Anti-angiogenesis therapy for treatment of ovarian cancer

Abstract

The present invention concerns anti-angiogenic therapies for the treatment of ovarian cancer. In general, the present invention concerns the treatment of diseases and pathological conditions with anti-VEGF antibodies. More specifically, the invention concerns the treatment of human patients susceptible to or diagnosed with cancer using anti-VEGF antibodies, preferably in combination with one or more additional anti-tumor therapeutic agents for the treatment of ovarian cancer.

Description

[0001] This application is a re-divisional application of the divisional application with the application date of February 22, 2011, Chinese application number 201910288567.6, and invention name “Anti-angiogenic therapy for treating ovarian cancer” (original application number 201180020417.9).

[0002] Related applications

[0003] This application claims priority to and the benefit of U.S. Provisional Application Serial No. 61 / 439,819, filed February 4, 2011; U.S. Provisional Application Serial No. 61 / 360,059, filed June 30, 2010; U.S. Provisional Application Serial No. 61 / 351,231, filed June 3, 2010; and U.S. Provisional Application Serial No. 61 / 307,095, filed February 23, 2010, the specifications of which are incorporated herein in their entirety.

[0004] Material preservation

[0005] The following hybridoma cell line has been deposited with the American Type Culture Collection (ATCC), Manassas, VA, USA, under the terms of the Budapest Treaty: Antibody Designation ATCC Number Deposit Date A4.6.1 ATCC HB-10709 March 29, 1991 Technical Field

[0006] In general, the present invention relates to the treatment of human diseases and pathological conditions. More specifically, the present invention relates to anti-angiogenic therapy, either alone or in combination with other anti-cancer therapies, for the treatment of ovarian cancer. Background Art

[0007] Cancer remains one of the most deadly threats to human health. In the United States, cancer affects nearly 1.3 million new patients each year and is the second leading cause of death after heart disease, accounting for approximately 1 in 4 deaths. For women with ovarian and peritoneal cancer, standard primary systemic chemotherapy for women with advanced epithelial ovarian cancer and peritoneal primary cancer consists of a platinum and taxane combination chemotherapy, typically carboplatin and paclitaxel, following initial surgical diagnosis, staging, and cytoreduction. See, for example, McGuire WP, et al. Cyclophosphamide and cisplatin compared with paclitaxel and cisplatin in patients with stage III and stage IV ovarian cancer. N Eng J Med 334: 1-6, 1996; Piccart MJ, et al. Randomized intergroup trial of cisplatin-paclitaxel versus cisplatin-cyclophosphamide in women with advanced epithelial ovarian cancer: three-year results. J Natl Cancer Inst 92:699-708, 20003; Alberts DS, et al. Improved therapeutic index of carboplatin plus cyclophosphamide versus cisplatin plus cyclophosphamide: final report by the Southwest Oncology Group of a phase III randomized trial in stages III and IVovarian cancer. J Clin Oncol 10: 706-17, 1992; du Bois A, et al. al.A Randomized clinical trial of cisplatin / paclitaxel versus carboplatin / paclitaxel as first-line treatment of ovarian cancer. J Natl Cancer Inst Sep. 3; 95. (17): 1320.-9.95: 1320, 2003; Ozols RF, et al.Phase III trial of carboplatin and paclitaxel compared with cisplatin and paclitaxel in patients with optimally resected stage III ovarian cancer: a Gynecologic Oncology Group study. J Clin Oncol 21:3194-200, 2003; and Swenerton K, et al. Cisplatin-cyclophosphamide versus carboplatin-cyclophosphamide in advanced ovarian cancer: a randomized phase III study of the National Cancer Institute of Canada Clinical trials Group. J Clin Oncol 10:718-26, 1992. Despite advances in patient management, this disease still has a high mortality rate for all gynecologic malignancies diagnosed in the United States. An estimated 25,580 new cases will be diagnosed in 2004, and 16,090 women will die from the disease. See, e.g., Jemal A, et al. Cancer statistics, 2004. CA Cancer J Clin 54:8-29, 2004. Improvements in primary treatment strategies are needed.

[0008] Angiogenesis is an important cellular event in which vascular endothelial cells proliferate, deplete, and reorganize to form new blood vessels from the existing vascular network. There is convincing evidence that the development of a vascular supply is crucial for normal and pathological proliferative processes (Folkman and Klagsbrun Science 235:442-447 (1987)). The transport of oxygen and nutrients, as well as the removal of catabolites, represents the rate-limiting step for most growth processes occurring in multicellular organisms.

[0009] Although the introduction of new blood vessels is considered the primary mode of tumor angiogenesis, recent data indicate that some tumors can grow by co-opting existing host blood vessels. The co-opted vasculature then recedes, leading to tumor regression, which is ultimately reversed by hypoxia-induced angiogenesis at the tumor margin. Holash et al. Science 284: 1994-1998 (1999).

[0010] One of the key positive regulators of both normal and abnormal angiogenesis is vascular endothelial growth factor (VEGF)-A. VEGF-A is part of a gene family that includes VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, and PlGF. VEGF-A primarily binds to two high-affinity receptor tyrosine kinases, VEGFR-1 (Flt-1) and VEGFR-2 (Flk-1 / KDR), the latter being the primary transmitter of VEGF-A's mitogenic signal in vascular endothelial cells. In addition, neuropilin-1 has been identified as a receptor for the heparin-binding VEGF-A isoform and may play a role in vascular development.

[0011] In addition to being an angiogenic factor in angiogenesis and vasculogenesis, VEGF, as a pleiotropic growth factor, exhibits a variety of biological effects in other physiological processes such as endothelial cell survival, vascular permeability and vasodilation, monocyte chemotaxis, and calcium influx. Ferrara and Davis-Smyth (1997), supra. In addition, studies have reported mitogenic effects of VEGF on a few non-endothelial cell types such as retinal pigment epithelial cells, pancreatic ductal cells, and Schwann cells. Guerrin et al. J. Cell Physiol. 164:385-394 (1995); Oberg-Welsh et al. Mol. Cell. Endocrinol. 126:125-132 (1997); Sondell et al. J. Neurosci. 19:5731-5740 (1999).

[0012] VEGF expression is upregulated in most malignancies, and VEGF overexpression is often associated with more advanced stages or worse prognosis in many solid tumors.

[0013] Because ovarian cancer remains one of the most deadly threats, patients are in need of additional cancer treatments. The present invention addresses these and other needs, as will become apparent upon reading the following disclosure. Summary of the Invention

[0014] Provided are uses of anti-VEGF antagonists for the treatment of ovarian cancer. For example, provided are uses of anti-VEGF antibodies for the effective treatment of women with newly diagnosed, previously untreated ovarian, fallopian tube, or primary peritoneal cancer or platinum-sensitive recurrent (or previously treated) ovarian, primary, peritoneal, or fallopian tube cancer. Provided are uses of bevacizumab in subjects (e.g., women) with newly diagnosed, previously untreated stage III (suboptimal and macroscopically optimal resected) and stage IV epithelial ovarian cancer, primary peritoneal cancer, or fallopian tube cancer. Data from a randomized phase III clinical trial of bevacizumab in combination with chemotherapy regimens are also provided (Example 1). Also provided are data from subjects (e.g., women) with newly diagnosed, high-risk stage I and IIa (clear cell carcinoma or grade 3 only) and stage IIb-IV epithelial ovarian, fallopian tube, or primary peritoneal cancer who underwent primary surgery and would not be considered for cytoreductive surgery before disease progression. Data from a randomized Phase III clinical trial in combination with chemotherapy regimens (Example 2) are also provided. Data from a placebo-controlled, randomized, multicenter Phase III study evaluating the efficacy and safety of bevacizumab (15 mg / kg, day 1, every 21 days) in combination with carboplatin (area under the curve [AUC] 4, day 1, every 21 days) and gemcitabine (1000 mg / m, day 1 and day 8, every 21 days) in women with platinum-sensitive recurrent epithelial ovarian cancer, primary peritoneal cancer, or fallopian tube cancer are also provided (Example 3). Such chemotherapy regimens include taxane therapy (e.g., paclitaxel or docetaxel), platinum-based chemotherapy (e.g., carboplatin) or gemcitabine, and combinations thereof. The success of this trial shows that providing an anti-VEGF antibody (e.g., bevacizumab) in combination with chemotherapy and continuing to provide the anti-VEGF antibody (e.g., bevacizumab) as maintenance therapy provides statistically significant and clinically meaningful benefit to patients with ovarian cancer. Results obtained in a clinical study using bevacizumab in both concurrent and maintenance therapy in human subjects with previously untreated and recurrent ovarian cancer showed positive efficacy (as assessed by progression-free survival (PFS)), particularly when compared to PFS data for treatment with chemotherapy agents alone. Subjects in the clinical trial who received bevacizumab in concurrent therapy with a combination of taxane therapy (e.g., paclitaxel or docetaxel) and platinum-based chemotherapy (e.g., carboplatin) or platinum-based chemotherapy (e.g., carboplatin) and gemcitabine and who received bevacizumab maintenance therapy had prolonged progression-free survival compared to subjects treated with taxane therapy (e.g., paclitaxel or docetaxel) and platinum-based chemotherapy (e.g., carboplatin) or platinum-based chemotherapy (e.g., carboplatin) and gemcitabine alone.

[0015] Thus, the present invention provides a method for treating a patient diagnosed with previously untreated or recurrent ovarian cancer, comprising administering to the patient a treatment regimen that combines at least one chemotherapy with administration of an effective amount of an anti-VEGF antibody, followed by maintenance therapy with the anti-VEGF antibody, wherein the patient's progression-free survival is prolonged with the treatment. The treatment regimen combining chemotherapy with administration of an anti-VEGF, followed by maintenance therapy with the anti-VEGF antibody, is effective in prolonging the patient's progression-free survival (PFS).

[0016] In certain embodiments, PFS is prolonged by about 1 month, 1.2 months, 2 months, 2.9 months, 3 months, 3.8 months, 4 months, 6 months, 7 months, 8 months, 9 months, 1 year, about 2 years, about 3 years, etc., compared to a control. In one embodiment, PFS is prolonged by about 2.9 to 3.8 months compared to a control (e.g., a treatment regimen of administering anti-VEGF followed by anti-VEGF maintenance therapy with combination chemotherapy). In one embodiment, PFS is prolonged by at least about 3.8 months compared to a control (e.g., a treatment regimen of administering anti-VEGF followed by anti-VEGF maintenance therapy with combination chemotherapy). In another embodiment, PFS is prolonged by about 2.3 months compared to a control (e.g., a treatment regimen of administering anti-VEGF followed by anti-VEGF maintenance therapy with combination chemotherapy). In one embodiment, PFS is prolonged by about 6 months compared to a control (e.g., a treatment regimen of administering anti-VEGF followed by anti-VEGF maintenance therapy with combination chemotherapy).

[0017] Any chemotherapeutic agent that exhibits anticancer activity can be used in accordance with the present invention. In certain embodiments, the chemotherapeutic agent is selected from the group consisting of alkylating agents, antimetabolites, folic acid analogs, pyrimidine analogs, purine analogs and related inhibitors, vinca alkaloids, epipodophyllotoxins, antibiotics, L-asparaginase, topoisomerase inhibitors, interferons, platinum coordination complexes, taxanes, anthracenedione substituted ureas, methylhydrazine derivatives, adrenocortical suppressants, adrenocortical steroids, progestins, estrogens, antiestrogens, androgens, antiandrogens, gemcitabine, and gonadotropin-releasing hormone analogs. In certain embodiments, the chemotherapeutic agent is, for example, a taxane, paclitaxel, docetaxel, paclitaxel protein-bound particles (e.g., ), gemcitabine, a platinum analog, carboplatin, or a combination thereof. Two or more chemotherapeutic agents, such as a taxane and a platinum analog or gemcitabine and a platinum analog, can be used in a mixture to be administered in combination with an anti-VEGF antibody. In one embodiment, it is carboplatin and paclitaxel. In one embodiment, it is carboplatin and docetaxel. In another embodiment, it is gemcitabine and carboplatin.

[0018] The clinical benefit of treatment according to the invention can be measured by, for example, duration of progression-free survival (PFS), time to treatment failure, objective response rate and duration of response.

[0019] The present invention also provides a kit. In one embodiment, a kit for treating previously untreated ovarian cancer in a human patient is provided, comprising a package comprising an anti-VEGF antibody composition and instructions for use of the anti-VEGF antibody composition in combination with taxane therapy and carboplatin, followed by anti-VEGF maintenance therapy, wherein the instructions describe that patients receiving taxane therapy, carboplatin therapy, and bevacizumab had a progression-free survival of 14.1 months and a hazard ratio of 0.717 (p-value <0.0001). In another embodiment, a kit for treating previously untreated ovarian cancer in a human patient is provided, comprising a package comprising an anti-VEGF antibody composition and instructions for use of the anti-VEGF antibody composition in combination with paclitaxel and carboplatin, followed by anti-VEGF maintenance therapy, wherein the instructions describe that patients receiving paclitaxel, carboplatin, and an anti-VEGF antibody had a progression-free survival of 18.3 months and a hazard ratio of 0.79. In certain embodiments, the kit comprises an anti-VEGF antibody having a heavy chain variable region comprising the following amino acid sequence: EVQLVESGGG LVQPGGSLRL SCAASGYTFTNYGMNWVRQA PGKGLEWVGW INTYTGEPTY AADFKRRFTF SLDTSKSTAY LQMNSLRAED TAVYYCAKYPHYYGSSHWYF DVWGQGTLVT VSS (SEQ ID No. 1) and a light chain variable region comprising the following amino acid sequence: DIQMTQSPSS LSASVGDRVT ITCSASQDIS NYLNWYQQKP GKAPKVLIYF TSSLHSGVPS RFSGSGSGTDFTLTISSLQP EDFATYYCQQ YSTVPWTFGQ GTKVEIKR (SEQ ID No. 2). In certain embodiments, the anti-VEGF antibody in the kit is bevacizumab. In certain embodiments, the kit is for use in patients with stage III or IV ovarian cancer.

[0020] Thus, the present invention features a method of instructing a human subject having cancer (e.g., ovarian cancer) to receive treatment with an anti-VEGF antibody, thereby extending the subject's progression-free survival, reducing the likelihood of cancer recurrence in the subject, or increasing the subject's likelihood of survival. In some embodiments, the method further comprises providing instructions to receive treatment with at least one chemotherapeutic agent. In some embodiments, the method further comprises providing instructions to receive treatment with at least two chemotherapeutic agents. In certain embodiments, treatment with the anti-VEGF antibody is performed concurrently and sequentially with treatment with the chemotherapeutic agent. In certain embodiments, the subject is treated as directed by the instructing method.

[0021] The present invention also provides a promotion method comprising promoting the administration of an anti-VEGF antibody to treat cancer (e.g., ovarian cancer) in a human subject. In some embodiments, the method further comprises promoting the administration of at least one chemotherapeutic agent. In certain embodiments of the present invention, the administration of the anti-VEGF antibody is performed concurrently and sequentially with the administration of the chemotherapeutic agent. Promotion can be performed by any available means. In some embodiments, the promotion is performed via a package insert accompanying a commercial formulation of the anti-VEGF antibody. The promotion can also be performed via a package insert accompanying a commercial formulation of the chemotherapeutic agent. Promotion can be performed by written or oral notification to a physician or health care provider. In some embodiments, the promotion is performed via a package insert, wherein the package insert provides instructions for receiving concurrent therapy with the anti-VEGF antibody and maintenance therapy with at least one chemotherapeutic agent and the anti-VEGF antibody. In some embodiments, the promotion is followed by treatment of the subject with the anti-VEGF antibody and one or more chemotherapeutic agents, followed by maintenance therapy with the anti-VEGF antibody.

[0022] The present invention provides a business method comprising marketing an anti-VEGF antibody for use in combination with at least one chemotherapeutic agent for treating cancer (e.g., ovarian cancer) in a human subject, followed by anti-VEGF maintenance therapy, thereby extending progression-free survival, reducing the likelihood of cancer recurrence in the subject, or increasing the likelihood of survival in the subject. In some embodiments, marketing is followed by treating the subject with the anti-VEGF antibody and the chemotherapeutic agent, followed by anti-VEGF maintenance therapy. In some embodiments, the method further comprises marketing two or more chemotherapeutic agents for use in combination with the anti-VEGF antibody, followed by anti-VEGF maintenance therapy. In some embodiments, marketing is followed by treating the subject with the anti-VEGF antibody and the chemotherapeutic agent, followed by anti-VEGF maintenance therapy.

[0023] Also provided is a business method comprising marketing a chemotherapeutic agent in combination with an anti-VEGF antibody, followed by anti-VEGF maintenance therapy, for treating cancer (e.g., ovarian cancer) in a human subject, thereby extending progression-free survival, reducing the likelihood of cancer recurrence in the subject, or increasing the likelihood of survival in the subject. In some embodiments, marketing is followed by treating the subject with a combination of a chemotherapeutic agent and an anti-VEGF antibody, followed by anti-VEGF maintenance therapy. Also provided is a business method comprising marketing two or more chemotherapeutic agents in combination with an anti-VEGF antibody, followed by anti-VEGF maintenance therapy, for treating cancer (e.g., ovarian cancer) in a human subject, thereby extending progression-free survival, reducing the likelihood of cancer recurrence in the subject, or increasing the likelihood of survival in the subject. In some embodiments, marketing is followed by treating the subject with a combination of a chemotherapeutic agent and an anti-VEGF antibody, followed by anti-VEGF maintenance therapy.

[0024] In each of the methods of the present invention, the anti-VEGF antibody may be replaced with a VEGF-specific antagonist, such as a VEGF receptor molecule or a chimeric VEGF receptor molecule described below. In certain embodiments, the anti-VEGF antibody is bevacizumab. The anti-VEGF antibody or antigen-binding fragment thereof may be a monoclonal antibody, a chimeric antibody, a fully human antibody, or a humanized antibody. Exemplary antibodies useful in the methods of the present invention include bevacizumab. In certain embodiments, the anti-VEGF antibody comprises a heavy chain variable region comprising the following amino acid sequence: EVQLVESGGGLVQPGGSLRL SCAASGYTFT NYGMNWVRQA PGKGLEWVGW INTYTGEPTY AADFKRRFTF SLDTSKSTAYLQMNSLRAED TAVYYCAKYP HYYGSSHWYF DVWGQGTLVT VSS (SEQ ID No. 1) and a light chain variable region comprising the following amino acid sequence: DIQMTQSPSS LSASVGDRVT ITCSASQDIS NYLNWYQQKP GKAPKVLIYFTSSLHSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ YSTVPWTFGQ GTKVEIKR (SEQ ID No. 2).

[0025] The antibody or antigen-binding fragment thereof may also be an antibody lacking an Fc portion, a F(ab')2, a Fab, or a Fv structure.

[0026] In one embodiment, treatment is a combination of a VEGF-specific antagonist (e.g., an anti-VEGF antibody) and at least one chemotherapeutic agent, followed by VEGF antagonist maintenance therapy. In one embodiment, treatment is a combination of a VEGF-specific antagonist (e.g., an anti-VEGF antibody) and two or more chemotherapeutic agents, followed by VEGF antagonist maintenance therapy.

[0027] Each method or purposes of the present invention can be practiced with respect to cancer treatment, including but not limited to carcinoma, lymphoma, blastoma, sarcoma and leukemia. More particular examples of such cancers include ovarian cancer, primary peritoneal cancer of the ovary, ovarian fallopian tube cancer, squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous cell carcinoma of the lung, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, liver cancer, bladder cancer, hepatic sarcoma (hepatoma), breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, renal cancer, vulvar cancer, thyroid cancer, liver cancer, gastric cancer, melanoma and various types of head and neck cancer. In some embodiments, the subject has previously untreated ovarian cancer. In some embodiments, the subject has newly diagnosed, previously untreated ovarian cancer. In some embodiments, the subject has newly diagnosed, previously untreated Stage III (sub optimally and macroscopic optimally debulked) or Stage IV epithelial ovarian primary peritoneal cancer or fallopian tube cancer. In some embodiments, the subject has platinum-sensitive recurrent epithelial ovarian cancer, primary peritoneal cancer, or fallopian tube cancer.

[0028] Each of the above aspects may further include monitoring the subject for cancer recurrence. Monitoring can be achieved, for example, by assessing progression-free survival (PFS) or overall survival (OS) or objective response rate (OR). In one embodiment, PFS is assessed after treatment initiation.

[0029] Depending on the type and severity of the disease, preferred dosages of anti-VEGF antibodies (e.g., bevacizumab) are described herein and may range from about 1 μg / kg to about 50 mg / kg, most preferably from about 5 mg / kg to about 15 mg / kg, including but not limited to 5 mg / kg, 7.5 mg / kg, 10 mg / kg, or 15 mg / kg. The frequency of administration will vary depending on the type and severity of the disease. For repeated administration over several days or longer, depending on the condition, treatment is continued until the cancer is treated or the desired therapeutic effect is achieved, as measured by methods described herein or known in the art. In one example, an anti-VEGF antibody of the invention is administered weekly, biweekly, or every three weeks at a dose ranging from about 5 mg / kg to about 15 mg / kg, including but not limited to 5 mg / kg, 7.5 mg / kg, 10 mg / kg, or 15 mg / kg. However, other dosage regimens may also be used. The progress of the therapy of the invention is readily monitored by conventional techniques and assays. In certain embodiments of the invention, anti-VEGF therapy is provided as maintenance therapy. In further embodiments, anti-VEGF therapy is provided for at least 14 months (including concurrent anti-VEGF therapy with chemotherapy and anti-VEGF maintenance therapy). In other embodiments, anti-VEGF therapy is provided for at least 12 months (including concurrent anti-VEGF therapy with chemotherapy and anti-VEGF maintenance therapy).

[0030] In further embodiments of each of the above aspects, the VEGF-specific antagonist (e.g., an anti-VEGF antibody) is administered locally or systemically (e.g., orally or intravenously). In other embodiments, one aspect of treatment is with the VEGF-specific antagonist as a monotherapy or as a monotherapy for the duration of the VEGF-specific antagonist treatment period (e.g., in an extended treatment period or maintenance therapy), as assessed by a clinician or as described herein. In certain embodiments, anti-VEGF maintenance therapy is administered at least from cycles 7 to 22. In other embodiments, anti-VEGF maintenance therapy is administered at least from cycles 7 to 18.

[0031] In other embodiments, treatment with a VEGF-specific antagonist is combined with other anti-cancer therapies, including but not limited to surgery, radiotherapy, chemotherapy, differentiation therapy, biological therapy, immunotherapy, angiogenesis inhibitors, cytotoxic agents, and anti-proliferative compounds. VEGF-specific antagonist treatment may also include any combination of the above-mentioned types of treatment regimens. In some embodiments, a chemotherapeutic agent and a VEGF-specific antagonist are administered concurrently, followed by anti-VEGF maintenance therapy. In some embodiments, two or more chemotherapeutic agents and a VEGF-specific antagonist are administered concurrently, followed by anti-VEGF maintenance therapy.

[0032] In embodiments comprising additional anti-cancer therapies, the subject can be further treated with the additional anti-cancer therapy before, during (e.g., simultaneously with), or after administration of the VEGF-specific antagonist. In one embodiment, the VEGF-specific antagonist, administered alone or in combination with an anti-cancer therapy, can be administered as a maintenance therapy.

[0033] The present application relates to the following embodiments.

[0034] 1. A method of treating a patient diagnosed with ovarian cancer, comprising administering to the patient a treatment regimen that combines chemotherapy with administration of an effective amount of an anti-VEGF antibody, followed by anti-VEGF maintenance therapy, wherein the chemotherapy of the treatment regimen comprises administration of at least one chemotherapeutic agent, and wherein the treatment regimen is effective to prolong progression-free survival of the patient.

