HK40013836A

HK40013836A - Anti-angiogenesis therapy for the treatment of ovarian cancer

Info

Publication number: HK40013836A
Application number: HK42020004057.4A
Authority: HK
Inventors: J‧杜邦; C‧伊尔
Original assignee: 霍夫曼-拉罗奇有限公司
Priority date: 2010-02-23
Filing date: 2020-03-11
Publication date: 2020-08-14

Description

Anti-angiogenic therapy for the treatment of ovarian cancer

The present application is a divisional application of an invention having an application date of 2011, 2/22, chinese application No. 201180020417.9, entitled "anti-angiogenesis therapy for treating ovarian cancer".

RELATED APPLICATIONS

The application claims united states provisional application serial No. 61/439,819 filed on 4/2/2011; U.S. provisional application serial No. 61/360,059 filed on 30/6/2010; U.S. provisional application serial No. 61/351,231 filed on 3/6/2010; and U.S. provisional application serial No. 61/307,095 filed on 23/2/2010, the specification of which is incorporated herein in its entirety.

Material preservation

The following hybridoma cell lines have been deposited with the American Type Culture Collection (ATCC), Manassas, Va., USA) according to the provisions of the Budapest treaty:

Technical Field

The present invention relates generally to the treatment of human diseases and pathological conditions. More particularly, the invention relates to anti-angiogenic therapies, either alone or in combination with other anti-cancer therapies, for the treatment of ovarian cancer.

Background

Cancer remains one of the most fatal threats to human health. Cancer affects nearly 130 million new patients each year in the united states and is the second cause of death after heart disease, accounting for approximately 1 of 4 deaths. For women with ovarian and peritoneal cancers, standard major systemic chemotherapy consists of chemotherapy with platinum and taxane combinations, usually carboplatin and paclitaxel, for women with advanced ovarian epithelial cancer and primary cancer of the peritoneum after initial surgical diagnosis, staging and cytoreduction. See, e.g., McGuire WP, et al, Cyclophosphosphingoid and cissplatin compounded with a paclitaxel and a cissplatin in tissues with stage III and stage IV over cancer. N Eng J Med334:1-6, 1996; piccrart MJ, et al, random interpolated tertiary of cispin-paclitaxel-cyclosporine in mouse with advanced temporal overview candidate viewer, thread-layer results, J Natl Cancer Inst 92:699-708, 20003; alberts DS, et al, improved thermal index of carboplatin ploycyclophosphamide vera cissplatin plus cyclophosphamide, final report by the south west ontology Group of a phase III random tertiary in classes III and IVovan cancer. J Clin Oncol10:706-17, 1992; du Bois A, et al.A random minor clinical trial of cissplatin/paclitaxel transplatin/paclitaxel active first-line transaction of overview cancer.J. Natl Cancer Inst Sep.3; 95.(17) 1320-9.95: 1320,2003; ozols RF, et al, phase III tertiary of carboplatin and paclitaxel with a probability in a probability with an optimal selected stage III over cancer, a Gynecology on Group study. J Clinonocol 21:3194-200, 2003; and Swenerton K, et al, Cisplatin-cyclophosphamide in advanced overhead Cancer a random Cancer III study of the National Cancer Institute of Cancer Clinical groups J Clin Oncol10: 718-. Despite advances in patient management, the disease still has a high mortality-to-case ratio for all gynecological malignancies diagnosed in the united states. It is estimated that 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. There is a need for improved primary treatment strategies.

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 compelling evidence that the development of vascular supply is critical for normal and pathological proliferative processes (Folkman and Klagsbrun Science 235:442-447 (1987)). The transport of oxygen and nutrients, as well as the elimination of catabolic products, represent the rate limiting steps for most growth processes occurring in multicellular organisms.

Although the introduction of new blood vessels is considered to be the primary mode of tumor angiogenesis, recent data indicate that some tumors can grow by recruiting existing host blood vessels. The recruited vasculature then regresses, resulting in tumor regression, eventually reverting to hypoxia-induced angiogenesis at the tumor margin. Holash et al science 284:1994-1998 (1999).

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 binds primarily to two high affinity receptor tyrosine kinases, VEGFR-1(Flt-1) and VEGFR-2(Flk-1/KDR), the latter being the major transmitters of the mitotic signals of VEGF-A vascular endothelial cells. In addition, neuropilin-1 (neuropilin-1) has been identified as a receptor for heparin-binding VEGF-A isoforms and may play a role in vascular development.

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 report mitogenic effects of VEGF on a few non-endothelial cell types such as retinal pigment epithelial cells, pancreatic ductal cells, and Schwann (Schwann) cells. Guerrin et al.J.CellPhysiol.164:385-394 (1995); Oberg-Welsh et al. mol.cell. Endocrinol.126:125-132 (1997); sondell et al.J.Neurosci.19:5731-5740 (1999).

VEGF expression is upregulated in most malignant tumors, and VEGF overexpression is often associated with more advanced stages or with poorer prognosis in many solid tumors.

Since ovarian cancer remains one of the most fatal threats, patients need additional cancer therapies. The present invention addresses these and other needs, as will be apparent upon reading the following disclosure.

Summary of The Invention

Use of an anti-VEGF antagonist for treating ovarian cancer is provided. For example, anti-VEGF antibodies are provided for use in 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 cancer, primary, peritoneal, or fallopian tube cancer. Providing bevacizumab in a subject (e.g., female) from previously untreated stage III (suboptimal and macroscopically optimally lumpectomy) and stage IV ovarian epithelial carcinoma, primary peritoneal carcinoma or fallopian tube carcinoma with a new diagnosisData from a randomized phase III clinical trial combined with a chemotherapeutic regimen (example 1). Also provided are bevacizumab (Bevacizumab) derived from subjects (e.g. females) with a new diagnosis, high risk of ovarian epithelial cancer, fallopian tube cancer or primary peritoneal cancer in stages I and IIa (clear cell carcinoma only or grade 3) and IIb-IV (which underwent initial surgery and did not take into account cytoreductive surgery before disease progression)Data from a randomized III clinical trial combined with a chemotherapeutic regimen (example 2). Also provided are methods for assessing and carboplatin (area under the curve [ AUC ] in females with platinum-sensitive recurrent ovarian epithelial, primary peritoneal, or fallopian tube cancer]4, day 1, every 21 days) and gemcitabine (1000mg/m, day 1 and day 8, every 21 days) with placebo-controlled, randomized, multicenter phase III study data for efficacy and safety of bevacizumab (15mg/kg, day 1, every 21 days) (example 3). Such chemotherapeutic regimens include taxane therapy (e.g., paclitaxel or docetaxel), platinum-based chemotherapy (e.g., carboplatin), or gemcitabine, and combinations thereof. The above-mentionedThe success of the trial shows that providing an anti-VEGF antibody (e.g., bevacizumab) in combination with chemotherapy and continuing to provide an anti-VEGF antibody (e.g., bevacizumab) as a maintenance therapy provides statistically significant and clinically meaningful benefits to ovarian cancer patients. Results obtained in clinical studies with bevacizumab in both concurrent and maintenance treatment in human subjects with previously untreated and recurrent ovarian cancer showed that efficacy (as assessed by Progression Free Survival (PFS)) was positive, especially compared to PFS data for chemotherapeutic treatment alone. Subjects receiving bevacizumab and bevacizumab maintenance therapy in concurrent treatment with a taxane therapy (e.g., paclitaxel or docetaxel) and a platinum-based chemotherapy (e.g., carboplatin) or a platinum-based chemotherapy (e.g., carboplatin) and gemcitabine combination in the clinical trial have prolonged progression-free survival compared to subjects treated with a taxane therapy (e.g., paclitaxel or docetaxel) and a platinum-based chemotherapy (e.g., carboplatin) alone or a platinum-based chemotherapy (e.g., carboplatin) and gemcitabine alone.

Accordingly, the present invention provides a method of treating a patient diagnosed with previously untreated or recurrent ovarian cancer, comprising subjecting the patient to a treatment regimen combining at least one chemotherapy with the administration of an effective amount of an anti-VEGF antibody, followed by administration of an anti-VEGF antibody for maintenance therapy, wherein with said treatment, the progression free survival of the patient is prolonged. Combining chemotherapy with a treatment regimen of administration of anti-VEGF followed by anti-VEGF maintenance therapy effectively extends the Progression Free Survival (PFS) of the patient.

In certain embodiments, the PFS is extended 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., as compared to a control. In one embodiment, PFS is extended by about 2.9 months to 3.8 months compared to a control (e.g., a treatment regimen with a combination chemotherapy and administration of anti-VEGF followed by administration of anti-VEGF maintenance therapy). In one embodiment, PFS is extended by at least about 3.8 months compared to a control (e.g., a treatment regimen with a combination chemotherapy and administration of anti-VEGF followed by administration of anti-VEGF maintenance therapy). In another embodiment, PFS is extended by about 2.3 months compared to a control (e.g., a treatment regimen with a combination chemotherapy and administration of anti-VEGF followed by administration of anti-VEGF maintenance therapy). In one embodiment, PFS is extended by about 6 months compared to a control (e.g., a treatment regimen with a combination chemotherapy and administration of anti-VEGF followed by administration of anti-VEGF maintenance therapy).

Any chemotherapeutic agent exhibiting anti-cancer activity may 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, adrenocorticosteroids, progestins, estrogens, antiestrogens, androgens, antiandrogens, gemcitabine, and gonadotropin-releasing hormone analogs. In certain embodiments, the chemotherapeutic agent is, e.g., a taxane, paclitaxel, docetaxel, paclitaxel protein-binding particles (e.g., paclitaxel, docetaxel, paclitaxel, or paclitaxel-conjugated particles)) Gemcitabine, platinum analogs, carboplatin, or combinations thereof. Two or more chemotherapeutic agents, such as a taxane and a platinum analog or gemcitabine and a platinum analog, may be used in the mixture to be administered in combination with the 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.

The clinical benefit of a treatment according to the invention can be measured by, for example, the duration of Progression Free Survival (PFS), the time to treatment failure, the objective response rate, and the duration of response.

The invention also provides a kit. In one embodiment, a kit is provided for treating previously untreated ovarian cancer in a human patient, comprising a package comprising an anti-VEGF antibody composition and instructions for using the anti-VEGF antibody composition in combination with taxane therapy and carboplatin, followed by anti-VEGF maintenance therapy, wherein the instructions recite a progression free survival of 14.1 months for patients receiving taxane therapy and carboplatin therapy and bevacizumab with a hazard ratio (hazard ratio) of 0.717(p value < 0.0001). In another embodiment, a kit is provided for treating previously untreated ovarian cancer in a human patient, comprising a kit of parts (packs) comprising an anti-VEGF antibody composition and instructions for using the anti-VEGF antibody composition in combination with paclitaxel and carboplatin, followed by anti-VEGF maintenance therapy, wherein the instructions recite that the progression free survival of patients receiving paclitaxel, carboplatin, and anti-VEGF antibody is 18.3 months with 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), the light chain variable region comprising the 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 with a patient having stage III or IV ovarian cancer.

Thus, the invention features a method of directing a human subject having cancer (e.g., ovarian cancer) to receive treatment with an anti-VEGF antibody by providing instructions to extend the progression free survival of the subject, reduce the likelihood of cancer recurrence in the subject, or increase the likelihood of survival of the subject. 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, the treatment with the anti-VEGF antibody is concurrent and sequential with the treatment with the chemotherapeutic agent. In certain embodiments, the subject is treated as directed by the method of guidance.

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

The present invention provides a commercial method comprising marketing an anti-VEGF antibody for use in treating cancer (e.g., ovarian cancer) in a human subject in combination with at least one chemotherapeutic agent, followed by an anti-VEGF maintenance therapy, thereby prolonging 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 treatment of the subject with an anti-VEGF antibody and a chemotherapeutic, 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 an anti-VEGF maintenance therapy. In some embodiments, marketing is followed by treatment of the subject with an anti-VEGF antibody and a chemotherapeutic, followed by administration of an anti-VEGF maintenance therapy.

Also provided is a commercial 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 prolonging 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 treatment of the subject with a combination of a chemotherapeutic and an anti-VEGF antibody, followed by anti-VEGF maintenance therapy. Also provided is a commercial 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 prolonging 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 treatment of the subject with a combination of a chemotherapeutic and an anti-VEGF antibody, followed by anti-VEGF maintenance therapy.

In each of the methods of the 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 as 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 invention include bevacizumabG6 antibody, B20 antibody, and fragments thereof. In certain embodiments, the anti-VEGF antibody has a heavy chain variable region comprising the 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 amino acid sequence: DIQMTQSPSS LSASVGDRVT ITCSASQDIS NYLNWYQQKP GKAPKVLIYFTSSLHSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ YSTVPWTFGQ GTKVEIKR (SEQ ID No. 2).

The antibody or antigen binding fragment thereof can also be an antibody lacking an Fc portion, F (ab') 2, Fab, or Fv structure.

In one embodiment, the 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, the 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.

Each of the methods or uses of the invention may 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, ovarian primary peritoneal cancer, ovarian fallopian tube cancer, squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, hepatic cancer, bladder cancer, hepatosarcoma (hepatoma), breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, kidney cancer, vulval cancer, thyroid cancer, hepatic carcinoma, 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 (suboptimal) and macroscopically optimally lumpectomized (macroepithelial refractory) or stage IV ovarian epithelial primary peritoneal or fallopian tube cancer. In some embodiments, the subject has platinum-sensitive recurrent ovarian epithelial carcinoma, primary peritoneal carcinoma, or fallopian tube carcinoma.

Each of the above aspects may further comprise monitoring the subject for cancer recurrence. Monitoring may 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 initiation of treatment.

Preferred dosages of anti-VEGF antibodies (e.g., bevacizumab) are described herein, depending on the type and severity of the disease, and may range from about 1 μ g/kg to about 50mg/kg, most preferably from about 5mg/kg to about 15mg/kg, including but not limited to 5mg/kg, 7.5mg/kg, 10mg/kg, or 15 mg/kg. The frequency of administration will vary with the type and severity of the disease. For repeated administrations over several days or longer, depending on the condition, the 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, the anti-VEGF antibody of the invention is administered once weekly, biweekly, or every three weeks at a dose ranging from about 5mg/kg to about 15mg/kg, including but not limited to 5mg/kg, 7.5mg/kg, 10mg/kg, or 15 mg/kg. However, other dosage regimens may 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 other embodiments, the anti-VEGF therapy is provided for at least 14 months (including anti-VEGF therapy and anti-VEGF maintenance therapy concurrently with chemotherapy). In other embodiments, the anti-VEGF therapy is provided for at least 12 months (including anti-VEGF therapy and anti-VEGF maintenance therapy concurrently with chemotherapy).

In other 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, an aspect of treatment is with a VEGF-specific antagonist in monotherapy or monotherapy for the duration of a 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, at least from cycle 7 to 22, the anti-VEGF maintenance therapy is administered. In other embodiments, at least from cycle 7 to 18, the anti-VEGF maintenance therapy is administered.

In other embodiments, treatment with a VEGF-specific antagonist is combined with another anti-cancer therapy, including, but not limited to, surgery, radiation therapy, chemotherapy, differentiation therapy, biological therapy, immunotherapy, angiogenesis inhibitors, cytotoxic agents, and anti-proliferative compounds. VEGF-specific antagonist therapy can also include any combination of the above types of therapeutic regimens. In some embodiments, the chemotherapeutic and the 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.

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

The present application relates to the following embodiments.

1. A method of treating a patient diagnosed with ovarian cancer, comprising subjecting the patient to a treatment regimen combining 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 embodiment 1, wherein the chemotherapeutic agent is a protein-bound particle of a taxane, paclitaxel, docetaxel, paclitaxel (e.g., paclitaxel, etc.)) A platinum analog, carboplatin, gemcitabine, or a combination thereof.

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

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

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

6. The method of embodiment 1, wherein said patient is diagnosed with recurrent ovarian cancer.

7. The method of embodiment 1, wherein said 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 embodiment 1, wherein said anti-VEGF antibody is a humanized antibody.

9. The method of embodiment 8, wherein the anti-VEGF antibody is a humanized a4.6.1 antibody or fragment thereof.

10. The method of embodiment 8, wherein said anti-VEGF antibody is bevacizumab.

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

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.

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

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

15. The method of embodiment 2, wherein paclitaxel is administered as shown in figure 1 or figure 2.

16. The method of embodiment 2, wherein the carboplatin is administered as shown in figure 1 or figure 2 or figure 11.

17. The method of embodiment 2, wherein docetaxel is administered as shown in figure 1.

18. The method of embodiment 2, wherein gemcitabine is administered as shown in figure 11.

19. The method of embodiment 2, wherein the anti-VEGF is administered as shown in figure 1 or figure 2 branch III or as described in example 3 branch II.

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

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

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

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

24. A method of instructing a human subject having ovarian cancer, the method comprising providing instructions to receive an anti-VEGF antibody in parallel with chemotherapy, followed by anti-VEGF maintenance therapy to treat the ovarian cancer, thereby prolonging progression free survival of the subject.

25. The method of embodiment 24, wherein said instructions further comprise instructions for performing a treatment with at least two chemotherapeutic agents.

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

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

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

29. The method of embodiment 26, wherein said promotion is by a package insert accompanying a commercial formulation of said anti-VEGF antibody.

30. The method of embodiment 26 wherein said promotion is by a package insert accompanying a commercial formulation of said chemotherapeutic agent.

31. The method of embodiment 26 wherein the promotion is by written notification to a physician or health care provider.

32. The method of embodiment 26 wherein the promotion is by oral notification to a physician or health care provider.

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

34. A commercial method comprising marketing an anti-VEGF antibody therapy concurrent with chemotherapy, followed by an anti-VEGF maintenance therapy, for treating ovarian cancer in a human subject, thereby prolonging progression-free survival of the subject.

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

36. The method of any one of embodiments 24, 26 or 34, wherein said chemotherapeutic agent is taxus cuspidataProtein-binding particles of alkanes, paclitaxel, docetaxel, paclitaxel (e.g., paclitaxel-coated particles)) Platinum analogs, carboplatin, gemcitabine, or combinations thereof.

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

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

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

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

41. The method of embodiment 2, wherein paclitaxel is administered as shown in figure 8.

42. The method of embodiment 2, wherein the carboplatin is administered as shown in figure 8.

43. The method of embodiment 2, wherein the anti-VEGF antibody is administered as shown in branch B of fig. 8.

44. A kit for treating previously untreated ovarian cancer in a human patient, comprising a package comprising an anti-VEGF antibody composition and instructions for using the anti-VEGF antibody composition in combination with paclitaxel and carboplatin followed by an anti-VEGF maintenance therapy, wherein the instructions recite that the progression free survival of a patient receiving paclitaxel, carboplatin, and anti-VEGF antibodies is 18.3 months with a hazard ratio of 0.79.

