour transplatation to the acceptor mouse. This model system allows (trastuzumab-sensitivity) breast cancer of research trastuzumab-resistance and trastuzumab-reply. Another animal model that is particularly suitable for testing the reagent that is used for the treatment of trastuzumab-resistance breast cancer is described in the U.S. Patent No. 6,632,979 of promulgating on October 14th, 2003, and it is complete to be disclosed in this and to be incorporated herein by reference clearly.

Another animal model that is suitable for studying tumour progression and transfer is by express the mouse breast cancer model that T cancer protein (polyoma middle T oncoprotein) (PyMT) causes in the middle of the polyoma in breast epithelium. Described PyMT tumour on histology with Her-2⁺Tumour is different, and stands from precancer or malignant stages to the tumor development that can obviously confirm, different phase that occurs with high-frequency to shift. Described PyMT tumour shows certain aggressive form with prognosis mala correlation human breast carcinoma in morphologic similitude, and therefore, provides fabulous model to be used for research and identifies the drug candidates that is used for treating described cancer. Referring to, such as Lin etc.,Am J.Pathol.，163(5)：2113-2126(2003)。

About the discussion of more animal models of metastatic breast cancer referring to, such as Heppner etc.,Breast Cancer Res..2(5)：331-334(2000)。

Can for example study metastatic melanoma in the substrain of the miniature pig of Sinclair (Sinclair Research Center, Inc.), the invasion and attack sexual type melanoma that described pig produces is very similar to people's homologue. This invasive melanoma has the unique property of spontaneous regression after shifting the phase fully, and thus, is particularly suitable for the generation of metastatic melanoma and the research of degeneration.

In addition, in the exploitation of melanoma model, often using mouse black-in tumor cell is B16, K1735 and Cloudman S91-M3 (with various subbreed). About the more details of the animal model that is suitable for studying the transfer in the melanoma referring to, such as Gattoni-Celli etc.,Pigment Cell Res.，6(6): 38-34 (1993) and Rusciano etc.,Invasion Metastasis，14(1-6)：349-361(1994-95)。

Can use animal model screening of the present invention to be used for the material that prevention or treatment soft tissue and/or bone are shifted, it is also effective in the treatment of primary tumor. Screen useful medicine and relate in certain dosage range animal model is used substances, and in the impact of the described material of different time point determinings for the state of the Secondary cases that exists and primary tumor.

In one embodiment, by animal is used substances in dosage range, and estimate described animal for the physiologic response of this compound screening test material over time. Use and can be orally, or by suitable injection, this depends on the chemical property of the compound that is evaluated. In some cases, described compound is fit to use with the confactor that can strengthen this compound effectiveness.

Except screening is used for the treatment of the medicine of disease or disease, method of the present invention also is used for studying validity or the mechanism of the effect of concrete medicine, and/or design is intended to prevent or cures described disease or sick therapeutic scheme (therapeutic regimen). For example, can be before described disease or sick morbidity, simultaneously, or treat described animal with concrete diet, daily exercise, radiotherapy, chemotherapy and/or in the combination of one or more compounds of this evaluation afterwards. Total autogenic therapy like this or therapeutic scheme may be than more effective with compounds for treating separately for resist the disease or disease.

Use the screenings of transgenic animals of the present invention can use any and related to cancer, be easy in animal model the easily phenomenon of assessment. Can by the independently effect of method monitoring test material as known in the art for primary tumor growth and soft tissue and bone transfer, comprise primary and Secondary cases terminal point. For example, can be by measuring tumor size, tumor incidence (number) and tropism (tropism) (position), with before the substances treatment, in the process and/or measure afterwards the endogenous TGF-β that is produced by tumour cell, with before the substances treatment, in the process and/or measure afterwards serum TG F-β level, histological score (histology scoring) and various imaging technique comprise little-computed tomography (micro-CT; μ CT) imaging comes the monitoring test material for the impact of primary or secondary tumors. Because in soft tissue, if there is not the time-consuming process of preparation and a large amount of histotomies of individual inspiration, little metastatic tumo(u)r is difficult to detect and is quantitative, and micro-CT is useful especially for the transfer of above-mentioned transfer and bone.

Micro-CT (the X ray microbedding is analysed photography) is nondestructive technology, is 2D and the 3D X-ray attenuation figure of several millimeters sample for generation of size. In order to use the micro-CT technology to make the lung imaging in vitro, lung can be immersed in ISOVIEW^TMIn the reagent (CT contrast preparation, iodine sugar (iodine sugar)). Then slowly inject soya-bean oil to remove contrast preparation from air flue (airway). Resolution ratio that can be different produces image. Therefore, the most of images that provide herein produce in 16-μ resolution ratio. This technology and histology adapt, and three-Wei software for display permission observer accepts or refuses agglomerate as possible tumour.

Another imaging technique that can carry out in vivo depends on the active bioluminescence imaging of luciferase (luciferase). Bioluminescence is known and the imaging technique that is widely used in the body. The non-invasive imaging of the cell of this technology permission expression luciferase protein and quantitative. The main luciferase that uses during this measures is from firefly (firefly), (phytonis pyralis). This enzyme (about 90 minutes) in external (about 3 minutes at 37 ℃) and body has the short half-life. Have than the long half-lift mutant also be commercial available. For the in-vivo imaging of tumour, use luciferase transfection tumor cell, such as breast tumor cell, and implant receptor, for example mouse. After the implantation, allow time enough to be used for tumour and form, luciferin is injected the animal of carrying tumour, for example in the peritonaeum of mouse. In the presence of luciferase, the bioluminescence that the reaction by luciferin, ATP and oxygen produces can be passed through the CCD camera.

About the description of the in-vivo imaging of metastatic cancer with fluorescin referring to, Hoffman for example,Cell Death and Differentiation，9：786-789(2002)。

Described substances is not concrete restriction, but the example comprises polypeptide, protein, peptide, non--the peptide organic molecule, synthetic compound, tunning and cell extract.

Candidate substances comprises many chemicals types, but they are organic molecules usually, preferably has greater than 50 and less than the little organic compound of about 2,500 daltonian molecular weight. Candidate agent comprises and protein structure interact necessary functional group, especially Hydrogenbond, and usually comprises at least amido, carbonyl, hydroxyl or carboxyl, in the preferred above-mentioned functional group at least two. Candidate agent usually comprises ring-type carbon or heterocycle structure and/or aromatic hydrocarbons structure or the polyaryl structure that replaces with one or more above-mentioned functional groups. Candidate agent also sees in the middle of the biomolecule, and it includes, but are not limited to: peptide, carbohydrate, aliphatic acid, steroids, purine, pyrimidine, derivative, analogue or its combination.

Comprise that from multiple source library synthetic or native compound obtains candidate substances. For example, many means can be used for the synthetic with orientation at random of multiple organic compound and biomolecule, comprise the expression of randomized oligonucleotides and oligopeptides. Optional, the library of the native compound of bacterium, fungi, plant and animal form of extract is available or produces easily. In addition, library and compound natural or synthetic generation are modified by chemistry, physics and the biochemistry means of routine easily, and can be used for producing combinatorial libraries (combinatorial libraries). Can be with the chemical modification that fix a point or at random of known pharmacological reagent, such as acidylate (acylation), alkanisation (alkylation), esterification (esterification), amidatioon (amidification) etc. to produce analogue.

Candidate substances comprises particularly, and is not limited to, antibody, as, anti--TGF-β antibody for example.

Separate, clone and several TGF-β 1 sequences that checked order. The tabulation of TGF-β 1 sequence is provided, and it may be suitable for using in implementing process of the present invention, for example producing the TGF-beta 1 antagonists, and with the Genbank accession number of these Serial relations:

People TGF-β 1 AA459172

Ox TGF-β 1 M36271

Precursor people TGF-β 1 E00973; X02812;

Sheep (Ovis) X76916; J05114; M38449;

TGF-β1 L36038 M55656

Pig TGF-β 1 M23703; X12373

Canidae TGF-β 1 L34956

Hamster TGF-β 1 X60296

Rat TGF-β 1 X52498

Muridae TGF-β 1 M13177

Antibody can be monospecific, bispecific or tri-specific or has more multiple specific herein. Multiple specific antibody can be specific (for example, F (ab ') to monomolecular different epi-positions₂Bispecific antibody) or can be specific for the epi-position on the different molecular. Design and the method for preparing multiple specific antibody are being known in the art. Referring to, for example, Millstein etc.,Nature，305：537-539 (1983); Kostelny etc.,J.Immunol.，148：1547-1553 (1992); With WO 93/17715. Three-specific antibody can be such as Tutt etc.,J.Immunol.，147：Prepare described in 60 (1991).

Particularly, bispecific antibody can use chemistry to connect preparation. Brennan etc.,Science，229：81 (1985) have described method, wherein complete antibody are cut to produce F (ab ') in the proteolysis mode₂Fragment. These fragments are reduced under the condition that dimercapto complexing agent sodium arsenite (sodium arsenite) exists so that the dimercapto stabilisation at ortho position and prevent from forming intermolecular disulphide. Then Fab ' the fragment that produces is converted into (TNB) derivative of sulfo-nitrobenzoate (thionitrobenzoate). Then a kind of by being converted into again Fab '-mercaptan with mercaptoethylmaine (mercaptoethylamine) reduction and mixing to form bispecific antibody with the another kind of Fab ' of equimolar amounts-TNB derivative with in Fab '-TNB derivative. The reagent of the bispecific antibody useful as selective immobilized enzyme that produces. In another embodiment, the Fab ' that directly reclaims from Escherichia coli-SH fragment can be in external chemical coupling to form bispecific antibody. Shalaby etc.,J.Exp.Med.，175：217-225(1992)。

Also described multiple directly from the preparation of recombinant cell culture thing and the technology of separating bispecific antibody. For example, used leucine zipper to produce bispecific antibody. Kostelny etc.,J.Immunol.， 148(5)：1547-1553 (1992). To be connected to from the leucine zipper peptide of Fos and Jun albumen the Fab ' part of two different antibodies by gene fusion. Described antibody morphism dimer (homodimer) is reduced to form monomer at hinge area, and then oxidation is to form antibody heterodimer (heterodimer). The method also can be used for producing the antibody morphism dimer. By Hollinger etc.,Proc.Natl.Acad.Sci.USA， 90：" bispecific antibody (diabody) " technology that 6444-6448 (1993) describes provides the interchangeable mechanism of preparation bispecific antibody fragment. Described fragment comprises weight-chain variable region (V_H), it is by joint and light-chain variable region (V_L) connect, described joint is too short so that do not allow to match between two territories on the same chain. Therefore, the V of a fragment_HAnd V_LThe V with the complementation of another fragment has in the territory_LAnd V_HThe territory pairing forms two antigen-binding sites thus. Also reported by utilizing list-chain Fv (sFv) dimer to prepare another strategy of bispecific antibody fragment. Referring to Gruber etc.,J.Immunol.， 152：5368 (1994). Optional, described bispecific antibody can be as at Zapata etc.,Protein Eng.， 8(10): " the linear antibody " that produces described in the 1057-1062 (1995).

Bispecific antibody comprises crosslinked or " allos coupling (heteroconjugate) " antibody. For example, can be with a kind of antibody in the allos bond and avidin (avidin) coupling, and another kind of antibody and biotin coupling. The allos binding antibody can use any easily cross-linking method preparation. Suitable crosslinking agent is known in the art, and is disclosed in U.S. Patent No. 4,676 together with many crosslinking technologicals, in 980.

Relate to other modification of antibody. For example, with respect to the effector function modified antibodies, may be required so that for example strengthen the validity of antibody in the treatment cancer. For example, cysteine residues can be imported the Fc district, form interchain disulfide bond thereby make in this district. The antibody of the homotype dimerization that produces like this can improve cell-lethal and the antibody-dependent cellular cytotoxicity (ADCC) of the complement of internalization (internalization) ability and/or increase-mediation. Referring to Caron etc.,J.Exp Med.，176：1191-1195 (1992) and Shopes,J.Immunol.，148：2918-2922 (1992). The antibody of homotype dimerization with Anti-tumor activity of enhancing also can use as at Wolff etc.,Cancer Research，53：The described isodigeranyl function of 2560-2565 (1993) (heterobifunctional) crosslinking agent prepares.

Developed multiple method for generation of antibody. Traditionally, antibody fragment derive from complete antibody proteolytic digestion (referring to, for example, Morimoto etc.,Journal of Biochemical and Biophysical Methods，24：107-117 (1992) and Brennan etc.,Science，229：81 (1985)). Yet these fragments and full length antibody and other antibody can directly produce by recombinant host cell now, and wherein the recombinant DNA technology of Application standard obtains the light chain of encoding antibody and the dna sequence dna of heavy chain. Can separate required dna sequence dna and check order by the cell that produces antibody such as hybridoma. Optional, can use nucleotides synthesizer or round pcr synthetic DNA. In case obtain, the DNA of just will encode light chain and heavy chain inserts and can copy in protokaryon or eucaryon host, the recombinant vector of expression and secretion heterologous polynucleotide. For example, can reclaim Fab '-SH fragment and chemical coupling to form F (ab ') from Escherichia coli₂Fragment (Carter etc.,Bio/Technology，10：163-167 (1992)). In another embodiment, use leucine zipper GCN4 to promote F (ab ')₂The assembling of molecule forms F (ab ')₂ According to another method, full length antibody or Fab or F (ab ')₂Fragment or other antibody can directly separate from the recombinant host cell culture. Can use for purposes of the invention many available and carriers known in the art. The selection that is fit to carrier will mainly depend on the nucleic acid size that is inserted into and will be with the concrete host cell of described carrier conversion.