[0035] 2. The method of embodiment 1, wherein the chemotherapeutic agent is a taxane, paclitaxel, docetaxel, protein-bound particles of paclitaxel (e.g. ), a platinum analog, carboplatin, gemcitabine, or a combination thereof.

[0036] 3. The method of embodiment 1, wherein the chemotherapy of the treatment regimen comprises administration of a taxane and carboplatin.

[0037] 4. The method of embodiment 1, wherein the chemotherapy of the treatment regimen comprises administration of carboplatin and gemcitabine.

[0038] 5. The method of embodiment 1, wherein the patient is diagnosed with previously untreated ovarian cancer.

[0039] 6. The method of embodiment 1, wherein the patient is diagnosed with recurrent ovarian cancer.

[0040] 7. The method of embodiment 1, wherein the anti-VEGF antibody binds to the same epitope as the monoclonal anti-VEGF antibody A4.6.1 produced by hybridoma ATCC HB 10709.

[0041] 8. The method of embodiment 1, wherein the anti-VEGF antibody is a humanized antibody.

[0042] 9. The method of embodiment 8, wherein the anti-VEGF antibody is a humanized A4.6.1 antibody or a fragment thereof.

[0043] 10. The method of embodiment 8, wherein the anti-VEGF antibody is bevacizumab.

[0044] 11. The method of embodiment 1, wherein the anti-VEGF antibody is bevacizumab and the chemotherapy of the treatment regimen comprises administration of capecitabine and paclitaxel or docetaxel.

[0045] 12. The method of embodiment 1, wherein the anti-VEGF antibody is bevacizumab and the chemotherapy of the treatment regimen comprises administration of carboplatin and gemcitabine.

[0046] 13. The method of embodiment 1, wherein the patient's progression-free survival is extended by at least about 3.8 months or more compared to another patient not treated with anti-VEGF maintenance therapy.

[0047] 14. The method of embodiment 8, wherein the anti-VEGF antibody has a heavy chain variable region comprising the following amino acid sequence: EVQLVESGGG LVQPGGSLRL SCAASGYTFT NYGMNWVRQAPGKGLEWVGW INTYTGEPTY AADFKPvRFTF SLDTSKSTAY LQMNSLRAED TAVYYCAKYP HYYGSSHWYFDVWGQGTLVT VSS (SEQ ID NO. 1) and a light chain variable region comprising the following amino acid sequence: DIQMTQSPSSLSASVGDRVT ITCSASQDIS NYLNWYQQKP GKAPKVLIYF TSSLHSGVPS RFSGSGSGTD FTLTISSLQPEDFATYYCQQ YSTVPWTFGQ GTKVEIKR (SEQ ID NO. 2).

[0048] 15. The method of embodiment 2, wherein paclitaxel is Figure 1 or Figure 2 Administer as indicated.

[0049] 16. The method of embodiment 2, wherein carboplatin is Figure 1 or Figure 2 or Figure 11 Administer as indicated.

[0050] 17. The method of embodiment 2, wherein docetaxel is Figure 1 Administer as indicated.

[0051] 18. The method of embodiment 2, wherein gemcitabine is Figure 11 Administer as indicated.

[0052] 19. The method of embodiment 2, wherein the anti-VEGF is Figure 1 or Figure 2 As shown in branch III or as described in Example 3 branch II.

[0053] 20. A kit for treating previously untreated ovarian cancer in a human patient, comprising packaging comprising an anti-VEGF antibody composition and instructions for use of the anti-VEGF antibody composition in combination with taxane therapy and carboplatin, followed by anti-VEGF maintenance therapy, wherein the instructions describe that patients receiving taxane therapy and carboplatin therapy and bevacizumab had a progression-free survival of 14.1 months and a hazard ratio of 0.717 (p-value < 0.0001).

[0054] 21. The kit of embodiment 20, wherein the anti-VEGF antibody has a heavy chain variable region comprising the following amino acid sequence: EVQLVESGGG LVQPGGSLRL SCAASGYTFT NYGMNWVRQAPGKGLEWVGW INTYTGEPTY AADFKRRFTF SLDTSKSTAY LQMNSLRAED TAVYYCAKYP HYYGSSHWYFDVWGQGTLVT VSS (SEQ ID No. 1) and a light chain variable region comprising the following amino acid sequence: DIQMTQSPSSLSASVGDRVT ITCSASQDIS NYLNWYQQKP GKAPKVLIYF TSSLHSGVPS RFSGSGSGTD FTLTISSLQPEDFATYYCQQ YSTVPWTFGQ GTKVEIKR (SEQ ID No. 2).

[0055] 22. The kit of embodiment 20, wherein the anti-VEGF antibody is bevacizumab.

[0056] 23. The kit of embodiment 20, wherein the patient has stage III or IV ovarian cancer.

[0057] 24. A method of instructing a human subject having ovarian cancer, the method comprising providing instructions for treating the ovarian cancer with an anti-VEGF antibody concurrently with chemotherapy, followed by anti-VEGF maintenance therapy, thereby prolonging progression-free survival in the subject.

[0058] 25. The method of embodiment 24, wherein the instructions further comprise providing instructions for administering treatment with at least two chemotherapeutic agents.

[0059] 26. A method of promoting the administration of an anti-VEGF antibody concurrently with chemotherapy followed by anti-VEGF maintenance therapy to treat ovarian cancer in a human subject, thereby prolonging progression-free survival in the subject.

[0060] 27. The method of embodiment 26, wherein the method further comprises promoting administration of at least two chemotherapeutic agents.

[0061] 28. The method of embodiment 26, wherein the promotion is via a package insert, wherein the package insert provides instructions for receiving the anti-VEGF antibody for cancer treatment.

[0062] 29. The method of embodiment 26, wherein the promotion is by way of a package insert accompanying a commercial formulation of the anti-VEGF antibody.

[0063] 30. The method of embodiment 26, wherein the promotion is by way of a package insert accompanying a commercial formulation of the chemotherapeutic agent.

[0064] 31. The method of embodiment 26, wherein the communication is performed by written notification to a physician or health care provider.

[0065] 32. The method of embodiment 26, wherein the communication is performed by verbally informing a physician or health care provider.

[0066] 33. The method of embodiment 26, wherein the promotion is followed by treatment of the subject with the anti-VEGF antibody and the chemotherapeutic agent, followed by anti-VEGF maintenance therapy.

[0067] 34. A business method comprising marketing an anti-VEGF antibody therapy concurrently with chemotherapy, followed by anti-VEGF maintenance therapy, for treating ovarian cancer in a human subject, thereby prolonging the subject's progression-free survival.

[0068] 35. The method of embodiment 34, wherein the marketing is followed by treatment of the subject with an anti-VEGF antibody concurrently with chemotherapy, followed by anti-VEGF maintenance therapy.

[0069] 36. The method of any one of embodiments 24, 26 or 34, wherein the chemotherapeutic agent is a taxane, paclitaxel, docetaxel, protein-bound particles of paclitaxel (e.g. ), platinum analogs, carboplatin, gemcitabine, or a combination thereof.

[0070] 37. The method of any one of embodiments 24, 26 or 36, wherein the anti-VEGF antibody is bevacizumab.

[0071] 38. The method of embodiment 1, wherein the chemotherapy of the treatment regimen comprises administration of paclitaxel and carboplatin.

[0072] 39. The method of embodiment 1, wherein said anti-VEGF antibody is bevacizumab and said chemotherapy of the treatment regimen comprises administration of paclitaxel and carboplatin.

[0073] 40. The method of embodiment 1, wherein the patient's progression-free survival is prolonged by at least about 2.3 months or more compared to another patient not treated with the anti-VEGF antibody.

[0074] 41. The method of embodiment 2, wherein paclitaxel is Figure 8 Administer as indicated.

[0075] 42. The method of embodiment 2, wherein carboplatin is Figure 8 Administer as indicated.

[0076] 43. The method of embodiment 2, wherein the anti-VEGF antibody is Figure 8 Branch B is shown as an example.

[0077] 44. A kit for treating previously untreated ovarian cancer in a human patient, comprising packaging comprising an anti-VEGF antibody composition and instructions for use of the anti-VEGF antibody composition in combination with paclitaxel and carboplatin followed by anti-VEGF maintenance therapy, wherein the instructions describe that patients receiving paclitaxel, carboplatin, and the anti-VEGF antibody had a progression-free survival of 18.3 months and a hazard ratio of 0.79.

[0078] Other features and advantages of the invention will be apparent from the detailed description, drawings, and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0079] Figure 1 The study design for ovarian cancer described in Example 1 is described.

[0080] Figure 2 Diagram depicting the study design of ovarian cancer trials using bevacizumab (BEV) or placebo and various chemotherapies.

[0081] Figure 3 Depicted from Figure 2 Selected adverse events from the indicated trials.

[0082] Figure 4 Depicted from Figure 2 Selected adverse events due to the treatment period of the indicated trials.

[0083] Figure 5 Describe the investigators' assessment Figure 2 Progression-free survival (PFS) for arm I, arm II, and arm III of the trial are shown.

[0084] Figure 6 depiction Figure 2 Shown are PFS values ​​for Arms I and III of the trial and the results using the CA-125 marker as a determinant of progression.

[0085] Figure 7 depiction Figure 2 Subgroup analysis of patients in arm III versus arm I of the trial shown.

[0086] Figure 8 The study design for the ovarian cancer trial described in Example 2 is described.

[0087] Figure 9 depiction Figure 8 Summary of progression-free survival (PFS) analyses for the indicated trials. "CP" corresponds to Figure 8 Clade A in CPB7.5+ corresponds to Figure 8 Branch B in.

[0088] Figure 10 Depicted from Figure 8 A graph showing the PFS results for the indicated trials. "CP" corresponds to Figure 8 Clade A in CPB7.5+ corresponds to Figure 8 Branch B in.

[0089] Figure 11 The study design for the ovarian cancer trial described in Example 3 is described. DETAILED DESCRIPTION

[0090] I. Definition

[0091] The term "VEGF" or "VEGF-A" is used to refer to the 165 amino acid human vascular endothelial growth factor and the related 121, 145, 189, and 206 amino acid human vascular endothelial growth factors, as described, for example, in Leung et al. Science, 246:1306 (1989); and Houck et al. Mol. Endocrin., 5:1806 (1991), as well as naturally occurring allelic forms and processed forms thereof. VEGF-A is part of a gene family that includes VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, and PlGF. VEGF-A primarily binds to two high-affinity receptor tyrosine kinases, VEGFR-1 (Flt-1) and VEGFR-2 (Flk-1 / KDR), the latter being the primary transmitter of the VEGF-A mitogenic signal in vascular endothelial cells. In addition, neuropilin-1 has been identified as a receptor for the heparin-binding VEGF-A isoform and may play a role in vascular development. The term "VEGF" or "VEGF-A" also refers to VEGF from non-human species such as mice, rats or primates. Sometimes, VEGF from a particular species is represented as follows, hVEGF for human VEGF and mVEGF for murine VEGF. The term "VEGF" is also used to refer to a truncated form of the polypeptide comprising amino acids 8-109 or 1-109 of human vascular endothelial growth factor comprising 165 amino acids. In this application, reference may be made to, for example, "VEGF (8-109)", "VEGF (1-109)" or "VEGF 165 " is used to identify any such form of VEGF. The amino acid positions of "truncated" native VEGF are numbered as shown in the native VEGF sequence. For example, amino acid position 17 (methionine) in truncated native VEGF is also position 17 (methionine) in native VEGF. Truncated native VEGF has comparable binding affinity to native VEGF for KDR and Flt-1 receptors.

[0092] An "anti-VEGF antibody" refers to an antibody that binds to VEGF with sufficient affinity and specificity. The selected antibody will typically have a binding affinity for VEGF, for example, the antibody may have a K between 100 nM and 1 pM. dThe antibody affinity can be determined by surface plasmon resonance-based assays (such as the BIAcore assay described in PCT Application Publication No. WO 2005 / 012359); enzyme-linked immunosorbent assays (ELISAs); and competition assays (e.g., RIAs). In certain embodiments, the anti-VEGF antibodies of the invention can be used as therapeutic agents to target and interfere with diseases or conditions in which VEGF activity is implicated. Furthermore, the antibodies can be subjected to other biological activity assays, such as those performed to assess their efficacy as therapeutic agents. Such assays are known in the art and depend on the antibody's target antigen and intended use. Examples include HUVEC inhibition assays; tumor cell growth inhibition assays (e.g., as described in WO 89 / 06692); antibody-dependent cellular cytotoxicity (ADCC) and complement-mediated cytotoxicity (CDC) assays (U.S. Pat. No. 5,500,362); and agonist activity or hematopoietic assays (see WO 95 / 27062). Anti-VEGF antibodies generally do not bind to other VEGF homologs, such as VEGF-B or VEGF-C, nor to other growth factors, such as PlGF, PDGF, or bFGF.

[0093] "VEGF antagonists" refer to molecules that neutralize, block, inhibit, eliminate, reduce, or interfere with VEGF activity, including its binding to one or more VEGF receptors. VEGF antagonists include anti-VEGF antibodies and antigen-binding fragments thereof, receptor molecules and derivatives that specifically bind to VEGF, thereby isolating it from binding to one or more receptors, anti-VEGF receptor antibodies, and VEGF receptor antagonists such as small molecule inhibitors of VEGFR tyrosine kinase.

[0094] "Native sequence" polypeptides include polypeptides having the same amino acid sequence as a polypeptide derived from nature. Thus, a native sequence polypeptide can have the amino acid sequence of a naturally occurring polypeptide from any mammal. Such native sequence polypeptides can be isolated from nature or can be produced by recombinant or synthetic means. The term "native sequence" polypeptide explicitly encompasses naturally occurring truncated or secreted forms (e.g., extracellular domain sequences), naturally occurring variant forms (e.g., alternatively spliced ​​forms), and naturally occurring allelic variants of the polypeptide.

[0095] By polypeptide "variant" is meant a biologically active polypeptide that has at least about 80% amino acid sequence identity with a native sequence polypeptide. Such variants include, for example, polypeptides in which one or more amino acid residues are added or deleted at the N-terminus or C-terminus of the polypeptide. Typically, a variant will have at least about 80% amino acid sequence identity, more preferably at least about 90% amino acid sequence identity, and even more preferably at least about 95% amino acid sequence identity with a native sequence polypeptide.

[0096] The term "antibody" is used herein in the broadest sense and includes monoclonal antibodies (including full-length or intact monoclonal antibodies), polyclonal antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments (see below) so long as they exhibit the desired biological activity.

[0097] Throughout this specification and claims, the numbering of immunoglobulin heavy chain residues herein is that of the EU index as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991), expressly incorporated herein by reference. "EU index as in Kabat" refers to the residue numbering of the human IgG1 EU antibody.

[0098] In one embodiment, according to the present invention, d ” or “K d The "value" is measured by a radiolabeled VEGF binding assay (RIA) using Fab-format antibodies and VEGF molecules as described in the following assay: the VEGF binding activity is measured by rinsing with a minimum concentration of 125 The solution binding affinity of the Fab for VEGF was measured by equilibrating the Fab with I-labeled VEGF (109) and then capturing the bound VEGF with an anti-Fab antibody-coated plate (Chen, et al., J Mol Biol 293:865-881 (1999)). In one example, to determine the assay conditions, microtiter plates (Dynex) were coated with 5 μg / ml of a capturing anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6) overnight and then blocked with 2% (w / v) bovine serum albumin in PBS at room temperature (about 23°C) for 2-5 hours. In a non-adsorbent plate (Nunc #269620), 100 pM or 26 pM [ 125I]-VEGF(109) was mixed with serially diluted Fab of interest (e.g., Fab-12 as described in Presta et al., Cancer Res. 57:4593-4599 (1997)). The Fab of interest was then incubated overnight; however, the incubation period could be extended to 65 hours to ensure equilibrium. Thereafter, the mixture was transferred to a capture plate and incubated at room temperature for 1 hour. The solution was then removed and the plate was washed with 0.1% The plates were washed eight times with PBS at 20°C. After the plates were dried, 150 μl / well of scintillation fluid (MicroScint-20; Packard) was added and the plates were counted for 10 minutes on a Topcount gamma counter (Packard). The concentration of each Fab that gave less than or equal to 20% of the maximal binding was selected for competitive binding assays. According to another embodiment, K d or K d The values ​​were determined by surface plasmon resonance using BIAcore TM -2000 or BIAcore TM -3000 (BIAcore, Inc., Piscataway, NJ) at 25°C using immobilized hVEGF (8-109) CM5 chip at approximately 10 response units (RU). Briefly, carboxymethylated dextran biosensor chips (CM5, BIAcore Inc.) were activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Human VEGF was diluted to 5 μg / ml (approximately 0.2 μM) with 10 mM sodium acetate, pH 4.8, and then injected at a flow rate of 5 μl / min until approximately 10 response units (RU) of coupled protein were obtained. Following injection of human VEGF, 1 M ethanolamine was injected to block unreacted groups. For kinetic measurements, 0.05% VEGF was injected at a flow rate of approximately 25 μl / min at 25°C. Fab was serially diluted two-fold (0.78 nM to 500 nM) in PBS (PBST) at 20°C. Association rates (k) were calculated by simultaneous fitting of association and dissociation sensorgrams using a simple one-to-one Langmuir binding model (BIAcore Evaluation Software version 3.2). on ) and dissociation rate (k off ). Equilibrium dissociation constant (K d ) with a ratio k off / k onCalculation. See, e.g., Chen, Y., et al., J Mol Biol 293:865-881 (1999). If the binding rate exceeds 10 according to the surface plasmon resonance assay above, 6 M -1 S -1 , the binding rate can be measured using a fluorescence quenching technique, i.e., fluorescence quenching on a spectrometer such as a stop-flow equipped spectrophometer (AvivInstruments) or a 8000 series SLM-Aminco TM The increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm band pass) of 20 nM anti-VEGF antibody (Fab form) in PBS, pH 7.2 at 25°C was measured in a spectrophotometer (ThermoSpectronic) using a stirred cuvette in the presence of increasing concentrations of the short form of human VEGF (8-109) or mouse VEGF.

[0099] A "blocking" antibody or antibody "antagonist" is an antibody that inhibits or reduces the biological activity of the antigen to which it binds. For example, a VEGF-specific antagonist antibody binds to VEGF and inhibits VEGF's ability to induce vascular endothelial cell proliferation or induce vascular permeability. In certain embodiments, a blocking antibody or antagonist antibody completely or substantially inhibits the biological activity of the antigen.

[0100] Unless otherwise indicated, the expression "multivalent antibody" is used throughout this specification to refer to an antibody that contains three or more antigen binding sites. For example, a multivalent antibody is engineered to have three or more antigen binding sites and is generally not a native sequence IgM or IgA antibody.

[0101] "Antibody fragments" comprise only a portion of an intact antibody, generally including the antigen binding site of the intact antibody, and thus retain the ability to bind to the antigen. Examples of antibody fragments encompassed by this definition include: (i) a Fab fragment having a VL, CL, VH, and CH1 domain; (ii) a Fab' fragment, which is a Fab fragment having one or more cysteine ​​residues at the C-terminus of the CH1 domain; (iii) an Fd fragment, which has a VH and CH1 domain; (iv) an Fd' fragment, which has a VH and CH1 domain and one or more cysteine ​​residues at the C-terminus of the CH1 domain; (v) an Fv fragment, which has the VL and VH domains of a single arm of an antibody; (vi) a dAb fragment (Ward et al., Nature 341:544-546 (1989)), which consists of a VH domain; (vii) isolated CDR regions; (viii) a F(ab')2 fragment, which is a bivalent fragment comprising two Fab' fragments linked by a disulfide bond at the hinge region; (ix) a single-chain antibody molecule (e.g., single-chain Fv; scFv) (Bird et al., Science 341:544-546 (1989)). 242:423-426 (1988) and Huston et al., PNAS (USA) 85:5879-5883 (1988)); (x) "diabodies", which have two antigen-binding sites and comprise a heavy-chain variable domain (VH) and a light-chain variable domain (VL) connected in the same polypeptide chain (see, e.g., EP 404,097; WO 93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993)); (xi) "linear antibodies", which comprise a pair of tandem Fd segments (VH-CH1-VH-CH1) that, together with complementary light chain polypeptides, form a pair of antigen-binding regions (Zapata et al., Protein Eng. 8(10):1057-1062 (1995) and U.S. Patent 5,641,870).

[0102] The term "monoclonal antibody" as used herein refers to an antibody obtained from a group of substantially homogeneous antibodies, i.e., each antibody individual constituting the group is identical, except for possible naturally occurring mutant forms that may exist in very small amounts. Monoclonal antibodies are highly specific, being directed against a single antigen. In addition, unlike typical polyclonal antibody preparations comprising different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" should not be interpreted as requiring the generation of antibodies by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention can be prepared by the hybridoma method initially described by Kohler et al. Nature 256:495, (1975), or can be prepared by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567). "Monoclonal antibodies" can also be isolated from phage antibody libraries using the techniques described in, for example, Clackson et al., Nature 352:624-628, (1991) or Marks et al., J. Mol. Biol. 222:581-597, (1991).

[0103] An "Fv" fragment is an antibody fragment that contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy chain variable domain (region) and one light chain variable domain (region) in tight association (the association may be covalent in nature, such as in scFv). It is in this configuration that the three CDRs of each variable domain (region) interact to form a V H -V L The dimer defines an antigen-binding site on its surface. The six CDRs or a subset thereof together confer antigen-binding specificity to the antibody. However, even a single variable domain (region) (or half an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind to an antigen, though typically with a lower affinity than the complete binding site.

[0104] As used herein, "antibody variable domain" refers to the portion of the light and heavy chains of an antibody molecule that contains the amino acid sequences of the complementarity determining regions (CDRs; i.e., CDR1, CDR2, and CDR3) and framework regions (FRs). H V refers to the heavy chain variable domain (region). L Refers to the light chain variable domain (region). According to the methods used in the present invention, the amino acid positions classified as CDRs and FRs can be defined according to Kabat (Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md., 1987 and 1991)). The amino acid numbering of antibodies or antigen-binding fragments also follows Kabat's amino acid numbering.

[0105] As used herein, the term "complementarity determining region (CDR; i.e., CDR1, CDR2, and CDR3) refers to the amino acid residues in an antibody variable domain (region) whose presence is essential for antigen binding. Each variable domain typically has three CDRs, identified as CDR1, CDR2, and CDR3. Each complementarity determining region can comprise amino acid residues from a "complementarity determining region" as defined by Kabat (i.e., approximately residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) of the light chain variable domain and residues 31-35 (H1), 50-65 (H2), and 95-102 (H3) of the heavy chain variable domain; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and / or residues from a "hypervariable loop" (i.e., approximately residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) of the light chain variable domain and residues 26-32 (H1), 53-55 (H2), and 96-101 (H3) of the heavy chain variable domain; Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). In some cases, a complementarity determining region can include amino acids from both a CDR region and a hypervariable loop as defined by Kabat. For example, CDRH1 of the heavy chain of antibody 4D5 includes amino acids 26-35.

[0106] "Framework regions" (hereinafter FR) refer to the residues in a variable domain other than the CDR residues. Each variable domain typically has four FRs, identified as FR1, FR2, FR3, and FR4. If the CDRs are defined according to Kabat, the light chain FR residues are located at approximately light chain residues 1-23 (LCFR1), 35-49 (LCFR2), 57-88 (LCFR3), and 98-107 (LCFR4), while the heavy chain FR residues are located at approximately heavy chain residues 1-30 (HCFR1), 36-49 (HCFR2), 66-94 (HCFR3), and 103-113 (HCFR4). If the CDRs comprise amino acid residues from a hypervariable loop, the light chain FR residues are located at approximately light chain residues 1-25 (LCFR1), 33-49 (LCFR2), 53-90 (LCFR3), and 97-107 (LCFR4), while the heavy chain FR residues are located at approximately heavy chain residues 1-25 (HCFR1), 33-52 (HCFR2), 56-95 (HCFR3), and 102-113 (HCFR4). In some cases, when the CDRs comprise amino acids from both the Kabat-defined CDRs and hypervariable loops, the FR residues will be adjusted accordingly. For example, when CDRH1 comprises amino acids H26-H35, the heavy chain FR1 residues are located at positions 1-25, and the FR2 residues are located at positions 36-49.