Other features and advantages of the invention will be apparent from the detailed description, the drawings, and the claims.

Brief Description of Drawings

FIG. 1 depicts the study design for ovarian cancer described in example 1.

Figure 2 depicts a study design of ovarian cancer trials using Bevacizumab (BEV) or placebo and various chemotherapies.

Figure 3 depicts selected adverse events from the trial shown in figure 2.

Figure 4 depicts selected adverse events resulting from the treatment period of the trial shown in figure 2.

Figure 5 depicts Progression Free Survival (PFS) of trial branch I, branch II and branch III shown in figure 2 as assessed by investigators.

FIG. 6 depicts PFS values for test branch I and branch III shown in FIG. 2 and results using the CA-125 marker as a progression of progression determiner.

Figure 7 depicts a subgroup analysis of patients in arm III and arm I of the trial shown in figure 2.

Figure 8 depicts the study design of the ovarian cancer assay described in example 2.

Figure 9 depicts a summary of the Progression Free Survival (PFS) analysis of the assay shown in figure 8. "CP" corresponds to branch A in FIG. 8. "CPB 7.5 +" corresponds to branch B in fig. 8.

Figure 10 depicts a graph of PFS results from the experiment shown in figure 8. "CP" corresponds to branch A in FIG. 8. "CPB 7.5 +" corresponds to branch B in fig. 8.

Figure 11 depicts the study design of the ovarian cancer assay described in example 3.

Detailed Description

I. Definition of

The term "VEGF" or "VEGF-A" is used to refer to 165 amino acids of human vascular endothelial cell growth factor and the related 121, 145, 189And 206 amino acids of human vascular endothelial cell growth factor, such as, for example, Leung et al science 246:1306 (1989); and Houck et al mol Endocrin, 5:1806(1991), and naturally occurring allelic 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 binds primarily to two high affinity receptor tyrosine kinases, VEGFR-1(Flt-1) and VEGFR-2(Flk-1/KDR), the latter being the major transmitters of the mitotic signals of VEGF-A vascular endothelial cells. In addition, neuropilin-1 has been identified as a receptor for heparin-binding VEGF-a isoforms (isofom) and may play a role in vascular development. The term "VEGF" or "VEGF-a" also refers to VEGF from non-human species such as mouse, rat, or primate. 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 truncated forms of the polypeptide comprising 165 amino acids from amino acid positions 8-109 or positions 1-109 of human vascular endothelial growth factor. In the present application, it is possible to use, for example, "VEGF (8-109)", "VEGF (1-109)" or "VEGF₁₆₅"to identify any such form of VEGF. The amino acid positions of a "truncated" native VEGF are numbered as indicated in the native VEGF sequence. For example, amino acid 17 (methionine) in truncated native VEGF is also amino acid 17 (methionine) in native VEGF. The truncated native VEGF has comparable binding affinity to the KDR and Flt-1 receptors as native VEGF.

An "anti-VEGF antibody" refers to an antibody that binds VEGF with sufficient affinity and specificity. The selected antibody will typically have binding affinity for VEGF, for example, the antibody may have a K between 100nM and 1pM_dValues bound to hVEGF. Antibody affinity can be determined by, for example, a surface plasmon resonance-based assay (such as the BIAcore assay described in PCT application publication No. wo 2005/012359); enzyme-linked immunosorbent assay (ELISA); and competition assays (e.g., RIA). In certain embodiments, the anti-VEGF antibodies of the invention are useful as therapeutic agents for targeting and interfering with diseases or conditions in which VEGF activity is implicated. Also, other biological activities may be performed on the antibodySex assays, such as biological activity assays performed to assess their efficacy as therapeutic agents. Such assays are known in the art and depend on the target antigen of the antibody and the intended use. Examples include HUVEC inhibition assays; tumor cell growth inhibition assays (as described, for example, in WO 89/06692); antibody-dependent cellular cytotoxicity (ADCC) and complement-mediated cytotoxicity (CDC) assays (U.S. patent 5,500,362); and agonist activity or hematopoietic assays (see WO 95/27062). anti-VEGF antibodies typically 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.

"VEGF antagonist" refers to a molecule that is capable of neutralizing, blocking, inhibiting, abrogating, reducing, or interfering 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 sequestering 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.

"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 may be isolated from nature, or may be produced by recombinant or synthetic means. The term "native sequence" polypeptide specifically encompasses naturally occurring truncated or secreted forms of the polypeptide (e.g., extracellular domain sequences), naturally occurring variant forms (e.g., alternatively spliced forms), and naturally occurring allelic variants.

By polypeptide "variant" is meant a biologically active polypeptide having at least about 80% amino acid sequence identity to the native sequence polypeptide. Such variants include, for example, polypeptides having one or more amino acid residues 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 to the native sequence polypeptide.

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.

Throughout the present 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 edition, Public Health service, National Institutes of Health, Bethesda, Md. (1991), which is expressly incorporated herein by reference. "EU index as in Kabat" refers to the residue numbering of the human IgG1EU antibody.

In one embodiment, "K" according to the invention_d"or" K_dThe value "is measured by a radiolabelled VEGF binding assay (RIA) using Fab-format antibodies and VEGF molecules as described in the following assays: by using the minimum concentration of the VEGF in the presence of a titration series of unlabelled VEGF¹²⁵I-labeling VEGF (109) equilibrates Fab and then the solution binding affinity of Fab for VEGF is measured by capturing the bound VEGF with anti-Fab antibody coated plates (Chen, et al, J MolBiol 293: 865-. In one example, to determine assay conditions, microtiter plates (Dynex) were coated with anti-Fab antibodies (Cappel Labs) overnight at 5. mu.g/ml capture in 50mM sodium carbonate (pH 9.6), followed by blocking with 2% (w/v) bovine serum albumin in PBS for 2-5 hours at room temperature (about 23 ℃). In a non-adsorption plate (Nunc #269620), 100pM or 26pM [ alpha ], [ beta ]¹²⁵I]VEGF (109) is mixed with serial dilutions of the Fab of interest (e.g.Fab-12 as in Presta et al, cancer Res.57:4593-4599 (1997)). Then keeping the temperature of the target Fab overnight; however, the incubation may continue for 65 hours to ensure equilibrium is reached. Thereafter, the mixture was transferred to a catch plate to be incubated at room temperature for 1 hour. Then the solution was removed and the product was diluted with 0.1%The plate was washed 8 times with 20 PBS. After the plates were dried, 150. mu.l/well scintillation fluid (MicroScint-20; Packard) was added and the plates were counted for 10 minutes on a Topcount Gamma counter (Packard). The concentration at which each Fab gives less than or equal to 20% of the maximum binding is selected for use in competitive binding assays. According to another embodiment, K_dOr K_dThe values are obtained by using BIAcore as a surface plasmon resonance assay^TM-2000 or BIAcore^TM-3000(BIAcore, inc., Piscataway, NJ) measured at 25 ℃ using an immobilized hVEGF (8-109) CM5 chip at about 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. mu.g/ml (about 0.2. mu.M) with 10mM sodium acetate pH 4.8 and then injected at a flow rate of 5. mu.l/min to obtain about 10 Response Units (RU) of conjugated protein. After injection of human VEGF, 1M ethanolamine was injected to block unreacted groups. For kinetic measurements, the mixture was injected at 25 ℃ at a flow rate of about 25. mu.l/min to a concentration of 0.05%20 two-fold serial dilutions of Fab (0.78nM to 500nM) in PBS (PBST). The binding rate (k) was calculated by simultaneous fitting of the binding and dissociation sensorgrams using a simple one-to-one Langmuir (Langmuir) binding model (BIAcore Evaluation Software version 3.2)_on) And dissociation rate (k)_off). Equilibrium dissociation constant (K)_d) At a ratio of k_off/k_onAnd (4) calculating. See, e.g., Chen, Y., et al, J Mol Biol 293:865-881 (1999). If the binding rate is more than 10 according to the above surface plasmon resonance assay⁶M^-1S^-1The rate of binding can then be determined using fluorescence quenching techniques, i.e.according to a spectrometer such as a spectrophotometer equipped with a flow-breaking device (Avivinstruments) or a 8000 series SLM-Aminco^TMSpectrophotometer (Thermospectronic)) In the measurement with stirred cuvettes, the fluorescence emission intensity of 20nM anti-VEGF antibody (Fab form) in PBS, pH 7.2, in the presence of increasing concentrations of human VEGF short form (8-109) or mouse VEGF at 25 ℃ (excitation 295 nM; increase or decrease in emission at 340nm, 16nm band pass (band pass)).

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

Unless otherwise indicated, the expression "multivalent antibody" is used throughout the specification to refer to an antibody comprising three or more antigen binding sites. For example, multivalent antibodies are engineered to have three or more antigen binding sites and are not typically native sequence IgM or IgA antibodies.

An "antibody fragment" comprises only a portion of an intact antibody, typically including the antigen-binding site of the intact antibody, and thus retains the ability to bind to the antigen. Examples of antibody fragments encompassed by this definition include: (i) a Fab fragment having VL, CL, VH and CH1 domains; (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 having VH and CH1 domains; (iv) an Fd' fragment having the VH and CH1 domains and one or more cysteine residues C-terminal to the CH1 domain; (v) fv fragments having VL and VH domains of an antibody single arm; (vi) dAb fragments (Ward et al, Nature 341:544-546(1989)) which consist of a VH domain; (vii) an isolated CDR region; (viii) f (ab')₂A fragment comprising a bivalent fragment of two Fab' fragments linked by a disulfide bond in the hinge region; (ix) single chain antibody molecules (e.g.single chain Fv; scFv) (Bird et al, Science 242: 423-; (x) "diabodies", having two antigen binding sites, comprising heavy chain variable domains linked in the same polypeptide chain(VH) and light chain variable domains (VL) (see, e.g., EP 404,097; WO 93/11161; and Hollinger et al, Proc. Natl. Acad. Sci. USA 90: 6444-; (xi) "Linear antibodies" comprising a pair of Fd segments (VH-CH1-VH-CH1) in tandem, together with a complementary light chain polypeptide, form a pair of antigen binding regions (Zapata et al, Protein Eng.8(10):1057-1062(1995) and U.S. Pat. No.5,641,870).

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutant forms that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigen. Furthermore, unlike polyclonal antibody preparations which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies to be used in accordance with the present invention can be prepared by the hybridoma method originally described by Kohler et al, Nature 256:495, (1975), or can be prepared by recombinant DNA methods (see, e.g., U.S. Pat. No.4,816,567). "monoclonal antibodies" can also be isolated from phage antibody libraries using techniques such as those described in Clackson et al, Nature 352: 624-.

An "Fv" fragment is an antibody fragment that comprises the entire antigen recognition and binding site. This region consists of a dimer of one heavy chain variable domain and one light chain variable domain that are tightly bound (the nature of the binding may be covalent, for example in a scFv). It is in this configuration that the three CDRs of each variable domain interact at V_H-V_LAn antigen binding site is defined on the surface of the dimer. Together, the six CDRs, or a subset thereof (subset), confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, but generally with a lower affinity than the entire binding site.

As used herein, "antibody variable domain" refers to that portion of the light and heavy chains of an antibody molecule that comprises the amino acid sequences of the complementarity determining regions (CDRs; i.e., CDR1, CDR2, and CDR3) and the Framework Region (FR). V_HRefers to the heavy chain variable domain. V_LRefers to the light chain variable domain. According to the method used in the present invention, the amino acid positions assigned to CDR and FR may 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 the antibody or antigen-binding fragment is also according to the amino acid numbering of Kabat.

As used herein, the term "complementarity determining regions (CDRs; i.e., CDR1, CDR2, and CDR3) refers to amino acid residues in the variable domain of an antibody whose presence is essential for antigen binding. Each variable domain typically has three CDRs, identified as CDR1, CDR2, and CDR 3. Each CDRs may comprise amino acid residues from the "CDRs" as defined by Kabat (i.e., about 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 immunologicaltest, 5th Ed. public Health Service, National Institutes of Health, Bethesda, MD (1991)) and/or residues from the "hypervariable loop" (i.e., about 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 (H917J 917: 96H 917: Bio1: 901). In some cases, the complementarity determining regions may comprise amino acids from both the CDR regions and the hypervariable loops as defined by Kabat. For example, the CDRH1 of the heavy chain of antibody 4D5 comprises amino acids 26-35.

The "framework region" (hereinafter FR) refers to residues other than CDR residues in the variable domain. Each variable domain typically has four FRs, identified as FR1, FR2, FR3, and FR 4. If the CDRs are defined according to Kabat, the light chain FR residues are located at about light chain residues 1-23(LCFR1), 35-49(LCFR2), 57-88(LCFR3), and 98-107(LCFR4), and the heavy chain FR residues are located at about heavy chain residues 1-30(HCFR1), 36-49(HCFR2), 66-94(HCFR3), and 103-113(HCFR 4). If the CDRs contain amino acid residues from hypervariable loops, the light chain FR residues are located at about light chain residues 1-25(LCFR1), 33-49(LCFR2), 53-90(LCFR3), and 97-107(LCFR4), and the heavy chain FR residues are located at about heavy chain residues 1-25(HCFR1), 33-52(HCFR2), 56-95(HCFR3), and 102-113(HCFR 4). In some cases, where the CDR comprises amino acids from both the CDR and the hypervariable loop as defined by Kabat, the FR residues will be adjusted accordingly. For example, when CDRH1 comprises amino acids H26-H35, heavy chain FR1 residues are located at positions 1-25 and FR2 residues are located at positions 36-49.

The "Fab" fragment comprises the variable and constant domains of the light chain and the variable and first constant domains of the heavy chain (CH 1). F (ab')₂Antibody fragments comprise a pair of Fab fragments, which are generally covalently linked near their carboxy termini by a hinge cysteine between them. Other chemically conjugated forms of antibody fragments are also known in the art.

"Single chain Fv" or "scFv" antibody fragments comprise the V of an antibody_HAnd V_LDomains, wherein the domains are present on a single polypeptide chain. Generally, Fv polypeptides are described at V_HAnd V_LFurther included between the domains is a polypeptide linker that enables the scFv to form the desired structure for binding to the antigen. 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.

The term "diabodies" refers to small antibody fragments having two antigen-binding sites, which fragments are in the same polypeptide chain (V)_HAnd V_L) Comprising a linked heavy chain variable domain (V)_H) And a light chain variable domain (V)_L). By using linkers that are too short to allow pairing between the two domains on the same chain, these domains are forced to pair with the complementary domains of the other chain, thereby creating two antigen binding sites. Diabodies are described more fully in, for example, EP 404,097; WO 93/11161; hollinger et al, Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993).

Expression (a)"Linear antibody" refers to the antibodies described in Zapata et al, Protein Eng,8(10):1057-1062 (1995). Briefly, these antibodies comprise a pair of Fd segments (V) in tandem_H-C_H1-V_H-C_H1) The segments, together with the complementary light chain polypeptide, form an antigen-binding region pair. Linear antibodies can be bispecific or monospecific.

Monoclonal antibodies specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, and the remaining portion of the chain is identical with or homologous to corresponding sequences 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. Pat. No.4,816,567; Morrison et al Proc. Natl.Acad.Sci.USA 81:6851-6855), (1984).

"humanized" forms of non-human (e.g., murine) antibodies refer to chimeric antibodies that contain minimal sequences derived from non-human immunoglobulins. For the most part, humanized antibodies are those in which residues from a hypervariable region of a human immunoglobulin (recipient antibody) are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. In some instances, Fv Framework Region (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues. In addition, humanized antibodies may comprise residues not found in the recipient antibody or in the donor antibody. These modifications are made to further improve the performance of the antibody. Typically, the humanized antibody will comprise substantially all of at least one, and typically two, variable regions in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. The humanized antibody optionally further comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For more details see Jones et al, Nature 321:522-525 (1986); riechmann et al, Nature 332: 323-; presta, curr, Op, struct, biol.2:593-596 (1992).

"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 antibodies specifically excludes humanized antibodies comprising non-human antigen binding residues. Human antibodies can be generated using a variety of techniques known in the art. In one embodiment, the human antibody is selected from a phage library expressing human antibodies (Vaughan et al, Nature Biotechnology14: 309-. Human antibodies can also be generated 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 was observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. Such methods are described, for example, in U.S. patent nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126, respectively; 5,633,425, respectively; 5,661,016, and the following scientific publications: marks et al, Bio/Technology 10:779-783 (1992); lonberget al, Nature 368: 856-; morrison, Nature 368:812-13 (1994); fishwild et al, Nature Biotechnology14:845-51 (1996); neuberger, Nature Biotechnology14: 826 (1996); lonberg and Huszar, Intern.Rev.Immunol.13:65-93 (1995). Alternatively, human antibodies can be prepared by immortalization of human B lymphocytes that produce antibodies to the target antigen (such B lymphocytes can be recovered from the individual, or can be immunized in vitro). See, e.g., 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.

An "affinity matured" antibody refers to an antibody that has one or more alterations in one or more CDRs of the antibody that result in an improvement in the affinity of the antibody for an antigen as compared to a parent antibody without the 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) describe affinity maturation by VH and VL domain shuffling. The following documents describe random mutagenesis of CDR and/or framework residues: barbas et al, Proc. Nat. Acad. Sci. USA 91: 3809-; schier et al, Gene 169: 147-; 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).

A "functional antigen-binding site" of an antibody refers to a site that is capable of binding a target antigen. The antigen binding affinity of the antigen binding site does not have to be as strong as the parent antibody from which the antigen binding site is derived, but the ability to bind antigen must be measurable using any of a variety of methods known for assessing binding of antibodies to antigens. Furthermore, the antigen binding affinity of each antigen binding site of a multivalent antibody herein need not be identical in amount. 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 assay, target antigens are mixed with multimeric antibodies in different ratios and the average molecular weight of the complexes is calculated, assuming different numbers of functional binding sites. These theoretical values were compared to the actual experimental values obtained to assess the number of functional binding sites.

An antibody having the "biological characteristics" of a given antibody refers to an antibody that possesses one or more biological characteristics that distinguish the given antibody from other antibodies that bind the same antigen.

To screen for Antibodies that bind to an epitope on an antigen to which an antibody of interest binds, a conventional cross-blocking assay can be performed, such as that described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, EdHarlow and David Lane (1988).

"ZiyiA diabody "refers to an antibody that has greater binding affinity for an antigen from a first mammalian species than for a homolog of the antigen from a second mammalian species. Typically, a species-dependent antibody "specifically binds" to a human antigen (i.e., has no more than about 1x 10^-7M, preferably not more than about 1x 10^-8M, most preferably not more than about 1x 10^-9Binding affinity (K) of M_d) But has a binding affinity for a homolog of the antigen from the second non-human mammalian species that is at least about 50-fold, or at least about 500-fold, or at least about 1000-fold weaker than its binding affinity for a human antigen. The species-dependent antibody may be of various types as defined above, but is typically a humanized or human antibody.