Usually, will contain the replicon of the species that are derived from suitable host cell and the recombinant vector of control sequence and be used as the parental generation carrier, be used for making up concrete carrier of the present invention. Described carrier usually carries origin of replication (origin of replication site) and can provide the flag sequence of Phenotypic Selection as the main chain part in the cell that transforms. Origin of replication is nucleotide sequence, and it can make carrier copy in the host cell of one or more selections. Usually, in cloning vector, this sequence is a kind of sequence that described carrier and host chromosome DNA are irrespectively copied, and comprises origin of replication or autonomously replicating sequence. For various bacteria, yeast and virus, such sequence is known. For most of gramnegative bacteriums, be suitable from the origin of replication of plasmid pBR322.

The cloning and expression carrier can comprise Select gene, but the selected marker of being also referred to as. The such albumen (a) of common Select gene coding for antibiotic or other toxin is for example given, the resistance of ampicillin (ampicillin), neomycin (neomycin), methotrexate or tetracycline (tetracycline), (b) complement auxotrophy, or (c) provide the important nutrients that can not provide in the complex medium, for example, the gene of the D-alanine racemase of coding bacillus. An example of selection scheme utilizes medicine to suppress the growth of host cell. Those cells that successfully transform with heterologous gene produce the albumen of giving drug resistance and survive thus selection mode (selection regimen). The example that is suitable for the plasmid vector of Escherichia coli conversion is pBR322. PBR322 contains the gene of coding ampicillin (Amp) and tetracycline (Tet) resistance and provides thus simple method for the identification of the cell that transforms. The derivative of pBR322 or other microorganism plasmid or bacteriophage also can be used as the parental generation carrier. The example of pBR322 derivative of be used for expressing antibody specific is at Carter etc., and U.S. Patent No. 5,648 is described in detail in 237.

In addition, contain the replicon of suitable host microorganism and the phage vector of control sequence and can be used as the conversion carrier relevant with these hosts. For example, bacteriophage can be used for preparing recombinant vector such as λ GEM.TM.-11, and described recombinant vector can be used for transforming responsive host cell such as Escherichia coli LE392.

In a preferred embodiment, the method from the time separately antibody light-expression of chain and weight-chain part. Particularly, the method preferably comprises with encode respectively light chain and heavy chain two translation unit transformed host cells independently; Cultured cell under suitable condition separates the generation of light chain and heavy chain thus so that light chain and heavy chain are expressed in a sequential manner from the time; With allow light chain and heavy chain to be assembled into the antibody of function.

Of this embodiment preferred aspect, from the time separately the expression of light chain and heavy chain be by utilizing two different promoters controlling respectively light chain and heavy chain to realize that wherein different promoters is activated under different condition. For example, the DNA of coding light chain and heavy chain can be mixed single plasmid vector but be divided into two translation units, wherein each is controlled by different promoters. A promoter (for example, first promoter) can be composing type or induction type, and another promoter (for example, second promoter) is induction type. Thereby, when the host cell cultivated under the condition that is being suitable for activating a promoter (for example, first promoter) with described carrier conversion, only have a chain (for example, light chain) to be expressed. Then, after article one chain (for example, light chain) is expressed one required period, condition of culture is changed into those (for example be suitable for activating another promoter, the and individual promoter) condition, and therefore induce the expression of second chain (for example, heavy chain). In a preferred embodiment, at first expressing light chain then is heavy chain. In another embodiment, at first expressing heavy chain then is light chain.

Particularly, according to a preferred embodiment, recombinant vector comprises at least two translation units, and one is used for that light-chain is expressed and another is used for the expression of weight-chain. And two translation units of light chain and heavy chain are under the control of different promoters. Promoter is the sequence of not translating that its expression is controlled in the upstream (5 ') that is positioned at coded sequence (usually within about 100 to 1000bp) beginning. Described promoter falls into two types usually, induction type and composing type. Inducible promoter is such promoter, and under their control, in response to some variations of condition of culture, for example variation of nutraceutical existence or shortage or temperature or pH causes being increased by the level of transcribing of DNA.

For the purpose of this embodiment, first promoter that can use composing type or inducible promoter on time to express as control article one-chain, and use inducible promoter as second promoter of the follow-up second of control-chain expression. In preferred embodiments, first promoter and second promoter all are to be subject to the strict inducible promoter of regulating. A lot of promoters of being identified by multiple possible host cell are known. Always source DNA separates the promoter sequence of selecting and inserts carrier of the present invention by digestion with restriction enzyme. Optional, can synthesize the promoter sequence of selection. Can use the promoter of natural promoter sequence and many allos to instruct amplification and/or the expression of target gene. Yet allogeneic promoter is preferred, because they compare with natural target polypeptides promoter that common permission is more transcribed and the yield of the target gene of higher expression.

The suitable promoter of using with prokaryotic hosts comprises that phoA promoter, beta-lactamase and Lac operon system, tryptophan (trp) promoter systems and hybrid promoter are such as tac or trc promoter. Yet it also is suitable that other promoter (such as other known bacterium or phage promoter) of function is arranged in bacterium. Their nucleotide sequence is open, thereby thereby make those skilled in the art be connected (Siebenlist etc. by using joint or conjugant operationally they and the target light chain of encoding to be connected translation unit with heavy chain with the restriction site that any needs are providedCell，20: 269 (1980)). Preferred promoter is phoA, tacI, tacII, lpp, lac-lpp, lac, ara, trp, trc and T7 promoter. The preferred promoter that is used for this invention is phoA promoter and tacII promoter. The promoter that function is arranged in eukaryotic host cell is known in the art, for example, and as in U.S. Patent No. 6,331, described in 415. The example of these promoters can comprise that those are derived from the promoter of polyomavirus (polyoma), adenovirus (Adenovirus) 2 or simian virus (Simian Virus) 40 (SV40).

Each translation unit of recombinant vector of the present invention contains the necessary additional non-translated sequence of the gene that gives full expression to insertion. These basic sequences of recombinant vector are known in the art, and comprise, for example, are positioned at 5 ' Shine-Dalgarno district of initiation codon and are positioned at transcription terminator (for example, the λ t of 3 ' end of translation unit₀)。

Each translation unit of recombinant vector also comprises the burst part, the secretion that it instructs the chain polypeptide of expression to pass film. Usually, secretory signal sequence can be the part of carrier, or it can be the part of the target polypeptides DNA that inserts described carrier. The secretory signal sequence of selecting for the present invention should be by host cell the secretory signal sequence of identifying and process (namely being cut by signal peptidase). For nonrecognition and process the prokaryotic host cell of the natural burst of heterologous polypeptide, burst for example is selected from that the burst of the protokaryon of lower group replaces: alkaline phosphatase (alkaline phosphatase), penicillase (penicillinase), Ipp or heat-staple enterotoxin 1 I (STII) targeting sequencing, LamB, PhoE, PelB, OmpA and MBP. In a preferred embodiment of the invention, the burst that is used for two translation units of expression system is STII burst or its variant. Preferably, the DNA of the described burst of coding is connected to 5 '-end of the DNA of coding light chain or heavy chain in reading frame, this produces fused polypeptide. In case be secreted into outside the cytoplasm of host cell, with enzyme signal peptide sequence downcut from mature polypeptide.

Of the present invention another preferred aspect, except the selection of time of expressing, the quantitative ratio of light chain and heavy chain expression of also regulating is so that the maximum production of the antibody of secretion and correct assembling. Such adjusting is finished by regulating simultaneously on the recombinant vector translation intensity of light chain and heavy chain. It is open in the U.S. Patent No.s such as Simmons 5,840,523 to be used for regulating a kind of method of translating intensity. In brief, the method is utilized the variant of translation initiation district (TIR) in the translation unit. For given TIR, can produce a series of amino acid or nucleotide sequence variant with the translation intensity of certain limit, the method for providing convenience is thus regulated this factor to obtain the required expression of concrete chain by the method. Can produce the TIR variant by conventional induced-mutation technique, the codon that described induced-mutation technique causes changing amino acid sequence changes, and is preferred although the silence in the nucleotide sequence changes (as described below). Change among the TIR can comprise, for example, and the number of Shine-Dalgarno sequence or the change at interval, and the change in the burst.

A kind of preferably for generation of the method for jump signal sequence be coded sequence begin produce " password word bank ", it does not change the amino acid sequence (that is, described variation is reticent) of burst. This can be by changing each codon the position of the 3rd nucleotides realize; In addition, some amino acid such as leucine, serine and arginine, have multiple first and second position that can increase complexity in the preparation in described storehouse. The method of this sudden change is at Yansura etc.,METHODS：A Companion to Methods in Enzymol.，4：Describe in detail among the 151-158 (1992).

Being used for the carrier clone of this paper or the suitable host cell of expression DNA is above-mentioned prokaryotes, yeast or more high eukaryotic cells. Suitable prokaryotes for this purpose comprise eubacteria (eubacteria), such as Gram-negative or gram-positive organism, for example, enterobacteriaceae (Enterobacteriaceae) is such as Escherichia (Escherichia) Escherichia coli (E.coli) for example, Enterobacter (Enterobacter), Erwinia (Erwinia), Klebsiella (Klebsiella), proteus (Proteus), Salmonella (Salmonella) is salmonella typhimurium (Salmonella typhimurium) for example, Serratia (Serratia) is serratia marcescens (Serratia marcescans) and Shigella (Shigella) for example, and Bacillus such as bacillus subtilis (B.subtilis) and bacillus licheniformis (B.licheniformis) are (for example, the DD 266 that publishes on April 12nd, 1989, disclosed bacillus licheniformis 41P in 710), pseudomonad such as pseudomonas aeruginosa (P.aeruginosa), and streptomyces (Streptomyces). A kind of preferred escherichia coli cloning host is that (ATCC 31 for Escherichia coli 294,446), although other bacterial strain such as Escherichia coli B, Escherichia coli X1776 (ATCC 31,537) and Escherichia coli W3110 (ATCC 27,325) are suitable. These examples are illustrative and nonrestrictive.

Except prokaryotes, the microorganism of eucaryon is suitable clone or expressive host for the carrier of encoding antibody such as filamentous fungi or yeast. In the middle of the host microorganism of low eucaryon such as grade, saccharomyces cerevisiae (Saccharomyces cerevisiae) or common Saccharomyces cerevisiae are the most frequently used host microorganisms. Yet many other genus and species and bacterial strain are normally available and be useful in this article, such as schizosaccharomyces pombe (Schizosaccharomyces pombe); Kluyveromyces (Kluyveromyces) host such as, for example, Kluyveromyces lactis (K.lactis), (ATCC 12 for Kluyveromyces fragilis (K.fragilis), 424), (ATCC 16 for Bulgarian kluyveromyces (K.bulgaricus), 045), (ATCC 24 for Brunswick kluyveromyces (K. wickeramii), 178), (ATCC 56 for K.waltii, 500), fruit bat kluyveromyces (K. drosophilarum) (ATCC 36,906), K.thermotolerans and kluyveromyces marxianus (K. marxianus); The West alpine yarrow mould (yarrowia) (EP 402,226); Pichia pastoris phaff (Pichia pastoris) (EP 183,070); Mycotoruloides (Candida); Trichoderma reesia (EP 244,234); Neuraspora crassa (Neurospora crassa); Permitted all so prosperous yeast of prosperous saccharomyces (Schwanniomyces) (Schwanniomyces occidentalis); With filamentous fungi such as, for example, Neurospora (Neurospora), Penicillium (Penicillium), Tolypocladium and aspergillus (Aspergillus) host such as aspergillus nidulans (A. nidulans) and aspergillus niger (A.niger).

The suitable host cell that is used for expression antibody also comprises without the vertebra cell such as plant and insect cell. Identified the insect host cell of many baculoviral bacterial strains and variant and corresponding license from host such as Spodoptera frugiperda (caterpillar), Aedes aegypti (Aedes aegypti) (mosquito), aedes albopictus (Aedes albopictus) (mosquito), Drosophila melanogaster (Drosophila melanogaster) (fruit bat) and silkworm (Bombyx mori). The multiple bacterial strain that is used for transfection can openly obtain, for example, the L-1 variant of autographa california (Autographa californica) NPV and the Bm-5 bacterial strain of BmSNPV, and such virus can be used as according to virus herein of the present invention, is particularly useful for transfection Spodoptera frugiperda cell. The plant cell cultures of cotton, corn, potato, soybean, petunia (petunia), tomato, tobacco also can be used as the host.

The example of useful mammalian host cell line is the monkey kidney CV1 clone (COS-7, ATCC CRL 1651) that transforms with SV40; The human embryonic kidney cell line (subclone with 293 or 293 cells of in suspension culture, growing (Graham etc.,J.Gen Virol.，36：59 (1977)); Baby hamster kidney cell (BHK, ATCC CCL 10); Chinese hamster ovary cell/-DHFR (CHO, Urlaub etc.,Proc.Natl.Acad. Sci.USA，77：4216 (1980)); Mouse Sai Ertuoli (sertoli) cell (TM4, Mather,Biol. Reprod.，23：243-251 (1980)); MK cells (CV1 ATCC CCL 70); African green monkey kidney cell (VERO-76, ATCC CRL-1587); Human cervical carcinoma cell (HELA, ATCC CCL 2); Canidae nephrocyte (MDCK, ATCC CCL 34); Buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442); Human pneumonocyte (W138, ATCC CCL 75); Human liver cell (Hep G2, HB 8065); MMT (MMT 060562, ATCC CCL51); The TRI cell (Mather etc.,Annals N.Y.Acad.Sci.， 383：44-68 (1982)); MRC 5 cells; FS4 cell and Bel7402 (Hep G2).