[0107] A "Fab" fragment comprises the variable and constant domains of the light chain and the variable and first constant domain (CH1) of the heavy chain. A F(ab')2 antibody fragment comprises a pair of Fab fragments, typically covalently linked near their carboxyl termini by a hinge cysteine ​​residue between them. Other chemical couplings of antibody fragments are also known in the art.

[0108] "Single-chain Fv" or "scFv" antibody fragments comprise the V H and V L domains, wherein these domains are present on one polypeptide chain. H With V L The domains further comprise a polypeptide linker that enables the scFv to form the desired structure for antigen binding. For a review of scFv, see Pluckthun, in The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, eds., Springer-Verlag, New York, pp. 269-315, 1994.

[0109] The term "diabody" refers to a small antibody fragment with two antigen-binding sites on the same polypeptide chain (V H and VL ) contains linked heavy chain variable domains (V H ) and light chain variable domain (V L By using a linker that is too short to allow pairing between the two domains on the same chain, these domains are forced to pair with the complementary domains of another chain, thereby creating two antigen-binding sites. Diabodies are more fully described in, for example, EP 404,097; WO 93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993).

[0110] The expression "linear antibodies" refers to the antibodies described in Zapata et al., Protein Eng, 8(10): 1057-1062 (1995). Briefly, these antibodies comprise a pair of tandem Fd segments (V H -C H 1-V H -C H 1), which together with the complementary light chain polypeptide form an antigen binding region pair. Linear antibodies can be bispecific or monospecific.

[0111] Monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical or homologous to the corresponding sequence in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to the corresponding sequence in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Patent No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855), (1984).

[0112] The "humanized" form of a non-human (e.g., mouse) antibody refers to a chimeric antibody that minimally comprises a sequence derived from a non-human immunoglobulin. To a great extent, a humanized antibody refers to an immunoglobulin in which the hypervariable region residues in a human immunoglobulin (recipient antibody) are replaced with hypervariable region residues from a non-human species (donor antibody) such as a mouse, rat, rabbit, or non-human primate with desired specificity, affinity, and ability. In some cases, the Fv framework region (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues. In addition, the humanized antibody may include residues that are not found in the recipient antibody or the donor antibody. These modifications are made in order to further improve the performance of the antibody. Typically, a humanized antibody comprises at least one, typically two, substantially entire variable regions, wherein all or substantially all hypervariable loops correspond to the hypervariable loops of a non-human immunoglobulin, and all or substantially all FR regions are FRs of human immunoglobulin sequences. The humanized antibody optionally further comprises at least a portion of an immunoglobulin constant region (Fc), typically a constant region of a human immunoglobulin. For more details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).

[0113] "Human antibody" refers to an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human and / or produced using any of the techniques disclosed herein for producing human antibodies. This definition of human antibody explicitly excludes humanized antibodies comprising non-human antigen-binding residues. Human antibodies can be produced using a variety of techniques known in the art. In one embodiment, human antibodies are selected from a phage library that expresses human antibodies (Vaughan et al., Nature Biotechnology 14:309-314 (1996); Sheets et al., Proc. Natl. Acad. Sci. 95:6157-6162 (1998); Hoogenboom and Winter, J. Mol. Biol. 227:381 (1991); Marks et al., J. Mol. Biol. 222:581 (1991)). Human antibodies can also be produced by introducing human immunoglobulin loci into transgenic animals (e.g., mice) in which endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed that closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This method is described in, for example, U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the following scientific publications: Marks et al., Bio / Technology 10:779-783 (1992); Lonberg et al., Nature 368:856-859 (1994); Morrison, Nature 368:812-13 (1994); Fishwild et al., Nature Biotechnology 14:845-51 (1996); Neuberger, Nature Biotechnology 14:826 (1996); Lonberg and Huszar, Intern. Rev. Immunol. 13:65-93 (1995). Alternatively, human antibodies can be prepared by immortalizing human B lymphocytes that produce antibodies against the target antigen (such B lymphocytes can be recovered from the individual or immunized in vitro). See, for example, Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol. 147(1): 86-95 (1991); and U.S. Pat. No. 5,750,373.

[0114] An "affinity matured" antibody is one that has one or more alterations in one or more CDRs of the antibody that result in an improvement in the antibody's affinity for the antigen compared to a parent antibody that does not have these alterations. Preferred affinity matured antibodies have nanomolar or even picomolar affinities for the target antigen. Affinity matured antibodies can be generated by procedures known in the art. Marks et al., Bio / Technology 10:779-783 (1992) describes affinity maturation by VH and VL domain shuffling. Random mutagenesis of CDR and / or framework residues is described in: Barbas et al., Proc. Nat. Acad. Sci. USA 91:3809-3813 (1994); Schier et al., Gene 169:147-155 (1995); Yelton et al., J. Immunol. 155:1994-2004 (1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995); Hawkins et al., J. Mol. Biol. 226:889-896 (1992).

[0115] A "functional antigen binding site" of an antibody refers to a site that is capable of binding to a target antigen. The antigen binding affinity of an antigen binding site need not be as strong as that of the parent antibody from which it was derived, but the ability to bind to an antigen must be measurable using any of the various methods known for assessing antibody binding to an antigen. In addition, the antigen binding affinity of each antigen binding site of a multivalent antibody herein need not be quantitatively identical. For multimeric antibodies herein, the number of functional antigen binding sites can be assessed using ultracentrifugation analysis, as described in U.S. Patent Application Publication No. 20050186208. According to this analytical method, the target antigen and the multimeric antibody are mixed in different proportions, and the average molecular weight of the complex is calculated, assuming different numbers of functional binding sites. These theoretical values ​​are compared with the actual experimental values ​​obtained to assess the number of functional binding sites.

[0116] An antibody having a "biological characteristic" of a specified antibody is one that possesses one or more biological characteristics that distinguish the specified antibody from other antibodies that bind the same antigen.

[0117] To screen for antibodies that bind to the epitope on the antigen to which the antibody of interest binds, conventional cross-blocking assays such as those described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988) can be performed.

[0118] A "species-dependent antibody" is an antibody that has a stronger binding affinity for an antigen from a first mammalian species than for a homolog of that antigen from a second mammalian species. Typically, a species-dependent antibody "specifically binds" to a human antigen (i.e., has an affinity of no more than about 1 x 10 -7 M, preferably no more than about 1 x 10 -8 M, most preferably no more than about 1 x 10 -9 The binding affinity (K d )), but has a binding affinity for a homolog of the antigen from a second non-human mammalian species that is at least about 50 times, or at least about 500 times, or at least about 1000 times weaker than its binding affinity for the human antigen. Species-dependent antibodies can be various types of antibodies as defined above, but are typically humanized antibodies or human antibodies.

[0119] When used herein, "antibody mutant" or "antibody variant" refers to an amino acid sequence variant of a species-dependent antibody in which one or more amino acid residues of the species-dependent antibody are modified. Such mutants must have less than 100% sequence identity or similarity with the species-dependent antibody. In one embodiment, the amino acid sequence of the antibody mutant has at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95% amino acid sequence identity or similarity with the amino acid sequence of the heavy or light chain variable domain of the species-dependent antibody. The identity or similarity of this sequence is defined herein as the percentage of amino acid residues in the candidate sequence that are identical (i.e., identical residues) or similar (i.e., amino acid residues from the same group based on common side chain properties, see below) to the residues of the species-dependent antibody after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percentage sequence identity. Extensions, deletions, or insertions within the antibody sequence outside the N-terminus, C-terminus, or variable domain (region) should not be considered to affect sequence identity or similarity.

[0120] In order to extend the half-life of an antibody or polypeptide comprising an amino acid sequence of the present invention, a salvage receptor binding epitope can be attached to an antibody (particularly an antibody fragment) as described, for example, in U.S. Patent No. 5,739,277. For example, a nucleic acid molecule encoding a salvage receptor binding epitope can be linked to a nucleic acid encoding a polypeptide sequence of the present invention in the same reading frame, such that the fusion protein encoded by the modified nucleic acid molecule comprises a salvage receptor binding epitope and a polypeptide sequence of the present invention. As used herein, the term "salvage receptor binding epitope" refers to an epitope in the Fc region of an IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4) that is responsible for extending the serum half-life of an IgG molecule in vivo (e.g., Ghetie et al., Ann. Rev. Immunol. 18:739-766 (2000), Table 1). Antibodies with substitutions in the Fc region and increased serum half-life are also described in WO 00 / 42072; WO 02 / 060919; Shields et al., J. Biol. Chem. 276:6591-6604 (2001); Hinton, J. Biol. Chem. 279:6213-6216 (2004). In another embodiment, serum half-life can also be extended by, for example, attaching other polypeptide sequences. For example, an antibody or other polypeptide useful in the methods of the present invention can be attached to serum albumin or a portion of serum albumin that binds to the FcRn receptor or serum albumin-binding peptide, such that serum albumin binds to the antibody or polypeptide. For example, such polypeptide sequences are disclosed in WO 01 / 45746. In one embodiment, the serum albumin peptide to be attached comprises the amino acid sequence DICLPRWGCLW. In another embodiment, the half-life of a Fab is extended by these methods. Serum albumin binding peptide sequences are also found in Dennis et al., J. Biol. Chem. 277: 35035-35043 (2002).

[0121] A "chimeric VEGF receptor protein" refers to a VEGF receptor molecule having an amino acid sequence derived from at least two different proteins (at least one of which is a VEGF receptor protein). In certain embodiments, the chimeric VEGF receptor protein is capable of binding to VEGF and inhibiting the biological activity of VEGF.

[0122] An "isolated" antibody is a polypeptide or antibody that has been identified and separated and / or recovered from a component of its natural environment. Contaminating components of its natural environment are substances that would interfere with the diagnostic or therapeutic use of the antibody and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In certain embodiments, the antibody is purified to (1) greater than 95% by weight of the antibody as determined by the Lowry method, and most preferably greater than 99% by weight, (2) a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence using a spinning cup sequenator, or (3) to homogeneity as determined by SDS-PAGE under reducing or non-reducing conditions and staining with Coomassie blue or silver. Isolated antibodies include antibodies in situ within recombinant cells, since at least one component of the antibody's natural environment will not be present. However, isolated antibodies will generally be prepared by at least one purification step.

[0123] "Fragment" refers to a portion of a polypeptide or nucleic acid molecule that preferably contains at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more of the full length of a reference nucleic acid molecule or polypeptide. The fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, or more nucleotides, or 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 190, 200 or more amino acids.

[0124] "Anti-angiogenic agent" or "angiogenesis inhibitor" refers to a small molecular weight substance, polynucleotide, polypeptide, isolated protein, recombinant protein, antibody, or conjugate or fusion protein thereof that directly or indirectly inhibits angiogenesis, vasculogenesis, or unwanted vascular permeability. It should be understood that anti-angiogenic agents include those that bind to and block the angiogenic activity of angiogenic factors or their receptors. For example, anti-angiogenic agents are antibodies or other antagonists of angiogenic agents as defined throughout the specification or known in the art, such as, but not limited to, antibodies to VEGF-A, antibodies to VEGF-A receptors (e.g., KDR receptor or Flt-1 receptor), VEGF-trap, anti-PDGFR inhibitors such as Gleevec, TM(Imatinib Mesylate). Anti-angiogenic agents also include natural angiogenesis inhibitors, such as angiostatin, endostatin, and the like. See, for example, Klagsbrun and D'Amore, Annu. Rev. Physiol. 53:217-39 (1991); Streit and Detmar, Oncogene 22:3172-3179 (2003) (e.g., Table 3 listing anti-angiogenic therapies in malignant melanoma); Ferrara & Alitalo, Nature Medicine 5(12):1359-1364 (1999); Tonini et al., Oncogene 22:6549-6556 (2003) (e.g., Table 2 listing known anti-angiogenic factors); Sato, Int. J. Clin. Oncol. 8:200-206 (2003) (e.g., Table 1 listing anti-angiogenic agents used in clinical trials).

[0125] A "maintenance" dose is herein one or more doses of a therapeutic agent administered to a patient during or after treatment. Typically, a maintenance dose is administered at certain treatment intervals, such as about once a week, about every two weeks, about every three weeks, or about every four weeks.

[0126] In one embodiment, the maintenance dose is as described herein. Figure 1 (Extended Therapy), Figure 2 or Figure 8 or Figure 11 of.

[0127] "Survival" refers to whether the patient remains alive, including progression-free survival (PFS) and overall survival (OS). Survival can be assessed using the Kaplan-Meier method, and any differences in survival can be calculated using the stratified log-rank test.

[0128] "Progression free survival (PFS)" refers to the time from treatment (or randomization) to the first disease progression or death. In one aspect of the invention, PFS can be assessed by Response Evaluation Criteria in Solid Tumors (RECIST). In one aspect of the invention, PFS can be assessed by CA-125 levels as a determinant of progression.

[0129] "Overall survival" refers to the period of time from the start of treatment or from initial diagnosis in which a patient remains alive, such as about 1 year, about 1.5 years, about 2 years, about 3 years, about 4 years, about 5 years, about 10 years, etc. In the studies underlying the invention, the event used for survival analysis was death from any cause.

[0130] "Extending survival" or "increasing the likelihood of survival" means that the PFS and / or OS of a treated patient is prolonged or improved relative to an untreated patient (i.e., relative to a patient not treated with a VEGF-specific antagonist, e.g., a VEGF antibody) or relative to a control treatment regimen (such as a treatment with a chemotherapeutic agent alone, such as those used in the standard treatment of ovarian cancer). For example, prolonged PFS refers to the time that a patient remains alive and cancer has not recurred for a certain period of time since the start of treatment or since initial diagnosis, compared to a control (e.g., a patient not treated with the same VEGF-specific antagonist). For example, such as about 1 month, 2 months, 2.3 months, 2.9 months, 3 months, 3.8 months, 4 months, 6 months, 7 months, 8 months, 9 months, 1 year, about 2 years, about 3 years, etc. In one embodiment, PFS is extended by about 2.9 months to about 3.8 months compared to a control. In one embodiment, PFS is extended by at least about 3.8 months compared to a control. In another embodiment, PFS is extended by about 2.3 months. In one embodiment, PFS is extended by about 6 months compared to a control. In certain embodiments, survival is monitored for at least about 1 month, 2 months, 4 months, 6 months, 9 months, or at least about 1 year, or at least about 2 years, or at least about 3 years, or at least about 4 years, or at least about 5 years, or at least about 10 years, etc. after initiation of treatment or after initial diagnosis.

[0131] The hazard ratio (HR) is a statistical definition of the rate of events. For the purposes of this invention, the hazard ratio is defined as representing the probability of an event in the experimental arm divided by the probability of an event in the control arm at any specific time point.

[0132] The "hazard ratio" in the progression-free survival analysis is the summary of the difference between the two progression-free survival curves and represents the reduction in the risk of death with treatment compared with control during the follow-up period.

[0133] The term "concurrent" is used herein to refer to the use of two or more therapeutic agents wherein at least some of the administration overlaps in time. Thus, concurrent administration includes dosing regimens in which the administration of one or more agents is interrupted followed by the administration of one or more other agents.

[0134] "Monotherapy" means a treatment regimen that includes only a single therapeutic agent to treat a cancer or tumor during the course of a treatment period. Monotherapy with a VEGF-specific antagonist means administering the VEGF-specific antagonist in the absence of other anticancer therapies during the treatment period.

[0135] "Maintenance therapy" means a treatment regimen given to reduce the likelihood of disease recurrence or progression. Maintenance therapy can be provided for any length of time, including extended periods up to the life of the subject. Maintenance therapy can be provided after initial therapy or in conjunction with initial or other therapy. The dosage used for maintenance therapy can vary and may include reduced doses compared to those used with other types of therapy.

[0136] In certain embodiments of the invention, at least 16 cycles of maintenance therapy are provided after completion of 5 cycles of chemotherapy concurrently with anti-VEGF therapy. In other embodiments, at least 12 cycles of maintenance therapy are provided after completion of 6 cycles of chemotherapy concurrently with anti-VEGF therapy. In one embodiment, maintenance therapy is provided as Figure 1 、 Figure 2 、 Figure 8 or Figure 11 Depicted in.

[0137] The terms "cancer" and "cancerous" refer to or describe a physiological condition in mammals that is typically characterized by unregulated cell growth. Included in this definition are benign and malignant cancers, as well as dormant tumors or micrometastases. Examples of cancer include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias. More specific examples of such cancers include ovarian cancer, primary peritoneal cancer of the ovary, ovarian fallopian tube cancer, platinum-sensitive recurrent epithelial ovarian cancer, primary peritoneal cancer, or fallopian tube cancer, squamous cell carcinoma, lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung), peritoneal cancer, hepatocellular carcinoma, gastric cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, liver cancer (liver cancer or hepatic carcinoma), bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, Cancers of the breast, ovary, and ovary are also included, as are B-cell lymphomas (including low-grade / follicular non-Hodgkin's lymphoma (NHL), small lymphocytic (SL) NHL, intermediate-grade / follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-cleaved cell NHL, bulky disease NHL, mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom's macroglobulinemia), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal blood vessel proliferation associated with phakomatoses, edema (such as that associated with brain tumors), and Meigs syndrome.

[0138] "Metastasis" refers to the spread of cancer from its primary site to other locations in the body. Cancer cells can break away from the primary tumor, infiltrate into lymph and blood vessels, circulate via the bloodstream, and grow in distant lesions (metastasis) in normal tissues elsewhere in the body. Metastasis can be local or distal. Metastasis is a continuous process that depends on whether tumor cells fall off from the primary tumor, spread via the bloodstream, and stop at a distant site. At the new site, the cells establish a blood supply and can grow to form a life-threatening mass. Stimulating and inhibitory molecular pathways within the tumor cells regulate this behavior, and the interaction between the tumor cells and the host cells in the distant site is also important.

[0139] "Subject" refers to a mammal, including but not limited to a human or non-human mammal, such as a cow, horse, dog, sheep, or cat. Preferably, the subject is a human. As used herein, a patient is also a subject. Typically, the subject is female.

[0140] For the methods of the present invention, the term "instructing" the subject means providing instructions regarding applicable therapies, medications, treatments, treatment regimens, etc. by any means, but preferably in writing, such as in the form of a package insert or other written promotional material.

[0141] For the purposes of the methods of the present invention, the term "promoting" means offering, advertising, selling, or describing a particular drug, drug combination, or therapeutic modality by any means, including in writing, such as in the form of a package insert. Promotion herein refers to the promotion of a therapeutic agent, such as a VEGF antagonist, e.g., an anti-VEGF antibody, or a chemotherapeutic agent for an indication, such as the treatment of ovarian cancer, where such promotion is approved by the Food and Drug Administration (FDA) as having demonstrated statistically significant therapeutic efficacy and an acceptable safety profile in a subject population.

[0142] The term "marketing" is used herein to describe the promotion, sale, or distribution of a product (e.g., a drug). Marketing specifically includes packaging, advertising, and any other commercial activities aimed at commercializing a product.

[0143] A "population" of subjects refers to a group of cancer subjects, such as in a clinical trial, or seen by an oncologist following FDA approval of a therapy for a particular indication, such as ovarian cancer.

[0144] The term "anti-cancer therapy" refers to a therapy useful in treating cancer. Examples of anti-cancer therapeutic agents include, but are not limited to, agents used in, for example, surgery, chemotherapeutic agents, growth inhibitors, cytotoxic agents, radiation therapy, anti-angiogenic agents, apoptotic agents, anti-tubulin agents, and other agents for treating cancer, such as anti-HER-2 antibodies, anti-CD20 antibodies, epidermal growth factor receptor (EGFR) antagonists (e.g., tyrosine kinase inhibitors), HER1 / EGFR inhibitors (e.g., erlotinib), ), platelet-derived growth factor inhibitors (such as Gleevec TM (Imatinib Mesylate)), COX-2 inhibitors (e.g., celecoxib), interferons, cytokines, antagonists (e.g., neutralizing antibodies) that bind to one or more of the following targets (ErbB2, ErbB3, ErbB4, PDGFR-β, BlyS, APRIL, BCMA or VEGF receptor, TRAIL / Apo2), and other biologically active and organic chemical agents, etc. The present invention also includes combinations thereof.

[0145] The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of cells and / or causes cell destruction. The term is intended to include radioactive isotopes (e.g., At 211 , I 131 , I 125 、Y 90 、Re 186 、Re 188 、Sm 153 、Bi 212 、P 32 and radioisotopes of Lu), chemotherapeutic agents, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and / or variants thereof.

[0146] "Chemotherapeutic agent" refers to a chemical compound that can be used to treat cancer. Examples of chemotherapeutic agents include chemical compounds that can be used to treat cancer. Examples of chemotherapeutic agents include alkylating agents, such as thiotepa and cyclophosphamide; alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodepa, carboquone, meturedepa, and uredepa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamine; annonaceous lactones, such as acetaminophen, benzophenone, benzophenone, benzophenone, benzophenone, benzophenone, benzophenone, benzophenone, benzophenone, benzophenone, benzophenone, benzophenone, benzophenone, benzophenone, benzophenone, benzophenone, benzophenone, benzophenone, benzophenone, benzophenone, benzophenone, benzophenone, benzophenone, benzophenone, benzophenone, benzophenone, benzophenone, benzophenone, benzophenone, benzophenone, benzophenone, benzophenone, benzophenone, benzophenone, benzophenone, benzophenone, benzophenone, benzophenone, benzophenone, benzophenone, benzophenone, benzophenone, benzophenone, benzophenone, benzophenone, etogenins (especially bullatacin and bullatacinone); camptothecins (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its synthetic analogues adozelesin, carzelesin, and bizelesin); cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin;Nitrogen mustards, such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard, mustard); nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics, such as the enediynes (e.g., calicheamicins, especially calicheamicin gamma and calicheamicin omega (see, e.g., Agnew (1994) Chem. Intl. Ed. Engl. 33:183-186); anthracyclines, including dynemicins A; bisphosphonates, such as clodronate; esperamicin; and neocarzinostatin chromophores and related chromoprotein enediyne antibiotic chromophores), aclacinomycin, actinomycin, anthramycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diaza-5-oxo-L-norleucine; Doxorubicin (including morpholinodoxorubicin, cyanomorpholinodoxorubicin, 2-pyrrolodoxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, terin), trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, propionate, epitiostanol, mepitiostane, testolactone; antiadrenal drugs such as aminoglutethimide, mitotane, and trilostane; folic acid supplements such as folinic acid; aceglatone;Aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defosfamide; demecolcine; diaziquone; elfornithine; elliptinium acetate acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids, such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; Polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verrucarin A, roridin A, and anguidin); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, such as Paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ), Cremophor, albumin-modified nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Illinois), and Doxetaxel ( -Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs, such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; Vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar, CPT-11) (a regimen that includes irinotecan with 5-FU and leucovorin); the topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid; capecitabine; combretastatin; leucovorin (LV); oxaliplatin, including the oxaliplatin regimen (FOLFOX); lapatinib PKC-α, Raf, H-Ras, EGFR (e.g., erlotinib ) and VEGF-A, inhibitors that reduce cell proliferation; and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing substances.