As 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 necessarily have less than 100% sequence identity or similarity to species-dependent antibodies. In one embodiment, the antibody mutant has an amino acid sequence that has at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, most preferably at least 95% amino acid sequence identity or similarity to the amino acid sequence of the heavy or light chain variable domain of the species-dependent antibody. Identity or similarity with respect to this sequence is defined herein as the percentage of amino acid residues in the candidate sequence that are identical (i.e., the same residues) or similar (i.e., amino acid residues from the same group according to common side chain characteristics, see below) to species-dependent antibody residues after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Neither N-terminal, C-terminal, nor extension, deletion, or insertion into the interior of antibody sequences outside of the variable domains should be considered to affect sequence identity or similarity.

To extend the half-life of an antibody or polypeptide comprising an amino acid sequence of the invention, a salvage receptor binding epitope can be attached to the antibody (particularly as described in, e.g., U.S. Pat. No.5,739,277), as described, e.g., in U.S. Pat. No.5,739,277Is an antibody fragment). For example, a nucleic acid molecule encoding a salvage receptor binding epitope can be linked in-frame to a nucleic acid encoding a polypeptide sequence of the invention such that the fusion protein encoded by the engineered nucleic acid molecule comprises the salvage receptor binding epitope and the polypeptide sequence of the invention. As used herein, the term "salvage receptor binding epitope" refers to an IgG molecule (e.g., IgG)₁、IgG₂、IgG₃Or IgG₄) The Fc region of (A) is an epitope responsible for extending the serum half-life of the IgG molecule in vivo (e.g., Ghetie et al, Ann. Rev. Immunol.18:739-766(2000), Table 1). Antibodies with substitutions in their 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 may also be extended, for example, by attaching other polypeptide sequences. For example, antibodies or other polypeptides useful in the methods of the invention can be attached to serum albumin or to that portion of serum albumin that binds to an FcRn receptor or a serum albumin binding peptide such that serum albumin binds to the antibody or polypeptide, e.g., such polypeptide sequences are disclosed in WO 01/45746. In one embodiment, the serum albumin peptide to be attached comprises the amino acid sequence diclprwgcllw. In another embodiment, the half-life of the Fab is extended by these methods. Serum albumin binding peptide sequences can also be found in Dennis et al, J.biol.chem.277: 35035-.

"chimeric VEGF receptor protein" refers to a VEGF receptor molecule having amino acid sequences 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 VEGF and inhibiting the biological activity of VEGF.

An "isolated" antibody refers to a polypeptide or antibody that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment refer to substances that would interfere with diagnostic or therapeutic uses of the antibody and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In certain embodiments, the antibody is purified (1) to more than 95% by weight, most preferably more than 99% by weight, of the antibody as determined by the Lowry method, (2) to an extent sufficient to obtain an N-terminal or internal amino acid sequence of at least 15 residues by using a rotor sequencer, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions and using coomassie blue or silver staining. 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, an isolated antibody will typically be prepared by at least one purification step.

"fragment" refers to polypeptides and portions of nucleic acid molecules that preferably contain 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.

An "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 inhibits, either directly or indirectly, angiogenesis (vasculogenesis), or unwanted vascular permeability. It is understood that anti-angiogenic agents include those agents that bind to and block the angiogenic activity of angiogenic factors or their receptors. For example, the anti-angiogenic agent is an antibody or other antagonist of an angiogenic agent as defined throughout the specification or known in the art, such as, but not limited to, an antibody to VEGF-A, an antibody to a VEGF-A receptor (e.g., a KDR receptor or a Flt-1 receptor), a VEGF-trap, an anti-PDGFR inhibitor such as Gleevec^TM(Imatinib Mesylate). Anti-angiogenic agents also include natural angiogenesis inhibitors such as angiostatin (angiostatin), endostatin (endostatin), and the like. See, e.g., Klagsbrun and D' Amore, Annu. Rev. physiol.53:217-39 (1991); streit and Detmar, Oncogene 22:3172-3179(2003) (for example, in malignant melanoma, anti-angiogenesis is mentionedTable 3 for the biotherapy); ferrara&Alitalo, Nature medicine 5(12): 1359-; tonnii 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).

A "maintenance" dose refers herein to one or more doses of a therapeutic agent administered to a patient during or after treatment. Typically, maintenance doses are administered at therapeutic intervals, for example, at intervals of about every week, about every two weeks, about every three weeks, or about every four weeks.

In one embodiment, the maintenance dose is as depicted in figure 1 (extended therapy), figure 2 or figure 8 or figure 11 herein.

"survival" (survival) refers to the patient remaining alive, including Progression Free Survival (PFS) and Overall Survival (OS). Survival can be assessed by the Kaplan-Meier method, and any difference in survival can be calculated using a hierarchical log-rank test.

"Progression Free Survival (PFS)" refers to the time from treatment (or randomization) to first disease Progression or death. In one aspect of the invention, PFS can be assessed by a solid tumor Response Evaluation Criteria (RECIST). In one aspect of the invention, PFS can be assessed by CA-125 levels as a determinant of progression.

"overall survival" means that the patient remains alive for a period of time, 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., since initiation of therapy or since initial diagnosis. In the study on which the present invention is based, the event used for the survival analysis is death of any cause.

By "extending survival" or "increasing the likelihood of survival" is meant that the PFS and/or OS of a treated patient is extended or increased relative to untreated patients (i.e., relative to patients not treated with a VEGF-specific antagonist, e.g., a VEGF antibody) or relative to a control treatment regimen, such as treatment with a chemotherapeutic agent alone, such as those used in standard therapy for ovarian cancer. For example, an extended PFS refers to a time during which a patient remains alive and has no recurrence of cancer, such as, for example, 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., from initiation of treatment or from initial diagnosis, as compared to a control (e.g., a patient not treated with the same VEGF-specific antagonist). 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., following initiation of therapy or following initial diagnosis.

Hazard Ratio (HR) is a statistical definition of the event ratio. For the purposes of the present invention, hazard ratio is defined to represent the probability of an event in the experimental partition divided by the probability of an event in the control partition at any particular point in time.

The "hazard ratio" in the progression free survival assay is a summary of the differences between the two progression free survival curves, representing a reduced risk of mortality for the treatment compared to the control over the follow-up period.

The term "concurrently" is used herein to refer to the use of two or more therapeutic agents, wherein at least some of the administrations overlap in time. Thus, concurrent administration includes a dosing regimen in which administration of one or more agents is discontinued followed by administration of one or more other agents.

By "monotherapy" is meant a treatment regimen that includes only a single therapeutic agent to treat the cancer or tumor during the course of the treatment period. Monotherapy with a VEGF-specific antagonist means that the VEGF-specific antagonist is administered during the treatment period in the absence of another anti-cancer therapy.

By "maintenance therapy" is meant a treatment regimen that is administered in order to reduce the likelihood of disease recurrence or progression. Maintenance therapy may be provided for any length of time, including extended periods of time up to the lifetime of the subject. Maintenance therapy can be provided after the initial therapy or in combination with the initial or additional therapy. The dosage for the maintenance therapy may vary and may include a reduced dosage compared to the dosage used for other types of therapy.

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

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, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific examples of such cancers include ovarian cancer, ovarian primary peritoneal cancer, ovarian fallopian tube cancer, platinum-sensitive recurrent ovarian epithelial cancer, primary peritoneal cancer, or fallopian tube cancer, squamous cell cancer, lung cancer (including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), peritoneal cancer, hepatocellular cancer, gastric cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, liver cancer (hepatic or renal carcinoma), bladder cancer, hepatoma (hepatoma), breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer (kidney or renal cancer), prostate cancer, vulval cancer, thyroid cancer, and various types of head and neck cancer, and B-cell lymphoma (including low-grade/follicular non-Hodgkin's lymphoma (NHL), Small Lymphocytic (SL) NHL, Intermediate grade/follicular NHL, intermediate grade diffuse NHL, high grade immunocytogenic NHL, high grade lymphoblastic NHL, high grade small non-nucleated NHL, storage disease (bulk disease) NHL, mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom's (Waldenstrom) 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 vascular proliferation associated with nevus (phakomatoess), edema (such as associated with brain tumors), and meggers (Meigs) syndrome.

"metastasis" refers to the spread of cancer from its primary site to other locations in the body. Cancer cells can detach from the primary tumor, penetrate into the lymph and blood vessels, circulate through the bloodstream, and grow in distant foci (metastases) in normal tissue elsewhere in the body. The transfer may be local or remote. Metastasis is a continuous process, depending on the shedding of tumor cells from the primary tumor, spreading through the bloodstream, and stopping at a distal site. At the new site, the cells establish a blood supply and can grow to form a life-threatening mass. Stimulatory and inhibitory molecular pathways within the tumor cell regulate this behavior, and interactions between the tumor cell and host cells in distal sites are also important.

"subject" refers to a mammal, including but not limited to a human or non-human mammal, such as a bovine, equine, canine, ovine, or feline. Preferably, the subject is a human. Herein, a patient is also a subject. Typically, the subject is female.

For the methods of the present invention, the term "instructing" a subject means providing instructions regarding applicable therapy, medication, treatment regimen, and the like, by any means, but preferably in writing, such as in the form of a package insert or other written promotional material.

For the methods of the present invention, the term "promoting" means providing, advertising, selling, or describing a particular drug, combination of drugs, or treatment modality by any means, including in writing, such as in the form of a package insert. Promotion refers herein to 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 ovarian cancer treatment, wherein such promotion is approved by the Food and Drug Administration (FDA) as having been demonstrated to be associated with statistically significant therapeutic efficacy and acceptable safety in a population of subjects.

The term "sale" is used herein to describe the promotion, sale, or distribution of a product (e.g., a medication). Sales specifically include packaging, advertising, and any commercial activity that commercializes a product.

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

The term "anti-cancer therapy" refers to a therapy useful in the treatment of cancer. Examples of anti-cancer therapeutics include, but are not limited to, e.g., surgery, chemotherapeutic agents, growth inhibitors, cytotoxic agents, agents used in radiotherapy, anti-angiogenic agents, apoptotic agents, anti-tubulin agents, and other agents for treating cancer, such as anti-HER-2 antibodies, anti-CD 20 antibodies, Epidermal Growth Factor Receptor (EGFR) antagonists (e.g., tyrosine kinase inhibitors), HER1/EGFR inhibitors (e.g., erlotinib)) Platelet derived growth factor inhibitors (e.g., 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 receptors, TRAIL/Apo2), and other biologically active and organic chemicals, and the like.

The term "cytotoxic agent" as used hereinRefers to a substance that inhibits or prevents cellular function and/or causes cellular destruction. The term is intended to include radioisotopes (e.g., At)²¹¹、I¹³¹、I¹²⁵、Y⁹⁰、Re¹⁸⁶、Re¹⁸⁸、Sm¹⁵³、Bi²¹²、P³²And radioactive isotopes 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.

"chemotherapeutic agent" refers to a chemical compound useful for the treatment of cancer. Examples of chemotherapeutic agents include chemical compounds useful for the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents (alkylating agents), such as thiotepa and thiotepaCyclophosphamide (cyclophosphamide); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines (aziridines), such as benzotepa (benzodepa), carboquone (carboquone), metoclopramide (meteredepa), and uretepa (uredepa); ethyleneimines and methylmelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimetalmamine; annonaceous acetogenins (especially bullatacin and bullatacin); camptothecin (camptothecin) (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin (adozelesin), carvelesin (carzelesin), and bizelesin (bizelesin) synthetic analogs); cryptophycins (especially cryptophycins 1 and 8); dolastatin (dolastatin); duocarmycins (including synthetic analogs, KW-2189 and CB1-TM 1); eiscosahol (eleutherobin); pancratistatin; sarcodictyin; spongistatin (spongistatin); nitrogen mustards, such as chlorambucil (ch)lorambucil), chlorambucil (chlorenaphazine), cholorophosphamide (cholephosphamide), estramustine (estramustine), ifosfamide (ifosfamide), mechlorethamine (mechlorethamine), mechlorethamine hydrochloride (mechlorethamine oxide hydrochloride), melphalan (melphalan), neozimine (novembichin), benzene mustard cholesterol (phenesterine), prednimustine (prednimustine), triamcinolone (trofosfamide), uracil mustard (uracil mustard); nitrosoureas such as carmustine (carmustine), chlorouretocin (chlorozotocin), fotemustine (fotemustine), lomustine (lomustine), nimustine (nimustine) and ramustine (ranimustine); antibiotics such as enediynes antibiotics (enediynes) (e.g., calicheamicins, especially calicheamicin γ 1I and calicheamicin ω I1 (see, e.g., Agnew (1994) Chem. Intl. Ed. Engl.33: 183-186)), anthracyclines (dynemicin), including dynemicin A, bisphosphonates (such as clodronate), epothilones (esperamicin), and neocarzinostatin (neocarzinostatin) and related chromogenes of chromenes, aclacinomycin (aclacinomycin), actinomycins (actinomycin), anthomycin (anthramycin), azaserines (azaserines), bleomycin (bleomycin), actinomycins (actinomycins), carinomycins (carvachromycins), adriamycin (streptomycins), adriamycin (monochromycin), adriamycin (monochromycin (doxorubicin (erythromycin (monochromycin), adriamycin), adr,Doxorubicin (doxorubicin) (including morpholino doxorubicin, cyanomorpholino doxorubicin, 2-pyrrol doxorubicin and deoxydoxorubicin), epirubicin (epirubicin), esorubicin (esorubicin), idarubicin (idarubicin), mariomycin (marcellomomycin), mitomycins such as mitomycin C, mycophenolic acid (mycophenolic acid), norramycin (nogalamycin), olivomycin (olivomycin), pelomycin (peplomycin), pofiomycin (potfiromycin), puromycin (puromycin), triiron doxorubicin (rubicin), doxorubicin (doxorubicinMycin (quelemycin), rodobicin (rodorubicin), streptonigrin (streptonigrin), streptozocin (streptozocin), tubercidin (tubicidin), ubenimex (ubenimex), zinostatin (zinostatin), zorubicin (zorubicin); antimetabolites such as methotrexate (methotrexate) and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine (fludarabine), 6-mercaptopurine (mercaptoprine), thiamiprine (thiamiprine), thioguanine (thioguanine); pyrimidine analogs such as ancitabine (ancitabine), azacitidine (azacitidine), 6-azauridine (azauridine), carmofur (carmofur), cytarabine (cytarabine), dideoxyuridine (dideoxyuridine), doxifluridine (doxifluridine), enocitabine (enocitabine), floxuridine (floxuridine); androgens such as carotinone (calusterone), dromostanolone propionate, epitioandrostanol (epitiostanol), mepiquitane (mepiquitane), testolactone (testolactone); anti-adrenal agents such as aminoglutethimide (aminoglutethimide), mitotane (mitotane), trilostane (trilostane); folic acid supplements such as folinic acid (folinic acid); acetoglucurolactone (acegultone); an aldophosphamide glycoside (aldophosphamideglycoside); aminolevulinic acid (aminolevulinic acid); eniluracil (eniluracil); amsacrine (amsacrine); bestrabuucil; bisantrene; edatrexate (edatraxate); desphosphamide (defosfamide); dimecorsine (demecolcine); diazaquinone (diaziqutone); elfornitine; ammonium etitanium acetate; epothilone (epothilone); etoglut (etoglucid); gallium nitrate; hydroxyurea (hydroxyurea); lentinan (lentinan); lonidamine (lonidamine); maytansinoids (maytansinoids), such as maytansine (maytansine) and ansamitocins (ansamitocins); mitoguazone (mitoguzone); mitoxantrone (mitoxantrone); mopidamol (mopidamol); diamine nitracridine (nitrarine); pentostatin (pentostatin); methionine mustard (phenamett); pirarubicin (pirarubicin); losoxantrone (losoxantrone); podophyllinic acid (podophyllic acid); 2-Ethyl hydrazide (e)thylhydrazide); procarbazine (procarbazine);polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane (rizoxane); rhizomycin (rhizoxin); sizofuran (sizofiran); helical germanium (spirogermanium); tenuazonic acid (tenuazonic acid); triimine quinone (triaziquone); 2,2' -trichlorotriethylamine; trichothecenes (trichothecenes), especially the T-2 toxin, verrucin A, rorodin A and snake-fish (anguidin); urethane (urethan); vindesine (vindesine); dacarbazine (dacarbazine); mannomustine (mannomustine); dibromomannitol (mitobronitol); dibromodulcitol (mitolactol); pipobromane (pipobroman); a polycytidysine; cytarabine (arabine) ("Ara-C"); cyclophosphamide (cyclophosphamide); thiotepa (thiotepa); taxoids, e.g. taxolPaclitaxel (paclitaxel) (Bristol-Myers Squibb Oncology, Princeton, N.J.),Without Cremophor, albumin engineered nanoparticle dosage forms of paclitaxel (American pharmaceutical Partners, Schaumberg, Illinois) anddocetaxel (doxetaxel) ((doxetaxel))-Poulenc ror, antonyy, France); chlorambucil (chlorambucil);gemcitabine (gemcitabine); 6-thioguanine (thioguanine); mercaptopurine (mercaptoprine); methotrexate (methotrexate); platinum analogs, such as cisplatin (cissplatin), oxaPlatinum (oxaliplatin) and carboplatin (carboplatin); vinblastine (vinblastine); platinum (platinum); etoposide (VP-16); ifosfamide (ifosfamide); mitoxantrone (mitoxantrone); vincristine (vincristine);vinorelbine (vinorelbine); oncostatin (novantrone); teniposide (teniposide); edatrexate (edatrexate); daunomycin (daunomycin); aminopterin (aminopterin); (xiloda); ibandronate (ibandronate); irinotecan (irinotecan) (Camptosar, CPT-11) (treatment regimens that include irinotecan with 5-FU and folinic acid); topoisomerase inhibitor RFS 2000; difluoromethyl ornithine (DMFO); retinoids (retinoids), such as retinoic acid (retinoic acid); capecitabine (capecitabine); combretastatin (combretastatin); leucovorin (LV); oxaliplatin (oxaliplatin), including oxaliplatin treatment regimen (FOLFOX); lapatinibPKC- α, Raf, H-Ras, EGFR (e.g., erlotinib)) And an inhibitor of VEGF-A that reduces cell proliferation; and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing.