Also cultivate in the conventional nutrient medium that is modified to the gene that is suitable for evoked promoter, selects transformant or the required sequence of amplification coding for generation of antibody with above-mentioned expression or cloning vector transformed host cell.

For generation of the prokaryotic of polypeptide of the present invention known in the art and be suitable for cultivating in the culture medium of host cell of selection and grow. The example of suitable culture medium comprises the luria fluid nutrient medium (LB) of the essential nutritional supplement of adding. In preferred embodiments, described culture medium also comprises the selective reagent of selecting based on the structure of expression vector, with the growth of the prokaryotic that optionally allows to contain described expression vector. For example, ampicillin is added culture medium and be used for the cell that ammonia benzyl-resistant gene is expressed in growth. Except carbon source, nitrogenous source and inorganic phosphorous sources, also can comprise with debita spissitudo being introduced separately into or as any essential fill-in of introducing with the mixture of another kind of fill-in or culture medium such as compound nitrogen source. Alternatively, described culture medium can comprise one or more reducing agents that is selected from lower group: glutathione (glutathione), cysteine (cysteine), cystamine (cystamine), TGA (thioglycollate), dithioerythritol (dithioerythritol) and dithiothreitol (DTT) (dithiothreitol).

Cultivate the host cell of protokaryon in suitable temperature. For example, for Escherichia coli Growth, preferably approximately 20 ℃ to about 39 ℃ of temperature ranges, more preferably about 25 ℃ to about 37 ℃, and even more preferably at about 30 ℃. The pH of culture medium can be any pH of about 5 to about 9, and this depends primarily on host organisms. For Escherichia coli, pH is preferably about 6.8 to about 7.4, and more preferably about 7.0.

Can in various culture mediums known in the art, cultivate the eukaryotic host cell for generation of antibody of the present invention. For example, commercially available culture medium is suitable for cultivating mammiferous eukaryotic host cell such as the Yi Geershi culture medium of Ham ' s F10 (Sigma), MEM (Minimal Essential Medium) ((MEM), Sigma), RPMI-1640 (Sigma) and Da Erbaikeshi improvement (Dulbecco ' s Modified Eagle ' s Medium) ((DMEM), Sigma). In addition, at Ham and Wallace,Meth.Enz.，58：44 (1979); Bames and Sato,Anal.Biochem.，102：255 (1980); U.S. 4,767,704; 4,657,866; 4,927,762 or 4,560,655; WO 90/03430; WO 87/00195; United States Patent (USP) Re.30, any culture medium of describing in 985 or U.S.5,122,469 can be used as the culture medium of host cell. Can as required following substances be added to any in these culture mediums: hormone and/or other growth factor (such as insulin, transferrins (transferrin) or EGF), salt (such as sodium chloride, calcium, magnesium and phosphate), buffer solution (such as HEPES), nucleosides (such as adenosine (adenosine) and thymidine (thymidine)), antibiotic (such as gentamicin (gentamycin)), trace element (being defined as the inorganic compound that usually exists with the final concentration in the micro-molar range) and glucose or the equivalent energy. Also can comprise any other essential fill-in by the known debita spissitudo of those skilled in the art. Condition of culture such as temperature, pH etc., is previous those conditions for selecting for the host cell of expressing, and will will be apparent for those of ordinary skill.

In case host cell grows into certain density, change condition of culture to promote the synthetic of albumen. If above-mentioned two-use inducible promoter in the promoter vector, express being suitable for activating under the condition of promoter inducible protein. In preferred embodiments, two promoters all are induction types. More preferably, double-promoter is respectively phoA and tacII. For example, can prepare carrier, wherein the phoA promoter is used for transcribing of control light chain, and the tacII promoter is used for transcribing of control heavy chain. In the phase I of inducing, in limit phosphorus type (phosphate-limiting) culture medium, cultivate the prokaryotic host cell that transforms with described phoA/tacII double-promoter carrier, be used for inducing the phoA promoter and expressing light chain. After the one required period of light chain expression, the isopropyl of capacity-beta-D-sulfo-synthesis (isopropyl-beta-D-thiogalactopyranoside) (IPTG) is added culture medium be used for inducing the tacII promoter and produce heavy chain.

In one aspect, if bacterial cell is used as host cell, just can in cytoplasm, express antibody. Useful several different methods is improved the generation of function antibody in Bacillus coli cells matter of solubility. For example, found that the coli strain that lacks the trxB gene strengthens the formation of disulfide bond in cytoplasm, and therefore can be used for promoting to have the expression of function antibody molecule in cytoplasm that correct disulfide bond forms. Proba etc.,Gene，159：203-207 (1995). But the Dispersal risk variant is replaced cysteine residues, so that described variant does not need to form disulfide bond in VH and VL; Therefore such antibody variants is sometimes referred to as " interior antibody (intrabody) ", can be in the reducing environment that is not suitable for effective disulfide bond formation, as preparing in the bacterial cytoplasm. Proba etc.,J.Mol.Biol.，275：245-253(1998)。

When using secretory signal sequence, the light chain of expression and heavy chain polypeptide are secreted into the pericentral siphon of host cell, and therefrom reclaim. Albumen reclaims the method destroy microorganisms that is usually directed to usually use such as osmotic shock (osmotic shock), sonication (sonication) or cracking. In case cell is broken, can remove cell fragment or whole cell by centrifugal or filtration. Can be further purified albumen, for example, by affine resin chromatography (affinity resin chromatography). Optional, albumen can be transported in the culture medium and therein separation. Can from that filter and concentrated culture and culture supernatant, reclaim cell for generation of being further purified of antibody. Can use method such as the polyacrylamide gel electrophoresis (PAGE) of common general knowledge further to separate with the Western Blot experiment and identify the antibody of expressing.

Can produce in a large number antibody by sweat. Various extensive feed supplements-batch fermentation step can be used for the production of recombinant protein. Large scale fermentation has at least 1000 liters output, preferably approximately 1,000 to 100,000 liter output. These fermentation tanks use agitator paddle or other suitable means to distribute oxygen and nutrients, especially glucose (preferred carbon source/energy). On a small scale fermentation is often referred at volume and is not more than about 100 liters and can be in about 1 fermentation that rises in the fermentation tank of about 100 liters range changing.

During the fermentation, usually under suitable condition, grown into required density at cell, for example, the OD of about 180-270₅₅₀Begin afterwards inducing of protein expression. Vector construction body according to adopting such as known in the art and top description, can use multiple inducer. Before inducing, can make the cell term growth. Usually the about 12-50 of inducing cell hour, but can use longer or shorter induction time.

In order further to improve herein production yield and the quality of antibody, can change various fermentation conditions. For example, for the correct assembling of secretory antibody and folding, can use overexpression companion (chaperone) albumen, come cotransformation host prokaryotic such as the additional carrier of Dsb albumen (DsbA, DsbB, DsbC, DsbD and or DsbG) or FkpA (having peptidyl prolyl (peptidylprolyl) cis of chaperone activity, trans-isomerase (isomerase)). Proved that described chaperone promotes the correct of heterologous protein that produces to fold and solubility in bacterial host cell. Chen etc.,J.Bio.Chem.， 274：19601-19605 (1999); United States Patent(USP) Nos. 6,083,715 and 6,027,888; Bothmann and Pluckthun,J.Biol.Chem.，275：17100-17105 (2000); Ramm and Pluckthun,J.Biol. Chem.，275：17106-17113 (2000); Arie etc.,Mol.Microbiol.，39：199-210(2001)。

For the proteolysis (as in prokaryotic host cell) of the heterologous protein that makes expression (especially proteolysis sensitivity those) minimizes, some host strain that can lack proteolytic enzyme is used for the present invention. For example, can modify the prokaryotic host cell bacterial strain in the gene of the known bacterialprotease of coding, to realize gene mutation, described protease such as Protease III, OmpT, DegP, Tsp, TonA, PhoA, Protease I, Protease Mi, Protease V, Protease VI and its combination. The bacterial strain of some e. coli protein enzyme defects be available and, for example, Joly etc.,Proc.Natl.Acad.Sci.USA， 95：2773-2777 (1998); United States Patent(USP) Nos. 5,264,365 and 5,508,192; Hara etc.Microbial Drug Resistance，2：Describe among the 63-72 (1996). Most preferably, it has the genotype that comprises Δ ptr or Δ prc prc-mortifier.

In some embodiments, prepare and used the immune conjugate (immunoconjugate) that comprises the antibody that is connected with cytotoxic agent. Preferably, described immune conjugate and/or with the antigen of its combination by cell internalizing, the treatment validity that this causes described immune conjugate to increase in the target cell that kills with its combination. In preferred embodiments, the nucleic acid in described cytotoxic agent target or the interference target cell.

Antibody and one or more little molecule toxins such as the bond of Calicheamicin (calicheamicin), maytansine (U.S. Patent No. 5,208,020), trichothene and CC1065, also are desired herein.

In a preferred embodiment of the present invention, antibody is combined (for example each antibody molecule about 1 is to about 10 maytansine molecules) with one or more maytansine molecules. Maytansine can, for example, be converted into May-SS-Me, its can be reduced to May-SH3 and with the antibody response of modifying (Chari etc.,Cancer Research，52：127-131 (1992)) to produce maytansinoid-antibody mediated immunity conjugate.

Another kind of interested immune conjugate comprises the antibody of being combined with one or more Calicheamicin molecules. Antibiotic Calicheamicin family can produce the double-stranded DNA fracture with Asia-picomole concentration. The analogue of spendable Calicheamicin includes, but not limited to γ₁ ^I、α ₂ ^I、α ₃ ^I, N-acetyl-γ₁ ^I, PSAG and θ^I ₁(Hinman etc.,Cancer Research，53：3336-3342 (1993) and Lode etc.,Cancer Research，58：2925-2928 (1998)). Also referring to, United States Patent(USP) Nos. 5,714,586; 5,712,374; 5,264,586 and 5,773,001.

It comprises diphtheria (diphtheria) A chain with fragment spendable enzyme activity toxin, diphtheria toxin non--in conjunction with active fragment, exotoxin (exotoxin) A chain (from pseudomonas aeruginosa), ricin (ricin) A chain, abrin (abrin) A chain, modeccin (modeccin) A chain, alpha sarcine (sarcin), tung oil tree (Aleurites fordii) albumen, caryophyllin (dianthin) albumen, dyers' grapes (Phytolaca americana) albumen (PAPI, PAPII and PAPS), balsam pear (momordica charantia) mortifier, curcin (curcin), crotin (crotin), sapaonaria officinalis mortifier, gelonin (gelonin), mitogellin, restrictocin (restrictocin), phenomycin (phenomycin), enomycin (enomycin) and tricothecene. Referring to, for example, the WO 93/21232 that on October 28th, 1993 published.

The present invention further contemplates that antibody and has the compound of nucleolytic activity (for example ribalgilase (ribonuclease) or DNA endonuclease such as deoxyribonuclease (deoxyribonuclease); DNase) immune conjugate that forms between.

Multiple radio isotope can be used for producing the antibody of radiation combination. Example comprises At²¹¹、I ¹³¹、I ¹²⁵、 Y ⁹⁰、Re ¹⁸⁶、Re ¹⁸⁸、Sm ¹⁵³、Bi ²¹²、P ³²Radio isotope with Lu.

Can use the bond of multiple bifunctional protein coupling agent Dispersal risk and cytotoxic agent; described albumen coupling agent such as N-succinyl-3-(2-dithio pyridine)-propionate (N-succinimidyl-3-(2-pyridyldithiol) propionate) (SPDP); succinyl-4-(N-Malaysia acyl methyl) cyclohexane-1-carboxylate (succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate); iminothiolane (IT); the dual-function derivative of imino-ester (imidoester) (such as dimethyl adipyl imino-ester HCL); active ester (such as two succinimide suberates (disuccinimidyl suberate)); aldehyde (such as glutaraldehyde (glutaraldehyde)); two-azido (bis-azido) compound (such as two (p-azido benzoyl base) hexamethylene diamine (bis (p-azidobenzoyl) hexanediamine)); two-diazo (bis-diazonium) derivative is (such as two-(p-diazobenzene formoxyl)-ethylenediamine (bis-(p-diazoniumbenzoyl)-ethylenediamine)); diisocyanate (diisocyanate) is (such as tolyl 2; (tolyene 2 for the 6-diisocyanate; 6-diisocyanate)) and two-active fluorine compounds (such as 1; 5-two fluoro-2; 4-dinitro benzene (1; 5-difluoro-2,4-dinitrobenzene)). For example, can be such as Vitetta etc.,Science， 238：1098 (1987) described preparation ricin immunotoxins. The 1-isothiocyanato benzyl of carbon-14-mark-3-methyl DTPA (1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid) is that representational chelating agent is used for the radioactive nucleus thuja acid is combined with antibody (MX-DTPA). Referring to WO 94/11026. Joint can be " joint that can cut off " that promotes that cytotoxic drug discharges in cell. For example, can use joint, peptase sensitiveness joint, the dimethyl joint of acid labile or contain disulphide joint (Chari etc.,Cancer Research，52：127-131(1992))。

Optional, for example can be by recombinant technique or the synthetic fusion that comprise antibody and cytotoxic agent for preparing of peptide.