[0147] The definition also includes antihormonal agents that act to modulate or inhibit the effects of hormones on tumors, such as antiestrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including Tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene; aromatase inhibitors that inhibit the aromatase enzyme that regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazole, aminoglutethimide, megestrol acetate, Exemestane, formestane, fadrozole, Vorozole, Letrozole and anastrozole; antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those that inhibit the expression of genes in signaling pathways involved in aberrant cell proliferation, such as, for example, PKC-α, Ralf, and H-Ras; ribozymes, such as inhibitors of VEGF expression (e.g., nucleic acids) and HER2 expression inhibitors; vaccines, such as gene therapy vaccines, e.g. vaccine, Vaccines and vaccine; rIL-2; Topoisomerase 1 inhibitors; rmRH; and any pharmaceutically acceptable salts, acids or derivatives thereof.

[0148] The term "cytokine" is a generic term for proteins released by one cell population that act as intercellular mediators on another cell. Examples of such cytokines include lymphokines, monokines, and traditional polypeptide hormones. Cytokines include growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and luteinizing hormone (LH); epidermal growth factor; hepatic growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-α and -β; Mullerian inhibitory substance; mouse gonadotropin-related peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factor such as NGF-α; platelet-derived growth factor; transforming growth factor (TGF), such as TGF-α and TGF-β; insulin-like growth factor-I and -II; erythropoietin (EPO); osteoinductive factor (OSF) factor); interferons, such as interferon-α, -β and -γ; colony stimulating factors (CSFs), such as macrophage CSF (M-CSF), granulocyte-macrophage CSF (GM-CSF) and granulocyte CSF (G-CSF); interleukins (ILs), such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; tumor necrosis factors, such as TNF-α or TNF-β; and other polypeptide factors, including LIF and kit ligand (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of native sequence cytokines.

[0149] As used herein, "growth inhibitory agent" refers to a compound or composition that inhibits cell growth in vitro and / or in vivo. Thus, a growth inhibitory agent may be an agent that significantly reduces the percentage of cells in S phase. Examples of growth inhibitory agents include agents that block cell cycle progression (at a position other than S phase), such as agents that induce G1 arrest and M phase arrest. Classical M phase blockers include the vincas (vincristine and vinblastine), and topoisomerase II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Agents that arrest G1 also spill over into S phase arrest, for example, DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C. For more information, see The Molecular Basis of Cancer, ed. Mendelsohn and Israel, Chapter 1, entitled "Cell cycle regulation, oncogenes, and antineoplastic drugs", Murakami et al., WB Saunders, Philadelphia (1995), especially page 13.

[0150] The term "prodrug" as used herein refers to a precursor or derivative form of a pharmaceutically active substance that is less cytotoxic to tumor cells than the parent drug and can be enzymatically activated or converted into the more active parent drug form. See, for example, Wilman, "Prodrugs in Cancer Chemotherapy", Biochemical Society Transactions, 14, pp. 375-382, 615th Meeting Belfast (1986) and Stella et al., "Prodrugs: A Chemical Approach to Targeted Drug Delivery", Directed Drug Delivery, Borchardt et al., eds., pp. 247-267, Humana Press (1985). Prodrugs of the present invention include, but are not limited to, phosphate / ester prodrugs, thiophosphate / ester prodrugs, sulfate / ester prodrugs, peptide prodrugs, D-amino acid modified prodrugs, glycosylated prodrugs, β-lactam prodrugs, optionally substituted phenoxyacetamide prodrugs or optionally substituted phenylacetamide prodrugs, 5-fluorocytosine and other 5-fluorouridine prodrugs that can be converted into more active, non-cytotoxic drugs. Examples of cytotoxic drugs that can be derivatized into prodrug forms for use in the present invention include, but are not limited to, those chemotherapeutic agents described above.

[0151] "Radiotherapy" or "radiotherapy" refers to the use of directed gamma or beta rays to induce sufficient damage to cells to limit the cells' ability to function normally or to destroy them altogether. It will be appreciated that many ways are known in the art to determine the dosage and duration of treatment. Typical treatments are given as a single administration, and typical dosages range from 10 to 200 units (Gray) per day.

[0152] "Reduce or inhibit" refers to the ability to cause an overall decrease of preferably 20% or more, more preferably 50% or more, and most preferably 75%, 85%, 90%, 95%, or more. Reduce or inhibit can refer to the symptoms of the disorder being treated, the presence or size of metastases or micrometastases, the size of the primary tumor, the presence or size of a tumor, or the size or number of blood vessels in an angiogenic disorder.

[0153] The term "intravenous infusion" refers to the introduction of a drug into a vein of an animal or human patient over a period of time greater than about 5 minutes, preferably 30-90 minutes, although according to the present invention, intravenous infusions may be administered for 10 hours or less.

[0154] The term "intravenous push" or "intravenous rapid injection" refers to the administration of a drug into a vein of an animal or human so that the body receives the drug within about 15 minutes or less, preferably 5 minutes or less.

[0155] The term "subcutaneous administration" refers to the introduction of a drug under the skin of an animal or human patient, preferably into a pocket between the skin and subcutaneous tissue, by relatively slow, sustained delivery from a drug container. The pocket can be created by pinching or pulling the skin away from the subcutaneous tissue.

[0156] The term "subcutaneous infusion" refers to the introduction of a drug under the skin of an animal or human patient, preferably into a pocket between the skin and subcutaneous tissue, by relatively slow, continuous delivery from a drug container over a period of time, including but not limited to 30 minutes or less, or 90 minutes or less. Optionally, the infusion can be performed by subcutaneous implantation of a drug delivery pump implanted under the skin of an animal or human patient, wherein the pump delivers a predetermined amount of drug over a predetermined period of time, such as 30 minutes, 90 minutes, or a period of time spanning the duration of the treatment regimen.

[0157] The term "subcutaneous bolus" refers to the administration of a drug under the skin of an animal or human patient, wherein bolus drug delivery is preferably less than about 15 minutes, more preferably less than 5 minutes, and most preferably less than 60 seconds. Administration is preferably into a pocket between the skin and subcutaneous tissue, wherein the pocket can be created by, for example, pinching or pulling the skin away from the subcutaneous tissue.

[0158] "Disorder" refers to any condition that would benefit from treatment with an antibody. This includes chronic and acute disorders or diseases, including those pathological conditions that predispose a mammal to the disorder in question. Non-limiting examples of disorders to be treated herein include cancer; benign and malignant tumors; leukemias and lymphoid malignancies; disorders of neurons, glia, astrocytes, hypothalamus and other glands, macrophages, epithelial, stromal, and blastocoel; and inflammatory, angiogenic, and immunological disorders.

[0159] The term "therapeutically effective amount" refers to an amount of a drug that is effective in treating a disease or condition in a mammal. In the case of cancer, a therapeutically effective amount of a drug can reduce the number of cancer cells; reduce the size of the tumor; inhibit (i.e., slow down to a certain extent, preferably prevent) the infiltration of cancer cells into surrounding organs; inhibit (i.e., slow down to a certain extent, preferably prevent) tumor metastasis; inhibit tumor growth to a certain extent; and / or alleviate one or more symptoms associated with the condition to a certain extent. Depending on the extent to which the drug can prevent the growth of existing cancer cells and / or kill existing cancer cells, it can be cytostatic and / or cytotoxic. For cancer therapy, in vivo efficacy can be measured, for example, by evaluating duration of survival, duration of progression-free survival (PFS), extension of progression-free survival (PFS), response rate (RR), duration of response, and / or quality of life.

[0160] "Treatment" or "treatment" refers to the therapeutic treatment of a subject in need of treatment, including subjects already suffering from a disorder.

[0161] "Preventative or preventative measures" are those intended to prevent a condition.

[0162] The term "label," as used herein, refers to a detectable compound or composition that is coupled directly or indirectly to a polypeptide. The label can itself be detectable (e.g., a radioisotope label or a fluorescent label) or, in the case of an enzyme label, can catalyze chemical alteration of a detectable substrate compound or composition.

[0163] II. Anti-VEGF Antibodies and Antagonists

[0164] Provided herein are uses of anti-VEGF antagonists for the treatment of ovarian cancer. Angiogenesis is one of the primary processes leading to invasion and metastasis of solid tumors. Angiogenic promoters such as vascular endothelial growth factor (VEGF) are released from tumor cells into the local microenvironment, triggering angiogenic signaling pathways. There is increasing evidence that angiogenesis plays a role in disease prognosis and possibly progression and outcome in ovarian cancer. See, for example, Yoneda J, et al., Expression of angiogenesis-related genes and progression of human ovarian carcinomas in nude mice. J Natl Cancer Inst 90:447-54, 1998; Nakanishi Y, et al. 16:256-62,1997; Gasparini G, et al. Prognostic and predictive value of tumor angiogenesis in ovarian carcinomas. Int J Cancer 69: 205-11, 1996; Hollingsworth HC, et al. al.Vascular endothelialgrowth factor expression in early stage ovarian carcinoma.Cancer 80:98-106,1997;Alvarez AA,et al.The prognostic significance of angiogenesis inepithelial ovarian carcinoma.Clin Cancer Res5:587-91,1999;Gasparini G.Therationale and future potential of angiogenesis inhibitors in neoplasia.Drugs58:17-38,1999;van Hinsbergh VW,et al.Angiogenesis and anti-angiogenesis:perspectives for the treatment of solid tumors.Ann Oncol 10Suppl 4:60-3,1999;Malonne H,et al.Mechanisms of tumor angiogenesis and therapeuticimplications:angiogenesis inhibitors.Clin Exp Metastasis 17:1-14,1999; FolkmanJ.Tumor angiogenesis:therapeutic implications.N Eng J Med 285:1182-6,1971; KimKJ,et al.Inhibition of vascular endothelial growth factor-inducedangiogenesis suppresses tumor growth in vivo.Nature 362:841-4,1993; and Luo JC,et al. al.,Differential inhibition of Fluid accumulation and tumor growth in two mouse ascites tumors by an anti vascular endothelial growth factor / permeability factor neutralizing antibody. Cancer Res 58:2594-600,1998. .

[0165] (i) VEGF antigen

[0166] The VEGF antigen to be used to generate antibodies can be, for example, the VEGF165 molecule as well as other isoforms of VEGF or fragments thereof containing the desired epitope. Other forms of VEGF that can be used to generate anti-VEGF antibodies of the invention will be apparent to those skilled in the art.

[0167] Human VEGF was first obtained by screening a cDNA library prepared from human cells using bovine VEGF cDNA as a hybridization probe. Leung et al. (1989) Science, 246:1306. One cDNA thus identified encodes a 165-amino acid protein that is over 95% homologous to bovine VEGF; this 165-amino acid protein is often referred to as human VEGF (hVEGF) or VEGF165. The mitogenic activity of human VEGF has been demonstrated by expressing human VEGF in mammalian host cells. Culture medium conditioned by cells transfected with human VEGF cDNA promotes the proliferation of capillary endothelial cells, whereas control cells do not. Leung et al. (1989) Science, supra.

[0168] Although vascular endothelial growth factor can be isolated and purified from natural sources and subsequently used for therapeutic purposes, the relatively low concentration of the protein in follicular cells and the high cost, both in terms of effort and expense, of recovering VEGF have proven commercially unviable. Consequently, further efforts have been made to clone and express VEGF via recombinant DNA technology. (See, e.g., Ferrara, Laboratory Investigation 72:615-618 (1995) and references cited therein).

[0169] Originating from alternative RNA splicing, VEGF is expressed in a variety of tissues as a variety of homodimeric forms (121, 145, 165, 189, and 206 amino acids per monomer). VEGF121 is a soluble mitogen that does not bind heparin; longer forms of VEGF bind heparin with increasing affinity. Heparin-bound forms of VEGF can be cleaved at the carboxyl terminus by plasmin to release a diffusible form of VEGF. The amino acid sequencing of the carboxyl terminal peptide identified after plasmin cleavage is Arg110-Ala111. As the amino-terminal "core" protein isolated as a homodimer, VEGF (1-110) binds to neutralizing monoclonal antibodies (such as antibodies called 4.6.1 and 3.2E3.1.1) and soluble forms of the VEGF receptor with similar affinity compared to the complete VEGF165 homodimer.

[0170] Several molecules structurally related to VEGF have also been identified, including placental growth factor (PIGF), VEGF-B, VEGF-C, VEGF-D, and VEGF-E. Ferrara and Davis-Smyth (1987) Endocr. Rev., supra; Ogawa et al. J. Biological Chem. 273:31273-31281 (1998); Meyer et al. EMBO J., 18:363-374 (1999). The receptor tyrosine kinase Flt-4 (VEGFR-3) has been identified as a receptor for VEGF-C and VEGF-D. Joukov et al. EMBO. J. 15: 1751 (1996); Lee et al. Proc. Natl. Acad. Sci. USA 93: 1988-1992 (1996); Achen et al. (1998) Proc. Natl. Acad. Sci. USA 95: 548-553. VEGF-C has been shown to be involved in the regulation of lymphangiogenesis. Jeltsch et al. Science 276: 1423-1425 (1997).

[0171] (ii) Anti-VEGF antibodies

[0172] Anti-VEGF antibodies useful in the methods of treating ovarian cancer of the present invention include any antibody or antigen-binding fragment thereof that binds to VEGF with sufficient affinity and specificity and is capable of reducing or inhibiting the biological activity of VEGF. Anti-VEGF antibodies generally do not bind to other VEGF homologs, such as VEGF-B or VEGF-C, or other growth factors, such as PlGF, PDGF, or bFGF.

[0173] In certain embodiments of the invention, the anti-VEGF antibodies include, but are not limited to, monoclonal antibodies that bind to the same epitope as the monoclonal anti-VEGF antibody A4.6.1 produced by hybridoma ATCC HB10709; the recombinant humanized anti-VEGF monoclonal antibody produced according to Presta et al. (1997) Cancer Res. 57:4593-4599. In one embodiment, the anti-VEGF antibody is "bevacizumab (BV)", also known as "rhuMAb VEGF" or It contains mutated human IgG1 framework regions and antigen-binding complementarity-determining regions from the mouse anti-hVEGF monoclonal antibody A.4.6.1, blocking human VEGF from binding to its receptor. Approximately 93% of bevacizumab's amino acid sequence (including most of the framework regions) is derived from human IgG1, and approximately 7% of the sequence is derived from the mouse antibody A4.6.1.

[0174] Bevacizumab and other humanized anti-VEGF antibodies are further described in U.S. Patent No. 6,884,879, issued February 26, 2005. Additional antibodies include the G6 or B20 series antibodies (e.g., G6-31, B20-4.1), as described in PCT Publication No. WO 2005 / 012359, PCT Publication No. WO 2005 / 044853, and U.S. Patent Application No. 60 / 991,302, the contents of which are expressly incorporated herein by reference. For additional antibodies, see U.S. Patent Nos. 7,060,269, 6,582,959, 6,703,020; 6,054,297; WO 98 / 45332; WO 96 / 30046; WO 94 / 10202; EP 0666868 Bl; U.S. Patent Application Publication Nos. 2006009360, 20050186208, 20030206899, 20030190317, 20030203409, and 20050112126; and Popkov et al., Journal of Immunological Methods 288: 149-164 (2004). Other antibodies include those that bind to a functional epitope on human VEGF comprising residues F17, M18, D19, Y21, Y25, Q89, I91, K101, E103, and C104 or comprising residues F17, Y21, Q22, Y25, D63, I83, and Q89.

[0175] In one embodiment of the invention, the anti-VEGF antibody has a heavy chain variable region comprising the following amino acid sequence:

[0176] EVQLVESGGG LVQPGGSLRL SCAASGYTFT NYGMNWVRQA PGKGLEWVGW INTYTGEPTYAADFKRRFTF SLDTSKSTAY LQMNSLRAED TAVYYCAKYP HYYGSSHWYF DVWGQGTLVT VSS (SEQ IDNO: 1)

[0177] and a light chain variable region comprising the following amino acid sequence:

[0178] DIQMTQSPSS LSASVGDRVT ITCSASQDIS NYLNWYQQKP GKAPKVLIYF TSSLHSGVPSRFSGSGSGTD FTLTISSLQP EDFATYYCQQ YSTVPWTFGQ GTKVEIKR (SEQ ID NO: 2).

[0179] The "G6 series antibodies" according to the present invention refer to anti-VEGF antibodies derived from the sequence of the G6 antibody or according to PCT Publication No. WO2005 / 012359. Figure 7

[0014] The present invention also provides a G6-derived antibody of any one of the following types: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, and 34, 35, the entire disclosures of which are incorporated herein by reference. See also PCT Publication No. WO2005 / 044853, the contents of which are expressly incorporated herein by reference. In one embodiment, the G6 series antibody binds to a functional epitope on human VEGF comprising residues F17, Y21, Q22, Y25, D63, I83, and Q89.

[0180] According to the present invention, a "B20 series antibody" refers to an anti-VEGF antibody derived from the sequence of the B20 antibody or a B20-derived antibody according to any of Figures 27-29 of PCT Publication No. WO2005 / 012359, the entire disclosure of which is incorporated herein by reference. See also PCT Publication No. WO2005 / 044853 and U.S. Patent Application 60 / 991,302, the contents of which are expressly incorporated herein by reference. In one embodiment, the B20 series antibody binds to a functional epitope on human VEGF comprising residues F17, M18, D19, Y21, Y25, Q89, I91, K101, E103, and C104.

[0181] According to the present invention, a "functional epitope" refers to an amino acid residue in an antigen that strongly contributes to antibody binding. Mutation of any of the contributing residues in the antigen (e.g., alanine or homolog mutation to wild-type VEGF) will disrupt antibody binding, resulting in a relative affinity ratio (IC50 mutant VEGF / IC50 wild-type VEGF) greater than 5 (see Example 2 of WO2005 / 012359). In one embodiment, the relative affinity ratio is determined by solution-bound phage display ELISA. Briefly, 96-well Maxisorp immunoplates (NUNC) are coated with the test antibody in Fab format at a concentration of 2 μg / ml in PBS overnight at 4°C and blocked with PBS, 0.5% BSA, and 0.05% Tween 20 (PBT) for 2 hours at room temperature. Serial dilutions of phage displaying hVEGF alanine point mutants (residues 8-109) or wild-type hVEGF (8-109) in PBT were first incubated on Fab-coated plates at room temperature for 15 minutes. The plates were then washed with PBS, 0.05% Tween 20 (PBST). Bound phage were detected using an anti-M13 monoclonal antibody-horseradish peroxidase (Amersham Pharmacia) conjugate diluted 1:5000 in PBT. Color was developed with 3,3',5,5'-tetramethylbenzidine (TMB, Kirkegaard & Perry Labs, Gaithersburg, MD) substrate for approximately 5 minutes, quenched with 1.0 M H3PO4, and read spectrophotometrically at 450 nm. The ratio of IC50 values ​​(IC50,ala / IC50,wt) represents the fold reduction in binding affinity (relative binding affinity).

[0182] (iii) VEGF receptor molecules

[0183] Two VEGF receptors have been identified: Flt-1 (also known as VEGFR-1) and KDR (also known as VEGFR-2). Shibuya et al. (1990) Oncogene 8:519-527; de Vries et al. (1992) Science 255:989-991; Terman et al. (1992) Biochem. Biophys. Res. Commun. 187:1579-1586. Each receptor has different specificities for each VEGF family member, but VEGF-A binds to both Flt-1 and KDR. Neuropilin-1 has been shown to be a selective VEGF receptor, capable of binding to the heparin-binding VEGF isoform (Soker et al. (1998) Cell 92:735-45). Both Flt-1 and KDR belong to the receptor tyrosine kinase (RTK) family. RTKs comprise a large family of transmembrane receptors with diverse biological activities. Currently, at least nineteen (19) unique RTK subfamilies have been identified. The receptor tyrosine kinase (RTK) family includes receptors that are crucial for the growth and differentiation of a variety of cell types (Yarden and Ullrich (1988) Ann. Rev. Biochem. 57: 433-478; Ullrich and Schlessinger (1990) Cell 61: 243-254). The intrinsic function of RTKs is activated upon ligand binding, leading to phosphorylation of the receptor and a variety of cellular substrates, which subsequently leads to a variety of cellular responses (Ullrich & Schlessinger (1990) Cell 61: 203-212). Thus, receptor tyrosine kinase-mediated signal transduction is initiated by extracellular interaction with specific growth factors (ligands), which is generally followed by receptor dimerization, stimulation of intrinsic protein tyrosine kinase activity, and receptor transphosphorylation. This creates binding sites for intracellular signal transduction molecules and leads to the formation of complexes with a range of cytoplasmic signaling molecules, driving appropriate cellular responses. (e.g., cell division, differentiation, metabolic effects, changes in the extracellular microenvironment) See Schlessinger and Ullrich (1992) Neuron 9:1-20. Structurally, both Flt-1 and KDR have seven immunoglobulin-like domains in the extracellular domain, a single transmembrane region, and a consensus tyrosine kinase sequence interrupted by a kinase insert domain. Matthews et al. (1991) Proc. Natl. Acad. Sci. USA 88:9026-9030; Terman et al. (1991) Oncogene 6:1677-1683.

[0184] VEGF receptor molecules or fragments thereof that specifically bind to VEGF can be used in the methods of the present invention to bind to and sequester the VEGF protein, thereby preventing it from signaling. In certain embodiments, the VEGF receptor molecule or VEGF-binding fragment thereof is a soluble form, such as sFlt-1. The soluble form of the receptor exerts an inhibitory effect on the biological activity of the VEGF protein by binding to VEGF, thereby preventing it from binding to its native receptor present on the surface of target cells. VEGF receptor fusion proteins are also included, examples of which are described below.

[0185] A chimeric VEGF receptor protein refers to a receptor molecule that has amino acid sequences derived from at least two different proteins (at least one of which is a VEGF receptor protein, such as flt-1 or KDR receptor) and is capable of binding to and inhibiting the biological activity of VEGF. In certain embodiments, the chimeric VEGF receptor protein of the present invention is composed of amino acid sequences derived from only two different VEGF receptor molecules; however, an amino acid sequence comprising one, two, three, four, five, six, or all seven Ig-like domains from the extracellular ligand-binding region of the flt-1 and / or KDR receptor can be linked to an amino acid sequence from another unrelated protein, such as an immunoglobulin sequence. Other amino acid sequences that can be combined with Ig-like domains will be apparent to those of ordinary skill in the art. Examples of chimeric VEGF receptor proteins include, for example, soluble Flt-1 / Fc, KDR / Fc, or Flt-1 / KDR / Fc (also known as VEGF Trap) (see, for example, PCT Application Publication No. WO 97 / 44453).

[0186] The soluble VEGF receptor proteins or chimeric VEGF receptor proteins of the present invention include VEGF receptor proteins that are not anchored to the cell surface via a transmembrane domain. Thus, soluble forms of VEGF receptors (including chimeric receptor proteins), while capable of binding and inactivating VEGF, do not contain a transmembrane domain and thus generally do not become associated with the cell membrane of cells in which the molecule is expressed.

[0187] III. Therapeutic Uses of Anti-VEGF Antibodies

[0188] The present invention encompasses anti-angiogenic therapy, a novel cancer treatment strategy that aims to inhibit the development of tumor blood vessels required to support tumor growth and provide nutrients. Because angiogenesis is involved in both primary tumor growth and metastasis, the anti-angiogenic therapy provided by the present invention can not only inhibit the neoplastic growth of tumors at the primary site, but also prevent the metastasis of tumors at secondary sites, thereby allowing the tumor to be attacked by other therapeutic agents. In addition, ovarian cancer is associated with high levels of circulating vascular endothelial growth factor (VEGF), a protein involved in tumor growth and spread. Studies of women with ovarian cancer have shown an association between high levels of VEGF and poor prognosis (Alvarez A et al. 1999 Clin Cancer Res.; 5: 587-591; Yamamoto S et al. 1997 Br J Cancer; 76: 1221-1227).