This definition also includes anti-hormonal agents such as antiestrogens and Selective Estrogen Receptor Modulators (SERMs) that act to modulate or inhibit the action of hormones on tumors, including for example tamoxifen (tamoxifen) (includingTamoxifen), raloxifene (raloxifene), droloxifene (droloxifene), 4-hydroxytamoxifene, trioxifene (trioxifene), naloxifene (keoxifene), LY117018, onapristone (onapristone), andtoremifene (toremifene); aromatase inhibitors which inhibit aromatase which regulates estrogen production in the adrenal gland, such as, for example, 4(5) -imidazole, aminoglutethimide,Megestrol acetate (megestrol acetate),Exemestane (exemestane), formestane (formestane), fadrozole (fadrozole),Vorozole (vorozole),Letrozole (letrozole) andanastrozole (anastrozole), antiandrogens, such as flutamide (flutamide), nilutamide (nilutamide), bicalutamide (bicalutamide), leuprolide (leuprolide), and goserelin (goserelin), and troxacitabine (1, 3-dioxolane nucleoside cytosine analogues), antisense oligonucleotides, particularly antisense oligonucleotides that inhibit gene expression in signaling pathways involving aberrant (atherant) cell proliferation, such as, for example, PKC- α, Ralf, and H-Ras, ribozymes, such as VEGF expression inhibitors (e.g., VEGF expression inhibitors)Nucleic acid) and inhibitors of HER2 expression; vaccines, such as gene therapy vaccines, e.g.A vaccine,A vaccine anda vaccine;rIL-2；a topoisomerase 1 inhibitor;rmRH; and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing.

The term "cytokine" is a generic term for proteins released by a population of cells that act on another cell as intercellular mediators, such as 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, liver growth factor, fibroblast growth factor, prolactin, placental lactogen, tumor necrosis factors- α and- β, Mullerian (Mullerian) inhibitory substances, mouse gonadotropin hormone-related peptides, inhibin, activin, vascular growth factor, integrins, Thrombopoietin (TPO), nerve growth factor such as NGF- α, platelet growth factor, transforming growth factor (CSF), such as TGF-CSF), and TGF- α, and TGF- β, endothelial growth factor, and cytokines such as IL-5631, TNF-7, and its natural cytokine-derived from a biological source, such as IL-7, TNF-gamma-interferon, or a protein, or a recombinant IL-derived from a recombinant factor, or a recombinant IL-derived from a mammalian cell, such as IL-derived from a TNF-derived from a.

"growth inhibitory agent" as used herein refers to a compound or composition that inhibits cell growth in vitro and/or in vivo. As such, the growth inhibitory agent may be one 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 outside the 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 (doxorubicin), epirubicin (epirubicin), daunorubicin (daunorubicin), etoposide (etoposide), and bleomycin (bleomycin). Those agents that block G1 also spill over into S phase arrest, for example, DNA alkylating agents such as tamoxifen (tamoxifen), prednisone (prednisone), dacarbazine (dacarbazine), mechlorethamine (mechlorethamine), cisplatin (cissplatin), methotrexate (methotrexate), 5-fluorouracil (5-fluorouracil), and ara-C. For more information see The Molecular Basis of Cancer, eds Mendelsohn and Israel, Chapter 1, entitled "cell regulation, oncogenes, and anti-inflammatory drugs", Murakami et al, WBSaunders, Philadelphia (1995), especially page 13.

The term "prodrug" as used herein refers to a precursor or derivative form of a pharmaceutically active agent that is less cytotoxic to tumor cells than the parent Drug (parent Drug) and is capable of being enzymatically activated or converted to a more active parent Drug form see, for example, Wilman, "Prodrugs in Cancer Chemotherapy," Biochemical society transduction, 14, pp.375-382,615th Meeting Belfast (1986) and Stella et al, "Prodrugs: AChemic administration to Targeted Drug Delivery," direct Drug Delivery, Borchardt et al, eds., 247-267, Humana Press (1985). Prodrugs of the invention include, but are not limited to, phosphate-containing Prodrugs, phosphorothioate-containing Prodrugs, sulfate-containing Prodrugs, peptide-containing Prodrugs, D-amino acid modified Prodrugs, glycosylated Prodrugs, β -phenoxy-containing Prodrugs, substituted phenoxyamide-containing Prodrugs, and optionally substituted phenyl acetamide-containing Prodrugs such as those that are useful as chemotherapeutic agents that are not limited to the cytotoxic Prodrugs of the invention but are optionally convertible to the more active drugs described herein as uridine-5 drugs.

"radiotherapy" or "radiotherapy" refers to the use of directed gamma rays or beta rays to induce sufficient damage to cells to limit their 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 one administration, while typical doses range from 10-200 units per day (Gray).

By "reduce or inhibit" is meant the ability to elicit an overall reduction of preferably 20% or more, more preferably 50% or more, and most preferably 75%, 85%, 90%, 95%, or more. Decreasing or inhibiting 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.

The term "intravenous infusion" refers to the introduction of a drug into the vein of an animal or human patient over a period of time in excess of about 5 minutes, preferably 30-90 minutes, although intravenous infusion may alternatively be administered for 10 hours or less in accordance with the present invention.

The term "bolus intravenous injection" or "bolus intravenous injection" refers to administration of a drug into a vein of an animal or human such that the body receives the drug in about 15 minutes or less, preferably 5 minutes or less.

The term "subcutaneous administration" refers to the introduction of a drug into the subcutaneous space of an animal or human patient, preferably within the pocket between the skin and the underlying tissue, by relatively slow, sustained delivery from a drug container. The pocket may be created by pinching or pulling the skin up and away from the subcutaneous tissue.

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

The term "subcutaneous bolus" refers to administration of a drug beneath the skin of an animal or human patient, wherein the bolus drug is delivered preferably in less than about 15 minutes, more preferably in less than 5 minutes, and most preferably in less than 60 seconds. Administration is preferably within a pocket between the skin and the subcutaneous tissue, wherein the pocket may be created by, for example, pinching or pulling the skin up and away from the subcutaneous tissue.

"disorder" refers to any condition that would benefit from treatment with an antibody. This includes chronic and acute conditions or diseases, including those pathological conditions that predispose a mammal to the condition in question. Non-limiting examples of conditions to be treated herein include cancer; benign and malignant tumors; leukemia and lymphoid malignancies; neuronal, glial, astrocytic, hypothalamic and other glandular, macrophage, epithelial, stromal and blastocoel disorders; and inflammatory, angiogenic and immunological disorders.

The term "therapeutically effective amount" refers to an amount of a drug effective to treat a disease or disorder in a mammal. In the case of cancer, a therapeutically effective amount of the drug may reduce the number of cancer cells; reducing the size of the tumor; inhibit (i.e., slow to some extent, preferably prevent) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent, preferably prevent) tumor metastasis; inhibit tumor growth to some extent; and/or to alleviate one or more symptoms associated with the condition to some extent. Depending on the extent to which the drug can prevent growth 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 assessing survival duration, Progression Free Survival (PFS) duration, prolongation of Progression Free Survival (PFS), Response Rate (RR), response duration, and/or quality of life.

"treatment" or "treating" refers to a therapeutic treatment of a subject in need of treatment, including subjects who have already suffered from a disorder.

"prophylactic or preventative measures" refer to those in which a disorder is to be prevented.

The term "label" as used herein refers to a detectable compound or composition that is directly or indirectly conjugated to a polypeptide. The label may itself be detectable by itself (e.g., a radioisotope label or a fluorescent label), or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable.

anti-VEGF antibodies and antagonists

Provided herein are uses of anti-VEGF antagonists for the treatment of ovarian cancer. Angiogenesis is one of the major processes leading to the invasion and metastasis of solid tumors. Release of angiogenic promoters such as Vascular Endothelial Growth Factor (VEGF) from tumor cells into the local microenvironment can trigger angiogenic signaling pathways. There is increasing evidence that angiogenesis plays a role in the prognosis and possible progression and prognosis of ovarian cancer disease. See, e.g., Yoneda J, et al, Expression of angiogenic-related genes and growth of human ovoarian carcinomas input mice J Natl Cancer Inst90:447-54, 1998; nakanishi Y, et al, the expression of a vascular endostrial growth factor and transformation growth factor-beta assortment with angiogenesis in epithelial anion cancer caner. int J Gynecol Pathol 16: 256. 1997; gasparini G, et al, Prognositic and predictive value, atomic emission biology in ovoarian carcinomas. int J Cancer 69: 205-; hollingsworth HC, et al, Tumor angiogenisis in advanced stage ovariancarccinoma, am J Pathol 147:33-41,1995; pocket PJ, et al, vacuum endothialgrowth factor expression in early stage overview cancer 80:98-106,1997; alvarez AA, et al, the cosmetic design of angiogenisis antibiotic, Clin Cancer Res5:587-91, 1999; gasparini G.Therational and future location of angiogenesis inhibitors in neoplasma. drugs58:17-38,1999; van Hinsbergh VW, et al, angiogenisis and anti-angiogenisis for the treatment of solid tumors, Ann Oncol10 Suppl 4:60-3,1999; mahonne H, et al, mechanics of tumor angiogenesis and therapeutics, angiogenesis inhibitors, clin Exp Metastasis 17:1-14,1999; FolkmanJ. Tumor angiogenesis. thermal assays. N Eng J Med 285: 1182-; KimKJ, et al, inhibition of vascular end growth factor-induced angiogenesis in vivo, Nature 362:841-4, 1993; and Luo JC, et al, Differential inhibition of fluid accumulation and growth in two molecular assays tumors by an anti variable approach factor/permeability factor neutral analyzing antibody. cancer Res 58:2594-600, 1998.

(i) VEGF antigens

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

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 such identified cDNA encodes a 165 amino acid protein having more than 95% homology to bovine VEGF; this 165 amino acid protein is commonly referred to as human VEGF (hVEGF) or VEGF 165. The mitogenic activity of human VEGF was verified by expression of human VEGF in mammalian host cells. Media conditioned by cells transfected with human VEGF cDNA promoted capillary endothelial cell proliferation, while control cells did not. Leung et al (1989) Science, supra.

Although vascular endothelial growth factor can be isolated and purified from natural sources for subsequent therapeutic use, the relatively low concentration of this protein in follicular cells and the high cost of recovering VEGF, both in terms of effort and expense, prove to be commercially ineffective. Thus, further efforts were made to clone and express VEGF via recombinant DNA techniques. (see, e.g., Ferrara, Laboratory Investigation 72: 615-618(1995) and references cited therein).

VEGF is expressed in various tissues as multiple homodimeric forms (121, 145, 165, 189, and 206 amino acids per monomer) resulting from alternative RNA splicing. VEGF121 is a soluble mitogen and does not bind heparin; longer forms of VEGF bind heparin with increasingly higher affinity. The heparin-bound form of VEGF can be cleaved by plasmin at the carboxy terminus to release the diffusible form of VEGF. The amino acid sequence of the carboxy-terminal peptide identified after plasmin cleavage was Arg110-Ala 111. The amino-terminal "core" protein, VEGF (1-110), isolated as a homodimer, binds neutralizing monoclonal antibodies (such as the antibodies designated 4.6.1 and 3.2 e3.1.1) and soluble forms of VEGF receptors with similar affinity compared to the intact VEGF165 homodimer.

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) was 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).

(ii) anti-VEGF antibodies

anti-VEGF antibodies useful in the methods of treating ovarian cancer of the invention include any antibody or antigen-binding fragment thereof that binds VEGF with sufficient affinity and specificity and is capable of reducing or inhibiting the biological activity of VEGF. anti-VEGF antibodies typically 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.

In certain embodiments of the invention, anti-VEGF antibodies include, but are not limited to, antibodies directed against the monoclonal anti-VEGF antibody a4.6.1 produced by hybridoma ATCC HB 10709; according to Presta et al (1997) Cancer Res.57: the recombinant humanized anti-VEGF monoclonal antibody generated by 4593-4599 can be combined with monoclonal antibody with same epitope. In one embodiment, the anti-VEGF antibody is "Bevacizumab (BV)", also known as "rhuMAb VEGF" orIt contains mutated human IgG1 framework region and antigen binding complementarity determining region from mouse anti-hVEGF monoclonal antibody A.4.6.1, blocking human VEGF from binding its receptor. Bevacizumab derives approximately 93% of the amino acid sequence (including most of the framework regions) from human IgG1 and approximately 7% of the sequence from the mouse antibody a4.6.1.

Bevacizumab and other humanized anti-VEGF antibodies are further described in U.S. patent No.6,884,879, published on 26/2/2005. Additional antibodies include antibodies of the G6 or B20 series (e.g., G6-31, B20-4.1), as described in PCT publication No. WO2005/012359, PCT publication No. WO2005/044853, and U.S. patent application 60/991,302, the contents of which are expressly incorporated herein by reference. For additional antibodies, see U.S. Pat. nos. 7,060,269, 6,582,959, 6,703,020; 6,054,297; WO 98/45332; WO 96/30046; WO 94/10202; EP0666868B 1; 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 that comprises residues F17, M18, D19, Y21, Y25, Q89, I91, K101, E103, and C104 or that comprises residues F17, Y21, Q22, Y25, D63, I83, and Q89.

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

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

and a light chain variable region comprising the amino acid sequence:

DIQMTQSPSS LSASVGDRVT ITCSASQDIS NYLNWYQQKP GKAPKVLIYF TSSLHSGVPSRFSGSGSGTD FTLTISSLQP EDFATYYCQQ YSTVPWTFGQ GTKVEIKR(SEQ ID NO：2)。

the "G6 series antibody" according to the present invention refers to an anti-VEGF antibody derived from the sequence of the G6 antibody or a G6-derived antibody according to any one of fig. 7, 24-26, and 34-35 of PCT publication No. wo2005/012359, the entire disclosure of which is 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, which comprises residues F17, Y21, Q22, Y25, D63, I83, and Q89.

The "B20 series antibody" according to the present invention refers to an anti-VEGF antibody derived from the sequence of the B20 antibody or a B20 derived antibody according to any one of fig. 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 that comprises residues F17, M18, D19, Y21, Y25, Q89, I91, K101, E103, and C104.

"functional epitope" according to the present invention refers to an amino acid residue in an antigen that strongly facilitates antibody binding. Mutations in any of the contributing residues in the antigen (e.g., alanine or homolog mutations to wild-type VEGF) disrupt antibody binding such that the relative affinity ratio of the antibody (IC50 mutant VEGF/IC50 wild-type VEGF) is greater than 5 (see example 2 of WO 2005/012359). In one embodiment, the relative affinity ratio is determined by a solution-binding phage display ELISA. Briefly, 96-well Maxisorp immunoplates (NUNC) were coated with the antibody to be tested in Fab form at a concentration of 2ug/ml in PBS overnight at 4 ℃ and blocked with PBS, 0.5% BSA, and 0.05% Tween20(PBT) for 2 hours at room temperature. Phage displaying hVEGF alanine spot mutants (residues 8-109 forms) or wild type hVEGF (8-109) were first incubated in PBT serial dilutions in Fab coated plates for 15 min at room temperature and the plates were washed with PBS, 0.05% Tween20 (PBST). Bound phage were detected with a 1:5000 dilution of anti-M13 monoclonal antibody horseradish peroxidase (Amersham Pharmacia) conjugate in PBT, developed with 3,3 ', 5, 5' -tetramethylbenzidine (TMB, Kirkegaard & Perry Labs, Gaithersburg, Md.) substrate for approximately 5 minutes, quenched with 1.0M H3PO4, and read spectrophotometrically at 450 nm. The ratio of the IC50 values (IC50, ala/IC50, wt) represents the fold reduction in binding affinity (relative binding affinity).

(iii) VEGF receptor molecules

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-; de Vries et al (1992) Science 255: 989-; terman et al (1992) biochem. biophysis. res. commun.187: 1579-1586. The specificity of each receptor for each VEGF family member varies, but VEGF-A binds both Flt-1 and KDR. Neuropilin-1 is shown to be a selective VEGF receptor, capable of binding heparin-binding VEGF isoforms (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 are identified. The family of Receptor Tyrosine Kinases (RTK) includes receptors that are essential 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 receptors and various cellular substrates, and subsequently 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), usually followed by receptor dimerization, stimulation of intrinsic protein tyrosine kinase activity and receptor transphosphorylation. Thereby creating binding sites for intracellular signaling molecules and resulting in complex formation with a range of cytoplasmic signaling molecules, driving the appropriate cellular response. (e.g., changes in cell division, differentiation, metabolic effects, extracellular microenvironment) see Schlesssinger 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 insertion domain. Matthews et al (1991) proc.natl.acad.sci.usa 88: 9026-; terman et al (1991) Oncogene 6: 1677-1683.

VEGF receptor molecules or fragments thereof that specifically bind VEGF can be used in the methods of the invention to bind to and sequester 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 the target cell. Also included are VEGF receptor fusion proteins, examples of which are described below.

Chimeric VEGF receptor proteins refer to receptor molecules having amino acid sequences derived from at least two different proteins, at least one of which is a VEGF receptor protein, such as the flt-1 or KDR receptor, and which are capable of binding to and inhibiting the biological activity of VEGF. In certain embodiments, the chimeric VEGF receptor proteins of the invention consist of amino acid sequences derived from only two different VEGF receptor molecules; however, amino acid sequences 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 may be linked to amino acid sequences from other unrelated proteins, such as immunoglobulin sequences. Other amino acid sequences in combination with an Ig-like domain will be apparent to one 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, e.g., PCT application publication No. WO97/44453).

Soluble or chimeric VEGF receptor proteins of the invention include VEGF receptor proteins that are not immobilized to the surface of a cell via a transmembrane domain. Thus, soluble forms of VEGF receptors (including chimeric receptor proteins), while capable of binding to and inactivating VEGF, do not contain a transmembrane domain and as such do not generally become bound to the cell membrane of the cell in which the molecule is expressed.

Therapeutic uses of anti-VEGF antibodies

The present invention encompasses anti-angiogenic therapy, a novel cancer treatment strategy that addresses the inhibition of tumor vascular development required to provide nutrition to support tumor growth. Because angiogenesis is involved in both primary tumor growth and metastasis, the anti-angiogenesis therapy provided by the present invention is capable of inhibiting not only the neoplastic growth of tumors at primary sites, but also preventing metastasis of tumors at secondary sites, thereby allowing the tumors 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 in women with ovarian Cancer have shown a correlation between high levels of VEGF and a poor prognosis (Alvarez A et al 1999Clin Cancer Res.; 5: 587. sup. 591; Yamamoto S et al 1997Br J Cancer; 76: 1221. sup. sup. -. sup. 1227. sup. -.

In particular, in one embodiment, the 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 in parallel with administration of an effective amount of an anti-VEGF antibody, followed by anti-VEGF maintenance therapy. In certain embodiments of the invention, the patient has stage III (suboptimal and macroscopically optimally lumpectomy) or stage IV ovarian epithelial primary peritoneal or fallopian tube cancer in other embodiments, the patient has stage I and IIa (clear cell carcinoma only or grade 3) or stage IIb-IV ovarian epithelial, fallopian tube or primary peritoneal cancer.

Combination therapy

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

In certain aspects, the 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 may be used in the combination treatment methods of the present invention. An exemplary and non-limiting list of contemplated chemotherapeutic agents is provided herein under the "definitions" or described herein.