In another embodiment, antibody and " acceptor " (such as streptavidin (streptavidin)) combination can be used for the pre-target of tumour (pretargeting), wherein to patient's administration of antibodies-receptors bind thing, then use fining agent (clearing agent) from circulation, to remove free bond, then use with cytotoxic agent (for example, radionuclide (radionuclide)) " part " of combination (for example, avidin).

Also antibody can be used for ADEPT by antibody is combined with the prodrug kinase, described prodrug kinase is converted into active resisting-the cancer medicine with prodrug (prodrug) (for example, peptide acidylate therapeutic agent is referring to WO81/01145). Referring to, for example, WO 88/07378 and U.S. Patent No. 4,975,278.

The enzyme component that is used for the immune conjugate of ADEPT comprises that any prodrug that can act in one way is with the enzyme of the more great-hearted toxic forms that is translated into it.

The enzyme that is used for the ADEPT method includes, but not limited to for the alkaline phosphatase (alkaline phosphatase) that phosphatic prodrug is converted into free drug (free drug); Be used for the prodrug of containing sulfate is converted into the aryl sulfatase (arylsulfatase) of free drug; Be used for nontoxic 5-flurocytosine is converted into the cytosine deaminase (cytosine deaminase) of anti--cancer medicine 5 FU 5 fluorouracil; Protease, such as Serratieae Proteases, thermolysin (thermolysin), subtilopeptidase A (subtilisin), carboxypeptidase (carboxypeptidase) and cathepsin (cathepsin) (such as cathepsin B and L), it is converted into free drug for the prodrug that will contain peptide; Be used for transforming the D-alanyl carboxypeptidase (D-alanylcarboxypeptidase) of the prodrug that contains D-49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor base; Be used for glycosylated prodrug is converted into the carbohydrate-lyases of free drug such as beta galactosidase (β-galactosidase) and neuraminidase (neuraminidase); Being used for will (β-medicine of lactam) deriving be converted into the beta-lactamase (β-lactamase) of free drug with beta-lactam; And penicillin amidase, as the medicine that is respectively applied to derive with benzene oxygen acetyl (phenoxyacetyl) or phenylacetyl group (phenylacetyl group) at their ammonia nitrogen is converted into ospen amidase or the Penicillin-G-amidases of free drug. Optional, can use the antibody with enzymatic activity, be also referred to as " abzyme " in this area, with prodrug of the present invention be converted into free active medicine (referring to, for example, Massey,Nature，328：457-458 (1987)). Can be used for abzyme is delivered to tumour cell colony by Dispersal risk as described herein-abzyme bond.

Can pass through methods known in the art, as use above-mentioned allos bi-functional cross-linking agent with the antibody covalent bond of described enzyme and this paper. Optional, can use recombinant DNA technology construction of fusion protein known in the art, described fusion comprises the antigen-land of antibody of the present invention at least, its partly be connected with the functional activity of enzyme of the present invention at least (referring to, for example, Neuberger etc.,Nature，312：604-608 (1984))。

Antibody herein can be used for increasing tumour and penetrates (penetration). In this case, required is that modified antibodies is to increase its serum half-life. This can, for example, (for example mix antibody by saving (salvage) receptor binding domain, sudden change by appropriate area in the antibody or peptide-labeled by epi-position is mixed, then merge with antibody at any end or middle, for example, synthetic by DNA or peptide) obtain. Referring to the WO 96/32478 that published on October 17th, 1996.

The rescue receptor binding domain forms the district usually, wherein will transfer to position like the antibody class from any one or a plurality of amino acid residue of one or two ring in Fc district. Even more preferably, will shift from three or more residues of one or two ring in Fc district. Also more preferably CH1, CH3 or the V of antibody removed and transferred to epi-position from (for example, IgG) the CH2 territory in Fc district_HDistrict, perhaps more than one district in the above-mentioned district. Optional, epi-position is removed and is transferred to the C of antibody from the CH2 territory in Fc district_LDistrict and/or V_LThe district.

The covalent modification of antibody is also included within the scope of the present invention herein. If they can be by chemical synthesis or adaptable words, enzyme process or chemical cleavage by antibody prepare. The covalent modification of other type of antibody imports molecule by the reaction by target amino acid residue and organic derivating agent of antibody, described derivating agent can with the side chain of selecting or N-or the reaction of C-terminal residue.

The representational covalent modification of polypeptide is in U.S. Patent No. 5,534, describes in 615. The covalent modification type of preferred antibody comprise with antibody and multiple non--polymer of protein for example, a kind of with U.S. Patent No. 4 in polyethylene glycol (polyethylene glycol), polypropylene glycol (polypropylene glycol) or the polyoxyalkylene (polyoxyalkylene), 640,835; 4,496,689; 4,301,144; 4,670,417; The mode of listing in 4,791,192 or 4,179,337 connects.

In yet another aspect, the present invention also relates to determine to be diagnosed as the mammal of cancer, people for example, patient's possibility is benefited from the method with the treatment of TGF-beta antagonist. The method comprises the steps:

(a) cancer cell that obtains from the patient of test is for the sensitiveness of the growth inhibition effect of TGF-beta;

(b) obtain the gene expression atlas of the cancer cell that obtains from the patient, and its gene expression atlas with the cancer cell that obtains from animal model is compared, described animal model is for replying with the treatment of TGF-beta antagonist; With

(c) if the cancer cell that obtains from the patient is insensitive for the growth inhibition effect of TGF-beta, and have to from the similar gene expression atlas of the cancer cell gene expression atlas of the described animal model gained that responds to described treatment, then the patient is accredited as the treatment that may benefit from the TGF-beta antagonist.

For purpose herein, " similar " refers to that expression map passes through the similar or mutual tracking aspect one or more of Explicit Expression pattern, described expression pattern has about 80% to 100% homogeneity in amount or other measurable expression parameter, this depends on experiment or the method for measuring gene expression atlas as described in detail later, more preferably have approximately 90 to 100%, and more preferably have about homogeneity of 95 to 100%. From the patient usually obtain by identical method or experiment with gene expression atlas from the cancer cell of animal model so that its relatively.

The production method of multiple TGF-beta antagonists and they is known in the art, and more antagonist and preparation method thereof at present (referring to for example, Dennis etc., U.S. Patent No. 5,821,227) just under development. The concrete TGF-beta antagonists of using is not restricted feature; Any effective TGF-beta antagonists can be used for method and composition of the present invention as defined herein, such as the example in definition provided herein.

A kind of desirable TGF-beta antagonists has the high affinity for TGF-β, be stable with external long-term use in vivo, and can distinguish in some way and relate to " pathology " TGF-β of causing or aggravating lysis and relate to " physiological " the TGF-β that in a plurality of tracts, keeps normal homeostasis (homeostasis) and cell function. Although understand described mechanism and be not use essential to the invention, but in one embodiment of the invention, if need TGF-β to keep normal homeostasis, then be activated in the part that generates the site, and promptly from cell, do not discharge with near receptors bind, and pathologic process is relevant with the widely activation of TGF-β, then the antagonist SR2F that does not have cell-surface conjunction territory as discussed above of relative volume large (bulky), can be difficult to approach " the physiological TGF-β " relevant with cell, but " pathology " TGF-β that can effectively neutralize. Yet, and do not mean that and limit the invention to any concrete mechanism.

If cancer is breast cancer, comprise primary and metastatic breast cancer, aforesaid method of prognosis also can comprise the step of the Her2 state of measuring the patient, wherein Her2⁺The patient usually but be not always may not reply for independent treatment with the TGF-beta antagonist or replys very poor.

If the patient may be from being benefited the treatment of TGF-beta antagonists, after the abovementioned steps may for use separately or with any chemotherapeutant of effective dose and/or the TGF-beta antagonists of cytotoxic agent and/or other form of therapy (comprising radiotherapy) coupling effective dose.

The method that gene expression atlas is analyzed (profiling) is known in the art, and usually based on the hybridization analysis of polynucleotides or polynucleotide sequence. The most normally used method that quantification of mrna is expressed in sample as known in the art comprise northern trace and in situ hybridization (Parker and Barnes,Methods in Molecular Biology，106：247-283 (1999)); The RNAse Protection (Hod,Biotechniques， 13：852-854 (1992)); And reverse transcriptase polymerase chain reaction (RT-PCR) (Weis etc.,Trends in Genetics，8：263-264 (1992)). Optional, can adopt the antibody that can identify the specific double-strand body, described duplex comprises DNA duplex, RNA duplex and DNA-RNA heteroduplex body or DNA-albumen duplex. The representational method of gene expression analysis based on order-checking comprises serial analysis of gene expression (SAGE), and measures (massively parallel signature sequencing) gene expression analysis (MPSS) by whole parallel characteristic sequence. In these methods any, or other method as known in the art, can be used for measuring the gene expression atlas of the tumour cell that is obtained by the patient, described patient such as people patient and as the animal of the cancer model that the TGF-beta antagonists is replied, such as mouse model. In people patient's situation, the source of tumour cell can be fresh, freezing or fixing and paraffin-embedded (paraffin-embedded) tissue sample, can and carry out gene expression analysis from this sample extraction mRNA.

Optional, also can use proteomics method to compare the expression map of people and contrast (for example mouse) cancer cell. The proteomics collection of illustrative plates is for example representative of the expression pattern of most protein in the cancerous tissue of biological sample. Described expression map can, for example, be expressed as mass spectrum, but comprise that also other is based on any physical chemistry of albumen or the expression of biochemical property. Therefore, described expression map can, for example, based on the difference in the electrophoretic property of albumen, as by two-Wei gel electrophoresis, for example by 2-D PAGE measure like that, and can be expressed as, for example the most of spots in two-Wei running gel. Proteomics method is known in the art, and, for example, in following textbook, describe:Proteome Research：New Frontiers in Functional Genomics (Principles and Practice),M.R.Wilkins etc., eds., Springer Verlag, 1007;2-D Proteome Analysis Protocols，Andrew L Link，editor， Humana Press，1999； Proteome Research：Two-Dimensional Gel Electrophoresis And Identification Methods (Principles and Practice),T.Rabilloud editor，Springer Verlag，2000； Proteome Research:Mass Spectrometry (Principles and Practice),P. James editor，Springer Verlag，2001； Introduction to Proteomics，D.C.Liebler editor，Humana Press，2002； Proteomics in Practice：A Laboratory Manual of Proteome Analysis，R.Westermeier etc., eds., John Wiley ﹠ Sons, 2002.

In yet another aspect, to do not reply or reply very poor patient's available combination therapy for treating with the treatment of TGF-beta antagonists, described combination treatment comprises the TGF-beta antagonists of using doses, when using separately, it does not have significant Anti-tumor effect, but can be effectively antitumor when one or more chemotherapeutants of itself and effective dose or cytotoxic agent and/or radiotherapy coupling.

In yet another aspect, the present invention relates to by the patient being used the treatment of TGF-beta antagonists and mammal, for example relevant osteoclasia or the bone loss of the tumor metastasis among the people patient of effective dose. Described osteoclasia or bone loss can result from a variety of causes, comprise primary and the Secondary cases cancer of infiltrating bone. Treatment comprises reverse (reversal) osteoclasia or bone loss, and the pathologic process of stop or slowing down osteoclasia or bone loss.

In yet another aspect, the invention provides the methods for the treatment of of the mammalian subject that is diagnosed as cancer, it comprises the combination of described patient being used TGF-beta antagonists and chemotherapeutant or the cytotoxic agent of effective dose, also treats with the radiotherapy of effective dose alternatively. The monitoring patient is for the reaction of described combination. Described method is so that the effective dose sum that the effective dose of described combination is lower than described TGF-beta antagonist and described chemotherapeutant or cytotoxic agent when separating administration as independent reagent. This cancer is preferably breast cancer, such as the breast cancer that shifts, or colorectal cancer. Described chemotherapeutant is preferably taxane.

In yet another aspect, the invention provides the methods for the treatment of of the mammalian subject that is diagnosed as cancer, it comprises TGF-beta antagonist and the radiocurable combination of described patient being used effective dose, also uses alternatively anti-angiogenic agent, such as the antibody of specific bond VEGF. Described method is so that the effective dose of described combination is lower than described TGF-beta antagonist and radiocurable effective dose sum when separating administration as independent reagent. Preferred described cancer is breast cancer, such as the breast cancer that shifts, or colorectal cancer.

In yet another aspect, the invention provides the methods for the treatment of of the mammalian subject that is diagnosed as cancer, it comprises uses the TGF-beta antagonist of effective dose and the combination of anti-angiogenic agent to described patient, also use alternatively chemotherapeutant or the cytotoxic agent of effective dose, and monitor described patient for the reaction of described combination. This anti-angiogenic agent is preferably the antibody of specific bond VEGF. In one aspect, described method is so that the effective dose of described combination is lower than described TGF-beta antagonist when separating administration as independent reagent and and the effective dose sum of described anti-angiogenic agent.

TGF-beta antagonist herein can use separately or in treatment with other composition coupling. For example, described antagonist can be used jointly with anti-other antibody with tumor associated antigen except TGF-beta, described antibody such as one or more with EGFR, ErbB2, ErbB3, ErbB4 or VEGF antigen, chemotherapeutant (cocktail that comprises chemotherapeutant), cytotoxic agent, resist-angiogenic agent, antibody that cell factor and/or growth inhibitor combine. Concrete required can be also suppresses other therapeutic combination of tumor growth with antibody and one or more. Optional, or in addition, the radiotherapy that the patient can accept to make up (for example outside radiation exposure (external beam irradiation) or treat with radiolabeled reagent such as antibody). The therapy of above mentioned these combinations comprises the administration (wherein comprising two or more reagent in identical or different formulations (formulation)) of combination, with minute other administration, in this case, antagonist uses to occur in and uses before the adjunct treatment and/or afterwards. The appropriate dose of growth inhibitor is the dosage that uses at present, and can reduce this dosage when the compound action with other reagent that uses with the TGF-beta antagonist (acting synergistically (synergy)).

