HK1131037B - Use of tumor therapy kit in preparing an antibody for vascular endothelial groeth factor and an antibody for human epithelial growth factor receptor type 2 - Google Patents

Description

Application of antibody aiming at vascular endothelial growth factor and antibody aiming at human epidermal growth factor type 2 receptor in preparation of kit for treating tumor

Technical Field

The present invention relates to combination therapy using anti-HER 2 and anti-VEGF antibodies. In particular, the invention relates to the use of such antibodies to treat breast cancer disease in patients who have failed prior therapy with an anti-VEGF antibody.

Background

Angiogenesis is involved in the pathogenesis of various disorders, including solid tumors, intraocular neovascular syndromes such as proliferative retinopathy or age-related macular degeneration (AMD), rheumatoid synovial fluid, and psoriasis (Folkman, J., et al, J. biol. chem. (J. chem. biol.) (267 (1992)) 10931-. In the case of solid tumors, neovascularization allows the tumor cells to acquire a growth advantage and proliferative autonomy compared to normal cells. Thus, in breast cancer, as well as in some other tumors, a correlation between microvascular density in tumor sections and patient survival has been observed (Weidner, N., et al, N.Engl. J. Med. (New England journal of medicine) 324(1991) 1-6; Horak, E.R., et al, Lancet340(1992) 1120-.

Vascular Endothelial Growth Factor (VEGF) is involved in the regulation of normal and abnormal angiogenesis and neovascularization associated with tumor and intraocular disorders (Ferrara, N., et al, Endocr. Rev. (endocrine review) 18(1997) 4-25; Berkman, R.A., et al, J.Clin. Invest. (J. Clin. Res.) (J. Clin. Res. 91(1993) 153; Brown, L.F., et al, Human Pathol. (Human pathology) 26(1995) 86-91; Brown, L.F., et al, Cancer Res. (Cancer research) 53(1993) 4727. Buckin. 4735; Matter, J., et al, Brit. J. Cancer (J. Cancer J. Cantor. J. Cantor. 1996) 73 (931) 934; and Dvorak, H.F. et al, am.J. Pathol. 146. 1039) (1039). anti-VEGF neutralizing antibodies inhibit growth in mice of various human tumor cell lines (Kim, K.J., et al, Nature 362(1993) 841-844; Warren, R.S., et al, J.Clin.Invest (J.Clin. Clin. J.Clin. Clin. 95(1995) 1789-1797; Borgstrom, P, et al, Cancer Res. 56(1996) 4032-4039; and Melnyk, O.et al, Cancer Res. 56(1996) 921-924). WO94/10202, WO98/45332, WO2005/00900 and WO00/35956 refer to antibodies directed against VEGF. Humanized monoclonal antibody bevacizumab (under the trademark Bevacizumab)Sold) is an anti-VEGF antibody for tumor therapy and is the only anti-angiogenic approved for cancer therapy (WO 98/45331).

HER2 is a member of the human epidermal growth factor receptor family and inhibits protein kinase activity in its cytoplasmic domain. HER2 is overexpressed in tumor cells and is associated with a very low prognosis and survival. Therefore, HER2 is a valuable target for breast cancer therapy. Antibodies against HER2 are known from: takai, N., et al, Cancer 104(2005) 2701-; yeon, C.H., et al, invest.New Drugs 23(2005) 391-409; wong, W.M., et al, Cancer practice (practice therein) 7(1999) 48-50; albanell, J., et al, Drugs Today (Barc) 35(1999) 931-46.

Trastuzumab(under the trademark VISITE)Sold) is a recombinant humanized anti-HER 2 monoclonal antibody for the treatment of metastatic breast cancer with HER2 overexpressed/HER 2 gene amplification. Preclinical studies have shown that the antibody has in vivo and in vitro anti-tumor activity. Furthermore, in mouse models, an additional or synergistic enhancement of the anti-tumor activity of trastuzumab was observed in combinations of various anti-tumor agents. In clinical studies, prolongation of survival was observed in patients with metastatic breast cancer overexpressing HER 2.

According to WO98/45331, the utility of an anti-VEGF antibody in the prevention or treatment of disease can be increased by administering the antibody continuously or in combination with another agent effective for those purposes, such as an antibody capable of binding to the HER2 receptor. WO2005/012531 describes that antibodies that can be used in combination with anti-VEGF antibodies in the treatment of colorectal, metastatic breast and renal cancers (e.g.,) And/or anti-ErbB antibodies (e.g.,a combination of antibodies. According to WO2005/063816, an anti-VEGF antibody can be combined with an anti-ErbB antibody in the treatment of metastatic breast cancer. WO2005/00090 and WO2003/077841 also disclose the combination of anti-VEGF antibodies with anti-ErbB 2 antibodies for tumor therapy.

Clinical oncologists agree that failure of cancer therapy is not necessarily caused by growth of the primary tumor, which is usually surgically treated, but rather by metastatic spread to different organs. Regression of primary tumors by different cytotoxic drugs is not itself always indicative of anti-metastatic activity. In contrast, enhanced metastasis was observed in response to some anti-Cancer drugs (Geldof, A.A., et al, Anticancer Res. 8(1988) 1335-1339; Murphy, S.B., J.Clin.Oncol. (J.Clin.Oncol.) (J.Oncol.) -11 (1993) 199-201; and De Larco, J.E., et al, Cancer Res. (Cancer Res.) 61(2001) 2857-2861). Clearly, there is a need to develop therapies that not only target primary tumors but also inhibit metastasis.