[0189] Specifically, in one embodiment, the present invention provides a method of treating a patient diagnosed with (optionally newly diagnosed) previously untreated ovarian cancer, comprising subjecting the patient to a treatment regimen that combines at least chemotherapy concurrently with the administration of an effective amount of an anti-VEGF antibody, followed by anti-VEGF maintenance therapy. In certain embodiments of the present invention, the patient has stage III (sub optimally and macroscopic optimally debulked) or stage IV epithelial ovarian primary peritoneal or fallopian tube cancer. In other embodiments, the patient has stage I and IIa (clear cell carcinoma or grade 3 only) or stage IIb-IV epithelial ovarian, fallopian tube, or primary peritoneal cancer. In another embodiment, the present invention provides a method of treating a patient diagnosed with recurrent or previously treated ovarian cancer, comprising subjecting the patient to a treatment regimen that combines at least chemotherapy concurrently with the administration of an effective amount of an anti-VEGF antibody, followed by anti-VEGF maintenance therapy.

[0190] Combination therapy

[0191] The present invention features the use of at least one VEGF-specific antagonist in combination with one or more other anti-cancer therapies, followed by anti-VEGF maintenance therapy. Examples of anti-cancer therapies include, but are not limited to, surgery, radiation therapy (radiotherapy), biological therapy, immunotherapy, chemotherapy, or combinations of these therapies. In addition, cytotoxic agents, anti-angiogenic agents, and anti-proliferative agents can be used in combination with VEGF-specific antagonists.

[0192] In certain aspects, the present invention provides a method of treating ovarian cancer by administering an effective amount of an anti-VEGF antibody and one or more chemotherapeutic agents to a patient susceptible to or diagnosed with previously untreated ovarian cancer or recurrent ovarian cancer. A variety of chemotherapeutic agents can be used in the combination treatment methods of the present invention. An exemplary and non-limiting list of chemotherapeutic agents encompassed by the present invention is provided herein under "Definitions" or described herein.

[0193] In one example, the invention features the use of a VEGF-specific antagonist and one or more chemotherapeutic agents (e.g., a mixture) or any combination thereof. In certain embodiments, the chemotherapeutic agent is, for example, a taxane, paclitaxel, docetaxel, or a protein-bound particle of paclitaxel (e.g., ), platinum analogs, carboplatin, gemcitabine, or a combination thereof. In one embodiment, the chemotherapeutic agent is carboplatin and paclitaxel or docetaxel. In another embodiment, the chemotherapeutic agent is carboplatin and gemcitabine. Combination administration includes simultaneous administration, use of separate formulations or a single drug formulation, and sequential administration in either order, wherein preferably there is a period of time for all active agents to simultaneously exert their biological activities, followed by maintenance therapy with a VEGF-specific antagonist, such as Figure 1 、 Figure 2 ,or Figure 8 or Figure 11 The preparation and dosing schedule of such chemotherapeutic agents may be used according to the manufacturer's instructions or determined empirically by a skilled practitioner. The preparation and dosing schedule of chemotherapy is also described in Chemotherapy Service Ed., M.C. Perry, Williams & Wilkins, Baltimore, Md. (1992). The chemotherapeutic agent may be administered before or after the VEGF-specific antagonist, or may be administered simultaneously with it. In certain embodiments of the present invention, the dosing schedule and amount are as follows: Figure 1 、 Figure 2 or Figure 8 or Figure 11 Listed in.

[0194] In some other aspects, other therapeutic agents useful for combination tumor therapy with the antibodies of the present invention include antagonists of other factors involved in tumor growth such as EGFR, ErbB2 (also known as Her2), ErbB3, ErbB4, or TNF. Sometimes, it may be beneficial to also administer one or more cytokines to the patient. In one embodiment, the anti-VEGF antibody is co-administered with a growth inhibitor. For example, the growth inhibitor can be administered first, followed by the VEGF antibody. However, simultaneous administration or administration of the VEGF antibody first is also contemplated. Suitable dosages of the growth inhibitor are those currently used and may be reduced due to the combined action (synergy) of the growth inhibitor and the anti-VEGF antibody.

[0195] The formulations herein may also contain more than one active compound as necessary for the specific indication being treated, preferably with complementary activities that do not adversely affect each other. For example, it may be desirable to further provide in a formulation an antibody that binds to EGFR, VEGF (e.g., an antibody that binds to a different epitope on VEGF), VEGFR, or ErbB2 (e.g., ) or another antibody used in an oncology indication. Alternatively, or in addition, the composition may include a cytotoxic agent, a cytokine, a growth inhibitory agent, and / or a small molecule VEGFR antagonist. Suitably, such molecules are present in combination in amounts effective for their intended purpose.

[0196] In certain embodiments, VEGF antagonists (e.g., anti-VEGF antibodies) are used to treat ovarian cancer. In certain embodiments, VEGF antagonists (e.g., anti-VEGF antibodies) are combined with carboplatin and paclitaxel, followed by anti-VEGF maintenance therapy. In certain embodiments, VEGF antagonists (e.g., anti-VEGF antibodies) are combined with cisplatin and paclitaxel, followed by anti-VEGF maintenance therapy. In certain embodiments, VEGF antagonists (e.g., anti-VEGF antibodies) are combined with carboplatin and docetaxel, followed by anti-VEGF maintenance therapy. In certain embodiments, VEGF antagonists (e.g., anti-VEGF antibodies) are combined with carboplatin and gemcitabine, followed by anti-VEGF maintenance therapy.

[0197] In certain aspects, other therapeutic agents useful for combination cancer therapy with the antibodies of the invention include other anti-angiogenic agents. Many anti-angiogenic agents have been identified and are known in the art, including those listed by Carmeliet and Jain (2000). In one embodiment, the anti-VEGF antibodies of the invention are used in combination with another VEGF antagonist or VEGF receptor antagonist, such as a VEGF variant, a soluble VEGF receptor fragment, an aptamer capable of blocking VEGF or VEGFR, a neutralizing anti-VEGFR antibody, a low molecular weight inhibitor of VEGFR tyrosine kinase, and any combination thereof. Alternatively, or in addition, two or more anti-VEGF antibodies may be co-administered to a patient.

[0198] For the prevention or treatment of disease, the appropriate dosage of the VEGF-specific antagonist will depend on the type of disease to be treated (as defined above), the severity and course of the disease, whether the VEGF-specific antagonist is administered for preventive or therapeutic purposes, prior therapy, the patient's clinical history and response to the VEGF-specific antagonist, and the judgment of the attending physician. The VEGF-specific antagonist can be appropriately administered to the patient at one time or over a series of treatments. In a combination therapy regimen, the VEGF-specific antagonist of the present invention and one or more anti-cancer therapeutic agents are administered in a therapeutically effective amount or a synergistic amount. As used herein, a therapeutically effective amount refers to an amount of the composition of the present invention that results in a reduction or inhibition of cancer as described above, or a co-administered amount of a VEGF-specific antagonist and one or more other therapeutic agents. A therapeutically synergistic amount refers to the amount of the VEGF-specific antagonist and one or more other therapeutic agents necessary to synergistically or significantly reduce or eliminate the conditions or symptoms associated with a particular disease or to prolong progression-free survival.

[0199] VEGF-specific antagonists and one or more other therapeutic agents can be administered simultaneously or sequentially in an amount and for a time sufficient to reduce or eliminate the occurrence or recurrence of tumors, dormant tumors, or micrometastases. VEGF-specific antagonists can be administered as maintenance therapy to prevent or reduce the likelihood of tumor recurrence or to prolong progression-free survival of patients.

[0200] As will be appreciated by those of ordinary skill in the art, appropriate dosages of chemotherapeutic agents or other anticancer agents will generally be approximately those already employed in clinical therapy (e.g., administering the chemotherapeutic agent alone or in combination with other chemotherapeutic agents). Variations in dosage are likely to occur depending on the condition being treated. The physician administering the treatment will be able to determine the appropriate dosage for the individual subject.

[0201] In addition to the above treatment options, patients may undergo radiation therapy.

[0202] In certain embodiments, the VEGF antibody administered is an intact naked antibody. However, the VEGF antibody may be conjugated to a cytotoxic agent. In certain embodiments, the conjugated antibody and / or its bound antigen is internalized by the cell, resulting in increased therapeutic efficacy of the conjugate in killing the cancer cells to which it binds. In one embodiment, the cytotoxic agent targets or interferes with nucleic acids in cancer cells. Examples of such cytotoxic agents include maytansinoids, calicheamicins, ribonucleases, and DNA endonucleases.

[0203] The present invention also features a method of instructing a human subject having ovarian cancer to receive treatment with an anti-VEGF antibody, thereby extending progression-free survival, reducing the likelihood of cancer recurrence in the subject, or increasing the likelihood of survival. In some embodiments, the method further comprises providing instructions to receive treatment with at least one chemotherapeutic agent, followed by anti-VEGF maintenance therapy. In some embodiments, the method further comprises providing instructions to receive two or more chemotherapeutic agents, followed by anti-VEGF maintenance therapy. Treatment with the anti-VEGF antibody can be performed concurrently with treatment with the chemotherapeutic agent. In certain embodiments, the subject is treated as directed by the method. Treatment of ovarian cancer by administering the anti-VEGF antibody, with or without chemotherapy, can continue until cancer recurrence or death.

[0204] In certain embodiments of the invention, the patient is treated with at least 16 cycles of anti-VEGF therapy following concurrent therapy with a chemotherapeutic agent.In other embodiments of the invention, the patient is treated with at least 12 cycles of anti-VEGF therapy following concurrent therapy with a chemotherapeutic agent.

[0205] The present invention further provides a method of promoting the administration of an anti-VEGF antibody for the treatment of ovarian cancer in a human subject. In some embodiments, the method further comprises promoting the administration of at least one chemotherapeutic agent followed by anti-VEGF maintenance therapy. In some embodiments, the method further comprises promoting the administration of two or more chemotherapeutic agents followed by anti-VEGF maintenance therapy. Administration of the anti-VEGF antibody can be performed concurrently with administration of the chemotherapeutic agent. Promotion can be performed by any available means. In some embodiments, promotion is provided via a package insert accompanying a commercial formulation of the anti-VEGF antibody. Promotion can also be provided via a package insert accompanying a commercial formulation of the chemotherapeutic agent. Promotion can be provided by written or oral communication to a physician or healthcare provider. In some embodiments, promotion is provided via a package insert providing instructions for receiving ovarian cancer therapy with the anti-VEGF antibody. In yet another embodiment, the package insert includes some or all of the results from Example 1, Example 2, or Example 3. In some embodiments, promotion is followed by treatment of the subject with the anti-VEGF antibody with or without the chemotherapeutic agent.

[0206] The present invention provides a business method comprising marketing an anti-VEGF antibody for use in treating ovarian cancer in a human subject, thereby extending the subject's progression-free survival time, reducing the likelihood of cancer recurrence in the subject, or increasing the likelihood of survival in the subject. In some embodiments, the method further comprises marketing a chemotherapeutic agent for use in combination with the anti-VEGF antibody, followed by anti-VEGF maintenance therapy. In some embodiments, the marketing is followed by treating the subject with the anti-VEGF antibody with or without the chemotherapeutic agent, followed by anti-VEGF maintenance therapy. In some embodiments, the method further comprises marketing two or more chemotherapeutic agents for use in combination with the anti-VEGF antibody, followed by anti-VEGF maintenance therapy. In some embodiments, the marketing is followed by treating the subject with the anti-VEGF antibody with or without the chemotherapeutic agent, followed by anti-VEGF maintenance therapy.

[0207] Also provided is a business method comprising marketing a chemotherapeutic agent in combination with an anti-VEGF antibody for treating ovarian cancer in a human subject, thereby extending the time the subject survives without progression, reducing the likelihood that the subject's cancer will recur, or increasing the likelihood that the subject will survive. In some embodiments, marketing is followed by treating the subject with a combination of the chemotherapeutic agent and the anti-VEGF antibody, followed by anti-VEGF maintenance therapy. Also provided is a business method comprising marketing two or more chemotherapeutic agents in combination with an anti-VEGF antibody, followed by anti-VEGF maintenance therapy, for treating ovarian cancer in a human subject, thereby extending the time the subject survives without progression, reducing the likelihood that the subject's cancer will recur, or increasing the likelihood that the subject will survive. In some embodiments, marketing is followed by treating the subject with a combination of the chemotherapeutic agent and the anti-VEGF antibody, followed by anti-VEGF maintenance therapy.

[0208] IV. Dosage and Duration

[0209] VEGF-specific antagonist compositions will be formulated, dosed, and administered in a manner consistent with good medical practice. Factors considered in this context include the specific condition being treated, the specific subject being treated, the individual patient's clinical condition, the cause of the condition, the site of delivery of the agent, the method of administration, the schedule of administration, and other factors known to medical practitioners. The "therapeutically effective amount" of the VEGF-specific antagonist to be administered will be determined by such considerations and is the minimum amount necessary to prevent, ameliorate, or treat, or stabilize cancer; prolong the time to progression (progression-free survival duration); or treat or prevent the development or recurrence of a tumor, dormant tumor, or micrometastasis. VEGF-specific antagonists are not required but can optionally be formulated with one or more agents currently used to prevent or treat cancer or the risk of developing cancer. The effective amount of such other agents depends on the amount of VEGF-specific antagonist present in the formulation, the type of condition or treatment, and the other factors discussed above. These are generally used in the same dosages and by the same route of administration as previously used, or at approximately 1-99% of the previously employed dosage.

[0210] Depending on the type and severity of the disease, about 1 μg / kg to 100 mg / kg (e.g., 0.1 mg / kg-20 mg / kg) of VEGF-specific antagonist is administered to the patient as an initial candidate dose, whether, for example, by one or more separate administrations or by continuous infusion. A typical daily dose range can be about 1 μg / kg to about 100 mg / kg or more, depending on the factors described above. Particularly desirable doses include, for example, 5 mg / kg, 7.5 mg / kg, 10 mg / kg, and 15 mg / kg. For repeated administration over several days or longer, depending on the condition, treatment is continued until the cancer is treated, as measured by methods described above or known in the art. However, other dosage regimens may be useful. In one example, if the VEGF-specific antagonist is an antibody, the antibody of the invention is administered once every week, every two weeks, or every three weeks at a dose range of about 5 mg / kg to about 15 mg / kg, including but not limited to 5 mg / kg, 7.5 mg / kg, 10 mg / kg, or 15 mg / kg. The progress of the therapies of the invention can be readily monitored by conventional techniques and assays.

[0211] In other embodiments, such dosing regimens are used in combination with chemotherapy regimens (including but not limited to one or more chemotherapeutic agents) as first-line therapy for treating previously untreated ovarian cancer, followed by maintenance therapy. In other embodiments, such dosing regimens are used in combination with chemotherapy regimens (including but not limited to one or more chemotherapeutic agents) as second-line therapy for treating recurrent ovarian cancer, followed by maintenance therapy. Further information on appropriate dosages is provided in the Examples below.

[0212] The duration of treatment will continue for as long as the physician dictates or until the desired therapeutic effect (e.g., those described herein) is achieved. In certain embodiments, VEGF-specific antagonist therapy continues for a period of 1 month, 2 months, 4 months, 6 months, 8 months, 10 months, 1 year, 2 years, 3 years, 4 years, 5 years, or up to the lifetime of the subject. In certain embodiments, anti-VEGF therapy continues for at least 16 cycles following concurrent anti-VEGF therapy with a chemotherapeutic agent. In other embodiments, anti-VEGF therapy continues for at least 12 cycles following concurrent anti-VEGF therapy with a chemotherapeutic agent.

[0213] The VEGF-specific antagonists of the present invention are administered to a subject (e.g., a human patient) according to known methods, such as intravenous administration (e.g., bolus injection or continuous infusion over a period of time), by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intraarticular, intrasynovial, intrathecal, oral, topical, or inhalation routes. Topical administration is particularly desirable if VEGF antagonism is associated with widespread side effects or toxicity. Ex vivo strategies can also be used for therapeutic applications. Ex vivo strategies involve transfecting or transducing cells obtained from a subject with a polynucleotide encoding a VEGF antagonist. The transfected or transduced cells are then returned to the subject. The cells can be any of a wide variety of types, including but not limited to hematopoietic cells (e.g., bone marrow cells, macrophages, monocytes, dendritic cells, T cells, or B cells), fibroblasts, epithelial cells, endothelial cells, keratinocytes, or muscle cells.

[0214] For example, if the VEGF-specific antagonist is an antibody, the antibody is administered by any suitable means, including parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal, and (if local immunosuppressive therapy is desired) intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Additionally, it is suitable to administer the antibody by pulse infusion, particularly using decreasing doses of the antibody. Preferably, the dosage is administered by injection, and most preferably, the dosage is administered by intravenous or subcutaneous injection, depending in part on whether the administration is short-term or long-term.

[0215] In another example, when the condition or tumor location permits, the VEGF-specific antagonist compound is administered locally, for example, by direct injection, and the injection can be repeated periodically. The VEGF-specific antagonist can also be delivered systemically / systemically to a subject after surgical resection of a tumor or directly to tumor cells, such as a tumor or tumor bed, to prevent or reduce local recurrence or metastasis (e.g., dormant tumors or micrometastases).

[0216] Alternatively, an inhibitory nucleic acid molecule or polynucleotide containing a nucleic acid sequence encoding a VEGF-specific antagonist can be delivered to appropriate cells in a subject. In certain embodiments, the nucleic acid can be directed to the tumor itself.

[0217] Nucleic acids can be introduced into cells by any means appropriate for the vector employed. Many such methods are known in the art (Sambrook et al., supra, and Watson et al., Recombinant DNA, Chapter 12, 2d edition, Scientific American Books, 1992). Examples of gene delivery methods include liposome-mediated transfection, electroporation, calcium phosphate / DEAE dextran, gene guns, and microinjection.

[0218] V. Pharmaceutical Formulations

[0219] Therapeutic formulations of the antibodies used in accordance with the present invention are prepared for storage in the form of lyophilized formulations or aqueous solutions by mixing the antibody having the desired purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations employed and include buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl alcohol or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) (c) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as TWEEN®. TM 、PLURONICS TMor polyethylene glycol (PEG). Preferred lyophilized anti-VEGF antibody formulations are described in WO 97 / 04801, which is expressly incorporated herein by reference. Optionally, the formulation contains a pharmaceutically acceptable salt, typically sodium chloride, and preferably at approximately physiological concentrations. Optionally, the formulations of the present invention may contain a pharmaceutically acceptable preservative. In some embodiments, the concentration of the preservative ranges from 0.1-2.0%, typically v / v. Suitable preservatives include those known in the pharmaceutical art. Benzyl alcohol, phenol, m-cresol, methylparaben, and propylparaben are examples of preservatives. Optionally, the formulations of the present invention may include a pharmaceutically acceptable surfactant at a concentration of 0.005-0.02%.

[0220] Typically, bevacizumab is supplied in 100 mg and 400 mg preservative-free, single-use vials to deliver 4 ml or 16 ml of bevacizumab (25 mg / ml) for therapeutic use. A 100 mg dose is prepared in 240 mg of dehydrated α,α-trehalose, 23.2 mg of sodium phosphate (monobasic, monohydrate), 4.8 mg of sodium phosphate (dibasic, anhydrous), 1.6 mg of polysorbate 20, and Water for Injection, USP. A 400 mg dose is prepared in 960 mg of dehydrated α,α-trehalose, 92.8 mg of sodium phosphate (monobasic, monohydrate), 19.2 mg of sodium phosphate (dibasic, anhydrous), 6.4 mg of polysorbate 20, and Water for Injection, USP.

[0221] Also see the bevacizumab label.

[0222] The formulations herein may also contain more than one active compound as necessary for the specific indication being treated, preferably with complementary activities that do not adversely affect each other. For example, it may be desirable to further provide in a formulation an antibody that binds to EGFR, VEGF (e.g., an antibody that binds to a different epitope on VEGF), VEGFR, or ErbB2 (e.g., Alternatively, or in addition, the composition may comprise a cytotoxic agent, a cytokine, a growth inhibitory agent, and / or a small molecule VEGFR antagonist. Suitably, such molecules are present in combination in amounts effective for the intended purpose.

[0223] The active ingredient can also be encapsulated in microcapsules (e.g., hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively) prepared, for example, by coacervation techniques or by interfacial polymerization, colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or macroemulsions. Such techniques are disclosed, for example, in Remington's Pharmaceutical Sciences, 16th edition, Osol, A., ed. (1980).

[0224] Sustained-release formulations can be prepared. Suitable examples of sustained-release formulations include solid hydrophobic polymer semipermeable matrices containing the antibody, which are in the form of shaped articles, such as films or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γ-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT®, and the like. TM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate) and poly-D-(-)-3-hydroxybutyric acid. Although polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid can release molecules for more than 100 days, some hydrogels release proteins for shorter periods of time. When encapsulated antibodies are maintained in vivo for a long time, they may denature or aggregate due to exposure to a humid environment at 37°C, resulting in loss of biological activity and possible changes in immunogenicity. Rational stabilization strategies can be designed based on the relevant mechanisms. For example, if the aggregation mechanism is found to be through the formation of intermolecular SS bonds via thiol-disulfide exchange, stabilization can be achieved by modifying the sulfhydryl groups, lyophilizing from acidic solutions, controlling humidity, using appropriate additives, and developing specific polymer matrix compositions.

[0225] Formulations to be used for in vivo administration should be sterile. This can be readily accomplished by filtration through sterile filtration membranes.

[0226] VI. Therapeutic Efficacy

[0227] A major advantage of the treatments of the present invention is the ability to produce significant anti-cancer effects in human patients without causing significant toxicity or adverse effects, resulting in an overall benefit for the patient. The efficacy of the treatments of the present invention can be measured by assessing various endpoints commonly used in cancer treatment, including, but not limited to, tumor regression, reduction in tumor weight or size, time to progression, duration of survival, progression-free survival, overall response rate, duration of response, and quality of life. Because the anti-angiogenic agents of the present invention target tumor vasculature and not necessarily the neoplastic cells themselves, they represent a unique class of anticancer drugs and, therefore, can employ unique measures and definitions of clinical response to the drugs. For example, a tumor reduction of greater than 50% in a two-dimensional analysis is a standard cutoff for declaring a response. However, the anti-VEGF antibodies of the present invention may cause inhibition of metastatic spread without shrinking the primary tumor, or may only exert a tumor-suppressing effect. Thus, other methods for determining the efficacy of anti-angiogenic therapy are optionally employed, including, for example, measuring plasma or urine markers of angiogenesis and measuring response via radiological imaging.

[0228] In another embodiment, the present invention provides a method for extending progression-free survival in human patients susceptible to or diagnosed with cancer. Time to disease progression is defined as the time from administration of the drug to disease progression or death. In a preferred embodiment, the present invention uses a combination treatment with an anti-VEGF antibody and one or more chemotherapeutic agents, followed by anti-VEGF maintenance therapy, to significantly extend progression-free survival by at least about 1 month, 2 months, 2.3 months, 2.9 months, 3 months, 3.8 months, preferably about 1 to about 6.1 months, compared to treatment without anti-VEGF antibody maintenance therapy. In one embodiment, the median PFS in months (95% CI) was extended by 3.8 months (0.717 (0.625, 0.824), one-sided p-value (log-rank) <0.001) in patients treated with bevacizumab and a taxane therapy (e.g., docetaxel or paclitaxel) and carboplatin, followed by anti-VEGF maintenance therapy, compared to controls. In another embodiment, the median difference in PFS in months (95% CI) between patients who received paclitaxel and carboplatin alone and patients who received paclitaxel, carboplatin, and an anti-VEGF antibody followed by anti-VEGF maintenance therapy was 2.3 months, HR=0.79, p-value (log-rank test) was 0.0010.