Use TGF-beta antagonist (with attached therapeutic agent) by suitable method, described method comprises outside stomach and intestine in (parenteral), subcutaneous (subcutaneous), the peritonaeum (intranasal) administration in (intrapulmonary) and nose in (intraperitoneal), the lung, and, if need topical therapeutic, can be through damage (intralesional) administration. The outer infusion (Parenteral infusion) of stomach and intestine comprises in muscle in (intramuscular), intravenous (intravenous), the artery in (intraarterial), the peritonaeum or subcutaneous administration. In addition, antagonist injects (pulse infusion) by pulse, specifically along with the decay of antagonist dosage is used suitably. Preferred administration gives by injection, most preferably intravenous or hypodermic injection, and this part ground depends on that administration is of short duration or long-term.

Antagonist composition will prepare in the mode that meets useful medical practice, quantitatively and use. The factor of considering in this context comprises scheduling and the known other factors of practitioner of the clinical condition of the disease specific that is treated, the concrete mammal that is treated, individual patient, the cause of disease, the position that antagonist is sent, the type of antagonist, the method for using, administration. Antagonist need not but alternatively be used at present prevention or treat one or more reagent of described illness formulated together. Other reagent of these of effective dose depends on the type of the antagonist that exists in the formulation and type and the other factors discussed above of amount, illness or treatment. Usually with identical dosage and the dosage of the before this usefulness of the method for administration of using in having as mentioned or about 1 to 99% use described material.

In order to prevent or treat disease, the antibody of appropriate dose (when independent use or with other reagent such as chemotherapeutant, cytotoxic agent, growth inhibitor or anti--angiogenic agent, or the antibody of aforesaid anti-not synantigen or cell factor is when being used in combination) will depend on type, the disease of type, the antagonist of disease to be treated seriousness and the course of disease, to use antagonist be for the clinical history that prevents purpose or therapeutic purposes, previous treatment, patient and to the judgment with the doctor in charge of replying of this antagonist. Once or in a series for the treatment of the patient is being used antagonist suitably. Type and seriousness according to disease, the antagonist (if especially it is antibody) of about 1 μ g/kg to 15mg/kg (for example 0.1mg/kg-10mg/kg) is the initial candidate dosage that the patient is used, and no matter for example be to divide other administration by one or many, or by continuous injection. Common daily dosage may be about 1 μ g/kg to 100mg/kg or more scope, and this depends on above mentioned factor. For for several days or longer time repeat administration, according to circumstances, continued treatment is until occur the required inhibition of disease symptoms.

The preferred dosage of antagonist, especially antibody will be at about 0.05mg/kg to the scope of about 10mg/kg. Therefore, can use one or more dosage among about 0.5mg/kg, 2.0mg/kg, 4.0mg/kg or the 10mg/kg (or its any combination) to the patient. Such dosage can be used off and on, for example weekly or use in per three weeks (for example so that the patient accept antibody about two to about 20, about six times dosage for example). Can use initial higher loading dose, then be one or more lower dosage. Representational quantitative scheme comprises the initial load dosage of using about 4mg/kg, then is the maintenance dose by week of about 2mg/kg antibody. Yet other quantitative scheme can be useful. The process of this treatment is easily monitored in method by routine and experiment.

Mix with optional physiologically acceptable carrier, excipient or stabilizing agent by the antagonist that will have required purity, the therapeutic dosage forms for preparing antagonist with the form aqueous solution, freeze-drying or other dry formulation is used for storing (Remington ' sPharmaceutical Sciences16th edition, Osol, A.Ed. (1980)). Acceptable carrier, excipient or stabilizing agent are nontoxic with used dosage and concentration to acceptor, and comprise buffer solution such as phosphate, citrate, histidine and other organic acid; Antioxidant comprises ascorbic acid and methionine; Anticorrisive agent is (such as octadecyl dimethyl benzene ammonio methacrylate (octadecyldimethylbenzyl ammonium chloride); Chloor-hexaviet (hexamethonium chloride); Benzalkonium chloride (benzalkonium chloride), benzethonium chloride (benzethonium chloride); Phenol, butyl or benzylated polyol; Alkyl parabens (alkyl parabens) is such as methyl or propyl para-hydroxybenzoate; Catechol (catechol); Resorcinol (resorcinol); Cyclohexanol (cyclohexanol); 3-amylalcohol (3-pentanol) and m-cresol (m-cresol)); Low-molecular-weight (less than about 10 residues) polypeptide; Protein is such as seralbumin, gelatin or immunoglobulin (Ig); Hydrophilic polymer such as polyvinylpyrrolidone (polyvinylpyrrolidone); Amino acid such as glycine, glutamine, asparagine, histidine, arginine or lysine; Monose, disaccharides and other carbohydrate comprise glucose, mannose or dextrin; Chelating agent such as EDTA; Carbohydrate such as sucrose, sweet mellow wine, trehalose or sorbierite (sorbitol); Salify equilibrium ion (salt-forming counter-ion) is such as sodium; Metal composite (for example, Zn-protein complex); And/or nonionic surface active agent such as TWEEN^TM、PLURONICS ^TMOr polyethylene glycol (PEG).

As mentioned above, formulation herein also can comprise more than one essential reactive compounds for the concrete indication that is treated, and is preferably those compounds of the complementary activity with adverse drug reactions. Described molecule is present in the combination suitably with effective amount for the expection purpose.

Also active component can be caught in the microcapsules of preparation, for example, by cohesion (coacervation) method or by at the delivery system of colloid (for example, liposome, albumin microsphere, microemulsion (microemulsion), milimicron particle (nano-particle) and nano-microcapsules (nanocapsule)) in or respectively interfacial polymerization (interfacial polymerization) in coarse emulsion (macroemulsion), for example, methylol (hydroxymethyl) cellulose or gelatin-microcapsules and poly--(methyl methacrylate) (poly-(methylmethacylate)) microcapsules. Described method is at Remington ' sPharmaceutical Sciences16th edition, Osol, open among the A.Ed. (1980). Particularly, can by as at Epstein etc.,Proc.Natl.Acad.Sci.USA，82：3688 (1985); Hwang etc.,Proc.Natl.Acad.Sci. USA，77：4030 (1980); With United States Patent(USP) Nos. 4,485, the method preparation of describing in 045 and 4,544,545 contains the liposome of antagonist. Have the liposome of circulation timei of increase in U.S. Patent No. 5,013, open in 556.

Can produce useful especially liposome with containing phosphatidyl-ethanolamine (phosphatidylethanolamine) lipid composition (PEG-PE) that phosphatid ylcholine (phosphatidylcholine), cholesterol (cholesterol) and PEG-derive by anti-phase method of evaporating. Liposome is extruded to obtain having the liposome of required diameter by the filter that limits pore size. Fab ' the fragment of antibody of the present invention can with such as Martin etc.,J.Biol.Chem.，257：Liposome described in the 286-288 (1982) is by disulphide mutual exchange reaction combination. Chemotherapeutant (such as Doxorubicin) is included in the liposome alternatively. Referring to Gabizon etc.,J. National Cancer Inst.，81(19)：1484(1989)。

The formulation that is used for using in the body must be aseptic. This realizes by the filtration through aseptic filter membrane easily.

Can prepare sustained release (Sustained-release) preparation. The suitable example of extended release preparation comprises the semi-permeable matrix of the solid hydrophobic polymer that contains antagonist, and its mesostroma is the form of formative substance, for example, and film or microcapsules. The example of sustained release matrix (for example comprises polyester (polyester), hydrogel, poly-(2-ethoxy-methacrylate) (poly (2-hydroxyethyl-methacrylate)) or poly-(vinyl alcohol) (poly (vinylalcohol))), polyactide (polylactide) (U.S. Patent No. 3,773,919), Pidolidone and the γ ethyl-copolymer of Pidolidone salt (glutamate), nondegradable ethylene-vinyl acetate (ethylene-vinyl acetate), degradable lactic acid-ethanol copolymer such as LUPRON DEPOT^TM(the injectable microballoon that is formed by lactic acid-ethanol copolymer and leuprorelin acetate) and poly--D-(-)-3-hydroxybutyrate (poly-D-(-)-3-hydroxybutyric acid). Surpass 100 days although polymer such as ethylene-vinyl acetate and lactic acid-ethanol discharge molecule, some hydrogel discharges albumen in the shorter time. When encapsulated antibody kept in vivo for a long time, as a result their changeabilities or the gathering that are exposed to moisture at 37 ℃, this caused bioactive loss and immunogenic possible variation. Can be reasonably tactful according to relevant mechanismic design for stability. For example, if discovery aggregation mechanism is to form by the intermolecular S-S key that sulfo--disulphide exchanges, just can be by modifying sulfhydryl residue, by the acid solution freeze-drying, the control water content obtains stability with suitable additive and exploitation specificity polymer matrix composition.

The present invention includes the cytotoxic chemotherapy of coupling and use soluble T GF-beta antagonists. Embodiment comprises use cell-cycle (cell-cycle) activating agent (for example, 5 FU 5 fluorouracil), and it organizes lacuna (compartment) what have an active circulating cells, such as show dose in marrow and the alimentary canal-restricted toxicity. Although understand described mechanism and be not and use essential to the inventionly, TGF-β remains on stem cell the state of static (quiescence). Be expected at one and take turns and use soluble T GF-beta antagonists after the chemotherapy and strengthen stem cells hyperplasia, and thus, recover hematopoiesis (Sitnicka etc.,Blood，88：82-88 (1996)). Except the independent result for the treatment of of TGF-beta antagonists, cause the toxicity of chemotherapeutant to reduce with the therapeutic alliance of soluble T GF-beta antagonists and chemotherapeutant.

The present invention also comprises with the treatment of soluble T GF-beta antagonists together with immunotherapy. Although understand described mechanism and be not use essential to the invention, the expection tumour to the secretion of immune mortifier limited validity for the immunotherapy that strengthens immunity identification and tumor destruction (de Visser and Kast,Leukemia，13：1188-1199 (1999)). TGF-β is the immunodepressant of tumour high-level secretory. Embodiment of the present invention together with immunotherapy (for example comprise coupling TGF-beta antagonists, Anti-tumor vaccine inoculation, the immunotherapy that adopts), its cause the TGF-beta antagonists anti--synergy between the immunotherapy validity of transfer effect and increase.

The further details of the present invention provides in following embodiment. Following embodiment only is intended to illustrate enforcement of the present invention rather than provides in the mode that limits. The open of all patents of quoting herein and scientific literature clearly incorporated into as a reference with its integral body.

Embodiment 1

The generation of monoclonal antibody 2G7 and 4A11 and sign

A. experimental procedure

I.ELISA measures

At 4 ℃, with 1 μ g/ml, in the carbonate buffer solution of pH 9.6, apply 18 hours with the experiment of 96-hole polystyrene is dull and stereotyped with the TGF-beta1 of the purifying in 100 μ l/ holes. Flat board through applying 22 ℃ of sealings one hour, is used 0.05%TWEEN20 among the PBS with 0.5% bovine serum albumin(BSA) (BSA) (being called BPBS) in the salt solution (PBS) of phosphate-buffering^TM(being called PBST) washing, and with the hybridoma supernatant of 100 μ l 22 ℃ of incubations one hour. Dull and stereotyped with PBST washing, and use with peroxidase-conjugated goat anti--mouse IgG detects the antibody (Iago, Burlingame, CA) of combination. Dull and stereotyped with the PBST washing, and with 100 μ l/ holes adding p-phenylenediamine dihydrochloride (o-phenylenediamine dihydrochloride) substrate. Stop reaction after 15 minutes, and at UVMAX^TMFlat bed reader (Molecular Devices, Palo Alto, CA) is upper measures optical density in 492nm.

The iodate of II.rTGF-beta1

By the chloramine-T of modifying^TM(CHLORAMINE T ^TM) (empirical formula: C₇H ₇SO ₂N NaCl(3H ₂O)) method (Greenwood etc.,Biochem.J.，89：114 (1963)) with the TGF-beta1 iodate of purifying. In brief, the 0.1mg/ml CHLORAMINE T that adds 20 μ l by three orders^TM, every minor tick 2-minute incubation, use the Na of 1mCi on ice¹The rTGF-beta1 of 25I mark 10 μ g purifying. Use sequentially adds the 50mM N-Acetyl Tyrosine, 1M KI (potassium iodine) of 20 μ l, then is that the 8M urea of 200 μ l is with reaction terminating. Use C18 post and trifluoroacetic acid/acetonitrile gradient by rTGF-beta1 the Na from dissociate of HPLC with iodate¹25I separates, and collection contains the fraction of main peak and is stored in-70 ℃ (specific activity 112 μ Ci/ μ g).