Summary of The Invention

The invention includes the use of an anti-HER 2 antibody and an anti-VEGF antibody for the manufacture of a medicament for treating a breast cancer disease in a patient who has failed prior therapy with an anti-VEGF antibody, said use comprising administering to said patient a therapeutically effective amount of an anti-HER 2 antibody and an anti-VEGF antibody.

In a preferred embodiment, the invention comprises the use of trastuzumab and bevacizumab for the manufacture of a medicament for the treatment of a breast cancer disease characterized by overexpression of the HER2 receptor protein in a patient who has failed prior treatment with an anti-VEGF antibody, such as bevacizumab, comprising administering to the patient a therapeutically effective amount of trastuzumab and bevacizumab.

The invention also includes a method of treating a breast cancer disease in a patient who has failed prior therapy with an anti-VEGF antibody, comprising administering to the patient a therapeutically effective amount of an anti-HER 2 antibody while continuing the anti-VEGF antibody therapy.

The invention also includes a method of treating a breast cancer disease characterized by overexpression of the HER2 receptor protein in a patient who has failed prior therapy with an anti-VEGF antibody, comprising administering to the patient a therapeutically effective amount of trastuzumab while continuing bevacizumab therapy.

The invention also includes a method for increasing the duration of survival of a patient having breast cancer disease who has failed prior therapy with an anti-VEGF antibody, comprising administering to the patient effective amounts of an anti-VEGF antibody and an anti-HER 2 antibody, whereby co-administration of the anti-VEGF antibody and the anti-HER 2 antibody effectively increases the duration of survival.

The invention also includes a method for increasing the progressive free survival of a patient having breast cancer disease who has failed prior therapy with an anti-VEGF antibody, the method comprising administering to the patient effective amounts of an anti-VEGF antibody and an anti-HER 2 antibody, whereby co-administration of the anti-VEGF antibody and the anti-HER 2 antibody effectively increases the duration of progressive free survival.

The invention also includes a method for treating a group of patients having breast cancer disease and who have failed prior therapy with an anti-VEGF antibody, the method comprising administering to the patient effective amounts of an anti-VEGF antibody and an anti-HER 2 antibody, whereby co-administration of the anti-VEGF antibody and the anti-HER 2 antibody effectively increases the response rate in the group of patients.

The invention also includes a method for increasing the duration of response in a patient having breast cancer disease who has failed prior therapy with an anti-VEGF antibody, comprising administering to the patient effective amounts of an anti-VEGF antibody and an anti-HER 2 antibody, whereby co-administration of the anti-VEGF antibody and the anti-HER 2 antibody effectively increases the duration of response.

The invention also includes a method of treating a patient having breast cancer disease who has failed prior therapy with an anti-VEGF antibody, comprising administering to the patient effective amounts of an anti-VEGF antibody and an anti-HER 2 antibody, whereby co-administration of the anti-VEGF antibody and the anti-HER 2 antibody results in a statistically significant and clinically meaningful improvement in the treated patient, as measured by duration of survival, progressive free survival, response rate, or duration of response.

The invention also includes a method for reducing metastasis in a patient having breast cancer disease who has failed prior therapy with an anti-VEGF antibody, the method comprising administering to the patient an effective amount of an anti-VEGF antibody and an anti-HER 2 antibody, whereby co-administration of the anti-VEGF antibody and the anti-HER 2 antibody effectively reduces metastasis.

The invention also includes a method for treating a group of patients having breast cancer disease and who have failed prior therapy with an anti-VEGF antibody, the method comprising administering to the patient effective amounts of an anti-VEGF antibody and an anti-HER 2 antibody, whereby co-administration of the anti-VEGF antibody and the anti-HER 2 antibody effectively reduces metastasis in the group of patients.

The present invention provides an article of manufacture (an article of manufacture) comprising a container, a composition within the container, the composition comprising an anti-VEGF antibody, and a package insert instructing a user of the composition to administer the anti-VEGF antibody and an anti-HER 2 antibody to a patient having a breast cancer disease who has failed prior therapy with the anti-VEGF antibody.

The invention also provides an article of manufacture comprising a container, a composition within the container comprising an anti-HER 2 antibody, and a package insert instructing a user of the composition to administer the anti-HER 2 antibody and an anti-VEGF antibody to a patient having breast cancer disease who has failed prior therapy with the anti-VEGF antibody.

The invention also provides a composition comprising an anti-HER 2 antibody and an anti-VEGF antibody effective for treating a breast cancer disease in a patient who has failed prior therapy with an anti-VEGF antibody. Preferably, the anti-HER 2 antibody is trastuzumab. Also preferably, the anti-VEGF antibody is bevacizumab.

