CN101405030B - Use of an antibody for vascular endothelial groeth factor and an antibody for human epithelial growth factor receptor type 2 in preparing kit for tumor therapy - Google Patents

Abstract

The present invention provides a method of treating a breast cancer disease in a patient who has failed prior treatment with an anti-VEGF antibody, comprising administering to the patient a therapeutically effective amount of an anti-HER2 antibody while continuing said anti-VEGF antibody therapy. The invention also provides corresponding articles of manufacture and pharmaceutical compositions.

Description

For the antibody of VEGF with for the application of antibody in the test kit of preparation treatment tumor of hEGF's 2 receptors

Invention field

The present invention relates to use the combined therapy of anti-HER2 and VEGF antibody.Particularly, the present invention relates to the application that such antibody is used for treating patient's failed in the formerly treatment of using VEGF antibody breast cancer disease.

Background of invention

The pathogeny of various diseases occurs to participate in blood vessel, it comprises solid tumor, ophthalmic neovascularity syndrome such as proliferating retinopathy or relevant degeneration of macula (AMD) of age, rheumatoid joint liquid, and psoriasis (Folkman, J., Deng, J.Biol.Chem. (biological The Chemicals) 267 (1992) 10931-10934; Klagsbrun, M., etc., Annu.Rev.Physiol. (physics's year summary) 53 (1991) 217-239; And Garner, A, Vascular diseases (angiopathy),: Pathobiology ofocular disease, A dynamic approach (ophthalmic diseases, the i.e. pathology of kinetic approach), (eds.) Garner and A, Klintworth, G K, second edition Marcel Dekker, New York, (1994), 1625-1710 page or leaf).In the situation of solid tumor, to compare with normal cell, neovascularity generates and allows described tumor cell to obtain growth vigor and propagation autonomy.Therefore, in breast carcinoma and in some other tumors, dependency (Weidner, N. between Total microvessel density in tumor biopsy and the patient survival have been observed, Deng, N.Engl.J.Med. (New England Journal of Medicine) 324 (1991) 1-6; Horak, E.R., etc., Lancet340 (1992) 1120-1124; And Macchiarini, P., etc., Lancet340 (1992) 145-146).

The regulation and control that normal and unusual blood vessel occurs and neovascularity generates that VEGF (VEGF) participation is relevant with the ophthalmic disease with tumor (Ferrara, N., etc., Endocr.Rev. (endocrine summary) 18 (1997) 4-25; Berkman, R.A., etc., J.Clin.Invest. (Journal of Clinical Investigation) 91 (1993) 153-159; Brown, L.F., etc., Human Pathol. (human pathology) 26 (1995) 86-91; Brown, L.F., etc., Cancer Res. (cancer research) 53 (1993) 4727-4735; Mattern, J., etc., Brit.J.Cancer (Britain's cancer magazine) 73 (1996) 931-934; And Dvorak, H.F., etc., Am.J.Pathol. (American Journal of Pathology) 146 (1995) 1029-1039).Anti-VEGF neutralizing antibody suppress various human tumor cells tie up to growth in the mice (Kim, K.J. is etc., Nature (nature) 362 (1993) 841-844; Warren, R.S., etc., J.Clin.Invest. (Journal of Clinical Investigation) 95 (1995) 1789-1797; Borgstrom, P, etc., Cancer Res. (cancer research) 56 (1996) 4032-4039; And Melnyk, O., etc., Cancer Res. (cancer research) 56 (1996) 921-924).WO94/10202, WO98/45332, WO2005/00900 and WO00/35956 mention the antibody for VEGF.Humanized monoclonal antibody bevacizumab is (with trade mark

Sell) be the VEGF antibody for oncotherapy, and be that approval resists-blood vessel propellant (WO98/45331) for the unique a kind of for the treatment of of cancer.

HER2 is a member of human epidermal growth factor acceptor family, and at its Cytoplasm domain Profilin kinase activity.HER2 is overexpression in tumor cell, and relevant with survival with extremely low prognosis.Therefore, HER2 is the valuable target of breast cancer treatment.Antibody for HER2 is known from following: Takai, and N., etc., Cancer (cancer) 104 (2005) 2701-2708; Yeon, C.H., etc., Invest.New Drugs (novel drugs research) 23 (2005) 391-409; Wong, W.M., etc., Cancer Pract. (wherein putting into practice) 7 (1999) 48-50; Albanell, J., etc., Drugs Today (medicine today) is .35 (1999) 931-46 (Barc).

Trastuzumab is (with trade mark Sale) is used for the treatment of the recombinant humanized anti-HER 2 monoclonal antibody that HER2 crosses the metastatic breast cancer of expression/HER2 gene amplification.Pre-clinical research shows that this antibody has in the body and anti tumor activity in vitro.In addition, in mouse model, in the combination of various anti-tumor agents, observe additional or collaborative enhancing of trastuzumab anti-tumor activity.In clinical research, cross the prolongation of observing survival among the expression metastatic breast cancer patient at HER2.

According to WO98/45331, VEGF antibody in prevention or treatment the effectiveness in the disease can by continuously or with to the effective another kind of medicament of those purposes if improve with this antibody of antibody combined administration of HER2 receptors bind.WO2005/012531 described in the treatment of colorectal carcinoma, metastatic breast cancer and renal carcinoma can with VEGF antibody (for example,

) and/or anti-ErbB antibody (for example,

The antibody of combination.According to WO2005/063816, in the treatment of metastatic breast cancer, VEGF antibody can make up with anti-ErbB antibody.The combination that WO2005/00090 and WO2003/077841 also disclose VEGF antibody and anti-ErbB 2 antibodies is used for oncotherapy.