[0229] VII. Antibody Generation (i) Polyclonal Antibodies

[0230] Polyclonal antibodies are preferably raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant. It may be useful to conjugate the relevant antigen to a protein that is immunogenic in the species to be immunized, such as keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor, using bifunctional or derivatizing agents such as maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine ​​residues), N-hydroxysuccinimide (conjugation through lysine residues), glutaraldehyde, succinic anhydride, SOCl2, or R1N=C=NR (wherein R and R1 are different hydrocarbon groups).

[0231] Animals are immunized against antigens, immunogenic conjugates, or derivatives by mixing, for example, 100 μg or 5 μg of protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites. One month later, the animals are boosted with 1 / 5-1 / 10 of the initial amount of peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites. After 7-14 days, the blood of the animals is collected and the antibody titer of the serum is determined. The animals are boosted until the titer reaches a plateau. Preferably, the animals are boosted with the same antigen but with different proteins and / or conjugates obtained by coupling with different cross-linking agents. Conjugates can also be prepared as protein fusions in recombinant cell culture. Similarly, coagulants such as alum are appropriately used to enhance the immune response.

[0232] (ii) Monoclonal antibodies

[0233] There are various methods in the art for preparing monoclonal antibodies herein. For example, monoclonal antibodies can be produced using the hybridoma method originally described by Kohler et al., Nature, 256:495 (1975), or can be produced by recombinant DNA methods (U.S. Patent No. 4,816,567).

[0234] In the hybridoma method, a mouse or other suitable host animal, such as a hamster or macaque, is immunized as described above to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization. Alternatively, lymphocytes can be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103, Academic Press, 1986).

[0235] The hybridoma cells thus prepared are seeded and cultured in an appropriate culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridomas will typically contain hypoxanthine, aminopterin, and thymidine (HAT medium), which prevent the growth of HGPRT-deficient cells.

[0236] Preferred myeloma cells are those that fuse efficiently, support the selected antibody-producing cells to stably and at high levels produce antibodies, and are sensitive to culture media such as HAT culture media. Among them, preferred myeloma cell lines are murine myeloma lines, such as those from the MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center (Salk Institute Cell Distribution Center, San Diego, California USA) and SP-2 or X63-Ag8-653 cells available from the American Type Culture Collection (American Type Culture Collection, Rockville, Maryland USA). Human myeloma and mouse-human heteromyeloma cell lines can also be used to generate human monoclonal antibodies (Kozbor, J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63, Marcel Dekker, Inc., New York, 1987).

[0237] The culture medium in which the hybridoma cells are growing can be assayed for the production of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

[0238] After hybridoma cells that produce antibodies with the desired specificity, affinity, and / or activity are identified, the clones can be subcloned by limiting dilution procedures and cultured by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103, Academic Press, 1986). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. Alternatively, hybridoma cells can be cultured in vivo in animals as ascites tumors.

[0239] The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0240] The DNA encoding the monoclonal antibody is easy to separate and sequenced using conventional operating procedures (e.g., by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of the monoclonal antibodies). Hybridoma cells are a preferred source of this type of DNA. Once separated, the DNA can be placed in an expression vector, which is then transfected into a host cell that does not otherwise generate immunoglobulin proteins, such as Escherichia coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells, to obtain the synthesis of the monoclonal antibody in recombinant host cells. The recombinant production of antibodies is described in more detail below.

[0241] In another embodiment, antibodies or antibody fragments can be isolated from phage antibody libraries constructed using the techniques described in McCafferty et al., Nature, 348: 552-554 (1990). Clackson et al., Nature, 352: 624-628 (1991) and Marks et al., J. Mol. Biol., 222: 581-597 (1991) describe the isolation of mouse and human antibodies using phage libraries, respectively. Subsequent publications describe the generation of high-affinity (nM range) human antibodies by chain shuffling (Marks et al., Bio / Technology 10: 779-783 (1992)), as well as combinatorial infection and in vivo recombination as strategies for constructing very large phage libraries (Waterhouse et al., Nuc. Acids Res. 21: 2265-2266 (1993)). Thus, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for isolation of monoclonal antibodies.

[0242] The DNA also can be modified, for example, by substituting the coding sequences for human heavy and light chain constant domains in place of the homologous murine sequences (U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851 (1984)), or by covalently linking the immunoglobulin coding sequence to all or part of the coding sequence for a non-immunoglobulin polypeptide.

[0243] Typically, such non-immunoglobulin polypeptides are substituted for the constant domains of an antibody, or they are substituted for the variable domains of one antigen-binding site of an antibody to create a chimeric bivalent antibody comprising one antigen-binding site with specificity for one antigen and another antigen-binding site with specificity for a different antigen.

[0244] (iii) Humanized antibodies and human antibodies

[0245] Humanized antibodies have one or more amino acid residues from non-human sources that are imported therein. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from the "import" variable domain. Humanization can be performed essentially following the method of Winter and colleagues (Jones et al., Nature, 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)), i.e., rodent CDR or CDR sequence are substituted for the corresponding sequence of a human antibody. Therefore, such "humanized" antibodies are chimeric antibodies (U.S. Patent No. 4,816,567), in which a region substantially smaller than the entire human variable domain is substituted with a corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted with residues from similar sites in rodent antibodies.

[0246] The selection of human variable domains, including light and heavy chains, for preparing humanized antibodies is very important for reducing antigenicity. According to the so-called "best-fit" method, the variable domain sequence of a rodent antibody is used to screen the entire library of known human variable domain sequences. The human sequence closest to the rodent sequence is then selected as the human framework (FR) of the humanized antibody (Sims et al., J. Immunol., 151: 2296 (1993); Chothia et al., J. Mol. Biol., 196: 901 (1987)). Another method uses a specific framework derived from the consensus sequence of all human antibodies of a specific light or heavy chain subgroup. The same framework can be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89: 4285 (1992); Presta et al., J. Immunol., 151: 2623 (1993)).

[0247] More importantly, the antibody maintains high affinity for antigens and other favorable biological properties after humanization. To achieve this goal, according to a preferred method, humanized antibodies are prepared by using a three-dimensional model of the parent and humanized sequence to analyze the parent sequence and various conceptual humanized products. Three-dimensional immunoglobulin models are usually available and are familiar to those skilled in the art. Computer programs that illustrate and display the possible three-dimensional conformational structures of selected candidate immunoglobulin sequences can also be obtained. Checking these displayed images can analyze the possible effects of residues in the functioning of the candidate immunoglobulin sequence, i.e., analyze the residues that affect the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected from the receptor and the input sequence and combined to obtain desired antibody characteristics, such as improved affinity for the target antigen. Generally, CDR residues are directly and most substantially involved in the impact on antigen binding.

[0248] Humanized anti-VEGF antibodies and affinity matured variants thereof are described, for example, in US Pat. No. 6,884,879, issued Feb. 26, 2005.

[0249] It is now possible to generate transgenic animals (e.g., mice) that are able to generate a complete repertoire of human antibodies upon immunization in the absence of endogenous immunoglobulin production. For example, homozygous deletion of the antibody heavy chain joining region (JH) gene in chimeric and germline mutant mice has been described, resulting in complete inhibition of endogenous antibody production. Transferring the human germline immunoglobulin gene array in such germline mutant mice will result in the generation of human antibodies upon antigen attack. See, for example, Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90: 2551 (1993); Jakobovits et al., Nature, 362: 255-258 (1993); Bruggermann et al., Year in Immuno., 7: 33 (1993); and Duchosal et al., Nature, 355: 258 (1992).

[0250] Alternatively, phage display technology (McCafferty et al., Nature 348:552-553 (1990)) can be used to generate human antibodies and antibody fragments in vitro from repertoires of immunoglobulin variable domain (V) genes from unimmunized donors. According to this technology, the antibody variable domain genes are cloned into the major or minor coat protein genes of filamentous phages such as M13 or fd in a frame-matched manner and displayed as functional antibody fragments on the surface of the phage particles. Because the filamentous phage particles contain a single-stranded DNA copy of the phage genome, selection based on the functional properties of the antibody also results in selection of genes encoding antibodies that display those properties. In this way, phages mimic some of the characteristics of B cells. Phage display can be performed in a variety of forms, for review see, for example, Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology 3:564-571 (1993). Several sources of V gene segments can be used for phage display. Clackson et al., Nature 352:624-628 (1991) isolated a large number of different antibodies from a small random combinatorial library of V genes derived from the spleens of immunized mice. V gene repertoires can be constructed from unimmunized human donors and antibodies to a wide variety of antigens (including self-antigens) can be isolated by essentially following the techniques described in Marks et al., J. Mol. Biol. 222:581-597 (1991) or Griffith et al., EMBO J. 12:725-734 (1993). See also U.S. Patent Nos. 5,565,332 and 5,573,905.

[0251] As described above, human antibodies can also be generated by in vitro activated B cells (see US Pat. Nos. 5,567,610 and 5,229,275).

[0252] Human monoclonal anti-VEGF antibodies are described in U.S. Patent No. 5,730,977, issued March 24, 1998.

[0253] (iv) Antibody fragments

[0254] A variety of techniques have been developed for generating antibody fragments. Traditionally, these fragments have been derived by proteolytic digestion of intact antibodies (see, for example, Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992); and Brennan et al., Science, 229:81 (1985)). However, these fragments can now be generated directly by recombinant host cells. For example, antibody fragments can be isolated from the phage antibody library discussed above. Alternatively, Fab'-SH fragments can be directly recovered from Escherichia coli and chemically coupled to form F(ab')2 fragments (Carter et al., Bio / Technology 10:163-167 (1992)). According to another method, F(ab')2 fragments can be isolated directly from recombinant host cell cultures. Other techniques for generating antibody fragments will be apparent to skilled practitioners. In other embodiments, the selected antibody is a single-chain Fv fragment (scFv). See WO 93 / 16185.

[0255] (vi) Other amino acid sequence modifications

[0256] Amino acid sequence modifications of the antibodies described herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibody may be prepared by introducing appropriate nucleotide changes into the antibody nucleic acid or by peptide synthesis. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues within the antibody amino acid sequence. Any combination of deletions, insertions, and substitutions may be performed to obtain the final construct, provided that the final construct possesses the desired properties. Amino acid changes may also alter post-translational processing of the antibody, such as by changing the number or position of glycosylation sites.

[0257] A method that can be used to identify certain residues or regions in an antibody as preferred mutagenesis positions is called "alanine scanning mutagenesis," as described in Cunningham and Wells, Science 244: 1081-1085 (1989). Here, a residue or a group of target residues (e.g., charged residues, such as arginine, aspartic acid, histidine, lysine, and glutamic acid) are identified and replaced with neutral or negatively charged amino acids (most preferably alanine or polyalanine) to affect the interaction of the amino acid with the antigen. The amino acid positions that exhibit functional sensitivity to substitution are then refined by introducing more or other variants at or to the substitution site. Thus, although the site into which the amino acid sequence variation is introduced is predetermined, the nature of the mutation itself does not need to be predetermined. For example, in order to analyze the consequences of a mutation at a given site, alanine scanning mutagenesis or random mutagenesis is performed at the target codon or region, and the expressed antibody variants are screened for desired activity.

[0258] Amino acid sequence insertions include amino and / or carboxyl terminal fusions ranging in length from one residue to polypeptides comprising hundreds or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue or antibodies fused to a cytotoxic polypeptide. Other insertion variants of the antibody molecule include fusions of enzymes (e.g., for ADEPT) or polypeptides that extend the serum half-life of the antibody at the N- or C-terminus of the antibody.

[0259] Another type of variant is an amino acid substitution variant. These variants have at least one amino acid residue in the antibody molecule replaced with a different residue. The sites of greatest interest for substitutional mutagenesis include the hypervariable regions, but FR changes are also contemplated.

[0260] Substantial modification of the biological properties of the antibody is accomplished by selecting substitutions that differ significantly in their effect on maintaining: (a) the structure of the polypeptide backbone in the region of substitution, for example as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Amino acids can be grouped according to the similarity of their side chain properties as follows (Allehninger, Biochemistry, 2nd edition, pp. 73-75, Worth Publishers, New York, (1975)):

[0261] (1) Non-polar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M)

[0262] (2) Uncharged, polar: Gly(G), Ser(S), Thr(T), Cys(C), Tyr(Y), Asn(N), Gln(Q)

[0263] (3) Acidic: Asp (D), Glu (E)

[0264] (4) Basic: Lys(K), Arg(R), His(H)

[0265] Alternatively, naturally occurring residues can be grouped based on common side-chain properties as follows:

[0266] (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;

[0267] (2) Neutral and hydrophilic: Cys, Ser, Thr, Asn, Gln;

[0268] (3) Acidic: Asp, Glu;

[0269] (4) Basic: His, Lys, Arg;

[0270] (5) Residues that affect chain orientation: Gly, Pro;

[0271] (6) Aromatic: Trp, Tyr, Phe.

[0272] Non-conservative substitutions will entail exchanging a member of one of these classes for a member of another class.

[0273] Any cysteine ​​residue not involved in maintaining the correct conformation of the antibody may also be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent abnormal cross-linking. Conversely, cysteine ​​bond(s) may be added to the antibody to improve its stability (particularly when the antibody is an antibody fragment such as an Fv fragment).

[0274] A particularly preferred class of substitutional variants involves replacing one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). In general, the resulting variants selected for further development will have improved biological properties relative to the parent antibody from which they were generated. A convenient method for generating such substitutional variants involves affinity maturation using phage display. Briefly, several hypervariable region sites (e.g., 6-7 sites) are mutated to generate all possible amino acid substitutions at each site. The antibody variants thus generated are displayed in a monovalent form on filamentous phage particles as fusions with the M13 gene III product packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g., binding affinity) as disclosed herein. In order to identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues that contribute significantly to antigen binding. Alternatively or additionally, it may be beneficial to analyze the crystal structure of the antigen-antibody complex to identify contact points between the antibody and human VEGF. Such contact residues and adjacent residues are candidate sites for substitution according to the techniques detailed herein. Once such variants are generated, the panel of variants is screened as described herein, and antibodies with superior properties in one or more relevant assays can be selected for further development.

[0275] Another type of amino acid variant of an antibody alters the original glycosylation pattern of the antibody. Alteration means deleting one or more carbohydrate moieties found in the antibody and / or adding one or more glycosylation sites that are not present in the antibody.

[0276] Glycosylation of antibodies is typically either N-linked or O-linked. N-linked refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (wherein X is any amino acid except proline) are recognition sequences for enzymatic attachment of a carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these two tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, but 5-hydroxyproline or 5-hydroxylysine may also be used.

[0277] Addition of glycosylation sites to the antibody can be conveniently accomplished by altering the amino acid sequence to include one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). The alteration can also be made by adding or substituting one or more serine or threonine residues to the sequence of the original antibody (for O-linked glycosylation sites).

[0278] If the antibody contains an Fc region, the carbohydrates attached thereto may be modified. For example, U.S. Patent Application US2003 / 0157108 A1 (Presta, L.) describes antibodies having a mature carbohydrate structure lacking fucose attached to the Fc region of the antibody. See also US2004 / 0093621 A1 (Kyowa Hakko Kogyo Co., Ltd.). WO 03 / 011878 (Jean-Mairet et al.) and U.S. Patent No. 6,602,684 (Umana et al.) describe antibodies having bisecting N-acetylglucosamine (GlcNAc) among the carbohydrates attached to the Fc region of the antibody. WO 97 / 30087 (Patel et al.) reports antibodies having at least one galactose residue among the oligosaccharides attached to the Fc region of the antibody. See also WO 98 / 58964 (Raju, S.) and WO 99 / 22764 (Raju, S.) for antibodies having altered carbohydrates attached to their Fc regions.

[0279] It may be desirable to modify the antibodies of the present invention in terms of effector function, for example, to enhance the antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) of the antibody. This can be achieved by introducing one or more amino acid substitutions into the Fc region of the antibody. Alternatively or in addition, cysteine ​​residues can be introduced into the Fc region to allow the formation of interchain disulfide bonds in the region. The homodimeric antibodies thus produced may have improved internalization capacity and / or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al., J. Exp. Med. 176: 1191-1195 (1992) and Shopes, B., J. Immunol. 148: 2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumor activity can also be prepared using heterobifunctional cross-linkers as described in Wolff et al., Cancer Research 53:2560-2565 (1993). Alternatively, antibodies can be engineered to have dual Fc regions, thereby enhancing complement lysis and ADCC capabilities. See Stevenson et al., Anti-Cancer Drug Design 3:219-230 (1989).

[0280] WO 00 / 42072 (Presta, L.) describes antibodies with improved ADCC function in the presence of human effector cells, wherein the antibodies comprise amino acid substitutions in their Fc regions. Preferably, the antibodies with improved ADCC comprise substitutions at positions 298, 333, and / or 334 (Eu residue numbering) in the Fc region. Preferably, the altered Fc region is a human IgG1 Fc region comprising or consisting of substitutions at one, two, or three of these positions. Such substitutions are optionally combined with substitutions that improve C1q binding and / or CDC.

[0281] Antibodies with altered C1q binding and / or complement-dependent cytotoxicity (CDC) are described in WO 99 / 51642, U.S. Patent No. 6,194,551 Bl, U.S. Patent No. 6,242,195 Bl, U.S. Patent No. 6,528,624 Bl, and U.S. Patent No. 6,538,124 (Idusogie et al.), wherein the antibodies comprise amino acid substitutions at one or more of amino acids 270, 322, 326, 327, 329, 313, 333, and / or 334 (Eu numbering) in their Fc regions.

[0282] In order to extend the serum half-life of an antibody, a salvage receptor binding epitope can be incorporated into the antibody (particularly an antibody fragment), as described, for example, in U.S. Patent No. 5,739,277. As used herein, the term "salvage receptor binding epitope" refers to an epitope in the Fc region of an IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4) that is responsible for extending the serum half-life of the IgG molecule in vivo.

[0283] Antibodies with improved binding to the neonatal Fc receptor (FcRn) and extended half-life are described in WO 00 / 42072 (Presta, L.) and US 2005 / 0014934 A1 (Hinton et al.). These antibodies comprise an Fc region having one or more substitutions therein that improve Fc region binding to FcRn. For example, the Fc region may have a substitution at one or more of positions 238, 250, 256, 265, 272, 286, 303, 305, 307, 311, 312, 314, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, 428, or 434 (Eu numbering). Preferred Fc region-containing antibody variants with improved FcRn binding comprise amino acid substitutions at one, two or three of positions 307, 380 and 434 (Eu residue numbering) of the Fc region thereof. In one embodiment, the antibody has a 307 / 434 mutation.

[0284] Engineered antibodies with three or more (preferably four) functional antigen binding sites are also contemplated (US Patent Application US2002 / 0004587 A1, Miller et al.).

[0285] Nucleic acid molecules encoding amino acid sequence variants of antibodies can be prepared by a variety of methods known in the art, including but not limited to isolation from natural sources (in the case of naturally occurring amino acid sequence variants), or by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared antibody variant or non-variant version.

[0286] (v) Immunoconjugates

[0287] The invention also relates to immunoconjugates comprising an antibody described herein conjugated to a cytotoxic agent, such as a chemotherapeutic agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).

[0288] Chemotherapeutic agents that can be used to generate such immunoconjugates have been described above. Enzymatically active toxins and fragments thereof that can be used include diphtheria toxin A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, α-sarcin, Aleurites fordii toxin, dianthin toxin, Phytolaca americana toxin (PAPI, PAPII and PAP-S), Momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis toxin, scutellaria baicalensis ... officinalis inhibitors, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the trichothecenes. A variety of radionuclides are available for the production of radioconjugated antibodies. Examples include 212 Bi, 131 I. 131 In, 90 Y and 186 Re.

[0289] Conjugates of antibodies and cytotoxic agents can be prepared using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate hydrochloride), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis(p-diazoniumbenzoyl)ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionucleotides to antibodies. See WO 94 / 11026.

[0290] In another embodiment, antibodies can be coupled to a "receptor" (such as streptavidin) for tumor pre-targeting, wherein the antibody-receptor conjugate is administered to the patient, followed by clearance of unbound conjugate from the circulation using a clearing agent, and then administration of a "ligand" (e.g., avidin) coupled to a cytotoxic agent (e.g., radionucleotide).

[0291] (vi) Immunoliposomes

[0292] The antibodies disclosed herein can also be formulated into immunoliposomes. Liposomes containing antibodies can be prepared by methods known in the art, such as Epstein et al., Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA 77:4030 (1980); and U.S. Patent Nos. 4,485,045 and 4,544,545. Liposomes with extended circulation time are disclosed in U.S. Patent No. 5,013,556.

[0293] Particularly useful liposomes can be generated by reverse phase evaporation using a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). The liposomes are squeezed through a filter having a set pore size to produce liposomes with the desired diameter. Fab' fragments of the antibodies of the present invention can be coupled to the liposomes via a disulfide exchange reaction as described in Martin et al., J. Biol. Chem. 257: 286-288 (1982). Optionally, a chemotherapeutic agent (such as doxorubicin (doxorubicin)) can be included in the liposomes. See Gabizon et al., J. National Cancer Inst. 81 (19): 1484 (1989).

[0294] VIII. Products and Kits

[0295] In another embodiment of the present invention, an article of manufacture comprising a substance useful for treating the conditions described above is provided. The article of manufacture comprises a container, a label, and a package insert. Suitable containers include, for example, bottles, vials, syringes, and the like. The container can be made of a variety of materials, such as glass or plastic. The container contains a composition effective for treating the condition and can have a sterile access port (for example, the container can be an intravenous solution bag or vial with a stopper pierceable by a hypodermic needle). At least one active agent in the composition is an anti-VEGF antibody. A label affixed to or associated with the container indicates that the composition is used to treat the selected condition. The article of manufacture can further comprise a second container containing a pharmaceutically acceptable buffer, such as phosphate-buffered saline, Ringer's solution, and dextrose solution. It can also include other substances desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes. Additionally, the article of manufacture comprises a package insert with instructions for use, including, for example, instructions for the user of the composition to administer the anti-VEGF antibody composition and a chemotherapeutic agent, such as a taxane, paclitaxel, docetaxel, or protein-bound particles of paclitaxel (e.g., ), platinum analogs, carboplatin, gemcitabine, or a combination thereof, followed by anti-VEGF maintenance therapy. The package insert may optionally contain some or all of the results found in Example 1 or Example 2 or Example 3.

[0296] VEGF-specific antagonists can be packaged alone or in combination with other anti-cancer therapeutic compounds into a kit. The kit may include optional components that facilitate administration of the unit dose to a patient, such as vials for reconstitution of the powder form, syringes for injection, custom IV delivery systems, inhalers, etc. In addition, the unit dose kit may contain instructions for preparing and administering the composition. In certain embodiments, the instructions include instructions for use, including, for example, instructing the user of the composition to administer the anti-VEGF antibody composition and a chemotherapeutic agent, such as a taxane, paclitaxel, docetaxel, or a protein-bound particle of paclitaxel (e.g., ), platinum analogs, carboplatin, gemcitabine, or a combination thereof, followed by anti-VEGF maintenance therapy. The instructions may optionally contain some or all of the results found in Example 1, Example 2, or Example 3. The kit can be manufactured as a single-use unit dose for one patient, for multiple uses for a particular patient (at a constant dose or where the potency of the various compounds can vary as treatment progresses); or, the kit can contain multiple doses suitable for administration to multiple patients ("bulk packaging"). The kit components can be assembled in cartons, blister packs, bottles, tubes, and the like.

[0297] The following examples are intended only to illustrate the practice of the present invention and are not provided by way of limitation.The entire disclosures of all patent and scientific literature cited herein are expressly incorporated by reference.