In one example, the invention features a VEGF-specific antagonist in combination with one or more chemotherapeutic agents: (a)E.g., a mixture) or any combination thereof. In certain embodiments, the chemotherapeutic agent is a protein-binding particle such as a taxane, paclitaxel, docetaxel, paclitaxel (e.g., paclitaxel-) Platinum analogs, carboplatin, gemcitabine, or combinations thereof. In one embodiment, the chemotherapeutic agent is carboplatin and paclitaxel or docetaxel. In another embodiment, the chemotherapeutic agent is carboplatin and gemcitabine. Combined administration includes simultaneous administration, use of separate formulations or a single pharmaceutical formulation, and sequential administration in either order, where it is preferred that all active agents exert their biological activity simultaneously for a period of time, followed by maintenance therapy with a VEGF-specific antagonist, e.g., as outlined in fig. 1, fig. 2, or fig. 8 or fig. 11. The preparation and dosing regimen for such chemotherapeutic agents may be used in accordance with the manufacturer's instructions or determined empirically by the skilled practitioner. The preparation and dosing regimen 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 administration of the VEGF-specific antagonist, or may be administered concurrently therewith. In certain embodiments of the invention, the dosing schedules and amounts are as set forth in figure 1, figure 2, or figure 8 or figure 11.

In some other aspects, other therapeutic agents useful for combination tumor therapy with the antibodies of the 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, an anti-VEGF antibody is co-administered with the growth inhibitory agent. For example, the growth inhibitory agent may be administered first, followed by the VEGF antibody. However, simultaneous or prior administration of the VEGF antibody is also contemplated. Suitable dosages of growth inhibitory agents are those currently used and may be reduced by the combined action (synergy) of the growth inhibitory agent and the anti-VEGF antibody.

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

In certain embodiments, a VEGF antagonist (e.g., an anti-VEGF antibody) is used to treat ovarian cancer. In certain embodiments, a VEGF antagonist (e.g., an anti-VEGF antibody) is combined with carboplatin and paclitaxel, followed by an anti-VEGF maintenance therapy. In certain embodiments, a VEGF antagonist (e.g., an anti-VEGF antibody) is combined with cisplatin and paclitaxel, followed by an anti-VEGF maintenance therapy. In certain embodiments, a VEGF antagonist (e.g., an anti-VEGF antibody) is combined with carboplatin and docetaxel, followed by anti-VEGF maintenance therapy. In certain embodiments, a VEGF antagonist (e.g., an anti-VEGF antibody) is combined with carboplatin and gemcitabine, followed by an anti-VEGF maintenance therapy.

In certain aspects, other therapeutic agents useful for combination cancer therapy with the antibodies of the invention include other anti-angiogenic agents. A number of 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 antibody of the invention is used in combination with another VEGF antagonist or VEGF receptor antagonist, such as VEGF variants, soluble VEGF receptor fragments, aptamers capable of blocking VEGF or VEGFR, neutralizing anti-VEGFR antibodies, low molecular weight inhibitors of VEGFR tyrosine kinases, and any combination thereof. Alternatively, or in addition, two or more anti-VEGF antibodies may be co-administered to the patient.

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

The VEGF-specific antagonist and the one or more additional therapeutic agents can be administered simultaneously or sequentially in amounts and for a time sufficient to reduce or eliminate the occurrence or recurrence of tumors, dormant tumors, or micrometastases. The VEGF-specific antagonist can be administered as a maintenance therapy to prevent or reduce the likelihood of tumor recurrence or to prolong progression-free survival of the patient.

As will be appreciated by those of ordinary skill in the art, suitable dosages of chemotherapeutic agents or other anti-cancer agents are generally up to date dosages that have been earlier adopted in clinical therapy (e.g., administration of chemotherapeutic agents alone or in combination with other chemotherapeutic agents). Dose variation may occur depending on the condition being treated. The physician administering the treatment will be able to determine the appropriate dosage for the individual subject.

Outside of the above treatment regimen, the patient may undergo radiation therapy.

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

The invention also features a method of directing a human subject having ovarian cancer to receive treatment with an anti-VEGF antibody by providing instructions to extend the time to progression free survival, reduce the likelihood of cancer recurrence in the subject, or increase the likelihood of survival in the subject. 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 an anti-VEGF antibody may be concurrent with treatment with a chemotherapeutic agent. In certain embodiments, the subject is treated as directed by the method of guidance. Treatment of ovarian cancer by administration of anti-VEGF antibodies with or without chemotherapy may continue until the cancer recurs or dies.

In certain embodiments of the invention, the patient is treated with at least 16 cycles of anti-VEGF therapy following concurrent therapy with the 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 the chemotherapeutic.

The invention further provides a method of promoting comprising promoting administration of an anti-VEGF antibody to treat ovarian cancer in a human subject. In some embodiments, the method further comprises promoting administration of at least one chemotherapeutic agent, followed by anti-VEGF maintenance therapy. In some embodiments, the method further comprises promoting administration of two or more chemotherapeutic agents followed by anti-VEGF maintenance therapy. Administration of the anti-VEGF antibody may be concurrent with administration of the chemotherapeutic agent. Promotion may be by any available means. In some embodiments, the promotion is by a package insert accompanying a commercial formulation of an anti-VEGF antibody. Promotion may also be by a package insert accompanying a commercial formulation of the chemotherapeutic agent. The promotion may be by written or oral notification to a physician or health care provider. In some embodiments, the promotion is by a package insert, wherein the package insert provides instructions to receive ovarian cancer therapy with an anti-VEGF antibody. In yet another embodiment, the package insert includes some or all of the results under example 1 or example 2 or example 3. In some embodiments, the promotion is followed by treatment of the subject with an anti-VEGF antibody with or without a chemotherapeutic agent.

The present invention provides a commercial method comprising marketing an anti-VEGF antibody for treating ovarian cancer in a human subject, thereby prolonging the time to progression free survival of the subject, reducing the likelihood of cancer recurrence in the subject, or increasing the likelihood of survival of 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, marketing is followed by treatment of the subject with an anti-VEGF antibody with or without a chemotherapeutic, 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 an anti-VEGF maintenance therapy. In some embodiments, marketing is followed by treatment of the subject with an anti-VEGF antibody with or without a chemotherapeutic, followed by anti-VEGF maintenance therapy.

Also provided is a commercial method comprising marketing a chemotherapeutic agent in combination with an anti-VEGF antibody for treating ovarian cancer in a human subject, thereby prolonging the time to progression free survival of the subject, reducing the likelihood of cancer recurrence in the subject, or increasing the likelihood of survival of the subject. In some embodiments, marketing is followed by treatment of the subject with a combination of a chemotherapeutic and an anti-VEGF antibody, followed by anti-VEGF maintenance therapy. Also provided is a commercial 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 prolonging the time to progression free survival of the subject, reducing the likelihood of cancer recurrence in the subject, or increasing the likelihood of survival of the subject. In some embodiments, marketing is followed by treatment of the subject with a combination of a chemotherapeutic and an anti-VEGF antibody, followed by anti-VEGF maintenance therapy.

Dose and duration

The 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 particular condition being treated, the particular subject being treated, the clinical condition of the individual patient, the cause of the condition, the site at which the agent is delivered, the method of administration, the schedule of administration, and other factors known to medical practitioners. A "therapeutically effective amount" of the VEGF-specific antagonist to be administered will be determined by such considerations, and is the prevention, amelioration, or treatment, or stabilization of the cancer; extended pre-progression time (progression-free survival duration); or the minimum amount necessary to treat or prevent the occurrence or recurrence of a tumor, dormant tumor, or micrometastases. The VEGF-specific antagonist is not required but optionally can 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 disorder or treatment, and other factors discussed above. These are generally used in the same dosages and routes of administration as previously used, or about 1-99% of the dosages previously employed.

Depending on the type and severity of the disease, about 1. mu.g/kg to 100mg/kg (e.g., 0.1mg/kg-20mg/kg) of the 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 dosage range may be from about 1. mu.g/kg to about 100mg/kg or more, depending on the factors described above. Particularly desirable doses include, for example, 5mg/kg, 7.5mg/kg, 10mg/kg, and 15 mg/kg. For repeated administrations over several days or longer, depending on the condition, the treatment is continued until the cancer is treated, as measured by the 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 weekly, biweekly, or every three weeks at a dose ranging from about 5mg/kg to about 15mg/kg, including but not limited to 5mg/kg, 7.5mg/kg, 10mg/kg, or 15 mg/kg. The progress of the therapy of the invention is readily monitored by conventional techniques and assays.

In other embodiments, such dosing regimens are used in combination with a chemotherapeutic regimen (including, but not limited to, one or more chemotherapeutic agents) as a first line therapy for treating previously untreated ovarian cancer, followed by maintenance therapy. In other embodiments, such dosing regimens are used in combination with a chemotherapeutic regimen (including, but not limited to, one or more chemotherapeutic agents) as a second line therapy for the treatment of recurrent ovarian cancer, followed by maintenance therapy. Further information on suitable dosages is provided in the examples below.

The duration of the treatment will continue as long as the order dictates or until the desired therapeutic effect (such as those described herein) is achieved. In certain embodiments, the VEGF-specific antagonist therapy is continued 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, the anti-VEGF therapy continues for at least 16 cycles after the anti-VEGF treatment concurrently with the chemotherapeutic. In other embodiments, the anti-VEGF therapy continues for at least 12 cycles after concurrent anti-VEGF treatment with the chemotherapeutic.

The VEGF-specific antagonists of the invention are administered to a subject (e.g., a human patient) according to known methods, such as intravenously (as a bolus injection or by continuous infusion over a period of time), by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intra-articular, 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 strategy may 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.

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. In addition, it is suitable to administer the antibody by pulse infusion, in particular with decreasing doses of the antibody. Preferably, the dosage is administered by injection, most preferably, the dosage is administered by intravenous or subcutaneous injection, depending in part on whether the administration is short-term or long-term.

In another example, the VEGF-specific antagonist compound is administered locally, e.g., by direct injection, as permitted at the site of the disorder or tumor, and the injection may be repeated periodically. VEGF-specific antagonists can also be delivered systemically to the subject or directly to tumor cells, e.g., a tumor or tumor bed, following surgical removal of the tumor, to prevent or reduce local recurrence or metastasis (e.g., dormant tumor or micrometastases).

Alternatively, an inhibitory nucleic acid molecule or polynucleotide comprising a nucleic acid sequence encoding a VEGF-specific antagonist can be delivered to an appropriate cell in the subject. In certain embodiments, the nucleic acid may be directed to the tumor itself.

The nucleic acid can be introduced into the cell by any means suitable for the vector employed. Many such methods are well 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 method, gene gun, and microinjection.

V. pharmaceutical formulation

Therapeutic formulations of antibodies for use in accordance with the present invention are prepared for storage by mixing the antibody of the desired purity with an optional pharmaceutically acceptable carrier, excipient or stabilizer (Remington's pharmaceutical Sciences 16th edition, Osol, a.ed. (1980)) in the form of a lyophilized formulation or an aqueous solution. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexane diamine chloride; benzalkonium chloride, benzethonium chloride; phenol, butanol or benzyl alcohol; hydrocarbyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) 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, expressly incorporated herein by reference. Optionally, the formulation contains a pharmaceutically acceptable salt, typically, for example, sodium chloride, and preferably at about 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 to 2.0%, typically v/v.Suitable preservatives include those known in the pharmaceutical arts. Benzyl alcohol, phenol, m-cresol, methyl paraben, and propyl paraben are examples of preservatives. Optionally, the formulation of the present invention may include a pharmaceutically acceptable surfactant at a concentration of 0.005-0.02%.

Typically, 4ml or 16ml of bevacizumab (25mg/ml) is delivered in 100mg and 400mg preservative-free, single use vials supplied with bevacizumab for therapeutic use 100mg of the product is formulated in 240mg of anhydro α -trehalose (dehydroate), 23.2mg of sodium phosphate (monobasic, monohydrate), 4.8mg of sodium phosphate (dibasic, anhydrous), 1.6mg of polysorbate 20, and water for injection, USP 400mg of the product is formulated in 960mg anhydro α -trehalose, 92.8mg of sodium phosphate (monobasic, monohydrate), 19.2mg of sodium phosphate (dibasic, anhydrous), 6.4mg of polysorbate 20, and water for injection, USP.

Also visible are the labels for bevacizumab.

The formulations herein may also contain more than one active compound as necessary for the particular indication being treated, preferably with complementary activities that do not adversely affect each other. For example, it may be desirable to further provide in one formulation an antibody that binds EGFR, VEGF (e.g., an antibody that binds a different epitope on VEGF), VEGFR, or ErbB2 (e.g.) The antibody of (1). 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.

The active ingredient may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (e.g., hydroxymethylcellulose or gelatin-microcapsules and poly (methylmethacylate) microcapsules, respectively), 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. eds (1980).

Sustained release formulations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., 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^TM(injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate) and poly-D- (-) -3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid are capable of releasing molecules for over 100 days, certain hydrogels release proteins for shorter periods of time. When the encapsulated antibodies are maintained in vivo for a long period of time, they may denature or aggregate by exposure to a humid environment at 37 ℃, resulting in a loss of biological activity and a possible change in immunogenicity. Rational stabilization strategies can be designed according to the relevant mechanisms. For example, if the aggregation mechanism is found to be intermolecular S-S bond formation via thiol-disulfide interchange, stabilization can be achieved by modifying sulfhydryl groups, lyophilizing from acidic solutions, controlling humidity, employing appropriate additives, and developing specific polymer matrix compositions.

Formulations for in vivo administration should be sterile. This can be easily achieved by filtration using sterile filtration membranes.

Therapeutic efficacy VI

The main advantage of the treatment of the present invention is the ability to produce significant anti-cancer effects in human patients without causing significant toxicity or adverse effects, such that the patients as a whole benefit from the treatment. The efficacy of the treatment of the invention can be measured by assessing various endpoints commonly used in cancer treatment, including but not limited to tumor regression, tumor weight or size reduction, 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 the tumor vasculature and not necessarily the neoplastic cells themselves, they represent a unique class of anti-cancer drugs and therefore can employ unique measurements and definitions of clinical response to the drug. For example, tumor shrinkage greater than 50% in a two-dimensional analysis is the standard cut-off for the declared response. However, the anti-VEGF antibody of the present invention may cause inhibition of metastatic spread without shrinkage of the primary tumor, or may exert only a tumor-inhibiting effect. Thus, other methods of determining the efficacy of anti-angiogenic therapy are optionally employed, including, for example, measuring plasma or urine markers of angiogenesis and measuring responses via radiological imaging.

In another embodiment, the invention provides a method for prolonging progression free survival of a human patient 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 of 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, as compared to treatment without anti-VEGF antibody maintenance therapy. In one embodiment, the median value of PFS in months (95% CI) in patients treated with bevacizumab and taxane therapy (e.g., docetaxel or paclitaxel) and carboplatin followed by anti-VEGF maintenance therapy is extended by 3.8 months (0.717(0.625,0.824), with a unilateral p-value (time series) <0.001)) compared to controls. In another embodiment, the median difference in PFS in months (95% CI) between patients receiving only paclitaxel and carboplatin and patients receiving paclitaxel, carboplatin, and anti-VEGF antibody, followed by anti-VEGF maintenance therapy is 2.3 months, HR 0.79, and p-value (time series test) 0.0010.

Antibody production

(i) Polyclonal antibodies

Polyclonal antibodies are preferably produced in animals by multiple subcutaneous (sc) passes) Or intraperitoneal (ip) injection of the relevant antigen and adjuvant. Using bifunctional or derivatizing reagents, e.g. maleimidobenzoyl sulphosuccinimide ester (coupled via cysteine residues), N-hydroxysuccinimide (coupled via lysine residues), glutaraldehyde, succinic anhydride, SOCl₂Or R₁N ═ C ═ NR (where R and R are₁Are different hydrocarbon groups) it may be useful to couple 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.

Animals are immunized against an antigen, immunogenic conjugate or derivative by mixing, for example, 100 or 5 μ g 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. After 1 month, animals were boosted with an initial amount of 1/5-1/10 of the peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites. After 7-14 days, blood was collected from the animals and the antibody titer of the serum was determined. Animals were boosted until the titer reached a plateau (plateau). Preferably, the animal is boosted with a conjugate of the same antigen but conjugated to a different protein and/or via a different cross-linking agent. Conjugates can also be prepared as protein fusions in recombinant cell culture. Also, a coagulant such as alum is suitably used to enhance the immune response.

(ii) Monoclonal antibodies

There are a variety of methods available in the art for preparing monoclonal antibodies herein. For example, monoclonal antibodies can be generated using the hybridoma method originally described by Kohler et al, Nature,256:495(1975), or can be generated by recombinant DNA methods (U.S. Pat. No.4,816,567).

In the hybridoma method, a mouse or other suitable host animal, such as a hamster or cynomolgus monkey, 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 may 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).

The hybridoma cells so prepared are seeded and cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused parent myeloma cells. For example, if the parental myeloma cells lack hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will contain hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent HGPRT-deficient cells from growing.

Preferred myeloma cells are those that fuse efficiently, support stable and high-level production of antibodies by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Among the preferred myeloma cell lines are murine myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute cell distribution Center (San Diego, Calif. 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 may 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).

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

Upon identification of hybridoma cells that produce antibodies with the desired specificity, affinity, and/or activity, the clones may be subcloned by limiting dilution procedures and cultured by standard methods (Goding, monoclonal antibodies: Principles and Practice, pp.59-103, Academic Press, 1986). Suitable media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells can be cultured in vivo in animals as ascites tumors.

Monoclonal antibodies secreted by subclones can be suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures, such as, for example, protein a-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

DNA encoding the monoclonal antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of the monoclonal antibody). Hybridoma cells are a preferred source of such DNA. Once isolated, the DNA may be placed into an expression vector, which is then transfected into a host cell that does not otherwise produce immunoglobulin proteins, such as an escherichia coli cell, simian COS cell, Chinese Hamster Ovary (CHO) cell, or myeloma cell, to obtain synthesis of monoclonal antibodies in the recombinant host cell. Recombinant production of antibodies is described in more detail below.

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-. Clackson et al, Nature,352: 624-. Subsequent publications describe the generation of high affinity (nM range) human antibodies by chain shuffling (Marks et al, Bio/Technology 10: 779-. As such, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for isolating monoclonal antibodies.

The DNA may also be modified, for example, by replacing homologous murine sequences with the coding sequences for the constant domains of the human heavy and light chains (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.

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 produce 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.

(iii) Humanized antibody and human antibody

Humanized antibodies have one or more amino acid residues introduced into them from a source other than human. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. Humanization can be performed essentially following the method of Winter and co-workers (Jones et al, Nature,321:522-525 (1986); Riechmann et al, Nature,332:323-327 (1988); Verhoeyen et al, Science,239:1534-1536(1988)), using rodent CDRs or CDR sequences in place of the corresponding sequences of a human antibody. Thus, such "humanized" antibodies are chimeric antibodies (U.S. Pat. No.4,816,567) in which substantially less than the entire human variable domain is replaced with the 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 replaced with residues from analogous sites in rodent antibodies.