III. antigen capture radiommunoassay

At 4 ℃, with IMMUNLON^TM 2″REMOVAWELL″ ^TMBar (Dynatech, Chantily, VA) in pH 9.6 carbonate with 5 μ g/ml goats anti--mouse IgG (Boehringer Mannheim) applies 18 hours. The hole is washed with PBST, with PBS (the being called PBSG) sealing that contains 0.1% gelatin, with the PBST washing, and with the hybridoma supernatant 22 ℃ of incubations 4 hours. The hole is washed with PBST, and add about 75, the 000CPM/ hole¹The 100 μ l of 25I-rTGF-beta1 contain the PBST solution of 0.1% gelatin, and 22 ℃ of incubations two hours. Dull and stereotyped with the PBST washing, and at GAMMAMASTER^TMThe upper quantitative combination of counter (LKB, Sweden)¹25I-rTGF-beta1。

The immunoprecipitation of IV.125I-rTGF-beta

The specificity of anti--TGF-beta monoclonal antibody is also by their immunoprecipitations¹Being derived from of 25I-rTGF-beta1 or pig is hematoblastic¹25I-TGF-beta2 (R ﹠ D Systems, Minneapolis, MN; Specific activity 103.4 μ Ci/ μ g) ability is estimated. With the monoclonal antibody of 2 μ g purifying with 5 * 10⁴CPM's¹25I-rTGF-beta1 or¹25I-TGF-beta2 was 22 ℃ of incubations two hours. In order to rabbit anti--albumin A-SEPHAROSE that mouse IgG (Boehringer Mannheim Biochemicals, Indianapolis, IN) applies^TM(bead-formed) that pearl forms also washs 3X with PBST subsequently based on gel filtration matrix (Repligen, Cambridge, MA) the precipitation immune complex of agarose. With the reproducibility sample buffer with described compound from albumin A-SEPHAROSE^TMThe gel filtration matrix based on agarose that pearl forms is separated, and in 12%SDS-polyacrylamide gel (SDS-PAGE) electrophoresis and be exposed to autoradiograph (autoradiography).

The compatibility of V.TGF-beta monoclonal antibody is measured

By Mariani etc.,J.Immunol.Methods，71：43 (1984) the solid phase radioimmunoassay experiment methods of describing are used for measuring the compatibility of TGF-beta-monoclonal antibody specific. In brief, at 4 ℃, with purifying anti--the TGF-beta monoclonal antibody is at IMMUNLON^TM 2″REMOVAWELL″ ^TMIn pH 9.6 carbonate buffer solutions, apply 18 hours on the bar. As mentioned above with hole washing and sealing. With among the 50 μ l PBSG 40, the 000CPM/ hole¹25I-rTGF-beta1 or pig¹25I-TGF-beta2 (R ﹠ D Systems) adds cold rTGF-beta1 or the pig TGF-beta2 of 2-times of serial dilution among the 50 μ l PBSG, and its scope is 2500 to 9.7ng/ holes. With the mixture that obtains 4 ℃ of incubations 18 hours. As mentioned above with hole washing and counting, and by Scatchard analyze (Munson and Pollard,Anal. Biochem.，107：220 (1980)) measure affinity costant, described Scatchard analyzes and obtains and Antoni and Mariani,J.Immunol.Meth.，83：The similar result of 61 (1985) nonlinear regression analysis.

VI. from the ascites fluid monoclonal antibody purification

Clone the parental generation Hybridoma Cell Culture thing of secretion positive antibody in above-mentioned experiment by restriction dilution, and with PRISTANE^TMPrimer pretreated (primed) Balb/c mouse (Potter etc.,JNCI， 49：305 (1972)) grow in the ascites fluid. Monoclonal antibody is purified to albumin A-SEPHAROSE from ascites fluid^TMPearl form based on the gel filtration matrix of agarose and at 0.1M acetic acid, 0.5 M NaCl, use among the pH 2.4 regulation method (Goding,J.Immunol.Methods，20：241 (1978)) wash-out and 4 ℃ of aseptic storage in PBS.

VII. the monoclonal antibody of external TGF-beta specific activity neutralization

It is that Mv-3D9 is (from the Mv1Lu subclone that the ermine lung fibroblast is used in external TGF-beta experiment, described Mv1Lu can be by American type culture collection (American Type Culture Collection), Manassas, VA obtains as ATCC No.CCL-64). In brief, with purifying anti--TGF-beta monoclonal antibody and contrast and rTGF-beta1, natural pig TGF-beta2 (R ﹠ D Systems) or rTGF-beta3 (Derynck etc.,Nature，316：701-705 (1985)) with the final concentration of 1000-2000pg/ml 4 ℃ of incubations 18 hours. These mixtures of 50 μ l are added 96-hole microtiter plate, then add 1 * 10⁴The Mv-3D9 cell, described in containing 50 μ l minimum essential mediums of 2mM glutamine and 5% hyclone, and at 37 ℃ at 5%CO₂Middle incubation 18-24 hour. With the hole with 1 μ Ci among the 20 μ l³H-thymidine pulsatile impact (pulse) and after 4 hours 37 ℃ of results, and in scintillation counter (scintillation counter) counting. Every kind of dilution of TGF-beta reference material³The TGF-beta that the sample that the inhibition percentage of H-thymidine picked-up is processed with negative control monoclonal antibody and the specific monoclonal antibody of TGF-beta-of pg/ml for calculating represents is active.

VIII. the isotype of monoclonal antibody is identified (isotyping)

Use PANDEX^TMThe fluorescent screening machine technology carries out the isotype of the reactive monoclonal antibody of TGF-beta1-to be identified. The granules of polystyrene of large mouse-anti-mouse IgG antiserum-coating is used in conjunction with being dispensed into PANDEX^TMMonoclonal antibody in the culture supernatant of 96-hole brassboard. Wash described plate and add the rat monoclonal of FITC-coupling anti--mouse isotype specific reagent (Becton Dickinson Monoclonal Center). Pass through PANDEX^TMThe fluorescence of the quantitative combination of fluorescent screening machine technology.

IX. epitope analysis

By Nakane and Kawaoi,J.Histochem.Cytochem.，22：1084 (1974) method resists-rTGF-beta1 monoclonal antibody and (HRP) coupling of horseradish peroxidase (horseradish peroxidase) purifying. The resisting of the flat board that rTGF-beta1-is applied and 50 μ g/ml purifying-rTGF-beta1 or negative control in PBS 22 ℃ of incubations 2 hours. Then will resist-dilution of the pre-mensuration of rTGF-beta monoclonal antibody-HRP bond adds dull and stereotyped and 22 ℃ of incubations 1 hour. Washing is dull and stereotyped and add substrate and quantitative reaction as mentioned above. With allos anti--the sealing percentage of rTGF-beta1 monoclonal antibody compares with the positive sealing contrast from body.

X. immunoblotting assay

Use the irreducibility sample buffer rTGF-beta1 electrophoresis in 12%SDS-PAGE of 1 μ g/ swimming lane to be had the reactivity of multiple monoclonal antibody of the dimeric forms of rTGF-beta1 with mensuration. Peptide is shifted trace on nitrocellulose paper and use the suitable monoclonal antibody of being combined with HRP to survey. Use insoluble substrate 4-chloro-1-naphthols (4-chloro-1-naphthol) (Kirkegaard and Perry, Gathersburg, MD) to manifest the antibody of combination. After 15 minutes by with distilled water thoroughly washing come cessation reaction and the Western blotting thing is dry and take a picture.

B. produce anti--TGF-beta1-and anti--specific monoclonal antibody of TGF-beta2-

In initial immunization protocol, with rTGF-beta1 (as by Derynck etc., Nature, described generation and purifying see above) by subcutaneous and endoperitoneal approach, use the former preparation of panimmunity, dosage and scheduling also to use complete and incomplete freund's adjuvant (Freund ' s adjuvant) to come immune Balb/c mouse. Immunity time-histories continuity 11 weeks of as many as. Several mouse measuring but low anti--rTGF-beta1 tires and replys, two in these mouse are put to death and their spleen are used for merging. From 1152 parental generation cultures, only detected for 84 positive resisting-the TGF-beta supernatant. Clone ten in these hybridomas and produce the monoclonal antibody of hanging down compatibility, it can not be used for exploitation determination method or purifying.

As interchangeable strategy, with 100-μ l DETOX^TMAdjuvant (RIBI ImmunoChem Res.Inc., Hamilton, MT) TGF-beta1 of 5 μ g/ agent purifying was expelled to one group of ten Balb/c female mice (Charles River Breeding Laboratories at the 0th, 3,7,10 and 14 day, Wilmington, MA) rear palmula in. The 17th day with sacrifice of animal, (inguinal) is with (popliteal) lymph node of popliteal section, and uses stainless steel mesh that lymphocyte is separated from knot matrix (node stroma) to remove their drainage groin. Collect from the lymphocyte suspension of whole ten mouse and use 50% Macrogol 4000 by method (Oi and Herzenberg, the in of regulationSelected Methods in Cellular Immunology，B.Mishel and S.Schiigi, eds., (W.J.Freeman Co., San Francisco, CA, 1980), p.351) with mouse myeloma cell line X63-Ag8.653 (Keamey etc.,J.Immunol.，123：1548 (1979)) merge. With the cell that merges with 2 * 10⁵The density of cells/well spreads into 1344 96-hole microtiter plates altogether, then the 1st day after merging carry out the HAT selection (Littlefield, J.W,Science，145：709(1964))。

Immobilized restructuring TGF-beta1 reacts in 1190 holes in the above-mentioned hole and the ELISA test. There are 18 to keep stable during amplification in these cultures, refrigeration (cryopreserve) described clone. These parental generation cultures are carried out isotype to be identified and has measured them and caught¹25I-rTGF-beta1 and in external and the ability of TGF-beta1 activity. From for and rTGF-beta1 and measure and carry out subsequently in 18 parental generation cultures that isotype identifies, two belong to IgG1 kappa isotype; The remaining IgG2b kappa isotype that belongs to. Find that the monoclonal antibody that only belongs to the IgG1 subclass shows that external rTGF-beta1 suppresses (neutralization) activity. Select three stable secretion height-compatibilities anti--hybridoma of TGF-beta monoclonal antibody. The characteristic of these antibody describes in detail below.

C. radiate the immunoprecipitation of the TGF-beta of iodate

Carry out immunoprecipitation experiment and measure the ability of three monoclonal antibodies with identification in solution and precipitation TGF-beta1. Autoradiograph shows anti--TGF-beta monoclonal antibody 2G7,4A11 and 12H5 immunoprecipitation equivalent¹25I-rTGF-beta1, and contrast monoclonal antibody 6G12 is negative. The band of immunoprecipitation has the obvious molecular weight of about 14.5kD. Use the competitive interactional compatibility that suppresses to test with between mensuration TGF-beta1 and each monoclonal antibody. Monoclonal antibody 2G7 and 4A11 have higher equally compatibility, and it is 1.2 * 10⁸The l/ mole.

Also carry out immunoprecipitation experiment and in solution, identify and precipitate the ability of TGF-beta2 to measure the monoclonal antibody of selecting. The autoradiograph demonstration, opposite with rTGF-beta1, only have antibody 2G7 immunoprecipitation 125I-TGF-beta2 to any measurable degree. The relatively demonstration of 4A11 and 12H5 and negative control does not almost have the specificity precipitation. These results are surprisingly intersected enclosed experiment and are shown that 4A11 and 2G7 can suppress each other the combination with people rTGF-beta1. Referring to table 1.

Table 1

In conjunction with monoclonal antibody	Mabs is to the intersection sealing percentage of TGF-beta1The sealing monoclonal antibody
					2G7	4A11	12H5	456 *
	2G7   4A11   12H5	100   96   28	74   100   12	32   19   100					1.9   1.5   3.4

^*Mab 456 is the control antibodies of reacting with CD4.

In a word, data show that by the epi-position of these two kinds of monoclonal antibodies identifications be different, but they or approach very much or in some way impact each other from the combination in a distant place. By immunoprecipitation and intersection-enclosed experiment, 12H5 looks like and visibly different epi-position, but also observes the situation of some sealings. This conclusion also obtain following in and the support of data.

D. use the immunoblotting assay of rTGF-beta1

Because the activity form of TGF-beta is homodimer, carries out Western blotting and whether identify this form to measure monoclonal antibody. Antibody 2G7,4A11 and 12H5 are reacting with TGF-beta1 dimer (non-reduced) form in the Western blotting indirectly. 2G7 provides than 4A11 or the much better than band of 12H5. And in immunoprecipitation experiment, control antibodies 6G12 is negative. In the direct Western trace with the HRP conjugate of these monoclonal antibodies, also observe the reactive pattern of this kind.

In a word, utilize complete and incomplete freund's adjuvant, in Balb/c and C3H mouse, use panimmunity method and dosage arrangement to carry out repeatedly implementing after the unsuccessful trial using scheme with the palmula immunity of the fusions coupling of draining lymph node for the destruction tolerance of rTGF-beta1. Usually, find the method for generation of immunogene a little less than these being had the very rapid answer of high-affinity, this and Dasch etc.,J.Immunol.，142：The experience of 1536-1541 (1989) is opposite, and the TGF-beta2 that the latter uses the ox bone of the purifying in the freund's adjuvant to derive produces the monoclonal antibody of TGF-beta1-and TGF-beta2-neutrality as the immunogene in the Balb/c mouse.

Whole three kinds of monoclonal antibodies are incorporated into rTGF-beta1 in Western blotting, ELISA, intersection-sealing and immunoprecipitation experiment. In anti--rTGF-beta antibody two kinds in external and rTGF-beta1 active, and only have in these two kinds a kind of in the experiment of ermine lung fibroblast in and TGF-beta2 and TGF-beta3 active. The TGF-beta1-neutrality antibody also seals the combination of rTGF-beta1 in the radioreceptor experiment of radiation iodate, and this neutralization that external rTGF-beta1 activity is described is attributable to receptor blockade.