Brief Description of Drawings

Figure 1 anti-tumor activity of combined trastuzumab and bevacizumab treatment on tumor growth after bevacizumab treatment failure. Tumor volume (mm)³) The average of (d) is plotted on the y-axis; days after tumor cell injection were plotted on the x-axis. Excipient (circles), trastuzumab at a 30mg/kg loading dose and 15mg/kg maintenance dose (squares), bevacizumab at 5mg/kg (triangles) for up to day 55 when treatment also included trastuzumab at 15 mg/kg.

Figure 2 effect of combined trastuzumab and bevacizumab treatment on lung metastases. The mean (ng/ml) of the human Alu DNA sequence was quantified from lung tissue using real-time PCR and plotted on the y-axis.

Detailed Description

According to the invention, the term "VEGF" refers to vascular endothelial cell growth factor (Switzerland-Prot No. P15692), alternatively spliced forms (see, e.g., Leung, D.W., et al, Science, 246(1989) 1306-1309; and Houck, K.A., et al, mol. Endocrin. (molecular Endocrinology) 5(1991)1806-1814) and active fragments, preferably the N-terminal fragments thereof.

According to the present invention, the term "anti-VEGF antibody" is an antibody that specifically binds to VEGF. Preferred humanized anti-VEGF antibodies or variant anti-VEGF antibodies herein are administered at no greater than about 1x 10^-8M and preferably no greater than about 5x10^-9The Kd value of M binds to human VEGF. Preferably the anti-VEGF antibody is a monoclonal antibody that binds to the same epitope as a recombinant humanized anti-VEGF monoclonal antibody produced according to Presta, l.g., et al, Cancer Res (Cancer research) 57(1997) 4593-4599. A preferred antibody is bevacizumab. anti-VEGF antibodies and methods for their preparation are described, for example, in US6,054,297, US2003/0190317, US6,632,926, US6,884,879, and US 2005/0112126.

Bevacizumab comprises a mutated human IgG1 framework region and an antigen binding complementarity determining region from a murine anti-hVEGF monoclonal antibody that blocks the binding of human VEGF to its receptor. About 93% of the amino acid sequence of bevacizumab, including most of the framework regions, is derived from human IgG1, and about 7% of the sequence is derived from murine antibody a4.6.1. Bevacizumab has a molecular mass of about 149,000 daltons and is glycosylated. Bevacizumab and its preparation method are described in EP 1325932.

HER2 is a 185-kDa growth factor receptor also known as neu and c-erbB-2(Slamon, D.J., et al, Science 235(1987) 177-182; Switzerland-Prot P04626), the function of which is associated with tumor transformation in human breast cancer cells. Overexpression of this protein has been identified in 20-30% of breast cancer patients, where it correlates with locally advanced disease, increased likelihood of tumor recurrence, and decreased patient survival. Over-expression of this protein may also be exhibited by as many as 30-40% of patients with gastric, endometrial, salivary gland, non-small cell lung, pancreatic, ovarian, peritoneal, prostate or colorectal cancer. anti-HER 2 antibodies and methods for their preparation are described, for example, in 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, WO99/31140 and WO 98/17797. In a preferred embodiment of the invention, the anti-HER 2 antibody is trastuzumab. Trastuzumab and its preparation are described in EP 0590058.

The term "overexpression" of the HER2 receptor protein is intended to mean an abnormal expression level of the HER2 receptor protein in cells from a tumor within a specific tissue or organ of a patient, relative to the expression level in normal cells from the tissue or organ. Patients with a cancer characterized by overexpression of the HER2 receptor can be determined by standard assays known in the art. Preferably, overexpression is measured in fixed cells of frozen or paraffin-embedded tissue sections using Immunohistochemical (IHC) detection. When combined with histological staining, the location of the targeted protein can be determined and its degree of expression within the tumor can be measured quantitatively and semi-quantitatively. Such IHC assays are known in the art and include Clinical Trial Assays (CTA), the commercially available LabCorp4D5 test, and the commercially available DAKO(DAKO, Carpinteria, Calif.). The latter test uses a specific range of cell staining from 0 to 3+ (0 being normal expression, 3+ representing the strongest positive expression) to identify cancers with overexpression of HER2 protein (see fig. 1)(trastuzumab) complete prescription information, 9 months 1998, GeneTechnology corporation (Genentech Inc.), san francisco, ca). Thus, patients suffering from a cancer characterized by overexpression of HER2 protein in the range of 1+, 2+, or 3+, preferably 2+ or 3+, more preferably 3+, will benefit from the treatment method of the invention.

The term "breast cancer disease" refers to the uncontrolled growth of abnormal breast cells. It includes ductal carcinoma in situ, invasive ductal carcinoma, lobular carcinoma in situ, invasive lobular carcinoma, medullary carcinoma, Paget's disease of the nipple, and metastatic breast cancer.