The choice of human variable domains, including light and heavy chains, used to make humanized antibodies is important for reducing antigenicity. The entire library of known human variable domain sequences is screened for the variable domain sequences of rodent antibodies according to the so-called "best-fit" method. The human sequence closest to the rodent sequence is then selected as the human Framework (FR) for the humanized antibody (Sims et al, J.Immunol.,151:2296 (1993); Chothia et al, J.mol.biol.,196:901 (1987)). Another approach uses a specific framework derived from the consensus sequence of all human antibodies of a specific subgroup of light or heavy chains. 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)).

More importantly, the antibodies retain high affinity for the antigen and other favorable biological properties after humanization. To achieve this, according to a preferred method, humanized antibodies are prepared by a method of analyzing the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are generally available and familiar to those skilled in the art. Computer programs are also available that illustrate and display the likely three-dimensional conformational structures of selected candidate immunoglobulin sequences. Examination of these display images enables analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that affect the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected from the recipient and import sequences and combined to obtain desired antibody characteristics, such as increased affinity for the target antigen. Generally, CDR residues are directly and most substantially involved in the effect on antigen binding.

Humanized anti-VEGF antibodies and affinity matured variants thereof are described, for example, in U.S. patent No.6,884,879, issued 2/26/2005.

It is now possible to generate transgenic animals (e.g., mice) that are capable of generating a complete repertoire of human antibodies upon immunization in the absence of endogenous immunoglobulin production. For example, it has been described that homozygous deletion of the antibody heavy chain joining region (JH) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of human germline immunoglobulin gene arrays in such germline mutant mice will result in the production of human antibodies upon antigen challenge. See, e.g., Jakobovits et al, Proc.Natl.Acad.Sci.USA,90:2551 (1993); jakobovits et al, Nature,362:255-258 (1993); bruggermann et al, Yeast in Immuno, 7:33 (1993); and Duchosal et al, Nature,355:258 (1992).

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 a repertoire of immunoglobulin variable domain (V) genes from an unimmunized donor. According to this technique, antibody variable domain genes are cloned in-frame into the major or minor coat protein genes of filamentous phage such as M13 or fd and displayed as functional antibody fragments on the surface of the phage particle. Because the filamentous phage particle contains a single-stranded DNA copy of the phage genome, selection based on the functional properties of the antibody also results in selection of the gene encoding the antibody displaying those properties. Thus, the phage mimics some of the properties of the B cell. Phage display can be performed in a variety of formats, for review see, e.g., Johnson, Kevin S. and Chiswell, David J., Current Opinion in structural Biology 3:564-571 (1993). Several sources of V gene segments are available for phage display. Clackson et al, Nature 352:624-628(1991) isolated a large number of different anti-orooxazolone antibodies from a random combinatorial library of small V genes derived from the spleen of immunized mice. Construction of V gene repertoires and isolation of antibodies against a large number of different antigens, including self-antigens, from non-immunized human donors can be performed essentially following the techniques described by Marks et al, J.mol.biol.222:581-597(1991) or Griffith et al, EMBO J.12:725-734 (1993). See also U.S. Pat. Nos. 5,565,332 and 5,573,905.

Human antibodies can also be generated by activating B cells in vitro, as described above (see U.S. Pat. nos. 5,567,610 and 5,229,275).

Human monoclonal anti-VEGF antibodies are described in U.S. Pat. No.5,730,977, published 24/3/1998.

(iv) Antibody fragments

Various techniques have been developed for generating antibody fragments. Traditionally, these fragments have been derived by proteolytic digestion of intact antibodies (see, e.g., Morimoto et al, Journal of Biochemical and biophysical methods 24:107-117 (1992); and Brennan et al, Science,229:81 (1985)). However, now it can be directly driven by heavyThe host cells of the group produce these fragments. For example, antibody fragments can be isolated from phage antibody libraries discussed above. Alternatively, Fab '-SH fragments can be recovered directly from E.coli and chemically coupled to form F (ab')₂Fragments (Carter et al, Bio/Technology 10: 163-. According to another method, F (ab') can be isolated directly from recombinant host cell cultures₂And (3) fragment. Other techniques for generating antibody fragments will be apparent to the skilled practitioner. In other embodiments, the antibody of choice is a single chain Fv fragment (scFv). See WO 93/16185.

(vi) Other amino acid sequence modifications

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 an antibody. Amino acid sequence variants of an antibody are prepared by introducing appropriate nucleotide changes into the antibody nucleic acid or by peptide synthesis. Such modifications include, for example, deletions from and/or insertions into and/or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions may be made to arrive at the final construct, so long as the final construct possesses the desired properties. Amino acid changes can also alter post-translational processing of the antibody, such as changing the number or position of glycosylation sites.

One method that can be used to identify certain residues or regions of an antibody that are preferred mutagenesis positions is referred to as "alanine scanning mutagenesis" as described by Cunningham and Wells, Science 244:1081-1085 (1989). Here, a residue or set of target residues (e.g., charged residues such as arginine, aspartic acid, histidine, lysine, and glutamic acid) is identified and replaced with a neutral or negatively charged amino acid (most preferably alanine or polyalanine) to affect the interaction of the amino acid with the antigen. Those amino acid positions exhibiting functional sensitivity to substitution are then refined by introducing more or other variants at or for the substitution site. Thus, although the site of introduction of an amino acid sequence variation is predetermined, the nature of the mutation itself need not be predetermined. For example, 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 the desired activity.

Amino acid sequence insertions include amino-and/or carboxy-terminal fusions ranging in length from one residue to polypeptides containing 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 insertional variants of the antibody molecule include fusion enzymes at the N-or C-terminus of the antibody (e.g., for ADEPT) or polypeptides that increase the serum half-life of the antibody.

Another class of variants are amino acid substitution variants. These variants have at least one amino acid residue in the antibody molecule replaced with a different residue. Sites of most interest for substitutional mutagenesis include hypervariable regions, but FR alterations are also contemplated.

Substantial modification of antibody biological properties is accomplished by selecting substitutions that differ significantly in their effectiveness in maintaining: (a) the structure of the polypeptide backbone of the surrogate region, e.g., as a folded sheet or helix conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the volume of the side chain. Amino acids can be grouped according to their similarity in side chain properties (A.L. Lehninger, Biochemistry, 2 nd edition, pp.73-75, Worth Publishers, New York, (1975)):

(1) non-polar: ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M)

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

(3) Acidic: asp (D), Glu (E)

(4) Basic: lys (K), Arg (R), His (H)

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

(1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral, hydrophilic: cys, Ser, Thr, Asn, Gln;

(3) acidic: asp and Glu;

(4) basic: his, Lys, Arg;

(5) residues that influence chain orientation: gly, Pro;

(6) aromatic: trp, Tyr, Phe.

Non-conservative substitutions will entail replacing one member of one of these classes for another.

Any cysteine residue not involved in maintaining the correct conformation of the antibody may also be substituted, typically with serine, to improve the oxidative stability of the molecule and prevent aberrant cross-linking. Conversely, cysteine bonds may be added to the antibody to improve its stability (particularly when the antibody is an antibody fragment such as an Fv fragment).

A particularly preferred class of surrogate 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 produced. One convenient method for generating such surrogate variants involves affinity maturation using phage display. Briefly, several hypervariable region sites (e.g., 6-7 sites) were mutated to generate all possible amino acid substitutions at each site. The antibody variants so produced are displayed in monovalent form on filamentous phage particles as fusions to 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. To identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues which contribute significantly to antigen binding. Alternatively, or in addition, 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 candidates 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.

Another class of amino acid variants of antibodies alters the original glycosylation pattern of the antibody. An alteration means the deletion of one or more carbohydrate moieties found in the antibody, and/or the addition of one or more glycosylation sites not present in the antibody.

Glycosylation of antibodies is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid except proline) are recognition sequences for enzymatic attachment of a carbohydrate module to an asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates potential glycosylation sites. 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.

The 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 may also be made by adding or replacing one or more serine or threonine residues to the sequence of the original antibody (for O-linked glycosylation sites).

If the antibody comprises an Fc region, the carbohydrate attached thereto may be altered. For example, U.S. patent application No. US 2003/0157108 a1(Presta, L.) describes antibodies with a mature carbohydrate structure lacking fucose attached to the Fc region of the antibody. See also US 2004/0093621a1(Kyowa Hakko Kogyo co., Ltd.). Antibodies having an aliquot of N-acetylglucosamine (GlcNAc) in the carbohydrate attached to the Fc region of the antibody are mentioned in WO 03/011878(Jean-Mairet et al) and U.S. Pat. No.6,602,684(Umana et al). Antibodies having at least one galactose residue in an oligosaccharide attached to the Fc region of an antibody are reported in WO 97/30087(Patel et al). For antibodies with altered carbohydrate attachment to their Fc region see also WO 98/58964(Raju, S.) and WO 99/22764(Raju, S.).

It may be desirable to modify an antibody of the invention with respect to effector function, e.g., to enhance 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 in the Fc region of the antibody. Alternatively, or in addition, cysteine residues may be introduced into the Fc region to allow for interchain disulfide bond formation in this region. The homodimeric antibody so 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-. Homodimeric antibodies with enhanced anti-tumor activity can also be prepared using heterobifunctional cross-linkers as described in Wolff et al, Cancer Research53: 2560-. Alternatively, antibodies can be engineered to have dual Fc regions, and thus can have enhanced complement lysis and ADCC capabilities. See Stevenson et al, Anti-Cancer Drug Design 3:219-230 (1989).

WO00/42072 (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 region. Preferably, antibodies with improved ADCC comprise substitutions at positions 298, 333, and/or 334 of the Fc region (Eu residue numbering). Preferably, the altered Fc region is a human IgG1Fc region comprising, replacing, or consisting of at one, two, or three of these positions. Such substitutions are optionally combined with substitutions that enhance C1q binding and/or CDC.

Antibodies with altered C1q binding and/or Complement Dependent Cytotoxicity (CDC) are described in WO 99/51642, U.S. patent No.6,194,551B1, U.S. patent No.6,242,195B1, U.S. patent No.6,528,624B1, and U.S. patent No.6,538,124 (idulogie et al). The antibody comprises an amino acid substitution at one or more of amino acids 270, 322, 326, 327, 329, 313, 333 and/or 334 (Eu residue numbering) of its Fc region.

To extend the serum half-life of the antibody, a salvage receptor binding epitope can be incorporated into the antibody (particularly an antibody fragment), as described, for example, in U.S. Pat. No.5,739,277. As used herein, the term "salvage receptor binding epitope" refers to an IgG molecule (e.g., IgG)₁、IgG₂、IgG₃Or IgG₄) Is responsible for extending the serum half-life of the IgG molecule in vivo.

Antibodies with improved binding to neonatal Fc receptor (FcRn) and extended half-life are described in WO00/42072 (Presta, L.) and US 2005/0014934a1(Hinton et al). These antibodies comprise an Fc region having one or more substitutions therein that improve the binding of the Fc region to FcRn. For example, the Fc region may have substitutions 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 residue 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 of their Fc region (Eu residue numbering). In one embodiment, the antibody has the 307/434 mutation.

Engineered antibodies having three or more (preferably four) functional antigen binding sites are also contemplated (U.S. patent application No. US 2002/0004587a1, Miller et al).

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

(v) Immunoconjugates

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 a fragment thereof), or a radioisotope (i.e., a radioconjugate).

Enzymatically active toxins and fragments thereof that may be used include diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin (ricin) A chain, abrin (abrin) A chain, modeccin A chain, α -sarcin (sarcin), Aleutites fordii (Aleurites fordii) toxic protein, carnation (dianthin) toxic protein, Phytolacca americana (Phytolacca americana) toxic protein (PaecPI, PAPII and PAP-S), Momordica charantia (radionuclide charantia) inhibitor, curcin (curcin), crotin (crotin), Saponaria officinalis (sapaonaria), gelonin (gelonin), trichothecin (trichothecin), various antibodies (trichothecin), and various antibodies including actinomycin), trichothecin (trichothecin), and various antibodies (trichothecin) including²¹²Bi、¹³¹I、¹³¹In、⁹⁰Y and¹⁸⁶Re。

conjugates of the antibody and cytotoxic agent may 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 hcl), 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, a ricin immunotoxin may be prepared as described in Vitetta et al Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelator for conjugating radionucleotides to antibodies. See WO 94/11026.

In another embodiment, the antibody may be conjugated 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, followed by administration of a "ligand" (e.g., avidin) conjugated to a cytotoxic agent (e.g., a radionucleotide).

(vi) Immunoliposomes

The antibodies disclosed herein can also be formulated as immunoliposomes. Liposomes containing antibodies can be prepared by methods known in the art, such as, for example, see 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. Pat. No.5,013,556.

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 extruded through a filter having a set pore size to produce liposomes having a desired diameter. Fab' fragments of the antibodies of the invention can be coupled to 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), is included in the liposomes. See Gabizon et al, J.national Cancer Inst.81(19):1484 (1989).

Article and kit

In another embodiment of the invention, an article of manufacture comprising a substance useful for treating the conditions described above is provided. Articles include containers, labels, and package inserts. Suitable containers include, for example, bottles, vials, syringes, and the like. The container can be made of various materials, such as glass orA plastic. The container contains a composition effective for treating the condition and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection 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 for use in treating a selected condition. The article of manufacture may further comprise a second container containing a pharmaceutically acceptable buffer, such as phosphate buffered saline, Ringer's solution, and dextrose solution. It may also include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles and syringes. In addition, the article of manufacture comprises a package insert with instructions for use, including, for example, instructing a user of the composition to administer to a patient an anti-VEGF antibody composition and a chemotherapeutic agent, e.g., a protein-binding particle of a taxane, paclitaxel, docetaxel, paclitaxel (e.g., paclitaxel-coated particles)) Platinum analogs, carboplatin, gemcitabine, or combinations 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.

The VEGF-specific antagonist may be packaged into a kit, alone or in combination with other anti-cancer therapeutic compounds. The kit may include optional ingredients to aid in administering unit doses to a patient, such as vials for reconstitution of powder form, syringes for injection, custom IV delivery systems, inhalers, and the like. In addition, the unit dosage kit can contain instructions for making and administering the composition. In certain embodiments, the instructions comprise instructions for use, including, for example, instructing a user of the composition to administer to a patient an anti-VEGF antibody composition and a chemotherapeutic agent, e.g., a protein-binding particle of a taxane, paclitaxel, docetaxel, paclitaxel (e.g., paclitaxel, etc.)) Platinum analog, carboplatin, gemcitabine, or a derivative thereofCombination, followed by anti-VEGF maintenance therapy. The instructions may optionally contain some or all of the results found in example 1 or example 2 or example 3. Kits can be manufactured in a single use unit dose for one patient, in multiple uses for a particular patient (at a constant dose or where the potency of each compound may vary as treatment progresses); alternatively, the kit may contain multiple doses ("bulk packs") suitable for administration to multiple patients. The kit components may be assembled in cartons, blister packs, bottles, tubes, and the like.

The following examples are intended only to illustrate the practice of the present invention and are not provided as limitations. The entire disclosure of all patent and scientific literature cited herein is expressly incorporated by reference.

Examples

Example 1: carboplatin and paclitaxel placebo vs carboplatin and paclitaxel plus concurrent bevacizumab followed by placebo, phase III trials in women with new diagnosis, previously untreated, stage III (suboptimal and macroscopically best massive resection) or stage IV ovarian epithelial carcinoma, primary peritoneal carcinoma or fallopian tube carcinoma, relative to carboplatin and paclitaxel plus concurrent and extended bevacizumab

Results from phase III randomized studies are presented to evaluate new treatment procedures for patients with gynecological international association of oncology (fig) III and IV ovarian epithelial cancer, primary peritoneal cancer or fallopian tube cancer (epithelial ovarian cancer, epithelial primary or fallopian tube cancer). The primary objective included determining whether the addition of 5 concurrent cycles of bevacizumab to 6 cycles of standard therapy (carboplatin and paclitaxel) [ arm II ] extended the duration of Progression Free Survival (PFS) compared to 6 cycles of standard therapy [ arm I ] alone in women with newly diagnosed stage III (with any overall residual disease) and stage IV ovarian epithelial cancer, primary peritoneal cancer or fallopian tube cancer; and determining whether the addition of 5 concurrent cycles of bevacizumab plus extended bevacizumab over 6 cycles of standard therapy (carboplatin and paclitaxel) in women with newly diagnosed stage III (with any overall residual disease) and stage IV ovarian epithelial cancer, primary peritoneal cancer or fallopian tube cancer extended progression free survival compared to 6 cycles of standard therapy [ branch I ].

GOG-0182-ICON5 is a 5-arm randomized clinical trial comprising standard therapies (carboplatin and paclitaxel) and four arms of investigation incorporating gemcitabine (gemcitgabine), topotecan (topotecan) and liposomal doxorubicin (lipoxaldoxorubin), either in combination or sequentially with paclitaxel and carboplatin. The major ovarian cancer clinical trial groups worldwide participated in this study. This international cooperation provides a unique opportunity to accumulate a large number of patients in a timely manner, which facilitates the simultaneous evaluation of multiple agents in a prospective randomized trial. With international participation, more than 1,200 patients accumulate each year, and the trial reaches its targeted accumulation target within four years of initiation.

While the results of GOG-0182-ICON5 helped to establish optimal chemotherapy for previously untreated patients with advanced ovarian and peritoneal primary cancers, next generation clinical trials explored 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 chemotherapeutic 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 biological agent administered in combination with a standard chemotherapeutic treatment, plus or minus a prolonged single agent, on outcome is evaluated in patients with advanced disease compared to standard chemotherapeutic treatment alone.