The clinical tumor scholar thinks that unanimously the growth that the failure for the treatment of of cancer needs not to be by primary tumo(u)r causes, but causes that by the transitivity diffusion to Different Organs described primary tumo(u)r is processed with operation usually.The differential cytotoxicity medicine is to not indication of antimetastatic activity always that disappears of primary tumo(u)r itself.On the contrary, in the reaction to some cancer therapy drugs, observe enhancing transfer (Geldof, A.A. is etc., Anticancer Res. (anticancer research) 8 (1988) 1335-1339; Murphy, S.B., J.Clin.Oncol. (Journal of Clinical Oncology) 11 (1993) 199-201; With De Larco, J.E., etc., Cancer Res. (cancer research) 61 (2001) 2857-2861).Clearly, the needs that have the therapy develop targeting primary tumo(u)r not only but also to suppress to shift.

Summary of the invention

The present invention includes Anti-HER 2 and VEGF antibody for the preparation of the application in the medicine that is used for treating the breast cancer disease among the patient failed in the formerly treatment with VEGF antibody, described application comprises Anti-HER 2 and the VEGF antibody to described patient's administering therapeutic effective dose.

In preferred embodiments, the present invention includes trastuzumab and bevacizumab for the preparation of the application in the medicine that is used for treating the breast cancer disease among the patient failed in the formerly treatment with VEGF antibody such as bevacizumab, described breast cancer disease is characterised in that the overexpression of HER2 receptor protein, and this application comprises trastuzumab and the bevacizumab to described patient's administering therapeutic effective dose.

The present invention also comprises the method for the treatment of breast cancer disease among the failed patient in the formerly treatment of VEGF antibody, and the method is included in when continuing described VEGF antibody treatment the Anti-HER 2 to described patient's administering therapeutic effective dose.

The present invention also comprises the method for the treatment of the breast cancer disease among the patient failed in the formerly treatment with VEGF antibody, described breast cancer disease is characterised in that the overexpression of HER2 receptor protein, and the method is included in when continuing bevacizumab treatment the trastuzumab to described patient's administering therapeutic effective dose.

The present invention also comprises the method for increasing the patient's who suffers from breast cancer disease of failure the survival persistent period in the formerly treatment of VEGF antibody, the method comprises VEGF antibody and the Anti-HER 2 of using effective dose to described patient, and described VEGF antibody and described Anti-HER 2 uses the persistent period that effectively increases survival jointly thus.

The present invention also comprises the method for freely surviving for increasing the progressivity of the patient who suffers from breast cancer disease of failure in the formerly treatment of VEGF antibody, the method comprises VEGF antibody and the Anti-HER 2 of using effective dose to described patient, and jointly using of described VEGF antibody and described Anti-HER 2 increases the persistent period that progressivity is freely survived effectively thus.

The present invention comprises that also being used for the treatment of one group suffers from breast cancer disease and failed patient's method in the formerly treatment of VEGF antibody, the method comprises VEGF antibody and the Anti-HER 2 of using effective dose to described patient, and described VEGF antibody and described Anti-HER 2 uses the response rate that effectively is increased in this patient group jointly thus.

The present invention also comprises the method for replying the persistent period for increasing the patient who suffers from breast cancer disease of failure in the formerly treatment of VEGF antibody, the method comprises VEGF antibody and the Anti-HER 2 of using effective dose to described patient, and described VEGF antibody and described Anti-HER 2 uses the persistent period that effectively increase is replied jointly thus.

The present invention also comprises treatment patient's who suffers from breast cancer disease of failure in the formerly treatment of VEGF antibody method, the method comprises VEGF antibody and the Anti-HER 2 of using effective dose to described patient, thus when by survival persistent period, the free survival of progressivity, response rate or the persistent period measurement of replying, jointly the using of described VEGF antibody and described Anti-HER 2 causes the patient's that is treated statistically significant and clinical significant improvement.

The present invention also comprises for reducing in the method for formerly treating the transfer among the failed patient who suffers from breast cancer disease with VEGF antibody, the method comprises VEGF antibody and the Anti-HER 2 of using effective dose to described patient, and effective minimizing of jointly using of described VEGF antibody and described Anti-HER 2 is shifted thus.

The present invention also comprises and is used for the treatment of one group of patient's the method for suffering from breast cancer disease and failure in the formerly treatment of VEGF antibody, the method comprises VEGF antibody and the Anti-HER 2 of using effective dose to described patient, and described VEGF antibody and described Anti-HER 2 uses the transfer that effectively reduces in this group patient jointly thus.

The invention provides goods (an article of manufacture), it comprises container, compositions in described container, said composition comprises VEGF antibody, and package insert, this package insert instructs the user of said composition described VEGF antibody and Anti-HER 2 to be administered to the patient who suffers from breast cancer disease of failure in the formerly treatment of described VEGF antibody.

The present invention also provides goods, it comprises container, compositions in described container, said composition comprises Anti-HER 2, and package insert, this package insert instructs the user of said composition described Anti-HER 2 and VEGF antibody to be administered to the patient who suffers from breast cancer disease of failure in the formerly treatment of described VEGF antibody.

The present invention also provides a kind of compositions, and said composition comprises Anti-HER 2 and VEGF antibody, and it is effective to treat the breast cancer disease among the patient failed in the formerly treatment with VEGF antibody.Preferably, described Anti-HER 2 is trastuzumab.Also preferably, described VEGF antibody is bevacizumab.