As used herein, the term "failure in prior treatment with an anti-VEFG antibody" or "treatment failure" refers to a tumor patient who failed to respond to prior treatment with an anti-VEGF antibody ("non-responder") or who initially responded to prior treatment but did not maintain the therapeutic response (called a "relapser"). Preferably, the term "failed prior treatment with an anti-VEFG antibody" refers to a relapser. Treatment failure (response (RE) and non-response (NR), respectively) is determined based on the medical judgment of the practitioner, as determined by results from clinical and laboratory data commonly used to assess patient treatment as known in the art. For example, such data may be obtained from clinical examinations, cytological and histological techniques, endoscopy and laparoscopy, ultrasound, CT, PET and MRI scans, chest X-ray and mammography, and measuring the concentration of tumor markers such as CEA, Cyfra, CA15-3, interleukin 8 and soluble HER 2. In this context, "treatment failure" is defined as a lack of clinical improvement. Alternatively, RECIST criteria can be used to determine tumor response (therase, P, et al, j. nat. cancer Institute 92(2000) 205-. In this context, "treatment failure" is defined as "incompletely responsive/stable disease" or "progressive disease".

According to these RECIST criteria, tumor responses of solid tumors (therase, p., et al, j.nat. cancer Institute 92(2000) 205-:complete Response (CR) or Partial Response (PR), Stable Disease (SD) and Progressive Disease (PD) (see table 1). In addition, European cancer Research and Treatment Organization (European Organization for Research and Treatment of cancer (EORTC)) relies on the 2-, [ 2 ], [¹⁸F]Fluorine-2-deoxyglucose positron emission tomography (FDG-PET) for measuring tumor metabolism and classification into 4 levels was proposed (Young H., et al, Eur.J. cancer (Eur. J. cancer J.) 35(1999)1773-1782 and Kellof, G.J., et al, Clin. cancer Res. (clinical cancer research) 11(2005) 2785-2808): complete Metabolic Response (CMR) or Partial Metabolic Response (PMR), Stable Metabolic Disease (SMD) and Progressive Metabolic Disease (PMD) (see table 2).

Table 1: CT-standard (according to RECIST) table 2: proposed FDG-PET Standard (according to EORTCC, see Young H., et al, Eur J Canc (Eur J.CANCE) 35(1999)1773-

Thus, preferably, according to the invention, the "Response (RE)" and "non-response (NR)" are determined using the RECIST and FDG-PET standards described above on the basis of data obtained by Computed Tomography (CT) and 2-, ", respectively¹⁸F]Fluorine-2-deoxyglucose positron emission tomography (FDG-PET) in combination (Kellof, G.J., et al, Clin.cancer Res. (clinical cancer research) 11(2005) 2785-. Thus, according to the invention, the Response (RE) and the non-response (NR) are preferably determined as follows:

response (RE):CR or PR was established by the CT-RECIST standard (Table 1), and at the same time CMR or PMR was established by FDG-PET (Table 2). Thus, Response (RE) means the following with respect to combined CT and POne of 4 cases of ET measurement: CR and CMR, PR and PMR, CR and PMR, and PR and CMR.

Non-response (NR):SD or PD was established by the CT-RECIST standard (Table 1), and at the same time SMD or PMD by FDG-PET (Table 2). Thus, the following 4 cases for combined CT and PET measurements represent No Response (NR): SD and SMD, SD and PMD, PD and SMD, and PD and PMD.

Typically the response is determined about 3 to 8 weeks, preferably about 6 weeks after initiation of treatment. Such a response determination is usually repeated at intervals of 4 to 8 weeks, preferably 6 to 8 weeks. When a significant Response (RE) is identified in the first determination, then a relapse (which means no Response (RE) after the first determination) may be determined earliest at the time of the second response determination.

In this context, the term "a patient who has failed prior therapy with an anti-VEGF antibody" refers to a patient in whom a No Response (NR) is established at the time of the first response determination ("non-responder") or a Response (RE) is established at the time of the first response determination, and a No Response (NR) is established at the time of the second or subsequent response determination ("relapser").

According to the present invention, the term "metastasis" refers to the metastasis of cancer cells from a primary tumor to one or more other sites in a patient resulting in a secondary tumor. Tumors formed by cells that have spread are called "metastases" or "metastases". The metastases contain cells similar to those in the original (primary) tumor. Methods of determining whether Cancer has metastasized are known in the art and include tumor marker detection, bone scanning, chest X-ray, Computed Tomography (CT), Computed Axial Tomography (CAT), Molecular Resonance Imaging (MRI), Positron Emission Tomography (PET), Single Photon Emission Computed Tomography (SPECT), Fluorescence Imaging (FI), and bioluminescence imaging (BLI) with tumor marker detection (see, e.g., Helms, m.w., et al, background microbiology 13(2006) 209-.

As used herein, the term "patient" preferably refers to a human in need of treatment for cancer, or a precancerous condition or lesion. However, the term "patient" may also refer to a non-human animal, preferably a mammal, such as, among others, dogs, cats, horses, cows, pigs, sheep, and non-human primates, which is in need of treatment.

The term "group" refers to a group of patients as well as a subgroup of patients.

The term "package insert" refers to instructions typically contained in commercial packages of therapeutic products, which may include information regarding the indications, use, dosage, administration, contraindications, and/or warnings regarding the use of the therapeutic products.