Bevacizumab is a recombinant humanized form of 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 extending the time to disease progression in patients with metastatic solid tumors. See, e.g., Presta LG, et al, manipulation of an anti-inflammatory growth hormone antibody for the therapy of solid tumors and other disorders. cancer Res 57: 4593. 9, 1997. Results of two single agent trials of bevacizumab for patients with recurrent primary cancers of ovarian epithelium and peritoneum have been published. See, e.g., Burger RA, et al, phase II tertiary of bevacizumab in persistence or recurrence epitope in cancer primary cancer a Gynecological Oncology Group study. J Clin Oncol25(33):5165-171, 2007; and Cannistra SA, et al, Phase II Study of Bevacizumab inPatients with Platinum Resistant innovative Cancer or Primary Peritroneal Serosuncor J clean Oncor 25(33) 5180-86.2007. GOG (GOG-0170-D) utilizes two common primary efficacy endpoints (co-primary efficacy endpoints): NCI RECIST standard clinical response and rate of progression free survival of at least 6 months. 62 participants received bevacizumab 15mg/kg every 21 days until clinical or radiographic evidence of disease progression or unacceptable toxicity. The primary disease is characteristic of patients with recurrent ovarian cancer, and approximately 43% of patients are considered primary platinum resistant. A 21% response rate was observed, while 40% had no progression for at least 6 months, with a median PFS of 4.7 months, compared to 1.8 months based on historical controls of negative phase II trials of previous cytotoxic agents in populations with similar clinical characteristics. Genentech AVF 2949 examined patients with higher risk conditions in terms of disease progression and potential for adverse events, only patients considered primary or secondary platinum resistance and who received 2 or 3 previous cytotoxicity protocols were admitted. These differences in enrollment conditions ultimately translate into higher levels of platinum resistance, a greater number of prior regimens, and a slightly worse performance state in the AVF population. 44 patients were treated with the same bevacizumab dose and schedule as used in GOG 170-D. 7 (16% >) responses were recorded, and 12 (27%) had no progress for at least 6 months.

In this study, two experimental arms were chosen to compare with standard cytotoxic chemotherapy of paclitaxel and carboplatin: one incorporated 5 cycles of bevacizumab (concurrent bevacizumab) and the other 16 cycles of bevacizumab (extended bevacizumab) after completing chemotherapy of paclitaxel and carboplatin.

Administration and dosage are shown in figures 1 and 2. Calvert equation for carboplatin (AUC) dosing:

total dose (mg) ═ target AUC (in mg/mL/min) ([ GFR (in mL/min) +25 ].

Statistical design of the primary endpoint study tested for PFS Hazard Ratio (HR) ≦ 0.77 (median PFS shift: 14.0 months (historically) → 18.2 months.) the primary assay compared the investigator's assessed baseline clinical characteristics of PFS patients to controls (assay 1-by RECIST (see, e.g., therase et al, J nature. cancer inst.,92:205 @, 16,2000), global clinical exacerbation (global clinical exacerbation), or CA-125; or by assay 2-RECIST or global clinical exacerbation, examining CA-125) for each bevacizumab arm, table 1. the baseline surgical-pathological characteristics of patients are table 2.

Patient enrollment was: the patient has a histological diagnosis of ovarian epithelial cancer (epithelial ovarian cancer), primary cancer of the peritoneum (epithelial primary cancer), or fallopian tube cancer; either in phase III (with any gross (macroscopic or palpable) residual disease) or in phase IV, surgically defined at the completion of the initial abdominal surgery and with appropriate tissue for histological evaluation. The minimum surgery required is abdominal surgery, which provides tissue for histological evaluation and the establishment and documentation of primary sites and stages, and the best effort for massive resection of tumors. If another procedure is performed, it should comply with the appropriate procedures for ovarian or peritoneal cancers as described in the GOG surgical protocols Manual (https:// www.gog.fccc.edu/manuals/pdf/surgman. pdf). However, the surgeon is not required to implement all of the items contained in this section of the GOG surgical procedure manual. Patients with stage III cancer in which the maximum diameter maximum of any residual tumor implant at the completion of this initial procedure is no greater than 1cm would be defined as "optimal"; all other cases will be defined as "suboptimal". Detectable disease in post-operative imaging studies is not required for inclusion.

Patients with the following histological epithelial cell types were enrolled: serous adenocarcinoma, endometrioid adenocarcinoma, mucinous adenocarcinoma, undifferentiated carcinoma, clear cell adenocarcinoma, mixed epithelial carcinoma, transitional cell carcinoma, malignant Brenner's tumor, or an otherwise unspecified adenocarcinoma (n.o.s.). However, the histological characteristics of the tumor must be consistent with the primary Mullerian epithelial adenocarcinoma. The patient may have coexisting carcinoma of the fallopian tubes in situ, as long as the primary origin of the invasive tumor is ovarian, peritoneal or fallopian tubes.

The patient must have the appropriate:

(1) bone marrow function: absolute Neutrophil Count (ANC) greater than or equal to 1,500/. mu.1, equates to an Adverse event cotoxicity Criteria (Common Toxicity criterion for Adverse Events) v3.0(CTCAE) grade 1. This ANC cannot be induced or inhibited by granulocyte colony stimulating factor.

(2) Platelets are greater than or equal to 100,000/μ 1. (CTCAE 0-1 grade).

(3) Renal function: creatinine <1.5x convention upper normal limit (ULN), CTCAE grade 1.

(4) Liver function:

(a) bilirubin is less than or equal to 1.5x ULN (CTCAE grade 1).

(b) SGOT and alkaline phosphatase less than or equal to 2.5 XULN (CTCAE grade 1).

(c) Nerve function: neuropathy (sensory and motor) is less than or equal to CTCAE grade 1.

(5) Blood coagulation parameters: such that the International Normalized Ratio (INR) is <1.5 (or range INR, typically between 2 and 3 if the patient is taking a stable dose of therapeutic warfarin for the purpose of controlling venous thrombosis, including pulmonary thromboembolism) and PTT <1.2x PT at the upper normal limit.

(6) Patients with a GOG performance status of 0,1, or 2.

(7) Patients must enter between 1 and 12 weeks after the initial surgery performed for the combined purposes of diagnosis, staging and cytopenia.

(8) Patients with detectable and undetectable disease were enrolled. The patient may or may not have symptoms associated with cancer.

(9) Patients who achieved the pre-entry requirements specified in subsection 7.0.

(10) The patient or guardian must sign an approved informed consent and an authorized license for the personal health information.

(11) The patient card in this trial may receive the lowest effective dose of ovarian estrogen +/-progestin replacement therapy (as indicated) at any time for controlling menopausal symptoms, but no progestin is taken for managing anorexia at the time of regimen-directed therapy or prior to disease progression.

Patients were not enrolled: patients currently diagnosed with either borderline ovarian epithelial tumors (previously referred to as "low malignant potential tumors") or recurrent invasive ovarian epithelial cancer, primary peritoneal cancer, or fallopian tube cancer (treated only with surgery), such as patients with la-grade or lb-grade ovarian epithelial cancer or fallopian tube cancer, are not enrolled. Patients with prior diagnoses of borderline tumors (surgical resection followed by the development of unrelated, new invasive ovarian epithelial, primary peritoneal or fallopian tube cancer) were enrolled, provided they did not receive prior chemotherapy for any ovarian tumor.

Patients who received prior radiation therapy to any part of the pelvis or abdomen were excluded. Prior radiation of localized cancers of the breast, head and neck, or skin is allowed, provided it is completed more than three years prior to enrollment, and the patient still has no recurrent or metastatic disease.

Patients who received prior chemotherapy (including neoadjuvant chemotherapy for their ovarian, primary peritoneal or fallopian tube cancer) for any abdominal or pelvic tumor were excluded. Patients may have received prior adjuvant chemotherapy for localized breast cancer, provided that it is completed more than three years prior to enrollment, and that the patient still has no recurrent or metastatic disease.

Patients who have received any targeted therapy (including but not limited to vaccines, antibodies, tyrosine kinase inhibitors) or hormonal therapy to treat their primary carcinoma of the ovarian epithelium or peritoneum.

Patients with a prior history of simultaneous primary endometrial cancer or primary endometrial cancer are excluded unless all of the following conditions are met: a stage not greater than I-B; no more than superficial myometrial invasion, no vascular or lymphatic invasion; there were no poorly differentiated subtypes, including papillary serous, clear cell or other FIGO grade 3 lesions.

Patients with other invasive malignancies were excluded, with any evidence of the presence of other cancers within the last 5 years or previous cancer treatments banned this regimen therapy, except for non-melanoma skin cancers and other specific malignancies described above.

Patients with acute hepatitis or active infection requiring parenteral antibiotics.

Patients with severe non-healing wounds, ulcers, or fractures. This included a history of 28-day intra-abdominal fistulas, gastrointestinal perforations, or intra-abdominal abscesses. Patients with secondary healed, granulated incisions were enrolled without evidence of surface dehiscence or infection, but required weekly wound examinations.

Patients with active bleeding or pathological conditions with a high risk of bleeding, such as known bleeding disorders, coagulopathies, or tumors involving major blood vessels.

The patient had a history or evidence of CNS disease at the time of physical examination, including a primary brain tumor, seizures uncontrolled by standard medical therapy, any brain metastases, or a history of cerebrovascular accidents (CVA, stroke), Transient Ischemic Attacks (TIA), or subarachnoid hemorrhage within the six months of treatment of this study.

Patients with clinically significant cardiovascular disease. This includes: uncontrolled hypertension, defined as systolic pressure >150mm Hg or diastolic pressure >90mm Hg; myocardial infarction or unstable angina <6 months prior to enrollment; congestive heart failure of New York Heart Association (NYHA) class II or greater; severe arrhythmia requiring medication. This excludes asymptomatic, ventricular rate-controlled atrial fibrillation; CTCAE grade 2 or greater peripheral vascular disease (transient ischemia without surgical treatment and without permanent defects (at least <24 hours)); history of CVA over six months.

Patients with known hypersensitivity to chinese hamster ovary cell products or other recombinant human or humanized antibodies.

Patients with clinically significant proteinuria. Urine proteins should be screened by the urine protein-creatinine ratio (UPCR). UPCR has been found to be directly related to the amount of protein excreted in 24 hour urine collection. See, e.g., Ginsberg JM, et al, Use of single void urea samples to estimate quality qualification protocol, N Engl J Med 309: 1543-; rodby RA, et al, The urea protein characterization as a predictor of 24-urea protein amplification in type1 biological protocols with a thiophosphory. The collagen studio group. am J Kidney Pis 26: 904-; schwab SJ, et al, quantification of protein by the use of protein-to-secretion rates in single urea samples, Arch Intern Med147: 943-; steinhauslin F, & Watters JP.quantity of proteinum in vegetable and mammalian tissues, acquisition of the proteinum/secretory ratio, ClinnNPhere 43:110-5, 1995; protein-affinity ratio for the qualitative assessment of protein from a random analysis sample, am JClin Pathol 100:419-24, 1993; and Zelmavitz T, et al, protein is stilluussesult for the screening and diagnosis of overkinetic chromatography, diabetes Care 21:1076-9, 1998. Specifically, a UPCR of 1.0 equates to 1.0 gram of protein in a 24 hour urine collection. Patients must have a UPCR <1.0 to allow participation in the study.

A patient having or expected to have an invasive procedure as defined below: bevacizumab/placebo therapy (cycle 2) with major surgery, open biopsy or major trauma within 28 days before day 1. Expected major surgery during the course of the study. This includes, but is not limited to, abdominal surgery (laparotomy or laparoscopy) prior to disease progression, such as colostomy or enterostomy reversal, interphase or secondary cytoreductive surgery, or secondary hook surgery. Core biopys (core biopsy) occurred within 7 days prior to day 1 of bevacizumab/placebo therapy (cycle 2).

Patients with grade 3 or 4 GOG performance.

Pregnant or lactating patients.

Patients under the age of 18 years.

Patients who received prior therapy with any anti-VEGF drug, including bevacizumab.

Patients with clinical symptoms or signs of gastrointestinal obstruction and who require parenteral hydration and/or nutrition.

Patients with other medical histories or conditions that the physician appears to exclude from study participation.

International standards recommended by the committee for solid tumor Response assessment Criteria (RECIST) will be used in this study to assess Response and progression. See, e.g., therase P, et al, New peptides to estimate the response to a Treatment in a solid mass, European Organization for Research and Treatment of Cancer, National Cancer Institute of the United States, National Cancer Institute of Cancer. JNTATL Cancer Institute 92:205 @, 16, 2000. Only the change in the maximum diameter of the tumor lesion (one-dimensional measurement) was used in RECIST criteria.

CA-125 as a biological marker of progressive disease: serum levels of CA-125, a tumor-associated glycoprotein antigen, are elevated in 80% of patients with ovarian epithelial cancer. See, e.g., Bast et al, N.Engl.J.Med.309:88307,1983. CA-125 is frequently monitored to test for response to therapy, the presence of residual disease, and as early evidence of relapse. However, CA-125 is not completely tumor specific, it can be elevated in a variety of benign conditions, such as endometriosis, uterine fibroids, and pelvic inflammation; this is particularly true in premenopausal women. In addition, CA-125 levels may be inconsistent with tumor response, with both false positive and false negative trends; the effect of biological agents on these inaccuracies is unclear. In any event, it has become standard practice for patients, and physicians consider the gradual rise of CA-125 after treatment to be evidence of recurrent or progressive disease, and will make treatment decisions based on CA-125. Current randomization trials will employ a conservative formula to define progressive disease based on a continuous increase in CA-125 (beyond the definition of other criteria in solid tumor treatment), but only after completion of initial chemotherapy. See, e.g., 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 following completion of cytotoxic chemotherapy if one of three conditions is reached: 1) patients with elevated CA-125 prior to treatment and normalization of CA-125 must show evidence that CA-125 is greater than or equal to twice the upper limit of normality at two times separated by at least one week; or 2) evidence that a patient with elevated CA-125 but never normalized prior to treatment must show CA-125 greater than or equal to twice the lowest value at two times separated by at least one week; or 3) patients with CA-125 in the normal range before treatment must show evidence that CA-125 is greater than or equal to twice the upper normal limit at two times separated by at least one week.

Results

The results of the study demonstrate that bevacizumab is effective as a first-line ovarian cancer in combination with chemotherapy and continued as a maintenance therapy. This combination is effective in prolonging the PFS. The safety preliminary assessment identified bevacizumab-related Adverse Events (AEs) recorded in previous studies. The primary analysis of PFS showed a median progression-free survival (month) of 10.3 (fig. 2 branch 1), compared to 14.1 months for branch 3 of fig. 2. The HR (95% CI) for the one-sided p-value (chronologically examined 0.08) of branch I of fig. 2 was 0.908(0.795,1.04) compared to 0.717(0.625,0.824) for the one-sided p-value (chronologically <0.001) of branch III of fig. 2. See fig. 5. The differences were statistically significant. Treatment regimens are generally better tolerated and adverse events (including GI perforation) are similar to previous bevacizumab studies. See fig. 3 and 4. This was the first anti-angiogenic therapy that exhibited benefit in this population. FIG. 6 illustrates branching using CA-125 as a progression determinant. CA-125 is an antigenic determinant on a high molecular weight glycoprotein that is recognized by a monoclonal antibody (OC-125) produced using an ovarian cancer cell line as an immunogen. CA125 was evaluated as a serum marker to monitor patients with ovarian epithelial cancer and other cancers. See, e.g., references Gvn Oncol 38:373,1990; gyn Oncol 38:181,1990; amer J Ob Gyn 160:667,1989; amer J Ob Gvn159:873,1988; amer J Ob Gvn159: 341,1988; ob Gvn 72:159,1988; and Gyn Oncol 36:299,1990 and described herein. FIG. 7 illustrates subgroup analysis of Branch I versus Branch III.

Table 1: baseline clinical characteristics

Table 2: baseline surgery-pathology characteristics

Example 2. One randomized, two-arm, multicenter gynecological cancer group trial with addition of bevacizumab to standard chemotherapy (carboplatin and paclitaxel) in patients with ovarian epithelial cancer

Results from a phase III randomization study (ICON7) are presented to evaluate the safety and efficacy of standard chemotherapy with bevacizumab added to carboplatin and paclitaxel. The primary endpoint was to determine whether the addition of bevacizumab to standard chemotherapy in women with newly diagnosed, histologically confirmed, high risk international association of gynecology and obstetrics (FIGO) stages I and IIa (grade 3 or clear cell carcinoma only) and FIGO IIb-IV (all grades and all histological types), ovarian epithelial cancer, fallopian tube cancer or primary peritoneal cancer, who underwent initial surgery (massive resection with reduced cell surgery or biopsy if the patient had a FIGO stage IV disease), and who did not consider reduced cell surgery before disease progression improved Progression Free Survival (PFS) compared to standard chemotherapy alone. Secondary endpoints include Overall Survival (OS), response rate, duration of response, biologically progress-free interval (defined as increasing CA125 or PFIBio), safety, and quality of life. ICON7 is a 2-arm randomized clinical trial comparing standard therapy (carboplatin and paclitaxel) with one arm of investigation incorporating bevacizumab in combination with paclitaxel and carboplatin (see figure 8). A total of 1528 enrolled women participated in the trial.

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 extending the time before disease progression in patients with metastatic solid tumors. See, e.g., Presta LG, et al, manipulation of an anti-inflammatory growth hormone antibody for the therapy of solid tumors and other disorders. cancer Res 57: 4593. 9, 1997.

Patient selection

ICON7 includes patients with newly diagnosed, histologically confirmed, high risk fino I and IIa (grade 3 or clear cell carcinoma only) and fino IIb-IV (all grades and all histological types) ovarian epithelial, fallopian tube, or primary peritoneal cancers who have undergone initial surgery (massive resection cytoreductive surgery or biopsy if the patient has a fino IV stage disease) and who will not consider cytoreductive surgery before disease progression. Patients with measurable and unmeasurable disease were enrolled. Patients were considered enrolled and enrolled in this trial if they met all of the admission criteria and none of the exclusion criteria described below:

patient admission criteria:

women aged 18 years or older

Histologically confirmed core biopsies from disease fractions as minimum requirements (cytology alone is not sufficient to make a diagnosis)

○ epithelial carcinoma of ovary

○ Primary peritoneal carcinoma (which must be of the papillary-serous histological type) or

○ fallopian tube carcinoma

And meet the criteria in Table 3

Patients with clear cell carcinoma at any stage are enrolled because of the poorer prognosis associated with this subtype. Patients with early stage ovarian epithelial or fallopian tube cancer previously treated with surgery alone are candidates in diagnosing abdomino-pelvic recurrence, provided that no further compartmental cytoreductive therapy is planned prior to disease progression.

For the purposes of this experiment, clear cell carcinoma was defined as > 50% of the clear cell component present or reported as clear cell carcinoma by local pathologists.

Table 3: criteria for histological inclusion

Grades refer to 1 (full differentiation), 2 (moderate differentiation) and 3 (poor differentiation)

Regardless of the stage of fig, patients with clear cell carcinoma were enrolled.

The patient should have undergone a surgical massive resection, performed by a surgeon experienced in ovarian cancer management, with the aim of maximum surgical cell reduction according to the collective declaration of the GCIG society. Bulk excision cannot be planned before disease progression.

○ has stage III and IV disease, with the precondition that patients with initial surgical massive resection inappropriate remain to be selected

■ the patient has a histological diagnosis and

■ boulder resection surgery not anticipating disease progression

○ patients should be able to begin systemic therapy within 8 weeks of the cytoreductive surgery if patients are randomized into the study arm, the first dose of bevacizumab must be omitted if the investigator decides to begin chemotherapy within 4 weeks of surgery.