The accompanying drawing summary

Fig. 1 after bevacizumab treatment failure combined trastuzumab and bevacizumab treatment to the anti-tumor activity of tumor growth.Gross tumor volume (mm ³) meansigma methods be plotted on the y-axle; Be plotted on the x-axle at the natural law behind the tumor cell injection.Excipient (circle) with the trastuzumab (square) of 30mg/kg loading dose and 15mg/kg maintenance dose, lasts till the 55th day the bevacizumab with 5mg/kg (triangle) when treatment also comprises trastuzumab with 15mg/kg.

The effect of Fig. 2 combined trastuzumab and bevacizumab treatment on lung metastasis.The meansigma methods (ng/ml) of people Alu DNA sequence is quantitative from lung tissue with PCR in real time, and is plotted on the y-axle.

Detailed Description Of The Invention

According to the present invention, term ＂ VEGF ＂ refers to vascular endothelial cell growth factor (Switzerland-Prot P15692), the alternative splicing form (referring to, for example, Leung, D.W., etc., Science (science), 246 (1989) 1306-1309; And Houck, K.A., etc., Mol.Endocrin. (molecular endocrinology) 5 (1991) 1806-1814) and active fragment, preferably its N end fragment.

According to the present invention, term " VEGF antibody " is the antibody with the VEGF specific binding.The preferred humanization VEGF antibody of this paper or variant VEGF antibody are to be not more than about 1 * 10 ^-8M and preferably be not more than about 5 * 10 ^-9The Kd value of M is in conjunction with people VEGF.Preferably described VEGF antibody is monoclonal antibody, and it is according to Presta, L.G., etc., the anti-VEGF monoclonal antibody of recombinant humanized that Cancer Res. (cancer research) 57 (1997) 4593-4599 produce is in conjunction with identical epi-position.Preferred antibody is bevacizumab.VEGF antibody and preparation method thereof, for example, at US6,054,297, US2003/0190317, US6,632,926, US6,884,879, and describe among the US2005/0112126.

Bevacizumab comprise human IgG1's framework region of sudden change and from the antigen of the anti-hVEGF monoclonal antibody in Mus source in conjunction with complementary determining region, described antibody hinders the combination of people VEGF and its receptor.The aminoacid sequence of bevacizumab about 93% comprises most of framework region, is to derive from the human IgG1, and about 7% sequence is to come from Mus source antibody A 4.6.1.Bevacizumab has about 149,000 daltonian molecular masses, and is glycosylated.Bevacizumab and preparation method thereof is described in EP1325932.

HER2 is the growth factor receptors of 185-kDa, also be called neu and c-erbB-2 (Slamon, D.J. is etc., Science (science) 235 (1987) 177-182; Switzerland-Prot P04626), the tumor in its function and the human breast cancer cell transforms relevant.The overexpression of this albumen identifies in the patient with breast cancer of 20-30%, and wherein it survives relevant with the probability of the increase of endemicity terminal illness, tumor recurrence and the patient of minimizing.Many overexpressions that also can show this albumen such as the patient who suffers from gastric cancer, carcinoma of endometrium, salivary-gland carcinoma, nonsmall-cell lung cancer, cancer of pancreas, ovarian cancer, peritoneal cancer, carcinoma of prostate or colorectal carcinoma of 30-40%.Anti-HER 2 and preparation method thereof, for example, at US6,054,297, WO89/06692, US6,953,842, US6,949,245, US6,399,063, US6,165,464, US6,054,297, US5,772,997, WO2003/087131, WO01/00245, WO01/00238, WO00/69460, WO00/52054 is described in WO99/31140 and the WO98/17797.In a preferred embodiment of the invention, described Anti-HER 2 is trastuzumab.Trastuzumab and preparation method thereof is described in EP0590058.

" overexpression " of term HER2 receptor protein be intended to refer to, with respect to the expression in the normal cell that comes self-organizing or organ, the HER2 receptor protein is from the unconventionality expression level in the cell of patient's specific tissue or intraorganic tumor.The patient who suffers from the cancer of the overexpression that is characterised in that described HER2 receptor can determine by standard test known in the art.Preferably, overexpression uses immunohistochemistry (IHC) detection to measure in the fixing cell of the tissue slice of freezing or paraffin-embedding.When histological stain is combined, can determines by the position of the albumen of targeting, and can measure its expression degree in tumor in quantitative and semi-quantitative ground.It is known that such IHC is determined in this area, and comprises that (CTA) measured in clinical trial, commercially available LabCorp4D5 detects, and commercially available DAKO (DAKO, Carpinteria, California).The cell dyeing (the 0th, normal expression, 3+ represents the strongest positive expression) of 0 to 3+ particular range is used in a rear test, with evaluation have HER2 albumen overexpression cancer (referring to

(trastuzumab) complete prescription information, in JIUYUE, 1998, Genentech, Inc. (US) 460 Point San Bruno Blvd, South San Francisco, CA, 94080 (Genentech Inc.), San Francisco, California).Therefore, suffer from and be characterised in that at 1+, 2+, or 3+, preferably 2+ or 3+, more preferably the patient of the cancer of the HER2 albumen overexpression in the 3+ scope will benefit from Therapeutic Method of the present invention.

Term " breast cancer disease " refers to the uncontrolled growth of abnormal milk glandular cell.It comprises ductal carcinoma in situ, invasive duct carcinoma, LCIS, invasive lobular carcinoma, medullary carcinoma, paget disease of nipple and metastatic breast cancer.