The cancer may be, for example, lung cancer, non-small cell lung (NSCL) cancer, bronchial (bronchoolvirolar) cell lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer (stomachcancer), gastric cancer (gastrotic cancer), colon cancer, breast cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, hodgkin's disease, carcinoma of the esophagus, carcinoma of the small intestine, carcinoma of the endocrine system, carcinoma of the thyroid gland, carcinoma of the parathyroid gland, carcinoma of the adrenal gland, sarcoma of soft tissue, carcinoma of the urethra, carcinoma of the penis, prostate cancer, carcinoma of the bladder, carcinoma of the kidney or urethra, renal cell carcinoma, carcinoma of the renal pelvis, mesothelioma, hepatocellular carcinoma, carcinoma of the gallbladder, chronic or acute leukemia, lymphocytic lymphomas, tumors of the Central Nervous System (CNS), tumors of the spine, brain stem glioma, A polymorphoglioblastoma, astrocytoma, schwannoma, ependymoma, medulloblastoma, meningioma, squamous cell tumor, pituitary adenoma, including refractory versions of any of the foregoing cancers, or a combination of one or more of the foregoing cancers. The precancerous condition or lesion includes, for example, the group consisting of: leukoplakia, actinic keratosis (actinic keratosis), precancerous colon or rectal polyps, gastric epithelial dysplasia, adenomatous dysplasia, hereditary non-polyp colon cancer syndrome (HNPCC), barrett's esophagus, bladder dysplasia, and precancerous cervical conditions. In a preferred embodiment, the cancer to be treated is a breast cancer disease. Also in a preferred embodiment, the cancer is characterized by overexpression of the HER2 receptor protein.

The invention includes a method of treating a breast cancer disease in a patient who has failed prior therapy with an anti-VEGF antibody, comprising administering to the patient a therapeutically effective amount of an anti-HER 2 antibody while continuing said anti-VEGF antibody therapy.

As used herein, unless otherwise indicated, the term "treating" means reversing, alleviating, inhibiting the development of, or preventing, partially or completely, tumor growth, tumor metastasis, or other cause of cancer or neoplastic cells in a patient. As used herein, the term "treatment" refers to the act of treating, unless otherwise indicated.

The phrase "method of treatment" or its equivalents, e.g., when used in reference to cancer, refers to an action method or process designed to reduce or eliminate the number of cancer cells in a patient or alleviate the symptoms of cancer. "method of treating cancer or another proliferative disorder" does not necessarily mean that the cancer cells or other disorder will actually be eliminated, the number of cells or disorder will actually be reduced, or the symptoms of the cancer or other disorder will actually be alleviated. Generally, methods of treating cancer will be performed with even a low likelihood of success, but are still considered a generally beneficial course of action given the patient's medical history and estimated survival prognosis.

The term "therapeutically effective amount" or "effective amount" means the amount of the test compound or combination that will elicit the biological or medical response of a tissue, system, animal or human that is being sought by the researcher, veterinarian, medical doctor or other clinician.

The invention also includes the use of an anti-HER 2 antibody and an anti-VEGF antibody for the manufacture of a medicament for treating a breast cancer disease in a patient who has failed prior therapy with an anti-VEGF antibody, comprising administering to the patient a therapeutically effective amount of an anti-HER 2 antibody while continuing said anti-VEGF antibody therapy. The antibodies may be administered separately or simultaneously.

The term "method of preparing a medicament" relates to the preparation of a medicament for the indications described herein and in particular for the treatment of tumors, tumor metastases or cancer in general. The term relates to the so-called "swiss-type" requirement form in the indicated indications.

In the context of the present invention, additional other cytotoxic, chemotherapeutic or anti-cancer agents, or compounds that enhance the effect of said agents, may be used in the anti-VEGF antibody plus anti-HER 2 antibody combination. Such agents include, for example: alkylating agents or agents with alkylating action, such as cyclophosphamide (CTX; e.g.,chlorambucil (CHL; e.g. benzene, butyric acid, butyricCisplatin (CisP; e.g. Chuanplatine injection)Busulfan (e.g. Marilan)Melphalan, carmustine (BCNU), streptozotocin, Trimetazidine (TEM), mitomycin C, etc.; antimetabolites, such as Methotrexate (MTX), etoposide (VP 16; e.g., Verbascum6-mercaptopurine (6MP), 6-thioguanine (6TG), cytarabine (Ara-C), 5-fluorouracil (5-FU), capecitabine (e.g., ionoda)) Dacarbazine (DTIC), etc.; antibiotics, such as actinomycin D, doxorubicin (DXR; e.g. adriamycinDaunorubicin (daunomycin), bleomycin, mithramycin, and the like; alkaloids such as vinca alkaloids like Vincristine (VCR), vinblastine, and the like; and other antineoplastic agents, such as paclitaxel (e.g.,and paclitaxel derivatives, cytostatics, glucocorticoids such as dexamethasone (DEX; for example,and corticosteroids such as prednisone, nucleosidase inhibitors such as hydroxyurea, amino acid depleting enzymes such as asparaginase, folinic acid and other folic acid derivatives, and similar, different antineoplastic agents. The following agents may also be used as additives: the amino-stiline (e.g.,actinomycin D, mechlorethamine (nitrogen mustard), streptozocin, cyclophosphamide, lomustine (CCNU), liposomal doxorubicin (e.g., liposomal doxorubicin hydrochloride)The gemcitabine (e.g.,the liposome of daunorubicin (e.g.,procarbazine, mitomycin, docetaxel (e.g.,aldesleukin, carboplatin, oxaliplatin, cladribine, camptothecin, CPT11 (irinotecan),10-hydroxy 7-ethyl-camptothecin (SN38), floxuridine, fludarabine, ifosfamide, idarubicin, sodium, interferon beta, interferon alpha, mitoxantrone, topotecan, leuprolide, megestrol, melphalan, mercaptopurine, plicamycin, mitotane, pemetrexed, pentostatin, pipobroman, plicamycin, tamoxifen, teniposide, testolactone, thioguanine, cetipipa, uramustine, vinorelbine, chlorambucil.