[0298] Example

[0299] Example 1: Phase III Trial of Carboplatin and Paclitaxel Plus Placebo Versus Carboplatin and Paclitaxel Plus Concurrent Bevacizumab Followed by Placebo in Women with Newly Diagnosed, Previously Untreated, Stage III (Suboptimal and Macroscopically Optimal Massectomized) or Stage IV Epithelial Ovarian, Primary Peritoneal, or Fallopian Tube Cancer

[0300] Results from a phase III randomized study are presented to evaluate a new treatment procedure for patients with International Federation of Gynecologic Oncology (FIGO) stage III and IV epithelial ovarian, peritoneal primary, or fallopian tube cancer. The primary objectives included determining whether the addition of 5 concurrent cycles of bevacizumab to 6 cycles of standard therapy (carboplatin and paclitaxel) [Arm II] prolonged the duration of progression-free survival (PFS) compared with 6 cycles of standard therapy alone [Arm I] in women with newly diagnosed stage III (with any gross residual disease) and stage IV epithelial ovarian, primary peritoneal, or fallopian tube cancer; and determining whether the addition of 5 concurrent cycles of bevacizumab to 6 cycles of standard therapy (carboplatin and paclitaxel) plus extension of bevacizumab for 16 cycles [Arm III] prolonged progression-free survival compared with 6 cycles of standard therapy [Arm I] in women with newly diagnosed stage III (with any gross residual disease) and stage IV epithelial ovarian, primary peritoneal, or fallopian tube cancer.

[0301] GOG-0182-ICON5 is a 5-arm, randomized clinical trial that includes standard of care (carboplatin and paclitaxel) and four investigational arms incorporating gemcitabine, topotecan, and liposomal doxorubicin (either in combination with paclitaxel and carboplatin or sequentially). Major ovarian cancer clinical trial groups worldwide participated in the study. This international collaboration provided a unique opportunity to accrue a large number of patients in a timely manner, facilitating the simultaneous evaluation of multiple agents in a prospective, randomized trial. With international participation, more than 1,200 patients were accrued annually, and the trial reached its targeted accrual goal within four years of its initiation.

[0302] While the results of GOG-0182-ICON5 help establish optimal chemotherapy for previously untreated patients with advanced primary cancers of the ovary and peritoneum, the next generation of clinical trials will explore the impact of molecular targeted therapies in combination with chemotherapy. In particular, growth factor signaling inhibitors and anti-angiogenic agents (as single agents and in combination with chemotherapy drugs) are currently being tested in women with these tumors. Many of these agents have been shown to have cytostatic effects and to be synergistic with chemotherapy in experimental models of human cancer. In this Phase III trial, the effect of an active biologic agent on outcomes was evaluated in patients with advanced disease in combination with standard chemotherapy, with or without extended single-agent administration, compared with standard chemotherapy alone.

[0303] Bevacizumab is a recombinant humanized form of a murine anti-human VEGF monoclonal antibody, called rhuMAb VEGF. Bevacizumab has entered clinical development as a single agent for inducing tumor growth inhibition in patients with solid tumors and in combination with cytotoxic chemotherapy for prolonging the time to disease progression in patients with metastatic solid tumors. See, for example, Presta LG, et al. Humanization of an anti-vascular endothelial growth factor monoclonal antibody for the therapy of solid tumors and other disorders. Cancer Res 57:4593-9, 1997. Results from two single-agent trials of bevacizumab in patients with recurrent ovarian epithelial and peritoneal primary cancers have been published. See, e.g., Burger RA, et al, Phase II trial of bevacizumab in persistent or recurrent epithelial ovarian cancer or primary peritoneal cancer: a Gynecologic Oncology Group study. J Clin Oncol 25(33):5165-171, 2007; and Cannistra SA, et al., Phase II Study of Bevacizumab in Patients with Platinum Resistant Ovarian Cancer or Primary Peritoneal Serous Cancer. J Clin Oncol 25(33):5180-86. 2007. GOG (GOG-0170-D) used two co-primary efficacy endpoints: clinical response according to NCI RECIST criteria and the proportion of patients who survived for at least 6 months without progression. 62 participants received 15 mg / kg bevacizumab every 21 days until clinical or radiographic evidence of disease progression or unacceptable toxicity. Primary disease characteristics were typical of patients with recurrent ovarian cancer, with approximately 43% of patients considered to be primarily platinum-resistant. A 21% response rate was observed, with 40% remaining progression-free for at least 6 months, and a median PFS of 4.7 months, compared to 1.8 months in historical controls based on negative phase II trials of previous cytotoxic agents in a population with similar clinical characteristics.Genentech AVF 2949 examined patients with a higher risk profile in terms of disease progression and potential for adverse events, allowing only patients who were considered primary or secondary platinum-resistant and who had received 2 or 3 prior cytotoxic regimens. These differences in inclusion criteria ultimately translated into a higher level of platinum resistance, a greater number of prior regimens, and a slightly worse performance status in the AVF population. 44 patients were treated with the same bevacizumab dose and schedule as used in GOG 170-D. Responses were recorded in 7 patients (16%>), and 12 patients (27%) remained progression-free for at least 6 months.

[0304] In this study, two experimental arms were selected for comparison with standard cytotoxic chemotherapy with paclitaxel and carboplatin: one incorporating five cycles of bevacizumab (concurrent bevacizumab), and the other with an additional 16 cycles of bevacizumab after completing chemotherapy with paclitaxel and carboplatin (extended bevacizumab).

[0305] Administration and dosage Figure 1 and 2 The Calvert equation for carboplatin (AUC) dosing is shown below:

[0306] Total dose (mg) = target AUC (in mg / mL / min) * [GFR (in mL / min) + 25].

[0307] The statistical design of the primary endpoint study was based on 90% power to detect a PFS hazard ratio (HR) ≤ 0.77 (median PFS change: 14.0 months (historically) → 18.2 months). The primary analysis compared investigator-assessed PFS for each bevacizumab arm with control (Analysis 1 - by RECIST (see, e.g., Therasse et al, J Natl. Cancer Inst., 92:205-16, 2000), global clinical deterioration, or CA-125; or by Analysis 2 - RECIST or global clinical deterioration, examining CA-125). The baseline clinical characteristics of the patients are shown in Table 1. The baseline surgical-pathological characteristics of the patients are shown in Table 2.

[0308] Eligible patients: Patients had a histological diagnosis of epithelial ovarian cancer, peritoneal primary carcinoma, or fallopian tube cancer; FIGO stage III (with any gross (macroscopic or palpable) residual disease) or FIGO stage IV, as defined surgically at the completion of the initial abdominal surgery and with appropriate tissue available for histological evaluation. The minimum surgical procedure required was abdominal surgery to provide tissue for histological evaluation and to establish and document the primary site and stage, as well as maximal tumor resection. If additional surgery was performed, it should have been consistent with appropriate surgery for ovarian or peritoneal cancer as documented in the GOG Surgical Procedure Manual (https: / / www.gog.fccc.edu / manuals / pdf / surgman.pdf). However, surgeons were not required to perform all items included in this section of the GOG Surgical Procedure Manual. Patients with stage III cancer were defined as "optimal" if any residual tumor implant was no greater than 1 cm in maximum diameter at the completion of this initial surgery; all other cases were defined as "suboptimal." Detectable disease on postoperative imaging studies was not required for inclusion.

[0309] Patients with the following histologic epithelial cell types were included: serous adenocarcinoma, endometrioid adenocarcinoma, mucinous adenocarcinoma, undifferentiated carcinoma, clear cell adenocarcinoma, mixed epithelial carcinoma, transitional cell carcinoma, malignant Brenner's tumor, or adenocarcinoma not otherwise specified (NOS). However, the histologic features of the tumor had to be consistent with primary Mullerian epithelial adenocarcinoma. Patients could have coexistent fallopian tube carcinoma in situ, as long as the primary origin of the invasive tumor was the ovary, peritoneum, or fallopian tube.

[0310] Patients must have appropriate:

[0311] (1) Bone marrow function: Absolute neutrophil count (ANC) greater than or equal to 1,500 / μl, equivalent to Common Toxicity Criteria for Adverse Events v3.0 (CTCAE) grade 1. This ANC cannot be induced or suppressed by granulocyte colony-stimulating factor.

[0312] (2) Platelet count greater than or equal to 100,000 / μl (CTCAE grade 0-1).

[0313] (3) Renal function: Creatinine <1.5x the conventional upper limit of normal (ULN), CTCAE grade 1.

[0314] (4) Liver function:

[0315] (a) Bilirubin less than or equal to 1.5x ULN (CTCAE grade 1).

[0316] (b) SGOT and alkaline phosphatase less than or equal to 2.5x ULN (CTCAE grade 1).

[0317] (c) Neurological function: Neuropathy (sensory and motor) less than or equal to CTCAE grade 1.

[0318] (5) Coagulation parameters: PT such that the international normalized ratio (INR) is <1.5 (or an INR within the range, usually between 2 and 3, if the patient is taking a stable dose of therapeutic warfarin for the control of venous thrombosis (including pulmonary thromboembolism)) and PTT is <1.2x the upper limit of normal.

[0319] (6) Patients with GOG performance status of 0, 1, or 2.

[0320] (7) Patients must be enrolled between 1 and 12 weeks after initial surgery performed for the combined purposes of diagnosis, staging, and cytoreduction.

[0321] (8) Patients with detectable and undetectable disease were enrolled. Patients may or may not have cancer-related symptoms.

[0322] (9) Patients who meet the pre-entry requirements specified in Section 7.0.

[0323] (10) The patient or guardian must sign an informed consent and authorization form for personal health information.

[0324] (11) Patients in this trial could receive the lowest effective dose of ovarian estrogen + / - progestin replacement therapy (as indicated) at any time for control of menopausal symptoms, but did not take progestins for management of anorexia during protocol-directed therapy or until disease progression.

[0325] Patients Not Eligible: Patients with a current diagnosis of borderline epithelial ovarian tumor (formerly called "tumors of low malignant potential") or recurrent invasive epithelial ovarian cancer, primary peritoneal cancer, or fallopian tube cancer (treated only with surgery) (such as patients with grade 1a or 1b low-grade epithelial ovarian cancer or fallopian tube cancer) are not eligible. Patients with a prior diagnosis of borderline tumor (surgically resected and subsequently developed an unrelated, new invasive epithelial ovarian cancer, primary peritoneal cancer, or fallopian tube cancer) are eligible, provided they have not received prior chemotherapy for any ovarian tumor.

[0326] Patients who had received prior radiation therapy to any part of the pelvis or abdominal cavity were excluded. Prior radiation to localized cancer of the breast, head and neck, or skin was allowed provided it was completed more than three years before enrollment and the patient remained free of recurrent or metastatic disease.

[0327] Patients who had received prior chemotherapy for any abdominal or pelvic tumor (including neoadjuvant chemotherapy for their ovarian, primary peritoneal, or fallopian tube cancer) were excluded. Patients could have received prior adjuvant chemotherapy for localized breast cancer if it had been completed more than three years before enrollment and the patient remained free of recurrent or metastatic disease.

[0328] Patients who have received any targeted therapy (including but not limited to vaccines, antibodies, tyrosine kinase inhibitors) or hormonal therapy for the management of their epithelial ovarian cancer or primary peritoneal cancer.

[0329] Patients with synchronous primary endometrial cancer or a previous history of primary endometrial cancer were excluded unless all of the following criteria were met: stage no greater than IB; no invasion beyond superficial myometrium, without vascular or lymphatic invasion; and no poorly differentiated subtypes, including papillary serous, clear cell, or other FIGO grade 3 lesions.

[0330] Patients with other invasive malignancies, any evidence of other cancers (or history) within the last 5 years, or previous cancer treatment that contraindicates the use of this regimen were excluded, with the exception of non-melanoma skin cancer and the other specified malignancies mentioned above.

[0331] Patients with acute hepatitis or active infection requiring parenteral antibiotics.

[0332] Patients with severe non-healing wounds, ulcers, or fractures. This includes a history of abdominal fistula, gastrointestinal perforation, or intra-abdominal abscess within 28 days. Patients with granulated wounds that healed by secondary intention without evidence of surface dehiscence or infection are eligible, but require weekly wound inspections.

[0333] Patients with active bleeding or pathological conditions that carry a high risk of bleeding, such as known bleeding disorders, coagulopathy, or tumors involving major blood vessels.

[0334] Patients had a history or evidence of CNS disease on physical examination, including primary brain tumor, seizures not controlled by standard medical therapy, any brain metastases, or a history of cerebrovascular accident (CVA, stroke), transient ischemic attack (TIA), or subarachnoid hemorrhage within six months of the first day of treatment in this study.

[0335] Patients with clinically significant cardiovascular disease. This includes: uncontrolled hypertension, defined as systolic blood pressure >150 mm Hg or diastolic blood pressure >90 mm Hg; myocardial infarction or unstable angina <6 months prior to enrollment; New York Heart Association (NYHA) class II or greater congestive heart failure; severe arrhythmias requiring medication. This does not include asymptomatic atrial fibrillation with controlled ventricular rate; CTCAE class 2 or greater peripheral vascular disease (transient ischemia (at least <24 hours) without surgical intervention and without permanent defects); and history of CVA within six months.

[0336] Patients with known hypersensitivity to Chinese hamster ovary cell products or other recombinant human or humanized antibodies.

[0337] Patients with clinically significant proteinuria should be screened for urine protein by urine protein-creatinine ratio (UPCR). UPCR has been found to directly correlate with the amount of protein excreted in a 24-hour urine collection. See, for example, Ginsberg JM, et al., Use of single voided urine samples to estimate quantitative proteinuria. N Engl J Med 309:1543-6, 1983; Rodby RA, et al., The urine protein tocreatinine ratio as a predictor of 24-hour urine protein excretion in type1diabetic patients with nephropathy. The Collaborative Study Group. Am J KidneyPis 26:904-9,1995; Schwab SJ, et al, Quantitation of proteinuria by the use of protein-to-creatinine ratios in single urine samples. Arch Intern Med147:943-4,1987; Steinhauslin F, & Wauters JP. Quantitation of proteinuria in kidneytransplant patients: accuracy of the urinary protein / creatinine ratio. ClinNephrol 43:110-5,1995;Wilson DM, & Anderson RL. Protein-osmolality ratio for the quantitative assessment of proteinuria from a random urinalysis sample. Am JClin Pathol 100:419-24, 1993; and Zelmanovitz T, et al., Proteinuria is stilluseful for the screening and diagnosis of overt diabetic nephropathy. DiabetesCare 21:1076-9, 1998.Specifically, a UPCR of 1.0 is equivalent to 1.0 gram of protein in a 24-hour urine collection. Patients must have a UPCR < 1.0 to be admitted to the study.

[0338] Patients who have or are expected to have an invasive procedure as defined below: Major surgery, open biopsy, or major trauma within 28 days prior to Day 1 of bevacizumab / placebo therapy (Cycle 2). Major surgery anticipated during the study. This includes, but is not limited to, abdominal surgery (laparotomy or laparoscopy) prior to disease progression, such as colostomy or enterostomy reversal, interval or secondary cytoreductive surgery, or second look surgery. Core biopsy within 7 days prior to Day 1 of bevacizumab / placebo therapy (Cycle 2).

[0339] Patients with grade 3 or 4 GOG performance.

[0340] Pregnant or breastfeeding patients.

[0341] Patients under the age of 18.

[0342] Patients who have received prior therapy with any anti-VEGF drug, including bevacizumab.

[0343] Patients with clinical symptoms or signs of gastrointestinal obstruction and requiring parenteral hydration and / or nutrition.

[0344] Patients with other medical histories or conditions that, in the opinion of their physician, preclude study participation.

[0345] In this study, the international standards recommended by the Response Evaluation Criteria in Solid Tumors (RECIST) committee will be used to evaluate response and progression. See, for example, Therasse P, et al. New guidelines to evaluate the response to treatment in solid tumors. European Organization for Research and Treatment of Cancer, National Cancer Institute of the United States, National Cancer Institute of Canada. J Natl Cancer Inst 92: 205-16, 2000. In the RECIST criteria, only the change in the maximum diameter (one-dimensional measurement) of the tumor lesion is used.

[0346] CA-125 as a biomarker of progressive disease: Serum levels of CA-125, a tumor-associated glycoprotein antigen, are elevated in 80% of patients with epithelial ovarian cancer. See, for example, Bast et al, N. Engl. J. Med. 309:88307, 1983. CA-125 is often monitored frequently to assess response to treatment, the presence of residual disease, and as early evidence of recurrence. However, CA-125 is not entirely tumor-specific and can be elevated in a variety of benign conditions, such as endometriosis, uterine fibroids, and pelvic inflammatory disease; this is particularly true in premenopausal women. Furthermore, CA-125 levels can be inconsistent with tumor response, with both false-positive and false-negative tendencies; the impact of biologic agents on these inaccuracies is unclear. Regardless, it has become standard practice for patients and physicians to consider a gradual increase in CA-125 after treatment as evidence of recurrence or progressive disease and to base treatment decisions on CA-125. Current randomized trials use a conservative formula that defines progressive disease based on serial increases in CA-125 (in addition to other standard definitions in the management of solid tumors), but only after completion of initial chemotherapy. See, for example, Guppy et al., Oncologists, 7:437043, 2002; Rustin et al, J. Clin. Oncol. 19:4054-7, 2001; Rustin, J. Clin. Oncol, 21:187-93, 2003; Rustin et al, Clin. Cancer Res. 10:3919-26, 2004; and Rustin et al, J Natl. Cancer Inst., 96:487-8, 2004. In one example, progression based on serum CA-125 can only be determined during the period after completion of cytotoxic chemotherapy if one of three conditions is met: 1) a patient with an elevated CA-125 prior to treatment and whose CA-125 normalized must show evidence of a CA-125 greater than or equal to two times the upper limit of normal at two times at least one week apart; or 2) a patient with an elevated CA-125 prior to treatment that never normalized must show evidence of a CA-125 greater than or equal to two times the nadir at two times at least one week apart; or 3) a patient with a CA-125 in the normal range prior to treatment must show evidence of a CA-125 greater than or equal to two times the upper limit of normal at two times at least one week apart.

[0347] result

[0348] The results of the study demonstrated that bevacizumab is effective when combined with chemotherapy as a first-line ovarian cancer treatment and continued as maintenance therapy. This combination is effective in prolonging PFS. The preliminary safety assessment identified adverse events (AEs) associated with bevacizumab that were not documented in previous studies. The primary analysis of PFS showed a median progression-free survival (months) of 10.3 ( Figure 2 Branch 1), with Figure 2 Compare that to Branch 3's 14.1 months. Figure 2 The HR (95% CI) of the one-sided p-value (log-rank test was 0.08) for branch I was 0.908 (0.795, 1.04), which was consistent with the Figure 2 The one-sided p-value (log order < 0.001) for branch III is 0.717 (0.625, 0.824). Figure 5 The difference was statistically significant. The treatment regimen was generally well tolerated, and adverse events (including GI perforation) were similar to those in previous bevacizumab studies. Figure 3 and Figure 4 This is the first anti-angiogenic therapy to show benefit in this population. Figure 6 The diagram shows a branching pattern using CA-125 as a progression determinant. CA-125 is an antigenic determinant on a high molecular weight glycoprotein recognized by a monoclonal antibody (OC-125) generated using an ovarian cancer cell line as an immunogen. CA 125 is assessed as a serum marker to monitor patients with epithelial ovarian cancer and other cancers. See, for example, Gvn Oncol 38:373, 1990; Gyn Oncol 38:181, 1990; Amer J Ob Gyn 160:667, 1989; Amer J Ob Gvn 159:873, 1988; Amer J Ob Gvn 159:341, 1988; Ob Gvn 72:159, 1988; and Gyn Oncol 36:299, 1990, and descriptions therein. Figure 7 Subgroup analysis of clade I relative to clade III is shown.

[0349] Table 1: Baseline clinical characteristics

[0350]

[0351]

[0352] Table 2: Baseline surgical-pathological characteristics

[0353]

[0354] Example 2. A randomized, two-arm, multicenter Gynecologic Cancer Intergroup trial of bevacizumab added to standard chemotherapy (carboplatin and paclitaxel) in patients with epithelial ovarian cancer

[0355] The result from a phase III randomized study (ICON7) was presented to assess the safety and efficacy of bevacizumab added to the standard chemotherapy of carboplatin and paclitaxel. The primary endpoint was to determine that there was newly diagnosed, confirmed through histology, high-risk International Federation of Gynecology and Obstetrics (FIGO) I and IIa phase (3 grades or only clear cell carcinoma) and FIGO IIb-IV phase (all grades and all histological types) ovarian epithelial cancer, fallopian tube cancer or primary peritoneal cancer, experienced initial surgery (massive resection cytoreduction surgery or biopsy, if the patient has FIGO IV phase disease), and whether bevacizumab will be added to standard chemotherapy compared with independent standard chemotherapy before disease progression to improve progression-free survival (PFS). Secondary endpoints included overall survival (OS), response rate, response duration, no biological progression interval (defined as improving CA 125 or PFI Bio), safety and quality of life. ICON7 is a 2-arm randomized clinical trial comparing standard therapy (carboplatin and paclitaxel) with the incorporation of bevacizumab, in combination with paclitaxel and carboplatin, in an investigational arm (see Figure 8 A total of 1,528 women were enrolled in the trial.

[0356] Bevacizumab is a recombinant humanized form of a murine anti-human VEGF monoclonal antibody, called rhuMAb VEGF. Bevacizumab has entered clinical development as a single agent for inducing tumor growth inhibition in patients with solid tumors and in combination with cytotoxic chemotherapy for prolonging the time to disease progression in patients with metastatic solid tumors. See, for example, Presta LG, et al. Humanization of an anti-vascular endothelial growth factor monoclonal antibody for the therapy of solid tumors and other disorders. Cancer Res 57:4593-9, 1997.

[0357] Patient selection

[0358] ICON7 includes patients with newly diagnosed, histologically confirmed, high-risk FIGO stage I and IIa (grade 3 or clear cell carcinoma only) and FIGO stage IIb-IV (all grades and all histological types) epithelial ovarian, fallopian tube, or primary peritoneal cancer who have undergone primary surgery (massive resection cytoreductive surgery or biopsy if the patient has FIGO stage IV disease) and who will not be considered for cytoreductive surgery before disease progression. Patients with measurable and non-measurable disease are eligible. Patients are considered eligible for enrollment in this trial if they meet all of the inclusion criteria and none of the exclusion criteria described below:

[0359] Patient admission criteria:

[0360] Women ≥18 years of age

[0361] Histologically confirmed with a core biopsy from the diseased portion as a minimum requirement (cytology alone is insufficient for diagnosis)

[0362] ○Ovarian epithelial cancer

[0363] ○ Primary peritoneal carcinoma (must be of papillary-serous histology) or

[0364] ○Fallopian tube cancer

[0365] ● and meet the standards in Table 3

[0366] Patients with clear cell carcinoma of any stage were eligible because of the poor prognosis associated with this subtype. Patients with early-stage epithelial ovarian or fallopian tube cancer previously treated with surgery alone were eligible at the time of diagnosis of abdominal-pelvic recurrence, as long as no further interval cytoreductive therapy was planned before disease progression.

[0367] For the purposes of this trial, clear cell carcinoma was defined as the presence of >50% clear cell component or reported as clear cell carcinoma by the local pathologist.

[0368] Table 3: Histological inclusion criteria

[0369]

[0370] Grades are 1 (well differentiated), 2 (moderately differentiated), and 3 (poorly differentiated).

[0371] E = Regardless of FIGO stage, patients with clear cell carcinoma were excluded.

[0372] ● Patients should have undergone surgical resection by a surgeon experienced in the management of ovarian cancer, with the goal of maximal surgical cytoreduction in accordance with the GCIG Society Consensus Statement. No planned surgical resection should have been performed before disease progression.