Embodiment 2

Humanized 2G7 antibody

The variable region of mouse monoclonal antibody 2G7 at first is cloned into the carrier that allows to produce mouse/people's chimeric Fab fragment. Use STRATAGENE^TMRNA extracts kit and separates total RNA according to the scheme of manufacturer from hybridoma. With described variable region by RT-PCR amplification, gel-purified and insert contain as previously mentioned people kappa constant region and people CH1 territory, based on the derivative of the plasmid of pUC119 (Carter etc.,Proc.Natl.Acad.Sci.(USA)，89：4285 (1992) and U.S. Patent No. 5,821,337). The Plasmid Transformation that obtains is entered coli strain 16C9 be used for expressing the Fab fragment. The growth of culture, protein expression induce with the purifying of Fab fragment as previously mentioned (Werther etc.,J.Immunol.，157：4986-4995 (1996); Presta etc.,Cancer Research，57：4593-4599(1997))。

The dna sequencing of chimerical clone allows the evaluation (Kabat etc. see above) of CDR residue. Use the oligonucleotides rite-directed mutagenesis, with all these 6 CDR districts importings be included in foregoing plasmid VX4 (Presta etc.,Cancer Research，57：4593-4599 (1997)) complete people's framework (VL kappa subclass I and VH subclass III). Expression and purifying are from the albumen of " CDR-exchanges (CDR-swap) " that produce as mentioned above. Carry out binding to compare two kinds of modification (version). In brief, with the TGF-beta extracellular domain (ECD of the every ml of 1 microgram; Such as generation as described in the WO 90/14357) at the carbonate buffer solution of 50mM, spending the night at 4 ℃ among the pH 9.6 applies NUNC MAXISORP^TMThen flat board uses ELISA diluent (0.5%BSA, 0.05% polysorbate TM20 (POLYSORBATE^TM20), PBS) room temperature sealing 1 hour. With the sample of serial dilution in ELISA diluent incubation 2 hours on flat board. After the washing, with biotinylated Muridae Anti-Human kappa antibody (ICN 634771), then use the horseradish peroxidase (Sigma) in conjunction with streptavidin and use 3,3 ', 5,5 '-tetramethyl benzidine (Kirkegaard ﹠ Perry Laboratories, Gaithersburg, MD) as substrate, detect thus the Fab fragment of combination. Read absorbance at 450nm. Compare with the combination of chimeric Fab fragment, the combination of CDR-exchange Fab reduces significantly.

In order to recover the combination of humanized Fab, use from CDR-exchange DNA and make up mutant as template. Use the model of computer-generations, design these and suddenly change people's framework region residue to be changed into their mouse homologue in the position that described variation may affect CDR conformation or antibody-antigen interface. Mutant is shown in table 2. (note all amino acid numbering such as Kabat etc., middle expression sees above) is about sequence, referring to Figure 19-22.

Table 2

The title of humanized 2G7FR sudden change

The mutant numbering	-framework region (FR) that TGF-beta consensus sequence (SEQ ID NO:6) compare anti-with the people replaces
		Modification 3	ArgH71Ala
Modification 4	ArgH71Ala，AlaH49Gly，
		Modification 5	ArgH71Ala，AlaH49Gly，PheH67Ala
Modification 6	ArgH71Ala，AlaH49Gly，LeuH78Ala
		Modification 709	ArgH71Ala，AlaH49Gly，ValH48Ile
Modification 710	ArgH71Ala，AlaH49Gly，IleH69Leu
		Modification
11	ArgH71Ala，AlaH49Gly，AsnH73Lys
		Modification 712	ArgH71Ala，AlaH49Gly，IleH69Leu，AsnH73Lys

Use modification 3 and 4 as the humanized Fab modification of intermediate with the numbering after obtaining to contain more. Have the modification 5 that changes AlaH49Gly, PheH67Ala and ArgH71Ala, it is in conjunction with seeming to have the combination that returns to original chimeric 2G7Fab fragment, and modification 709 and 11 is also like this. Expection modification 710 and 712 has similar combination for Chimeric fragment, but modification 712 has additional framework sudden change, and it is not because perhaps possibility that immunogenicity increases is required. Can with additional FR or CDR residue,, modify (for example replacing as follows: GlnL3Met, ArgL24Lys, ArgL54Leu, GluH35Ser) such as L3, L24, L54 and/or H35. May strengthen requiredly the replacement of stability be to replace methionine reducing oxidation with leucine or isoleucine, or the asparagine among the CDR becomes other residue and takes off-possibility of acid amides (de-amidation) with reduction. Optional, or in addition, humanized antibody can be affine sexually matured (see above) with further raising or improves its compatibility and/or other biologically active.

By the VL of chimeric 2G7 Fab and VH territory and humanized Fab modification 5,709 and 11 subclones are entered previously described pRK carrier for mammalian cell expression (Gorman etc.,DNA Prot.Eng.Tech.，2：3-10 (1990)) comes the plasmid of construction expression total length IgG. In brief, digest each Fab construct with cutting VL fragment with EcoRV and BlpI, its EcoRV/BlpI site (referring to Figure 23) of being cloned into plasmid pDR1 is to express completely light chain (VL-CL domain). In addition, digest each Fab construct with cutting VH fragment with PvuII and ApaI, its PvuII/ApaI site (referring to Figure 24) of being cloned into plasmid pDR2 is to express completely heavy chain (VH-CH1-CH2-CH3 territory).

For each IgG variant, by light chain expression plasmid and heavy chain expression plasmid be total to-are transfected into the human embryonic kidney cell line 293 that adenovirus transforms (Graham etc.,J.Gen.Virol.，36：59-74, (1977)) implement transient transfection (transient transfection). In brief, that day before transfection is 293 lysises, and is layered in the culture medium that contains serum. At second day, by the double-stranded DNA of light chain and heavy chain, together with PADVANTAGE^TMDNA (Promega, Madison, WI) prepares calcium phosphate precipitation together, and dropwise adds this flat board. Cell is incubated overnight at 37 ℃, then washs with PBS and in serum free medium, cultivated 4 days, gather in the crops conditioned medium this moment. Use albumin A-SEPHAROSE CL-4B^TMPearl form based on the gel filtration matrix of agarose from culture supernatant antibody purification, then buffering exchanges to the 10mM sodium succinate, 140mM NaCl, pH 6.0, and use CENTRICON-10  spin-on filter device (Amicon) concentrated. Measure protein concentrate by the absorbance of mensuration 280nm or by quantitative amino acid analysis.

Hu2G7 modification 5IgG is added to modify to help combination so that illustrate which CDR, and it is the sequence of kappa locus and do not lose activity which CDR can be returned to ethnic group, or for the stability of antibody. As shown in table 3, these be named as " heavy chain. light chain ", and provided amino acid difference between modification 5 and these modification.

Table 3

The title of humanized 2G7CDR sudden change

The mutant numbering	-CDR that TGF-beta modification 5 compare anti-with the people replaces
		Modification 5 (V5H.V5L)
H2N1.V5L	Identical with modification 5, except the Asn51 among the CDR H2 becomes Ile
		V5H.g1L2	Identical with modification 5, be the sequence of kappa locus L8/L9/L14/L15: YASSLQS (SEQ ID NO:8) except CDR L2 is responded as ethnic group

V5H.glL1glL2	Identical with modification 5, being responded as ethnic group except CDR L1 be that the sequence of kappa locus L8/L9: RASQGISSYLA (SEQ ID NO:7) and CDR L2 be responded as ethnic group is the sequence of kappa locus L8/L9/L14/L15: YASSLQS (SEQ ID NO:8)
		H2NI.glL1glL2	Identical with modification 5, being responded as ethnic group except CDR L1 be that the sequence of kappa locus L8/L9: RASQGISSYLA (SEQ ID NO:7) and CDR L2 be responded as ethnic group is that Asn51 among the sequence of kappa locus L8/L9/L14/L15: YASSLQS (SEQ ID NO:8) and the CDR H2 becomes Ile.

The kind that is used for CDR L1 is that the title of sequence is L8/L9, such as Cox etc.,Eur.J.Immunol.，24：Fig. 4 of 827-836 (1994) and Schable and Zachau,Biol.Chem.Hoppe-Seyler，374：Listed among Fig. 2 e of 1001-1022 (1993). For CDRL2, will plant is sequence called after L8/L9/L14/L15 (referring to Cox etc., see above, and Schable and Zachau, see above).

Carrying out in whole CDR ' s to the answer of the sequence of ethnic group (g1) kappa of system locus, is that revertant shows combination but only have the above-mentioned kind of listing. V5H.g1L2, it has the CDR L2 that the ethnic group of being returned to is the sequence of kappa locus, still is combined with TGF-beta and V5H.V5L. Two kinds of modification V5H.g1L1glL2 and H2NI.g1L1glL2, and H2NI.V5L, and combination does not occur in chimera.

Use mouse ' Mesangial (messangial) cell proliferation experiment test comparison antibody and several humanized antibody (V5H.V5L, V5H.glL2, H2NI.V5L, V5H.glL1glL2 and H2N1.glL1glL2). This scheme is as follows:

At the 1st day: Mesangial cell was layered on the 96-orifice plate in the culture medium (Dulbecco ' Eagle ' the s culture medium of s improvement and 3: 1 mixtures of the Ham`s F12 culture medium that adds 14mM HEPES buffer solution-95%-hyclone-5%) and grow overnight with mouse.

At the 2nd day: TGF-beta and three kinds of variable concentrations (100ng, 10ng and 1ng) and five kinds of dissimilar humanization TGF antibody (20 μ g/ml) diluted in serum free medium and add cell. With mouse TGF antibody with comparing (2G7).

At the 4th day: after 48 hours incubations, the reaction buffer of 20 μ l (is contained tetrazole compound 3-(4,5-dimethylthiazole-2-yl)-5-(3-carboxymethoxyl phenyl)-2-(4-sulfophenyl)-2H-tetrazolium (CELLTITER 96  AQUEOUS ONE SOLUTION REAGENT of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium), inner salt and electronics coupled reagent (phenazine ethosulfate) (Promega Inc.Cat number G3580)^TM) add each dull and stereotyped hole and allow and cultivated 2 hours. Measure absorbance (OD) at 490nm.

H2NI.V5L (20 μ g/ml) has blocked the cell that the TGF-beta by 1ng/ml induces fully and has suppressed, and this is the result identical with using the gomphosis mouse contrast. Similar with contrast, modification 5 (V5H.V5L) has also been blocked the cell inhibition.

Use 3T3cells to test multiple humanized antibody with respect to the activity of 2G7 in the multiple TGF-beta of neutralization, described cell is fibroblastic from the Switzerland's Development of Mouse Embryos that utilizes the external decomposition that excites of one of three kinds of TGF-beta, has then measured their propagation as activity. Humanized antibody H2NI.V5L is the contrast 2G7 antibody that surpasses far away on activity. The humanized antibody of other test, H2NI.glL2 (being returned to ethnic group is the CDR L2 of the sequence of kappa locus) and V5H.glL2 (being returned to ethnic group is the CDR L2 of the sequence of kappa locus), show comparable activity, and V5H.glL2 is that validity is minimum for all TGF-beta1 to-beta3.

In a word, humanized antibody V5H.V5L, V5H.glL2, H2NI.V5L, H2NI.glL2 and modification 709,710 and 711 are most preferred humanized modification, because them and chimeric antibody (chimH.chimL; The 2G7Fab fragment) comparably in conjunction with TGF-beta and/or in and TGF-beta or blocking-up suppressed by the external evoked cell of TGF-beta, and in the humanized antibody of all tests, having minimum framework variation, this will make the danger of the immune response in the patient body minimize. In addition, H2NI.V5L is particularly preferred antibody, because it is obviously outstanding aspect active in neutralization, and because the variation among the CDR H2 may have improved stability.

Embodiment 3

Tumor metastasis research in the mouse model of metastatic breast cancer

A.4T1 model

In first group of experiment, the 4T1 cell comes from from BALB/cfC₃The tumor of breast of the single spontaneous generation of H mouse. Primary 4T1 tumor cell injection is entered the mammary fat pad of immunocompetent BALB/c mouse. In a week after the injection, observe obvious primary tumor. This tumour spontaneous metastasis is to lung (approximately two weeks after the injection), liver and spleen (approximately three weeks after the injection) and bone (about 4 and 5 weeks after the injection).

With resisting-TGF-β antibody (2G7) treatment animal of 15mg/kg, 25mg/kg and 43mg/kg dosage. Injection after the cancer cell the 0th, 1,2 day with tested in the 1st and 2 weeks. As shown in Figure 1, reduce instantaneously the size of primary tumor with the treatment of 43-mg/kg dosage, and reduce the system level of VEGF. Find that 25-mg/kg dosage provides than the better result of 15-mg/kg dosage, and do not have marked difference (data of 15mg/kg and 25mg/kg dosage do not show) between the result who obtains with 25-mg/kg and 43-mg/kg dosage respectively.

Shown in Fig. 2,3 and 4, reduced the weight and volume of histological score (rank and the number of the leaf of impact (lobe)), Secondary cases lung neoplasm with the early treatment (5 weeks behind the injection cell) of anti--TGF-β antibody. Use following number range to determine histological score, wherein " % " is the percentage that contains the tissue of tumour cell, and " invasion " is whether found indication in blood vessel and/or lymph node of tumour cell:

Normally: it is minimum infiltrating (infiltration); %1-33; Without invasion.

The II level: moderate infiltrates; %34-66; Some invasions.

III level: the serious infiltration; %67-100; Many invasions.

Fig. 5 shows osteoclasia by the MicroCT image of relatively normal bone girder and bone transfer.

The quantitative analysis results of osteoclasia is presented at as in the following table 4.