In the context of the present invention, an anti-hormonal agent may be used in the anti-VEGF antibody plus anti-HER 2 antibody combination. As used herein, the term "anti-hormonal agent" includes natural or synthetic organic or peptide compounds that act to modulate or inhibit the action of hormones on tumors. Anti-hormonal agents include, for example: steroid receptor antagonists, antiestrogens such as tamoxifen, raloxifene, aromatase inhibiting 4(5) -imidazole, other aromatase inhibitors, 42-hydroxytamoxifene, trioxifene, keoxifene, LY117018, onapristone, and toremifene (e.g.,anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above; agonists and/or antagonists of glycoprotein hormones, such as Follicle Stimulating Hormone (FSH), Thyroid Stimulating Hormone (TSH), and Luteinizing Hormone (LH) and LHRH (gonadotropin-releasing hormone); LHRH agonist goserelin acetate, can(AstraZeneca) commercially available; the LHRH antagonist D-alaninamide (alaninamide) N-acetyl-3- (2-naphthyl) -D-alanyl-4-chloro-D-phenylalanyl-3- (3-pyridyl) -D-alanyl-L-seryl-N6- (3-pyridylcarbonyl) -L-lysyl-N6- (3-pyridylcarbonyl) -D-lysyl-L-leucyl-N6- (1-methylethyl) -L-lysyl-L-proline (for example,Ares-Serono); LHRH antagonist vinegarGanirelix acid; steroid antiandrogens cyproterone acetate (CPA) and megestrol acetate, and can be(Bristol-Myers Oncology) commercially available; the nonsteroidal antiandrogen flutamide (2-methyl-N- [4, 20-nitro-3- (trifluoromethyl) phenylpropionamide), can be(Schering company) commercially available; the non-steroidal antiandrogen nilutamide, (5, 5-dimethyl-3- [ 4-nitro-3- (trifluoromethyl-4' -nitrophenyl) -4, 4-dimethyl-imidazolidinedione); and antagonists of other non-permissive receptors such as RAR (retinoic acid receptor), RXR (retinoid X receptor), TR (thyroid receptor), VDR (vitamin-D receptor), and the like.

The use of the above cytotoxic and other anticancer agents in chemotherapeutic regimens is generally well characterized in cancer treatment technology, and their use here is in the consideration of monitoring tolerance and efficacy and in the consideration of controlling route of administration and dosage, with some adjustment. For example, the actual dose of cytotoxic agent may vary with the patient's cultured cellular response as determined by using tissue culture methods. Typically, the dosage will be reduced compared to the amount used in the absence of added other agents.

Typical dosages of effective cytotoxic agents may be within the range recommended by the supplier, and the dosage indicated by the in vitro response or the response in animal models, may be reduced by up to about an order of magnitude concentration or amount. Thus, the actual dosage will depend on the judgment of the practitioner, the condition of the patient, and the efficacy of the treatment method, based on the in vitro response of the primary cultured malignant cells or tissue cultured tissue sample, or the response observed in an appropriate animal model.

In the context of the present invention, other antiproliferative agents may be used in the anti-VEGF antibody plus anti-HER 2 antibody combination, which include, for example: inhibitors of the enzymatic enzyme nisin transferase and inhibitors of the receptor tyrosine kinase PDGFR, including those described in U.S. patent nos. 6,080,769; 6,194,438, respectively; 6,258,824, respectively; 6,586,447, respectively; 6,071,935, respectively; 6,495,564, respectively; 6,150,377, respectively; 6,596,735 and 6,479,513, and the compounds disclosed and claimed in international publication WO 01/40217.

In the context of the present invention, an effective amount of ionizing radiation may be carried out and/or a radiopharmaceutical may be used in addition to the anti-VEGF antibody plus anti-HER 2 antibody combination. The radiation source may be external or internal to the patient being treated. When the radiation source is external to the patient, the treatment is called External Beam Radiation Therapy (EBRT). When the radiation source is inside the patient, the treatment is called Brachytherapy (BT). The radioactive atoms used in the context of the present invention may be selected from the group including, but not limited to: radium, cesium-137, iridium-192, americium-241, gold-198, cobalt-57, copper-67, technetium-99, iodine-123, iodine-131, and indium-111. When the EGFR kinase inhibitor according to the present invention is an antibody, it is also possible to label said antibody with such a radioactive isotope.