When being used for this paper, term " failure in the formerly treatment of anti-VEFG antibody " or " treating unsuccessfully " refer to respond with the previous treatment of VEGF antibody tumor patient (" nonresponder ") but or the previous treatment therapeutic response of initial response do not have the tumor patient (being called " recidivist ") of maintenance.Preferably, term " failure in the formerly treatment of anti-VEFG antibody " refers to the recidivist.Treat unsuccessfully (being respectively to reply (RE) and nonreply (NR)) and determine that based on practitioner's medical judgment described medical judgment is to determine by clinical and result laboratory data from common assess patient treatment known in the art.For example, such data can be from clinical examination, cytology and histological techniques, splanchnoscopy and laparoscopy, ultrasonic, CT, PET and MRI scanning, thoracic cavity X-ray and mastography and measurement tumor marker are such as CEA, Cyfra, CA15-3, the concentration of interleukin 8 and solvable HER2 and obtaining.In this case, " treat unsuccessfully " shortage that is defined as clinical improvements.Alternatively, the RECIST standard can be used for determining tumor response (Therasse, P is etc., J.Nat.Cancer Institute (National Cancer Institute's magazine) 92 (2000) 205-216).In this case, " treat unsuccessfully " and be defined as " not exclusively replying/stable disease " or " progressivity disease ".

According to these RECIST standards, depending on gross tumor volume makes progress or (for example disappears, measure by CT), tumor response (Therasse with entity tumor, P., Deng, J.Nat.Cancer Institute (National Cancer Institute's magazine) 92 (2000) 205-216) be classified into 4 levels: reply (CR) fully or partly reply (PR), stable disease (SD) and progressivity disease (PD) (referring to table 1).In addition, European cancer research and treated tissue (European Organization for Research and Treatment ofCancer (EORTC)) depend on and pass through 2-[ ¹⁸F]-metabolism of the tumor that fluoro-1,5-anhydroglucitol positron emission tomography (FDG-PET) is measured and propose to be divided into classification (the Young H. of 4 levels, Deng, Eur.J.Cancer (European cancer magazine) 35 (1999) 1773-1782 and Kellof, G.J., Deng, Clin.Cancer Res. (Clinical Cancer Research) 11 (2005) 2785-2808): (CMR) replied in complete metabolism or (PMR) replied in the part metabolism, stablizes metabolic disease (SMD) and progressivity metabolic disease (PMD) (referring to table 2).

Table 1:CT-standard (according to RECIST) table 2: the FDG-PET standard of proposal is (according to EORTC, referring to Young H., Deng, Eur J Canc (European cancer magazine) 35 (1999) 1773-1782)

Therefore, preferably, according to the present invention, " replying (RE) " and " not replying (NR) " be use above-mentioned RECIST and FDG-PET standard based on such data and definite, described data communication device is crossed computerized tomography (CT) and 2-[ ¹⁸F]-combination of fluoro-1,5-anhydroglucitol positron emission tomography (FDG-PET) and obtain (Kellof, G.J., Deng, Clin.Cancer Res. (Clinical Cancer Research) 11 (2005) 2785-2808, with Young H., Deng, Eur.J.Canc. (European cancer research) 35 (1999) 1773-1782).Therefore,, reply (RE) and do not reply that (NR) is preferably following to be determined according to the present invention:

Reply (RE):CR or PR be by CT-RECIST standard places (table 1), and CMR or PMR establish (table 2) by FDG-PET simultaneously.Therefore, reply (RE) and mean a kind of in 4 kinds of situations that following CT about combination and PET measure: CR and CMR, PR and PMR, CR and PMR, and PR and CMR.

Do not reply (NR):SD or PD be by CT-RECIST standard places (table 1), and SMD or PMD establish (table 2) by FDG-PET simultaneously.Therefore, following 4 kinds of situations of measuring about CT and the PET of combination represent not reply (NR): SD and SMD, SD and PMD, PD and SMD, and PD and PMD.

Usually described replying after the treatment beginning determined in about 3 to 8 weeks, preferably determines in about 6 weeks.This replying determines that usually with 4 to 8 weeks preferably the interval in 6 to 8 weeks repeats.When for the first time determining to have identified significantly when replying (RE), so recurrence (it means not replying after determining for the first time (RE)) can reply for the second time when determining definite the earliest.

In this case, term " failed patient in the formerly treatment of VEGF antibody " refers to such patient, namely, in them, when first set reaction is determined, be asserted and do not reply (NR) (" not respondent ") or reply (RE) replying for the first time to be asserted in determining, and in replying for the second time or subsequently determined, be asserted and do not reply (NR) (" recidivist ").

According to the present invention, term " transfer " refers to that one or more other sites cancerous cell is from primary tumor to the patient are shifted and causes secondary tumors.Be called " metastatic tumor " or " transfer " by the plastidogenetic tumor that has spread.Described metastatic tumor comprises the cell similar to the cell in original (constitutional) tumor.Determine that the method whether cancer shifts is known in this area, and comprise that tumor marker detects, bone scanning, thoracic cavity X-line, computerized tomography (CT), computed axial tomography (CAT), molecular resonance imaging (MRI), positron emission tomography (PET), single photon emission computed tomography (SPECT), fluorescence imaging (FI), detect with bioluminescence imaging (BLI) and tumor marker (referring to, for example, Helms, M.W., Deng, Contributions tomicrobiology (microbiological contribution) 13 (2006) 209-231, and Pantel, K., Deng, J.Nat. Cancer Inst. (National Cancer Institute's magazine) 91 (1999) 1113-1124).