[0373] ○ Patients with stage III and IV disease in whom initial surgical resection was inadequate were still eligible provided that

[0374] ■Patients have a histological diagnosis and

[0375] ■ No major resection before disease progression is anticipated

[0376] o Patients should be able to start systemic therapy within 8 weeks of cytoreductive surgery. If patients are randomized to a study arm, the first dose of bevacizumab must be omitted if the investigator decides to start chemotherapy within 4 weeks of surgery.

[0377] o If the patient had two surgeries, e.g., an initial surgery (removal of what was thought to be a benign cyst) followed by a second gynecologic-oncology surgery (formal staging and maximal resection of the ovarian tumor), the date of the second surgery was recorded as the surgery date; the first systemic therapy was started within 8 weeks of this date. In the case of a diagnosis of ovarian cancer, the date of diagnosis was recorded as the date of the initial surgery.

[0378] ECOG performance status (PS) 0-2

[0379] Life expectancy > 12 weeks

[0380] Adequate bone marrow function (postoperative blood tests / calculations of all parameters) (within 28 days prior to randomization)

[0381] Absolute neutrophil count (ANC) ≥ 1.5 x 10 9 / l

[0382] Platelets (PLT) > 100 x 10 9 / l

[0383] ○Hemoglobin (Hb)>9g / dl (may be a blood transfusion offspring)

[0384] Adequate coagulation function (postoperative blood tests / calculations of all parameters) (within 28 days prior to randomization)

[0385] ○ Activated prothromboplastin time (APTT) ≤ 1.5 x ULN; or

[0386] International normalized ratio (INR) ≤ 1.5 (INR measurement is mandatory if the patient is receiving warfarin)

[0387] Adequate liver function (post-procedural blood tests / calculations for all parameters) (within 28 days prior to randomization)

[0388] ○ Serum bilirubin (BR) ≤ 1.5x ULN

[0389] Serum transaminases ≤ 2.5x ULN

[0390] Proteinuria dipstick <2+. If the dipstick is ≥2+, then the 24-hour urine sample must demonstrate ≤1 g of protein in 24 hours.

[0391] ●Adequate renal function, defined as serum creatinine ≤2.0 mg / dL or ≤177 μmol / L. Patient exclusion criteria:

[0392] Non-ovarian epithelial cancers, including malignant mixed Mullerian tumors

[0393] Borderline tumors (tumors with low malignant potential)

[0394] Planned intraperitoneal cytotoxic chemotherapy

[0395] Prior systemic anticancer therapy for ovarian cancer (eg, chemotherapy, monoclonal antibody therapy, tyrosine kinase inhibitor therapy, or hormonal therapy)

[0396] Surgery (including open biopsy) within four weeks of the expected first dose of bevacizumab (omission of bevacizumab from the first cycle of chemotherapy is permitted)

[0397] Any planned surgery during the 58-week period from the start of study treatment (54 weeks of treatment plus an additional 4 weeks to allow for bevacizumab washout)

[0398] Uncontrolled hypertension (record blood pressure measurements after the patient has rested for 5 minutes and in the seated position) (persistent elevation of BP >150 / 100 mmHg despite antihypertensive therapy)

[0399] Any previous radiation therapy to the abdomen or pelvis

[0400] Major traumatic injury within 4 weeks before the potential first dose of bevacizumab

[0401] History or clinical suspicion of brain metastases or spinal cord compression. In case of suspected brain metastases, a brain CT / MRI is mandatory (within 4 weeks before randomization). In case of suspected spinal cord compression, a spinal cord MRI is mandatory (within 4 weeks before randomization).

[0402] History or evidence of central nervous system (CNS) disease (during neurologic examination), unless inadequately treated with standard medical therapy (eg, uncontrolled seizures)

[0403] Previous cerebrovascular accident (CVA), transient ischemic attack (TIA), or subarachnoid hemorrhage (SAH) within 6 months before randomization

[0404] Fertile women of childbearing potential who are unwilling to use adequate contraception (oral contraceptives, intrauterine devices, or barrier methods combined with spermicidal gel or surgical sterilization) for the duration of the study and for at least 6 months thereafter

[0405] Pregnant or breastfeeding women

[0406] Previous exposure to mouse CA 125 antibodies

[0407] Participated in another clinical trial or was treated with any other investigational agent within 30 days prior to entering this trial

[0408] ● Malignancies other than ovarian cancer within 5 years prior to randomization, with the exception of appropriately treated cervical carcinoma in situ and / or basal cell skin cancer and / or early endometrial cancer as specified below. Patients may have received prior adjuvant chemotherapy for other malignancies such as breast or colorectal cancer if diagnosed more than 5 years prior and without subsequent evidence of recurrence

[0409] ● Patients with synchronous primary endometrial cancer or a past history of primary endometrial cancer were excluded unless all of the following criteria describing endometrial cancer were met

[0410] ○ Stage <Ib

[0411] ○ No more than superficial myometrial invasion

[0412] ○ No lymphovascular invasion

[0413] ○ Not poorly differentiated (ie, not grade 3 or papillary serous or clear cell)

[0414] Known hypersensitivity to bevacizumab, its excipients, or chemotherapy (including Cremophor)

[0415] Non-healing wounds, ulcers, or fractures. Patients with granulated incisions that healed by secondary intention without evidence of surface dehiscence or infection were eligible, but required three-week wound inspections.

[0416] History or evidence of thrombotic or bleeding disorders

[0417] Clinically significant cardiovascular disease, including

[0418] ○ Myocardial infarction or unstable angina within 6 months of randomization

[0419] ○ New York Heart Association (NYHA) grade 2 or higher congestive heart failure (CHF)

[0420] ○ Poorly controlled arrhythmias despite medical therapy (patients with atrial fibrillation who had rate control were included)

[0421] ○ Grade 3 or higher peripheral vascular disease (ie, symptomatic and interfering with activities of daily living [ADL] requiring repair or revision)

[0422] Current or recent (within 10 days before cycle 1) chronic use of aspirin >325 mg / day (low-dose aspirin (<325 mg / day) has not been shown to increase the risk of grade 3-4 bleeding when used with bevacizumab plus chemotherapy; therefore, prophylactic low-dose aspirin use in patients at risk for arterial thromboembolic events was not prohibited in this trial protocol)

[0423] Current or recent (within 10 days prior to cycle 1) use of full-dose oral or parenteral anticoagulants or thrombolytics for therapeutic purposes (except for line patency, in which case the INR must be maintained below 1.5)

[0424] Preexisting grade 2 or higher sensory or motor neuropathy

[0425] Evidence of any other disease, metabolic disorder, physical examination finding, or clinical laboratory finding that raises reasonable suspicion of a disease or condition that contraindicates the use of the investigational drug or places the patient at high risk for treatment-related complications

[0426] Tumor assessments were performed on patients in the study arms after 3 and 6 cycles of chemotherapy, and around 9 and 12 months in the first year, or after the 12th or 18th cycle of treatment, using the RECIST criteria. Tumor assessments were repeated every 6 months during the second and third years of the trial and thereafter as clinically indicated. These scans were performed regardless of whether the patient had optimal or suboptimal resection and whether there was measurable disease at the time of the baseline scan.

[0427] Patients were evaluated clinically and had CA 125 measured at the beginning of each chemotherapy cycle and then every 6 weeks during the first year of the trial. Patients were evaluated and had CA 125 measured every 3 months during the second and third years of the trial. Patients were evaluated clinically and had CA 125 measured every 6 months during the fourth and fifth years of the trial. Thereafter, the evaluations were repeated annually. Progression based solely on CA 125 criteria was verified with a CT scan. If this was negative, it was repeated if clinical progression was suspected.

[0428] Patients were followed for survival and subsequent ovarian cancer treatment every 6 months during the first 5 years of follow-up and annually thereafter after evidence of protocol-defined disease progression.

[0429] Regular physical examinations and routine blood tests were performed during treatment to monitor patient safety. Quality of life (QoL) was assessed using the EORTC QLQ C-30+OV-28 and EQ-5D questionnaires at the beginning of each chemotherapy cycle, every 6 weeks until the end of the first year, and then before the start of treatment for progression or every 3 months until the end of the second year. A separate QoL form was completed by all patients who were still alive 3 years after randomization. Adverse events and healthcare resource utilization were recorded during study treatment and follow-up.

[0430] result

[0431] The results of the study demonstrated that bevacizumab is effective for first-line ovarian cancer when combined with chemotherapy and continued as maintenance therapy for a total duration of 12 months. This combination is effective in prolonging progression-free survival (PFS). The primary analysis of PFS showed that the median PFS in the chemotherapy arm (CP) was 16.0 months, compared with 18.3 months in the chemotherapy plus bevacizumab arm (CPB7.5+), with a p-value (log-rank test) of 0.0010. The hazard ratio (HR) (95% CI) was 0.79 (0.68; 0.91). The difference was significant. The PFS analysis is summarized in Figure 9 and 10 .

[0432] Baseline characteristics were as follows:

[0433] Table 4: Baseline Characteristics - Demographics

[0434]

[0435] Table 5: Baseline Characteristics - History of Ovarian Cancer

[0436]

[0437]

[0438] Table 6: Baseline Characteristics - History of Ovarian Cancer

[0439]

[0440] Table 7: Baseline Characteristics - Ovarian Cancer Surgery

[0441]

[0442] A preliminary assessment of adverse events with bevacizumab was consistent with previous studies.

[0443] Table 8: Overview of Adverse Events (AEs)

[0444]

[0445]

[0446] Example 3. A Phase III, Multicenter, Randomized, Blinded, Placebo-Controlled Trial of Carboplatin and Gemcitabine Plus Bevacizumab in Patients with Platinum-Sensitive Recurrent Ovarian, Primary Peritoneal, or Fallopian Tube Cancer

[0447] Epithelial ovarian carcinoma (EOC) and its histological and clinical equivalents, primary peritoneal carcinoma (PPC) and fallopian tube cancer, occur in the United States at an incidence of approximately 25,000 cases per year and result in approximately 14,000 deaths per year. Because the disease tends to be asymptomatic in the early stages, most patients initially present with advanced (stage III or IV) disease. Although EOC, PPC, and fallopian tube cancer are sensitive to a variety of chemotherapeutic agents, particularly taxanes and platinum compounds, only 20%-30% of patients presenting with stage III or IV disease are alive at 5 years. Patients with platinum-sensitive recurrent cancer (defined as disease recurrence more than 6 months after completion of a platinum-based chemotherapy regimen) have a higher initial response rate to chemotherapy; however, these patients are not considered curable. Recently, the U.S. Food and Drug Administration (FDA) approved gemcitabine chemotherapy in combination with carboplatin for recurrent platinum-sensitive disease. Carboplatin and gemcitabine resulted in statistically significant progression-free survival (PFS) compared to carboplatin alone in patients with platinum-sensitive disease. See, e.g., Pfisterer, Plante M, Vergote I, et al. Gemcitabine plus Carboplatin compared with carboplatin in patients with platinum-sensitive recurrent ovarian cancer: an intergroup trial of the AGO-OVAR, the NCIC CTG, and the EORTC GCG. J. Clin Oncol 2006; 24: 4699707.

[0448] Angiogenesis appears to be an important factor in both the development and subsequent progression of EOC. Yoneda and colleagues (1998) demonstrated in a xenograft model of EOC that tumor growth rate is proportional to vascular density and that the development of malignant ascites (a feature associated with poor outcome in EOC) is associated with the expression of vascular endothelial growth factor (VEGF). See, for example, Yoneda J, Kuniyasu H, Crispens MA, et al. Expression of angiogenesis-related genes and progression of human ovarian carcinomas in nude mice. J Natl Cancer Inst. 1998 Mar 18; 90: 44754. Other studies have demonstrated an association between VEGF expression and microvessel density in EOC. In addition, studies have shown that the expression density of CD31 (a marker of vascular endothelium) detected by immunohistochemistry in EOC is inversely correlated with survival.

[0449] This example describes a placebo-controlled, randomized, multicenter phase III study evaluating 1000 mg / m2 of gemcitabine in women with platinum-sensitive recurrent epithelial ovarian, primary peritoneal, or fallopian tube cancer compared with carboplatin (area under the curve [AUC] 4 on day 1 every 21 days) and gemcitabine (1000 mg / m2). 2 The efficacy and safety of bevacizumab (15 mg / kg, day 1, every 21 days) in combination with 15 mg / kg of bevacizumab (15 mg / kg, day 1, every 21 days) were evaluated. Approximately 480 patients were enrolled over a period of approximately 2.5 years. Patients were randomized to carboplatin and gemcitabine and placebo or carboplatin and gemcitabine and bevacizumab. Additionally, at randomization, patients were stratified by platinum-sensitive disease (recurrence 6-12 months from the last platinum-based treatment vs. recurrence >12 months from the last platinum-based treatment) and cytoreductive surgery for recurrent epithelial ovarian cancer, primary peritoneal cancer, or fallopian tube cancer (those who underwent surgery versus those who did not).

[0450]

[0451] The research consists of two branches as shown below. Figure 11 .

[0452] Arm 1: Carboplatin (AUC 4 IV) and gemcitabine (1000 mg / m2) chemotherapy (6 cycles up to 20 cycles), followed by placebo

[0453] Arm 2: Avastin (15 mg / kg, 6 cycles up to 10 cycles) in combination with carboplatin and gemcitabine chemotherapy (6 cycles up to 10 cycles), followed by continuation of Avastin (15 mg / kg) alone until disease progression.

[0454] Carboplatin doses are calculated as target AUC to concentration x time according to the Calvert formula using estimated glomerular filtration rate (GFR); for example, for purposes herein, GFR is considered equivalent to creatinine clearance. Calvert formula for carboplatin (AUC) dosing

[0455] Total dose (mg) = target AUC (in mg / mL / min) x [GFR (in mL / min)25]

[0456] Creatinine clearance can be calculated according to conventional guidelines.

[0457] Patient selection

[0458] Patients with epithelial ovarian cancer, PPC, or fallopian tube cancer who have relapsed (first relapse) >6 months from platinum-based chemotherapy will be eligible for this study. Additional specific inclusion and exclusion criteria are listed below. Patient Inclusion Criteria:

[0459] Patients must meet the following criteria to be included in the study:

[0460] ●Sign the informed consent form

[0461] Age ≥18 years

[0462] Ovarian, primary peritoneal, or fallopian tube cancer with histologically documented recurrence >6 months after platinum-based chemotherapy

[0463] ● Patients must have recurrent epithelial ovarian cancer, primary peritoneal cancer, or fallopian tube cancer. This must be the first recurrence of epithelial ovarian cancer, primary peritoneal cancer, or fallopian tube cancer.

[0464] Examples of selected histologic cell types include: serous adenocarcinoma, endometrioid adenocarcinoma, mucinous adenocarcinoma, undifferentiated carcinoma, clear cell adenocarcinoma, transitional cell carcinoma, malignant Brenner's tumor, or adenocarcinoma not otherwise specified

[0465] No prior chemotherapy in the setting of relapse

[0466] Measurable disease according to modified RECIST with at least one lesion that is precisely measurable in at least one dimension (with the longest dimension recorded)

[0467] Each measurable lesion must be 20 mm by conventional techniques, CT, and magnetic resonance imaging (MRI), or 10 mm by spiral CT.

[0468] More than 28 days have passed since the previous radiation therapy or surgery and recovery from the previous radiation therapy or surgery

[0469] ECOG performance status 0 or 1

[0470] Use of effective contraception (for women of childbearing potential)

[0471] ● Ability to follow study and follow-up protocols

[0472] Patient exclusion criteria

[0473] Patients meeting any of the following criteria were excluded from study entry.

[0474] Disease-specific exclusions

[0475] Prior chemotherapy for recurrent ovarian, primary peritoneal, or fallopian tube cancer: Hormonal therapy (i.e., progesterone, estrogen, anti-estrogen, aromatase inhibitors) will not be considered a prior chemotherapy regimen. Patients participating in the study treatment are not allowed to have concomitant anti-neoplastic anti-hormonal therapy (including tamoxifen, aromatase inhibitors, etc.). Low-dose (physiologic) estrogen hormone replacement therapy (HRT) may be given.

[0476] ○ History of abdominal fistula, gastrointestinal perforation, or intra-abdominal abscess

[0477] ○ Patients with clinical symptoms or signs of GI obstruction or requiring parenteral hydration, parenteral nutrition, or tube feeding

[0478] o Patients with evidence of free abdominal air that cannot be explained by paracentesis or newer surgical procedures

[0479] General medical exclusions

[0480] ○ Life expectancy < 12 weeks

[0481] ○ Currently, recently (within 4 weeks of Cycle 1, Day 1), or planning to participate in an experimental drug study

[0482] ○Screening clinical laboratory values

[0483] Granulocyte count < 1500 / microL

[0484] Platelet count <100,000 / microL (<100,000 / microL)

[0485] Hemoglobin <8.5 g / dL (hemoglobin can be supported with blood transfusions or erythropoietin or other approved hematopoietic growth factors)

[0486] Serum bilirubin >2.0x upper limit of normal (ULN)

[0487] Alkaline phosphatase, aspartate aminotransferase (AST), and / or alanine aminotransferase (ALT) > 2.5 x ULN (AST, ALT > 5 x ULN for patients with liver metastases)

[0488] Serum creatinine ≥1.6

[0489] International normalized ratio (INR) > 1.5 and / or activated partial thromboplastin time (aPTT) > 1.5 x ULN (except in patients receiving anticoagulant therapy)

[0490] For patients taking full-dose warfarin, an INR in the range (usually between 2 and 3) and an aPTT < 1.2 x ULN

[0491] ○ History of other malignancies within 5 years of Day 1 of Cycle 1, excluding those with negligible risk of metastasis or death, such as inadequately controlled squamous cell carcinoma or basal cell carcinoma of the skin or carcinoma in situ of the cervix

[0492] ○ Any other disease, metabolic disorder, physical examination finding, or clinical laboratory finding that raises reasonable suspicion of a disease or condition that contraindicates use of the investigational drug or that could affect the interpretation of the results or place the patient at high risk for treatment complications

[0493] Bevacizumab-specific exclusion

[0494] ○ Systemic bevacizumab History of use of other VEGF or VEGF receptor targeting agents

[0495] Inadequately controlled hypertension (defined as systolic blood pressure >150 mmHg and / or diastolic blood pressure >100 mmHg while taking antihypertensive medication)

[0496] ○ Previous history of hypertensive crisis or hypertensive encephalopathy

[0497] ○ New York Heart Association class II or greater CHF

[0498] ○ History of myocardial infarction or unstable angina within 6 months before Day 1 of Cycle 1 (day of first bevacizumab / placebo infusion)

[0499] ○ History of stroke or TIA within 6 months prior to study enrollment

[0500] ○ Known CNS disease, excluding treated brain metastases

[0501] ■ Treated brain metastases are defined as no evidence of progression or bleeding after treatment and no need for dexamethasone at that time, as confirmed by clinical examination and brain imaging (MRI or CT) during screening. These metastases must not be located in the brainstem, midbrain, pons, medulla oblongata, or leptomeninges. Treatment of brain metastases may include whole brain radiation therapy (WBRT), radiosurgery (Gamma Knife, LINAC, or equivalent), or a combination deemed appropriate by the attending physician. Patients with CNS metastases treated by neurosurgical resection or brain biopsy performed within 3 months before Day 1 will be excluded.

[0502] ○ History of major vascular disease (eg, aortic aneurysm, aortic dissection)

[0503] ○ Recent peripheral arterial thrombosis within 6 months before Day 1 of Cycle 1

[0504] ○ History of hemoptysis (≥ 1 / 2 teaspoon of bright red blood per episode) within 1 month before day 1 of cycle 1

[0505] ○ Evidence of a bleeding diathesis or significant coagulopathy (in the absence of therapeutic anticoagulation)

[0506] ○ Major surgery, open biopsy, or major trauma within 28 days prior to Day 1 of Cycle 1 or anticipated need for major surgery during the study

[0507] ○ Core biopsy or other minor surgical procedure within 7 days prior to Day 1 of Cycle 1 to exclude vascular access device placement

[0508] ○ Severe non-healing wounds; active ulcers; or untreated fractures

[0509] ○ Proteinuria at screening, as demonstrated by a UPCR ≥ 1.0 at screening

[0510] ○ Known hypersensitivity to any component of bevacizumab

[0511] Pregnant (positive pregnancy test) or breastfeeding

[0512] ●Patients of reproductive potential must use effective contraception.

[0513] This study, OCEANS, enrolled a different patient population than Example 1 (GOG 0218) and Example 2 (ICON7); women with previously treated platinum-sensitive ovarian cancer were enrolled in this trial. Women with ovarian cancer may receive platinum-based chemotherapy as first-line treatment. The time between receiving the last dose of platinum-based chemotherapy and the disease returning (recurrence) is used to help determine the chemotherapy option used in the next first-line treatment. If the disease returns more than six months after completing the initial platinum-based chemotherapy, the woman has "platinum-sensitive" ovarian cancer. If the disease returns within six months of completing the initial platinum-based chemotherapy, the ovarian cancer is considered "platinum-resistant."

[0514] result

[0515] This Phase III study of bevacizumab plus chemotherapy in women with ovarian cancer met its primary endpoint. The purpose of the study was to evaluate the efficacy and safety of adding bevacizumab to standard chemotherapy, followed by extended use of bevacizumab alone until disease progression, compared to chemotherapy alone in previously treated women with ovarian cancer. The study showed that bevacizumab plus chemotherapy, followed by continued use of bevacizumab alone until disease progression, prolonged the time women with previously treated (recurrent) platinum-sensitive ovarian cancer lived without disease worsening (progression-free survival or PFS) compared to chemotherapy alone. PFS was defined as the time from randomization to disease progression or death from any cause (whichever occurred first) as determined by the investigators. The primary endpoint of PFS was assessed by study investigators. Measurable disease was assessed by investigators using modified RECIST (Therasse et al. 2000), for example, every 9 weeks throughout the study. See, e.g., Therasse P, Arbuck SG, Eisenhauser EA, et al. New guidelines to evaluate the response to treatment in solid tumors. J Natl Cancer Inst 2000; 92: 205-1. Secondary endpoints included overall survival (OS), response rate, duration of response, and safety. No new safety findings were observed, and adverse events were consistent with those observed in previous important trials of bevacizumab.

Claims

1. A method of treating a patient diagnosed with ovarian cancer, comprising administering to the patient a treatment regimen that combines chemotherapy with administration of an effective amount of an anti-VEGF antibody, followed by anti-VEGF maintenance therapy, wherein the chemotherapy of the treatment regimen comprises administration of at least one chemotherapeutic agent, and wherein the treatment regimen is effective to prolong progression-free survival of the patient.

2. The method of claim 1, wherein the chemotherapeutic agent is a taxane, paclitaxel, docetaxel, or a protein-bound particle of paclitaxel (e.g., ), a platinum analog, carboplatin, gemcitabine, or a combination thereof.

3. The method of claim 1, wherein the chemotherapy of the treatment regimen comprises administration of a taxane and carboplatin.

4. The method of claim 1, wherein the chemotherapy of the treatment regimen comprises administration of carboplatin and gemcitabine.

5. The method of claim 1, wherein the patient is diagnosed with previously untreated ovarian cancer.

6. The method of claim 1, wherein the patient is diagnosed with recurrent ovarian cancer.

7. The method of claim 1, wherein the anti-VEGF antibody binds to the same epitope as the monoclonal anti-VEGF antibody A4.6.1 produced by hybridoma ATCC HB 10709.

8. The method of claim 1, wherein the anti-VEGF antibody is a humanized antibody.

9. The method of claim 8, wherein the anti-VEGF antibody is a humanized A4.6.1 antibody or a fragment thereof.

10. The method of claim 8, wherein the anti-VEGF antibody is bevacizumab.

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