Radiation therapy is the standard treatment for controlling unresectable or inoperable tumors and/or tumor metastases. Improved results have been observed when radiation therapy is combined with chemotherapy. Radiation therapy is based on the principle that high doses of radiation delivered to the target area will result in the death of germ cells in tumor and normal tissues. Radiation dose regimes are usually defined in terms of absorbed radiation dose (Gy), time and fraction and must be carefully defined by the oncologist. The amount of radiation a patient receives will depend on various considerations, but the two most important considerations are the location of the tumor with respect to other critical structures or organs of the body, and the extent to which the tumor has spread. A typical course of treatment for a patient undergoing radiotherapy would be a treatment schedule lasting from 1 to 6 weeks, with a total dose between 10 and 80Gy administered to the patient, in a single daily fraction of about 1.8 to 2.0Gy for 5 days a week. In a preferred embodiment of the invention, there is a synergistic effect when a tumor in a human patient is treated with the combination therapy and radiation of the invention. In other words, the inhibition of tumor growth by means of the agents comprising the combination of the invention is enhanced when combined with radiation, optionally with other chemotherapeutic or anticancer agents. Parameters of adjuvant radiation therapy are, for example, contained in international publication WO 99/60023.

The antibodies are administered to the patient according to known methods, either intravenously as boluses (bolus), or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerebrospinal (intraspinal), subcutaneous, intraarticular, intrasynovial, or intrathecal routes. Intravenous or subcutaneous administration of the antibody is preferred.

The amount of anti-VEGF and anti-HER 2 antibody administered and the time of administration will depend on the type (race, sex, age, weight, etc.) and condition of the patient being treated and the severity of the disease or condition being treated.

The antibodies of the invention are used at a dose of about 1. mu.g/kg to 50mg/kg (e.g., 0.1-20mg/kg) of antibody, by one or more separate administrations, or by continuous infusion. A typical daily dose may range from about 1. mu.g/kg to about 100 mg/kg. In a preferred aspect, the antibody is administered every two to three weeks at a dose ranging from about 1mg/kg to about 15 mg/kg. The preferred dose of bevacizumab is once every 14 days with an IV infusion of 5mg/kg until disease progression is detected. The preferred dose of trastuzumab is a 4mg/kg loading dose administered over a 90 minute time period, followed by 2mg/kg weekly infusions administered over a 30 minute time period.

The invention also provides a kit comprising an anti-VEGF antibody and a package insert instructing a user of the composition to administer the anti-VEGF antibody and the anti-HER 2 antibody to a patient having breast cancer disease who has failed prior therapy with an anti-VEGF antibody. In a preferred embodiment, the kit container may further comprise a pharmaceutically acceptable carrier. The kit may further comprise a sterile diluent, which is preferably kept in a separate further container. The kit may further comprise a package insert comprising printed instructions directing the use of the combination therapy as a method of treating a breast cancer disease.

The invention also provides pharmaceutical compositions, in particular for the treatment of breast cancer diseases that have failed prior therapy with an anti-VEGF antibody, comprising an anti-HER 2 antibody and an anti-VEGF antibody. Such compositions optionally include a pharmaceutically acceptable carrier and/or excipient. In a preferred embodiment, the anti-VEGF antibody is bevacizumab and the anti-HER 2 antibody is trastuzumab.

The following experimental details are provided to assist in the understanding of the present invention, the true scope of which is set forth in the appended claims. It should be understood that the specific methods and results discussed are merely illustrative of the invention and are not to be construed as limiting the invention in any way.

Introduction to the word

This study examined the anti-tumor activity of bevacizumab and trastuzumab combination after failure of bevacizumab treatment alone in a human breast xenograft model. Other objectives of the study were to examine the effect of treatment on metastasis.

Detection medicament

Trastuzumab is provided as a 25mg/ml stock solution in histidine-HCl,. alpha. -trehalose (60mM), 0.01% pharmaceutically acceptable organic polymer carrier (Polysorb), pH6.0Bevacizumab is provided as a 25mg/ml stock solution in sodium phosphate, alpha-alpha trehalose (60mM), 0.01% pharmaceutically acceptable organic polymer carrier (Polysorb), pH6.0Both solutions were suitably diluted in PBS for injection.

Cell lines and culture conditions

The human breast cancer cell line KPL-4 was established from malignant pleural effusion of breast cancer patients with inflammatory skin metastases and overexpresses ErbB family receptors. (Kurebayashi, J., et al, Br. J. cancer (J. cancer) 79(1999)707-17) tumor cells were usually cultured in DMEM medium (PAA laboratory, Austria) supplemented with 10% fetal bovine serum (PAA) and 2mM L-glutamine (Gibco) at 37 ℃ in a water-saturated atmosphere of 5% CO 2. The passage of the cultures was performed with the trypsin EDTA1x (PAA), splitting 2 times/week. Cell passage P6 was used for in vivo studies.

Animal(s) production

SCID beige (C.B. -17) mice; 10-12 weeks old; weight 18-20g (Charles River, Sulzfeld, Germany) was maintained under conditions of no specific pathogen according to International guidelines (GV-Solas; Felasa; TierschG) with a 12 hour light/12 hour dark cycle per day. Upon arrival, animals were housed for 1 week in the animal facility's quarantine department for adaptation to new environments and for observation. Continuous health monitoring was performed on a regular basis. Diet food (Alltromin) and water (acidified pH2.5-3) were provided ad libitum.