When being used for this paper, term " patient " preferably refers to the people of the treatment of needs treatment cancer or precancerous condition or infringement.Yet term " patient " can also refer to the non-human animal, mammal preferably, and wherein such as Canis familiaris L., cat, horse, cow, pig, sheep and non-human primates, it needs treatment.

Term " group " refers to one group of patient and subgroup patient.

Term " package insert " refers to be generally comprised within the explanation in the commercial packing for the treatment of product, and it can comprise about indication, use, dosage, uses, the information of contraindication and/or the warning of using about described treatment product.

Described cancer can be, for example, pulmonary carcinoma, non-small cell lung (NSCL) cancer, bronchus (bronchioloalviolar) cell lung cancer, osteocarcinoma, cancer of pancreas, skin carcinoma, head or neck cancer, skin or intraocular melanoma, uterus carcinoma, ovarian cancer, rectal cancer, the anal region cancer, gastric cancer (stomachcancer), gastric cancer (gastric cancer), colon cancer, breast carcinoma, uterus carcinoma, carcinoma of fallopian tube, carcinoma of endometrium, cervical cancer, cancer of vagina, carcinoma vulvae, Hodgkin, esophageal carcinoma, carcinoma of small intestine, the hormonal system cancer, thyroid carcinoma, parathyroid carcinoma, adrenal carcinoma, soft tissue sarcoma, carcinoma of urethra, carcinoma of penis, carcinoma of prostate, bladder cancer, kidney or carcinoma of urethra, renal cell carcinoma, carcinoma of renal pelvis, mesothelioma, hepatocarcinoma, carcinoma of gallbladder, chronic or acute leukemia, the lymphocyte lymphatic cancer, central nervous system (CNS) tumor, tumor of spine, the brain stem glioma, multi-form glioblastoma, astrocytoma, schwannoma, ependymoma, medulloblastoma, meningioma, squamous cytoma, pituitary adenoma, the version rambunctious that comprises in the above-mentioned cancer any, or one or more combination in the above-mentioned cancer.Described precancerous condition or infringement comprise, for example, by the following group that forms: leukoplakia oris, actinic keratosis (actinic keratosis) (actinic keratosis (solarkeratosis)), precancerous colon or rectal polyp, Weishang skin dysplasia, the dysplasia of adenoma shape, hereditary nonpolyposis colon cancer syndrome (HNPCC), Bart's thunder esophageal disease, During Bladder Development is bad and pre-cancer disorder of cervix.In preferred embodiments, cancer to be treated is breast cancer disease.Also in preferred embodiments, described cancer is characterised in that the overexpression of HER2 receptor protein.

The present invention includes the method for the treatment of the breast cancer disease among the patient failed in the formerly treatment with VEGF antibody, the method is included in when continuing described VEGF antibody treatment, to the Anti-HER 2 of described patient's administering therapeutic effective dose.

When being used for this paper, except as otherwise noted, term " treatment " means tumor growth among the patient, neoplasm metastasis, or other cause cancer or tumor sexual cell partially or completely reverse, alleviate, hold back the development, perhaps prevent.When being used for this paper, except as otherwise noted, term " treatment " refers to the behavior for the treatment of.

Phrase " Therapeutic Method " or its equivalents, for example, when being used for cancer, the action method or the process that refer to be designed to reduce or eliminate the cancerous cell number among the patient or alleviate cancer symptoms." method for the treatment of cancer or another kind of proliferative disorders " needn't mean described cancerous cell or in fact other disease will be eliminated, and in fact cell number or disease will be reduced, and perhaps in fact the symptom of cancer or other disease will be alleviated.Usually, the method for the treatment of cancer will even be carried out with low successful probability, still, provides patient's medical history and the survival prediction of estimation, thinks that still it is a kind of substantially useful mechanism.

Term " treatment effective dose " or " effective dose " mean and will cause tissue, system, animal or human's biology or test-compound that medical science is replied or the amount of combination, and described tissue, system, animal or human are that researcher, veterinary, medical science doctor or other clinicists seek.

The present invention comprises that also Anti-HER 2 and VEGF antibody are for the preparation of the application in the medicine that is used for treating the breast cancer disease among the patient failed in the formerly treatment with VEGF antibody, this application is included in when continuing described VEGF antibody treatment, gives the Anti-HER 2 of described patient's administering therapeutic effective dose.Described antibody can separate or use simultaneously.

Term " method for preparing medicine " relates to for the preparation of indication as herein described and particularly is generally used for treating the medicine of tumor, neoplasm metastasis or cancer.This term relates to so-called " Switzerland-type " in described indication and requires form.

In the context of the present invention, Cytotoxic, the chemotherapeutical or anticancer agent of in addition other or the chemical compound that strengthens described pharmacy effect can be used in VEGF antibody and add in the Anti-HER 2 combination.Such medicament comprises, for example: and alkylating agent or the medicament with alkanisation, such as cyclophosphamide (CTX; For example,

Chlorambucil (CHL; For example

Cisplatin (CisP; For example, Platinol Busulfan (for example, Busulfan

Melphalan, carmustine (BCNU), streptozotocin, tretamine (TEM), ametycin etc.; Antimetabolite is such as methotrexate (MTX), etoposide (VP16; For example, VePesid

6-MP (6MP), 6-thioguanine (6TG), cytosine arabinoside (Ara-C), 5-fluorouracil (5-FU), capecitabine (for example, Xeloda

), dacarbazine (DTIC), etc.; Antibiotic, such as actinomycin D, doxorubicin (DXR; For example, amycin

Daunorubicin (daunomycin), bleomycin, radiance is mould etc.; Alkaloid is such as Vinca alkaloids such as vincristine (VCR), vinblastine etc.; And other antineoplastic agent, such as paclitaxel (for example,