Table 4

	The girder number	Little cantilever thickness	BV/TV	BS/BV	Mineral density
						Anti--TGF-β antibody	-2.8％ *	ns	-4.8％	ns	ns
+ cell	-7.2％ *	-22.5％	-28.2％	+28.6％	-15.9％
						Anti--TGF-β antibody+cell	+6.5％ **	+7.2％	+14.3％	-6.6％	+6.3％

^*With respect to tumor free control mice

^**Mouse with respect to the trouble tumour for the treatment of with control antibodies

+ cell=with the mouse of tumor cell injection

Resist-TGF-β antibody+cell=also use with tumor cell injection the cell of anti--TGF-β Antybody therapy.

The BV=bone volume

The TV=cumulative volume

The BS=bone surface

The result who provides in the table 4 shows some parameters that suppressed the osteoclasia of tumor of breast-induce with the early treatment of anti--TGF-β antibody (2G7) (5 weeks behind the injection tumour cell).

B. from Her2⁺The cell of tumor of breast

To inject the mammary fat pad of immunocompetent mouse from the epithelial cell of trastuzumab-sensitiveness (F2-1282) and trastuzumab-resistance (Fo5) tumor cell line. Respectively such as Fig. 6 and 7, shown in 8 and 9, with 25mg/kg dosage anti--treatment of TGF-β antibody (2G7) increased size and the VEGF of the system level of primary tumor the 0th day (same day of injection tumour cell), and do not rely on the trastuzumab-responsiveness of tumour.

Be different from the 4T1 epithelial cell, Her2⁺The synthetic high-caliber TGF-β of epithelial cell, its growth is subject to the inhibition of TGF-β, and poor growth all in vitro and in vivo. Produce non-surperficial lung neoplasm (image does not show) from the transfer of these cells, its generation and growth are not resisted-inhibition of TGF-β Antybody therapy.

The C.PymT tumour

This is the mouse model by the breast cancer that the expression of T cancer protein (PyMT) causes in the middle of the polyoma in the breast epithelium. To arrive from the primary tumor injection cell (200 ten thousand or 500 ten thousand cell) of PyMT tumour the mammary fat pad of acceptor mouse. Then the tumour that produces is gone down to posterity in a large amount of other mouse. The data declaration that shows among Figure 18 has reduced the primary tumor growth with the treatment of anti--TGF-β antibody (2G7).

Embodiment 4

Tumor metastasis research in the metastatic melanoma mouse model

B16-F10 and B16-BL6 metastatic melanoma subbreed are used in this research, and hypodermic injection enters immunocompetent mouse, and-TGF-β antibody (2G7) anti-to test is for primary and the melanomatous impact of Secondary cases. Particularly, C57Black 6 mouse are injected through subcutaneous with 500,000 tumour cells. Remove the primary tumor that forms at the 14th day, and put to death mouse at about the 28th day. Anti--TGF-β AC is about 30mg/kg. If known to directly injection is with B16-F10 subbreed importing bone, it can be settled in bone (being different from hypodermic result). The B16-BL6 subbreed is metastatic for lung.

Shown in Figure 10 (F10) and 11 (BL6), use the treatment of anti--TGF-β antibody 2G7 (approximately 30mg/kg) to increase the survival rate of suffering from melanomatous mouse.

Figure 12-the 15th, the multiple performance that the melanoma lung shifts comprises that CT analyzes and photoimaging.

Shown in Figure 16 and 17, use the treatment of anti--TGF-β antibody 2G7 in this model, to reduce significantly number and the incidence of disease of metastastic tumor of lung.

Notice that although do not use in this experiment, other subbreed of B16 is available, and can similarly use in the experiment. Described other subbreed comprises that for example, F0 (not shifting), F1 (hang down and shift; About 30%); And G3.26 (high metastatic).

The animal model of describing among the embodiment 3 and 4 has more effectiveness in the screening experiment of identifying new molecule, it may participate in or may for example restore the growth that (stromal recruitment) suppresses primary and/or secondary tumors by strengthening blood vessel generation and/or a matter, but the effectiveness of these experiments is not subjected to the restriction of the mechanism of action of the molecule of identifying.

Although the aforementioned specific embodiment that relates to is appreciated that the present invention is not like that limited. Those ordinarily skilled in the art will be expected and can carry out various modifications to disclosed embodiment, and not deviate from total viewpoint of the present invention. The intention modification that all are such is included within the scope of the present invention.

Claims (68)

1. screening technique, it comprises following steps: (1) uses multiple substances to the animal model of inhuman symimmunity activity being with or without in the situation of primary tumor, and this animal model suffers from least a soft tissue or bone shifts; (2) measure described substances to the impact of soft tissue or bone transfer, and if have primary tumor, described substances also measured to the primary tumor affects on the growth; (3) qualification test material, it suppresses the growth that soft tissue or bone shift, and if have primary tumor, described substances does not have harmful effect for the situation of primary tumor.

2. the process of claim 1 wherein that described soft tissue transfer is present in the tissue that is selected from lung or hepatic tissue.

3. claim 1 or 2 method, wherein said bone shifts and causes osteoclasia.

4. each method among the claim 1-3, wherein said animal model suffer from soft tissue and bone shifts.

5. each method among the claim 1-4, wherein said animal model suffers from primary tumor.

6. each method among the claim 1-4, wherein primary tumor is removed from described animal by operation.

7. each method among the claim 1-6, wherein said animal is rodent.

8. each method among the claim 1-7, wherein said animal is mouse or rat.

9. each method among the claim 1-8, wherein said animal is mouse.

10. each method among the claim 1-9, wherein said primary tumor is tumor of breast.

11. the method for claim 10, wherein said tumor of breast develops from the cell that is derived from spontaneous mouse breast cancer.

12. the method for claim 11, wherein said cell are the 4T1 mouse mastopathy cells.

13. the method for claim 10, wherein said primary breast tumour is Her2⁺, it is developed by epithelial cell, and described epithelial cell originates from Her2⁺Tumour or Her2⁺The passage cell of tumour.

14. the method for claim 13, wherein said primary breast tumour is the trastuzumab resistance tumor.

15. the method for claim 13, wherein said primary breast tumour are trastuzumab responsiveness tumours.

16. the method for claim 10, wherein said primary breast tumour is the PymT tumour, and it is developed by epithelial cell, and described epithelial cell originates from the passage cell of PymT tumour or PymT tumour.

17. each method among the claim 1-9, wherein said primary tumor is melanoma.

18. the method for claim 17, wherein said melanoma are the melanoma of B16 subbreed.

19. each method among the claim 1-18, the substances of wherein identifying are the antagonists of the molecule of secretion.

20. each method among the claim 1-19, the substances of wherein identifying is transformant growth factor-beta (TGF-beta) antagonist.

21. the method for claim 20, wherein said TGF-beta antagonist are the antibody of specific bond TGF-beta.

22. the method for claim 20 or 21, wherein said TGF-beta antagonist suppress bone and shift.

23. each method among the claim 20-22, wherein said TGF-beta antagonist reduces osteoclasia or bone loss.

24. each method among the claim 1-23, the substances of wherein described animal being used comprises known chemotherapeutant or cytotoxic agent.

25. the method for claim 24, wherein said chemotherapeutant or cytotoxic agent are taxanes.

26. the method for claim 24 or 25, wherein described animal is used two kinds of substances, wherein a kind of is the TGF-beta antagonist, and another kind is chemotherapeutant or cytotoxic agent, and measured the impact that coupling two kinds of substances shift soft tissue or bone, if and had primary tumor, these two kinds of substances of coupling would also be measured to the primary tumor affects on the growth.

27. the method for claim 26, the antibody that wherein said TGF-beta antagonist is specific bond TGF-beta and described chemotherapeutant or cytotoxic agent are taxanes.

28. each method among the claim 1-27 wherein also is exposed to described animal the radiotherapy of effective dose.

29. determine to be diagnosed as the method that the mammalian subject possibility of suffering from cancer is benefited from the treatment that utilizes the TGF-beta antagonist, it comprises:

(a) cancer cell that obtains from the patient of test is for the sensitiveness of the growth inhibition effect of TGF-beta;

(b) obtain the gene expression atlas of the cancer cell that obtains from the patient, and its gene expression atlas with the cancer cell that obtains from animal model is compared, described animal model is for replying with the treatment of TGF-beta antagonist; With

(c) if the cancer cell that obtains from the patient is insensitive for the growth inhibition effect of TGF-beta, and its have to from the similar gene expression atlas of the gene expression atlas of the cancer cell of the described animal model gained that responds to described treatment, then the patient is accredited as and may benefits from the treatment that utilizes the TGF-beta antagonist.

30. the method for claim 29, wherein said cancer is breast cancer.

31. the method for claim 29 or 30, wherein said cancer is metastatic breast cancer.

32. the method for claim 30 or 31, it also comprises the steps: to determine the Her2 state of the breast cancer cell that obtains from the patient, and if described cell be negative, the patient is accredited as may responds to the treatment that utilizes the TGF-beta antagonist.

33. each method among the claim 29-32, wherein said patient is the people.

34. each method among the claim 29-33, wherein said TGF-beta antagonist are the antibody of specific bond TGF-beta.

35. each method among the claim 29-34, wherein said patient has soft tissue and shifts.

36. the method for claim 35, wherein said soft tissue shift comprise that lung shifts and hepatic metastases at least a.

37. the method for claim 35 or 36, wherein said patient also has bone and shifts.

38. each method among the claim 29-37, wherein said patient shows osteoclasia or bone loss.

39. each method among the claim 29-38, it also comprises uses the TGF-beta antagonist of effective dose with the step for the treatment of cancer to the patient.

40. the method for claim 39, wherein said treatment is carried out after the exenterate primary breast cancer.

41. the method for claim 39 or 40, it also comprises uses the radiotherapy of the chemotherapeutant of effective dose or cytotoxic agent or effective dose with the treatment cancer to the patient.

42. the method for claim 41, wherein said chemotherapeutant is taxane.

43. each method among the claim 39-42, its also comprise to the patient use effective dose specific bond Her2 antibody with the treatment cancer.

44. the method for claim 43, wherein said antibody is trastuzumab.

45. each method among the claim 39-44, it also comprises uses the anti-angiogenic agent of effective dose with the treatment cancer to the patient.

46. the method for claim 45, wherein said anti-angiogenic agent are the antibody of specific bond VEGF.

47. the osteoclasia relevant with tumor metastasis or the method for bone loss in the treatment mammalian subject, it comprises the TGF-beta antagonist of the patient being used effective dose.

48. the method for claim 47, wherein said TGF-beta antagonist are the antibody of specific bond TGF-beta.

49. treatment is diagnosed as the method for the mammalian subject of suffering from cancer, it comprises the combination of the patient being used TGF-beta antagonist and chemotherapeutant or the cytotoxic agent of effective dose, and the monitoring patient is for the reaction of described combination, the effective dose sum that the effective dose of wherein said combination is lower than described TGF-beta antagonist and described chemotherapeutant or cytotoxic agent when separating administration as independent reagent.

50. the method for claim 49, wherein said cancer are breast cancer or colorectal cancer.

51. the method for claim 49 or 50, wherein said cancer are the breast cancer that shifts.

52. each method among the claim 49-51, wherein said chemotherapeutant is taxane.

53. each method among the claim 49-52 is wherein also treated the patient with the radiotherapy of effective dose.

54. treatment is diagnosed as the method for the mammalian subject of suffering from cancer, it comprises TGF-beta antagonist and the radiocurable combination of the patient being used effective dose, the effective dose sum that the effective dose of wherein said combination is lower than described TGF-beta antagonist and described radiotherapy when separating administration as independent reagent.

55. the method for claim 54, wherein said cancer is breast cancer.

56. the method for claim 54, wherein said cancer is colorectal cancer.

57. each method among the claim 54-56, it also comprises uses anti-angiogenic agent to the patient.

58. the method for claim 57, wherein said anti-angiogenic agent are the antibody of specific bond VEGF.

59. each method among the claim 54-58, wherein said TGF-beta antagonist are the antibody of specific bond TGF-beta.

60. treatment is diagnosed as the method for the mammalian subject of suffering from cancer, it comprises uses the TGF-beta antagonist of effective dose and the combination of anti-angiogenic agent to the patient, and the monitoring patient is for the reaction of described combination.

61. the method for claim 60, wherein said anti-angiogenic agent are the antibody of specific bond VEGF.

62. the method for claim 60 or 61, wherein said TGF-beta antagonist are the antibody of specific bond TGF-beta.

63. each method among the claim 60-62, it also comprises chemotherapeutant or the cytotoxic agent of the patient being used effective dose.

The effective dose sum that 64. each method among the claim 60-63, the effective dose of wherein said combination are lower than described TGF-beta antagonist and described anti-angiogenic agent when separating administration as independent reagent.

65. treatment is diagnosed as and suffers from cancer and do not reply or for the method for the very poor mammalian subject of replying of TGF-beta antagonists through pre-mensuration for the TGF-beta antagonists, it comprises TGF-antagonist and chemotherapeutant or cytotoxic agent or the radiocurable combination of the patient being used effective dose, and the monitoring patient is for the reaction of described combination.

66. the method for claim 65, wherein said cancer is breast cancer.

67. the method for claim 65 or 66, wherein said chemotherapeutant is taxane.

68. kit, the container that it comprises the antibody that contains the specific bond VEGF contains the container of the antibody of specific bond TGF-beta, and with the specification of two kinds of antibody of effective dose coupling with the cancer in the treatment mammalian subject.

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