In vivo tumor growth inhibition study

Tumor cells were harvested from culture flasks (Greiner TriFlask) (trypsin-EDTA) and transferred to 50ml of medium, washed once and resuspended in PBS. After another washing step with PBS and filtration (cell filter; Falcon100 μm), the final cell titer was adjusted to 0.75X10⁸And/ml. The tumor cell suspension was carefully mixed with a transfer pipette to avoid cell aggregation. Anesthesia was performed using a Stephens inhalation unit for small animals with a pre-incubation chamber (plexiglas), individual mouse nasal mask (silicon) and Isoflurane (Pharmacia-Upjohn, germany) in a closed circulatory system. Two days prior to injection, the animals were shaved of their hair. For intramammary fat pad (i.f.m.p.) injections, cells were injected in 20 μ l volume apposition (orthopicaily) into the right penultimate inguinal mammary fat pad of each anesthetized mouse. For orthotopic transplantation, the cell suspension is injected through the skin under the nipple.Tumor cell injection corresponds to the first day of the experiment.

Monitoring

Animals were controlled daily to detect clinical signs of adverse effects. For monitoring throughout the experiment, the body weight of the animals was recorded twice weekly and the tumor volume was measured twice weekly by calipers. Primary tumor volumes were calculated according to the NCI method (TW: 1/2ab2, where a and b are the major and minor diameters of the tumor size in mm, Teicher, b., Anticancer drug development guide, Humana press 5(1997) 92). Calculated values are reported as mean and standard deviation.

Treatment of animals

When the tumor volume is about 100mm³Tumor bearing mice were randomized (n-10 per group). Each group was closely matched prior to treatment, which began 20 days after tumor cell injection. Vehicle group (group 1) received 10ml/kg of PBS buffer intraperitoneally once a week. Trastuzumab (group 2) was administered intraperitoneally at a loading dose of 30mg/kg, followed by a dose of 15mg/kg (maintenance dose) once a week. anti-VEGF antibody bevacizumab was administered intraperitoneally at a dose of 5mg/kg twice weekly (group 3). On day 40, group 3 treatment was switched to a combination treatment of bevacizumab (5mg/kg twice weekly, intraperitoneally) and trastuzumab (15mg/kg weekly, intraperitoneally).

Assessment of metastasis

The spread of tumor cells to the lungs was determined in the sacrificed animals. Transfer was measured according to Schneider, T., et al, Clin. exp. Metastasis (clinical trials for transfer) 19(2002) 571-. In summary, lung tissue was collected and human Alu sequences were quantified by real-time PCR. Higher human DNA levels quantified by real-time PCR correspond to higher transfer levels.

Results

The effect of treatment on primary tumor growth is shown in figure 1 and table 3. Tumors in the vehicle group (group 1) grew rapidly and mice were sacrificed 38 days after tumor cell injection due to tumor ulceration and development of clinical symptoms. Monotherapy with trastuzumab (group 2) did not exert a significant effect on tumor volume, and therefore mice were sacrificed on day 44. Bevacizumab treatment significantly inhibited tumor growth; however, tumors started to regrow approximately at day 44. Bevacizumab and trastuzumab combination therapy starting on day 55 resulted in complete inhibition of tumor growth during the duration of the experiment (99 days) and treatment was well tolerated.

Table 3:anti-tumor activity of combined trastuzumab and bevacizumab treatment on tumor growth after bevacizumab treatment failure (data of fig. 1). Reported in mm³Mean tumor volume in units and Standard Deviation (SD).

The effect of treatment on lung metastasis is shown in figure 2 and table 4. Trastuzumab and bevacizumab combinations resulted in a sharp decrease in metastasis after bevacizumab treatment failure. At day 99, the levels of human Alu sequences (associated with tumor cell invasion into secondary tissues) were significantly lower in animals treated with combination therapy compared to vehicle-treated animals sacrificed at day 28 and compared to trastuzumab-treated animals sacrificed at day 44. This surprising effect on metastasis is in contrast to the effect observed with other cytotoxic agents (Geldof, A.A., et al, Anticancer Res. 8 (1988)) 1335-1339; Murphy, J.Clin.Oncol. (J.Clin.Oncol.) (J.11 (1993)199-201, and De Larco, J.E., et al, Cancer Res. (Cancer Res. 61 (2001)) 2857-2861).

Table 4: the treatment of pulmonary metastasis. The AluDNA was quantified by real-time PCR for each animal and recorded

Statistical significance of combination therapy

^*p＝0.001 ^**p＝<0.001

Claims (3)

1. Use of therapeutically effective amounts of an anti-HER 2 antibody and an anti-VEGF antibody for the manufacture of a kit for treating a breast cancer disease characterized by overexpression of the HER2 receptor protein in a patient who has failed prior treatment with the anti-VEGF antibody, wherein the anti-VEGF antibody is bevacizumab, wherein the anti-HER 2 antibody is trastuzumab.

2. The use of claim 1, wherein the patient is a human.

3. Use according to any one of claims 1 to 2, wherein the kit is for reducing metastasis.