And paclitaxel derivant, cytostatic agent, glucocorticoid is such as dexamethasone (DEX; For example, With corticosteroid such as prednisone, the nucleosidase inhibitor is such as hydroxyurea, aminoacid exhausts enzyme such as asparaginase, folinic acid and other folic acid derivatives, and similar, different antineoplastic agents.Following medicament also can (for example, be used as additive: amifostine

Actinomycin D, chlormethine (mechlorethamine) (chlormethine (nitrogen mustard)), streptozocin, cyclophosphamide, lomustine (CCNU), Mycocet (for example, Doxil

Gemcitabine (for example, Daunorubicin liposome (for example,

Procarbazine, mitomycin, docetaxel are (for example,

Aldesleukin, carboplatin, oxaliplatin, cladribine, camptothecine, CPT11 (irinotecan), 10-hydroxyl 7-Ethyl-camptothecin (SN38), floxuridine, fludarabine, ifosfamide, idarubicin, U.S. sodium, interferon beta, interferon-ALPHA, mitoxantrone, hycamtin, leuproside, megestrol, melphalan, mercaptopurine, plicamycin, mitotane, pegaspargase, pentostatin, pipobroman, plicamycin, tamoxifen, teniposide, testolactone, thioguanine, match TEPA, uracil mustard, vinorelbine, chlorambucil.

In situation of the present invention, antihormone can be used in VEGF antibody and add in the Anti-HER 2 combination.When being used for this paper, term " antihormone " comprises natural or synthetic regulation and control or the inhibitory hormone organic or peptide compounds to the effect of tumor that works.Antihormone comprises, for example: steroid receptor antagonist, estrogen antagonist such as tamoxifen, raloxifene, aromatase inhibition 4 (5)-imidazoles, other aromatase inhibitor, 42-trans-Hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (for example

Antiandrogen such as flutamide, nilutamide, bicalutamide, leuproside, and goserelin; And above-mentioned any pharmaceutical salts, acid or derivant; The agonist of glycoprotein hormones and/or antagonist, such as FSH (FSH), thyrotropin (TSH), and lutropin (LH) and LHRH (gonadotropin releasing hormone); LHRH agonist goserelin acetate can

(AstraZeneca) be purchased; Lhrh antagonist D-aminopropanamide (alaninamide) N-acetyl group-3-(2-naphthyl)-D-alanyl-4-chloro-D-phenylalanyl-3-(3-pyridine radicals)-D-alanyl-L-seryl-N6-(3-pyridine radicals carbonyl)-L-lysyl-N6-(3-pyridine radicals carbonyl)-(1-Methylethyl)-L-lysyl-L-PROLINE (for example for D-lysyl-L-leucyl-N6-

Ares-Serono); The lhrh antagonist ganirelix acetate; Steroid androgen antagonist cyproterone acetate (CPA) and megestrol acetate can (Bristol-MyersOncology) be purchased; On-steroidal androgen antagonist flutamide (2-methyl-N-[4,20-nitro-3-(trifluoromethyl) Phenylpropionamide) can

(Schering company) is purchased; On-steroidal androgen antagonist nilutamide, (5,5-dimethyl-3-[4-nitro-3-(trifluoromethyl-4 '-nitrobenzophenone)-4,4-dimethyl-imidazolidimedione); With the antagonist of other nonpermissive receptor, such as RAR (retinoic acid receptors), RXR (retinoid X receptor), TR (thryoid receptor), the antagonist of VDR (Vitamin D receptor) etc.

Above-mentioned cytotoxicity and the common feature of the application of other anticarcinogen in the chemotherapy scheme fully are technology of cancer treatment, and being applied in of they is that the consideration neutralization that is in monitoring toleration and effect is controlled in the consideration of route of administration and dosage here, has some adjustment.For example, the actual dose of cytotoxic agent can change along with replying by the definite patient's of using-system cultural method cultured cells.Usually, amount used during with other medicament of not having interpolation is compared, and dosage will reduce.

The exemplary dosage of effective cell toxicity medicine can be in the scope that supplier recommends, and by vitro responses or reply indicated dosage in animal model, can reduce the about order of magnitude concentration of as many as or amount.Therefore, based on malignant cell or the vitro responses of tissue sample of tissue culture or the replying of observing in suitable animal model of former culture, actual dose will depend on doctor's judgement, patient's the patient's condition and the effect of Therapeutic Method.

In situation of the present invention, other antiproliferative agents can be used in VEGF antibody and add in the Anti-HER 2 combination, and it comprises, for example: the inhibitor of enzyme process farnesyl protein transferring enzyme and the inhibitor of receptor tyrosine kinase PDGFR, be included in U.S. Patent number 6,080,769; 6,194,438; 6,258,824; 6,586,447; 6,071,935; 6,495,564; 6,150,377; 6,596,735 and 6,479,513, and the chemical compound that discloses and require among the international publication WO01/40217.

In situation of the present invention, can carry out the ionizing radiation of effective dose and/or except VEGF antibody adds the Anti-HER 2 combination, can use radiopharmaceuticals.Radioactive source can be the patient who is treated outside or inner.When described radioactive source when the patient is outside, this treatment is called outer light beam radiotherapy (EBRT).When radioactive source when the patient is inner, treatment is called brachytherapy (BT).The radioactive atoms that is used for situation of the present invention can be selected from and include but not limited to following group: radium, caesium-137, iridium-192, americium-241, gold-198, cobalt-57, copper-67, technetium-99, iodo-123, iodine-131, and indium-111.When foundation EGFR inhibitors of kinases of the present invention is antibody, also be possible with described antibody with such radioactive isotope labelling.

Radiotherapy is be used to the standard care of controlling unresectable or inoperable tumor and/or neoplasm metastasis.When radiotherapy and chemotherapy combination, observed the result of improvement.Radiotherapy is based on such principle, that is, the ray that is delivered to the high dose of target area will cause the death of the sexual cell in tumor and the normal structure.The radiological dose scheme defines about the roentgendosis (Gy), time and the fraction that absorb usually, and must be defined in earnest by oncologist.The amount of the ray that the patient accepts will depend on various considerations, but two kinds of most important considerations be tumor about other key structure of body or the position of organ, and the degree of tumor diffusion.About typical treatment course for the treatment of of carrying out radiocurable patient should be the treatment time table that continues 1 to 6 time-of-week, 10 and 80Gy between accumulated dose be administered to the patient, the daily fraction of single about 1.8 to 2.0Gy, 5 days weeks.In a preferred embodiment of the invention, when the tumor among the people patient is treated with combined therapy of the present invention and radiation, there is potentiation.In other words, when with radiation combination, during randomly with other chemotherapy or anticarcinogen combination, the medicament mode by comprising combination of the present invention is strengthened the inhibition of tumor growth.The radiocurable parameter of assisting a ruler in governing a country for example, is included among the international publication WO99/60023.

Described antibody is administered to the patient in accordance with known methods, (bolus) uses by intravenous as bolus, perhaps by at stage continuous infusion sometime, by intramuscular, in the intraperitoneal, cerebrospinal fluid (intracerobrospinal), subcutaneous, intraarticular, in the synovial membrane, or intrathecal route is used.Preferred intravenous or subcutaneous administration antibody.

The amount that anti-VEGF and Anti-HER 2 are used and the time of using will depend on the patient who is treated type (ethnic group, sex, the age, body weight, etc.) and the seriousness of the patient's condition and the disease that is treated or the patient's condition.

(for example, 0.1-20mg/kg) antibody is by using that one or many separates, or by continuous infusion to 50mg/kg for about 1 μ g/kg for the dosage that uses antibody of the present invention.Typical daily dosage may be at about 1 μ g/kg in about 100mg/kg scope.Aspect preferred, per two to three weeks of described antibody use, with the dosage in about 1mg/kg arrives about 15mg/kg scope.The preferred dose of bevacizumab is with per 14 days of IV infusion 5mg/kg once, until detect progression of disease.The preferred dose of trastuzumab is the load doses of the 4mg/kg that uses 90 minutes time cycle, and the infusion of the weekly 2mg/kg that uses 30 minutes time cycle subsequently.

The present invention also provides the test kit that comprises VEGF antibody and package insert, and the user of described package insert indication said composition is administered to described VEGF antibody and described Anti-HER 2 the patient who suffers from breast cancer disease of failure in the formerly treatment of VEGF antibody.In preferred embodiments, described kit containers may further include pharmaceutical carrier.Described test kit may further include sterile diluent, and it preferably is kept in the independently other container.Described test kit may further include package insert, and it comprises that guidance is with the indication of described combined therapy as the printing of the method for breast cancer disease.

The present invention also provides pharmaceutical composition, is used in particular for treating failed breast cancer disease in the formerly treatment of VEGF antibody, and described pharmaceutical composition comprises Anti-HER 2 and VEGF antibody.Such compositions randomly comprises pharmaceutical carrier and/or excipient.In preferred embodiments, described VEGF antibody is bevacizumab, and described Anti-HER 2 is trastuzumab.

Provide following experiment particulars with auxiliary the understanding of the present invention, real scope of the present invention proposes in accompanying claim.Should be appreciated that the concrete method and the result that discuss just illustrate the present invention, do not think to limit by any way the present invention.

Introduction

This research has been checked in people's mammary gland xenograft models at the independent rear bevacizumab of bevacizumab treatment failure and the anti-tumor activity of trastuzumab combination.Other purpose of this research is the effect of check treatment to shifting.

Test agents

Trastuzumab is with at histidine-HCl, α-alpha trehalose (60mM), and 0.01% medicinal organic polymer carrier (Polysorb), the liquid storage of the 25mg/ml among the pH6.0 provides

Bevacizumab is with at sodium phosphate, α-alpha trehalose (60mM), and 0.01% medicinal organic polymer carrier (Polysorb), the liquid storage of the 25mg/ml among the pH6.0 provides

Two kinds of solution all suitably are diluted in and are used for injection among the PBS.

Cell line and condition of culture

MCF-7 KPL-4 sets up from the malignant pleural exudation liquid with patient with breast cancer of inflammatory cutaneous metastatic, and overexpression ErbB family receptors.(Kurebayashi, J., Deng, Br.J.Cancer (Britain's cancer magazine) 79 (1999) 707-17) tumor cell is cultivated in water saturated 5%CO2 atmosphere at 37 ℃ in the DMEM culture medium (PAA laboratory, Austria) in having replenished 10% hyclone (PAA) and 2mM L-glutaminate (Gibco) usually.Going down to posterity of culture carried out with trypsin EDTA1x (PAA), 2 times/week of division.Passage P6 is used for research in the body.

Animal

SCID beige (C.B.-17) mice; Age in 10-12 week; Body weight 18-20g (Charles River, Sulzfeld, Germany) is according to international guidelines (GV-Solas; Felasa; TierschG) remain under the condition without specific pathogen 12 hours cycles every day illumination/12 hour dark.After arrival, animal was raised in cages for 1 week at the quarantine departments of animal mechanism, to shake down and to be used for observing.Carry out continuous health monitoring on conventional basis.Diet food (Alltromin) and water (acidify pH2.5-3) arbitrarily provide.

Tumor growth in vivo suppresses research

Tumor cell collected from culture bottle (Greiner TriFlask) (trypsin-EDTA), and transferring in the 50ml culture medium, washing are once and be resuspended among the PBS.At another washing step and the filtration (cell filter with PBS; Falcon100 μ m) afterwards, the final cell titre is adjusted to 0.75x10 ⁸/ ml.The tumor cell suspension is carefully mixed with transfer pipette, to avoid cell aggregation.Use the Stephens inhalation part that is used for toy to anaesthetize, it has the pre-raising chamber (plexiglas) in the blood circulation of sealing, individual mice nasal surface tool (silicon) and Isoflurane (Pharmacia-Upjohn, Germany).In injection a few days ago, the hair with animal shaves off.For fat pad in the breast (i.f.m.p.) injection, the volume coordination (orthotopically) of cell with 20 μ l is expelled in the right side penult inguinal mammary fat pad of every anesthetized mice.For homotopic transplantation, with cell suspension by injecting at inframammillary skin.Tumor cell injection is corresponding to the first day of experiment.

Monitoring

Control animal every day, to detect the clinical symptoms of detrimental effect.For the monitoring in whole experimentation, the body weight of animal records weekly twice, and gross tumor volume is measured weekly twice by clamp.Former generation gross tumor volume according to the NCI method (TW=1/2ab2, wherein a and b are major diameter and the minor axis of tumor size, unit is mm, Teicher, B., Anticancer drug development guide (anticarcinogen development guides), Humana publishing house 5 (1997) 92) calculate.Value of calculation is recorded as meansigma methods and standard deviation.

The treatment of animal

As the about 100mm of gross tumor volume ³The time, will be with the mice randomization (every group of n=10) of tumor.Before treatment every group is closely mated, it is beginning in 20 days behind tumor cell injection.Vehicle group (group 1) intraperitoneal is accepted 10ml/kg PBS buffer, and is weekly.Trastuzumab (group 2) is used with the loading dose intraperitoneal of 30mg/kg, and is then weekly with the dosage of 15mg/kg (maintenance dose).With the VEGF antibody bevacizumab with the dosage intraperitoneal of 5mg/kg give with, twice weekly (group 3).At the 40th day, group 3 treatment is converted to the combined therapy of bevacizumab (5mg/kg, weekly twice, intraperitoneal) and trastuzumab (15mg/kg, once in a week, intraperitoneal).

The assessment of shifting

Determine that in the animal of putting to death tumor cell is to the diffusion of pulmonary.Transfer is according to Schneider, T., etc., Clin.Exp.Metastasis (clinical experiment of transfer) 19 (2002) 571-582 measure.In general, collect lung tissue, and by the quantitative people's Alu sequence of PCR in real time.By the quantitative higher higher transfer level of people's dna level correspondence of PCR in real time.

The result

Treatment is presented among Fig. 1 and the table 3 effect of former generation tumor growth.Tumor growth in vehicle group (group 1) is rapid, and because the development of tumor ulcer and clinical symptoms, mice is put to death in 38 days behind tumor cell injection.Use the single therapy (group 2) of trastuzumab that gross tumor volume is not applied significant effect, and therefore mice was put to death at the 44th day.Suppress significantly tumor growth with bevacizumab treatment; Yet tumor began to regrow about the 44th day greatly.Bevacizumab and trastuzumab combined therapy (99 days) in the Therapy lasted process beginning in the 55th day cause fully inhibition to tumor growth, and treatment is fine tolerance.

Table 3:The trastuzumab of combination and bevacizumab treatment are to the anti-tumor activity (data of Fig. 1) of tumor growth after the bevacizumab treatment failure.Reported with mm ³Mean tumour volume and standard deviation (SD) for unit.

Treatment is presented among Fig. 2 and the table 4 effect that lung shifts.The combination of trastuzumab and bevacizumab causes the rapidly reduction shifted after bevacizumab treatment failure.Compare with the animal of the excipient treatment of putting to death at the 28th day, with compare with the animal of the trastuzumab treatment of putting to death at the 44th day, at the 99th day, the level of people's Alu sequence (relevant to the intrusion in the secondary tissue with tumor cell) was significantly lower in the animal of processing with combined therapy.This to the astonishing effect of shifting and the effect of observing with other cytotoxicity medicament (Geldof, A.A. is etc., Anticancer Res. (anticancer research) 8 (1988) 1335-1339; Murphy, J.Clin.Oncol. (Journal of Clinical Oncology) 11 (1993) 199-201, and De Larco, J.E., etc., Cancer Res. (cancer research) 61 (2001) 2857-2861) opposite.

Table 4: the effect that treatment is shifted lung.Pass through the quantitative AluDNA of PCR in real time for every animal, and record

The significance,statistical of combined therapy

^*p＝0.001 ^**p＝<0.001

Claims (3)

1. the Anti-HER 2 for the treatment of effective dose and VEGF antibody are for the preparation of the application in the test kit of the breast cancer disease that is characterised in that HER2 receptor protein overexpression among the treatment patient, described patient is failed in the formerly treatment with VEGF antibody, wherein said VEGF antibody is bevacizumab, and wherein said Anti-HER 2 is trastuzumab.

2. the application of claim 1, wherein said patient is the people.

3. each application of claim 1 to 2, wherein said test kit are used for reducing to be shifted.

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