○ if the patient has two procedures, for example, an initial procedure (removal of what is considered to be a benign cyst) followed by a second gynecological-oncology procedure (formal staging and maximum bulk removal of the ovarian tumor), the second procedure date is recorded as the procedure date, the first systemic treatment is initiated within 8 weeks of this date.

ECOG Performance State (PS)0-2

Life expectancy >12 weeks

Appropriate bone marrow function (for post-operative blood examination/calculation of all parameters) (within 28 days before randomization)

○ Absolute Neutrophil Count (ANC) ≥ 1.5x 10⁹/l

○ Platelets (PLT)>100x 10⁹/l

○ hemoglobin (Hb) >9g/dl (can be the post-transfusion)

Appropriate clotting function (for post-operative blood examination/calculation of all parameters) (within 28 days before randomization)

○ activated thromboplastin time (APTT) is less than or equal to 1.5x ULN, or

○ International Standard ratio (INR). ltoreq.1.5 (measurement of INR is mandatory if the patient is undergoing warfarin treatment)

Appropriate liver function (post-operative blood examination/calculation of all parameters) (within 28 days before randomization)

○ serum Bilirubin (BR) is less than or equal to 1.5x ULN

○ serum transaminase is less than or equal to 2.5x ULN

Proteinuria urine test paper <2 +. If the urine test paper is more than or equal to 2+, then 24 hours of urine must demonstrate less than or equal to 1g protein in 24 hours

Appropriate renal function, defined as serum creatinine ≦ 2.0mg/dl or ≦ 177 μmol/l patient exclusion criteria:

non-ovarian epithelial cancers, including malignant mixed Mullerian tumors

Borderline tumors (tumors of low malignant potential)

Planning of intraperitoneal cytotoxic chemotherapy

Prior systemic anti-cancer therapy (e.g. chemotherapy, monoclonal antibody therapy, tyrosine kinase inhibitor therapy or hormone therapy) for ovarian cancer

Surgery (including open biopsy) within 4 weeks before the expected first dose of bevacizumab (allowing the first cycle of chemotherapy to omit bevacizumab)

Any planned surgery during the 58 week period since study treatment initiation (54 weeks treatment plus 4 weeks to allow bevacizumab clearance)

Uncontrolled hypertension (recording blood pressure measurements of patients after 5 minutes rest and sitting position) (prolonged elevation of BP >150/100mmHg despite antihypertensive therapy)

Any prior radiotherapy directed to the abdomen or pelvis

There was a significant traumatic injury within 4 weeks before the potential first dose of bevacizumab

History or clinical suspicion of brain metastases or spinal cord compression. In the case of suspected brain metastases, brain CT/MRI was mandatory (within 4 weeks before randomization). In cases where spinal cord compression is suspected, spinal cord MRI is mandatory (within 4 weeks prior to randomization)

History or evidence of Central Nervous System (CNS) diseases (at neurological examination) unless treated inappropriately with standard medical therapy, e.g. uncontrolled seizures

Previous cerebrovascular accident (CVA), Transient Ischemic Attack (TIA) or subarachnoid hemorrhage (SAH) within 6 months prior to randomization

Fertile women with pregnancy potential reluctant to use appropriate contraception (oral contraceptive, intrauterine contraceptive or barrier contraceptive in combination with spermicidal gel or surgical infertility) for the duration of the study and for at least 6 months thereafter

Pregnant or lactating women

Prior exposure to mouse CA125 antibody

Participation in another clinical trial, or treatment with any other investigational agent, within 30 days before entry into the trial

Malignancy other than ovarian cancer within 5 years prior to randomization, except for appropriately treated cervical carcinoma in situ and/or basal cell skin carcinoma and/or early endometrial carcinoma as specified below. The patient may have received prior adjuvant chemotherapy for other malignancies, such as breast or colorectal cancer, if diagnosed 5 years ago and there is no evidence of subsequent relapse

Excluding patients with a history of simultaneous primary endometrial cancer or past primary endometrial cancer, unless all criteria for endometrial cancer described below are met

○ stage < Ib

○ not exceeding superficial myometrial invasion

○ non-lymphatic vascular invasion

○ are not poorly differentiated (i.e., are not grade 3 or papillary serous or clear cells)

Known hypersensitivity to bevacizumab and its excipients or chemotherapy (including cremophor)

Non-healing wounds, ulcers or fractures. Patients with secondary healed incisions, no evidence of surface dehiscence or infection, but requiring wound examination once every three weeks

History or evidence of thrombotic or hemorrhagic disorders

Clinically significant cardiovascular disease, including

○ randomization of myocardial infarction or unstable angina pectoris within 6 months

○ New York Heart Association (NYHA) stage 2 Congestive Heart Failure (CHF)

○ poor control of cardiac arrhythmias despite medication (patients with atrial fibrillation with heart rate control enrolled)

○ grade ≥ 3 peripheral vascular disease (symptomatic and interfering with daily activities [ ADL ] requiring repair or correction)

Current or recent (within 10 days prior to cycle 1 treatment) chronic aspirin use >325 mg/day (low dose aspirin (<325 mg/day) did not appear to increase the risk of grade 3-4 bleeding when used with bevacizumab plus chemotherapy, so use of prophylactic low dose aspirin in patients at risk for arterial thromboembolic events was not prohibited in this protocol)

The current or recent (within 10 days prior to cycle 1 treatment) use of full-dose oral or parenteral anticoagulants or thrombolytic agents for therapeutic purposes (except for line patent), in which case the INR must be maintained below 1.5)

Presence of grade 2 or more sensory or motor neuropathy

Evidence of any other disease, metabolic dysfunction, physical examination findings, or clinical laboratory findings that give reasonable doubt prohibiting the use of investigational drugs or placing patients in diseases or conditions at high risk for treatment-related complications

Tumor assessments were performed by CT or MRI scans on patients in the study arm after 3 and 6 cycles of chemotherapy, and around 9 and 12 months in the 1 st year, or after 12 th or 18 th cycle of treatment, measured using RECIST criteria. Tumor assessments were repeated every 6 months during the 2 nd and 3 rd years of the trial, and thereafter performed according to clinical instructions. These scans are performed regardless of whether the patient has had the best or suboptimal massive resection and regardless of whether there is measurable disease at the time of the limit scan.

Patients were clinically evaluated and CA125 measured at the beginning of each chemotherapy cycle and then every 6 weeks during trial year 1. Patients were evaluated and CA125 measured every 3 months in trial years 2 and 3. Patients were evaluated clinically and measured for CA125 every 6 months in 4 th and 5th years. Thereafter, the evaluation was repeated every year. The progress based solely on the CA125 standard was verified with CT scans. If this is negative, it is repeated when clinical progression is suspected.

Following evidence of disease progression as defined by the protocol, patient survival and subsequent ovarian cancer treatment were followed annually every 6 months and thereafter during the first 5 years of patient follow-up.

Periodic physical examinations and routine blood tests are performed during treatment to monitor patient safety. Quality of life (QoL) was assessed at the beginning of each chemotherapy cycle, every 6 weeks before the end of year 1 and then using the EORTC QLQ C-30+ OV-28 and EQ-5D questionnaires for treatment progressing before the beginning or every 3 months by the end of year 2. Another QoL table was completed by all patients who were still alive 3 years after randomization. Adverse events and medical resource usage were recorded during study treatment and follow-up.

Results

The results of the study demonstrate that bevacizumab is effective for first-line ovarian cancer when combined with chemotherapy and continued as a 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 a median PFS value of 16.0 months in the chemotherapy arm (CP) compared to 18.3 months for the chemotherapy plus bevacizumab arm (CPB7.5+) with a p-value (time series 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 fig. 9 and 10.

The baseline characteristics were as follows:

table 4: baseline characteristics-demographics

Table 5: baseline characteristics-ovarian cancer history

Table 6: baseline characteristics-ovarian cancer history

Table 7: baseline characteristics-ovarian cancer surgery

Preliminary assessments of adverse events for bevacizumab were consistent with previous studies.

Table 8: overview of Adverse Events (AE)

Example 3. One 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

Epithelial Ovarian Carcinoma (EOC) and its histological and clinical equivalents, Primary Peritoneal Carcinoma (PPC) and fallopian tube carcinoma, occur in the united states at an incidence of approximately 25,000 cases per year and result in approximately 14,000 deaths per year. Since the disease tends to be asymptomatic in the early stages, most patients initially present with advanced (stage III or IV) disease. While EOC, PPC, and fallopian tube cancer are sensitive to a variety of chemotherapeutic agents, particularly taxanes and platinum compounds, only 20% > -30% > patients presenting with stage III or IV disease will survive 5 years. Patients with platinum-sensitive recurrent cancer (defined as disease recurrence more than 6 months since 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 diseases. Carboplatin and gemcitabine resulted in statistically significant Progression Free Survival (PFS) in patients with platinum-sensitive disease compared to carboplatin alone. See, e.g., Pfister, Plante, Vergate I, et al, Gemcitabine plus Carboplatin compounded with Carboplatin linkage with platinum-sensitive receptor over cancer 2006 an intergroup trial of the AGO-OVAR, the NCIC CTG, and the EORTC GCG.J. Clin Oncol 2006; 24:4699707.

Angiogenesis appears to be an important factor in both the development and subsequent progression of EOC. Yoneda and colleagues (1998) demonstrated that tumor growth rate is directly proportional to vascular density in xenograft models of EOC, and that the development of malignant ascites (a feature of EOC associated with poor outcome) is associated with Vascular Endothelial Growth Factor (VEGF) expression. See, e.g., Yoneda J, Kuniyasu H, Crispens MA, et al, expression of angiogenisis-related genes and growth of human ovarian carcinomas in nuclear die. J NatlCancer Inst.1998Mar 18; 90:44754. Other studies demonstrated the correlation of VEGF expression in EOC with microvascular density. In addition, studies have shown that the density of CD31 (a marker of vascular endothelium) expression in EOC by immunohistochemical detection correlates inversely with survival.

This example describes a placebo-controlled, randomized, multicenter phase III study evaluated with platinumCarboplatin in women with susceptibility to recurrent ovarian epithelial, primary peritoneal, or fallopian tube cancer (area under the curve [ AUC [)]4, day 1, every 21 days) and gemcitabine (1000 mg/m)²Day 1 and day 8, every 21 days) efficacy and safety of combined bevacizumab administration (15mg/kg, day 1, every 21 days). Approximately 480 patients were enrolled during a period of approximately 2.5 years. Patients were randomized into carboplatin and gemcitabine and placebo or carboplatin and gemcitabine and bevacizumab. In addition, at the time of randomization, patients were treated with platinum-sensitive disease (6-12 months relapse since last platinum-based treatment versus 6-12 months relapse since last platinum-based treatment>12 months recurrence) and stratification by cytoreductive surgery (performed versus non-performed surgery) for recurrent ovarian epithelial, primary peritoneal, or fallopian tube cancer.

The study consisted of two branches as shown below. See also fig. 11.

Branch 1: carboplatin (AUC 4IV) and gemcitabine (1000mg/m2) chemotherapy (6 cycles up to 20 cycles), followed by placebo

And branch 2: avastin (15mg/kg, 6 cycles up to 10 cycles), combined with carboplatin and gemcitabine chemotherapy (6 cycles up to 10 cycles), followed by continued use of Avastin alone (15mg/kg) until disease progression.

Carboplatin dose was calculated as the target AUC to concentration x time according to Calvert's 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 the total dose (mg) versus target AUC (in mg/mL/min) x [ GFR (in mL/min) 25] creatinine clearance can be calculated according to conventional guidelines.

Patient selection

Patients with ovarian epithelial, PPC, or fallopian tube cancer who had relapsed for >6 months from platinum-based chemotherapy (first relapse) would be enrolled in the study. Other specific admission and exclusion criteria are listed below. Patient admission criteria:

patients must meet the following criteria to be enrolled in the study:

sign the informed-emotion and synonymy table

Age ≥ 18 years

Histological recording ovarian, primary peritoneal, or fallopian tube cancers that recur >6 months after platinum-based chemotherapy

The patient must have recurrent ovarian epithelial carcinoma, primary peritoneal carcinoma, or fallopian tube carcinoma. This must be the first recurrence of ovarian epithelial, primary peritoneal, or fallopian tube cancer.

● examples of selected histological cell types include: serous adenocarcinoma, endometrioid adenocarcinoma, mucinous adenocarcinoma, undifferentiated carcinoma, clear cell adenocarcinoma, transitional cell carcinoma, malignant Brenner's tumor, or other unspecified adenocarcinoma

Prior chemotherapy in the absence of recurrent background

Measurable disease according to modified RECIST, with at least one lesion precisely measurable in at least one dimension (recording longest dimension)

Each measurable lesion must be 20mm when measured by conventional techniques, CT and Magnetic Resonance Imaging (MRI) or 10mm when measured by spiral CT.

Greater than 28 days since radiation therapy or surgery and recovery from radiation therapy or surgery before

ECOG Performance State 0 or 1

Use of effective contraceptive means (for women with pregnancy potential)

Ability to comply with research and follow-up procedures

Patient exclusion criteria

Patients meeting any of the following criteria will be excluded from study entry.

● disease-specific exclusion

○ recurrent ovarian cancer, primary peritoneal carcinoma, or fallopian tube carcinoma prior chemotherapy treatment hormone therapy (i.e., progesterone, estrogen, antiestrogen, aromatase inhibitor) was not considered a prior chemotherapy regimen.

○ history of abdominal fistulas, gastrointestinal perforations, or intra-abdominal abscesses

○ patients with clinical symptoms or signs of GI obstruction or requiring parenteral hydration, parenteral nutrition, or tube feeding

○ patients with evidence of abdominal free air that could not be explained by paracentesis or a new surgical procedure

● general medical exclusion

○ Life expectancy <12 weeks

○ currently, recently (within 4 weeks of day 1 of cycle 1), or scheduled to participate in experimental drug studies

○ screening clinical laboratory values

■ granulocyte count <1500/μ L

■ platelet count <100,000/μ L

■ hemoglobin <8.5g/dL (hemoglobin can be supported by transfusion or erythropoietin or other approved hematopoietic growth factors)

■ serum bilirubin >2.0x Upper Limit of Normal (ULN)

■ alkaline phosphatase, aspartate Aminotransferase (AST), and/or alanine Aminotransferase (ALT) >2.5x ULN (for patients with liver metastasis, AST, ALT >5x ULN)

■ serum creatinine is more than or equal to 1.6

■ International Normalized Ratio (INR) >1.5 and/or activated partial thromboplastin time (aPTT) >1.5xULN (except for patients receiving anticoagulant therapy)

■ for patients taking full dose warfarin, INR is in the range (usually between 2 and 3) and aPTT <1.2xULN

○ has history of other malignancies within 5 years from day 1 of cycle 1, except for those with negligible risk of metastasis or death, such as improperly controlled squamous cell carcinoma of the skin or basal cell carcinoma or cervical carcinoma in situ

○ to give a ban on the use of investigational drugs or any other disease, metabolic dysfunction, physical examination findings, or clinical laboratory findings that may affect interpretation of results or put a patient in reasonable doubt of a disease or condition at high risk of treatment complications

● bevacizumab specific exclusion

○ System BevacizumabOr history of use of other VEGF or VEGF receptor targeting agents

○ improperly controlled hypertension (defined as systolic blood pressure >150mmHg and/or diastolic blood pressure >100mmHg when taking antihypertensive medication)

○ prior history of hypertensive crisis or hypertensive encephalopathy

○ New York Heart Association class II or greater CHF

○ history of myocardial infarction or unstable angina 6 months prior to cycle 1 day 1 (day of first bevacizumab/placebo infusion)

○ history of stroke or TIA within 6 months prior to study enrollment

○ known CNS disorders, except for the treatment of good brain metastases

■ good brain metastases were defined as no evidence of progression or bleeding after treatment and no dexamethasone was required at that time, as confirmed by clinical examination and brain imaging (MRI or CT) during screening.

○ history of major angiopathy (e.g. aortic aneurysm, aortic dissection)

○ recent peripheral arterial thrombosis in 6 months before day 1 of cycle 1

○ had a history of hemoptysis in 1 month before day 1 of cycle 1 (bright red blood equal to or greater than 1/2 teaspoons per episode)

○ (in the absence of therapeutic anticoagulation) evidence of hemorrhagic diathesis or major coagulopathy

○ Large surgery, open biopsy, or major trauma or research procedure is expected to be required during 28 days before cycle 1 day 1

○ core biopsy or other minor surgical procedure precluded vascular access device placement within 7 days prior to cycle 1 day 1

○ severe non-healing wounds, active ulcers, or untreated fractures

○ proteinuria occurred during screening, as evidenced by UPCR ≧ 1.0 at screening

○ is known to be hypersensitive to any of the components of bevacizumab

○ pregnancy (Positive pregnancy test) or lactation

● patients with pregnancy potential must use effective means of contraception.

This study, OCEANS, enrolled a different patient population than example 1(GOG 0218) and example 2(ICON 7); women with previously treated platinum-sensitive ovarian cancer enrolled in the trial. Women with ovarian cancer may receive platinum-based chemotherapy as first-line therapy. The time between receiving the last dose of platinum-based chemotherapy and the recurrence (recurrence) of the disease is used to help determine the chemotherapeutic options used in the next line of treatment. Women have "platinum-sensitive" ovarian cancer if the disease recurs six months after completion of the initial platinum-based chemotherapy. Ovarian cancer is considered "platinum-resistant" if it recurs within six months of completion of the initial platinum-based chemotherapy.

Results

This phase III study of bevacizumab plus chemotherapy in women with ovarian cancer reached its primary endpoint (primary endpoint). The objective of the study was to evaluate the efficacy and safety of adding bevacizumab to standard chemotherapy, followed by prolonged use of bevacizumab alone until disease progression, compared to chemotherapy alone, in previously treated women with ovarian cancer. Studies have shown that bevacizumab plus chemotherapy, followed by continued use of bevacizumab alone until disease progression, extends the time women with previously treated (recurrent) platinum-sensitive ovarian cancer survive without disease progression (progression-free survival or PFS) compared to chemotherapy alone. PFS is defined as the time from randomization to disease progression or death (whichever occurs first) by whatever cause is determined by the investigator. The primary endpoint of PFS was assessed by the study investigator. Measurable disease was assessed by investigators using modified RECIST (thersase et al 2000), for example every 9 weeks throughout the study. See, e.g., therase P, Arbuck SG, Eisenhauser EA, et al, New Guidelinest to said response to treatment in solid tumors.J Natl Cancer Inst 2000; 92:205-1. Secondary endpoints included Overall Survival (OS), response rate, response duration, and safety. No new safety findings were observed and adverse events were consistent with those observed in previous important trials of bevacizumab.

Sequence listing

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Claims

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

3. A method of instructing a human subject having ovarian cancer, the method comprising providing instructions to receive an anti-VEGF antibody in parallel with chemotherapy, followed by anti-VEGF maintenance therapy to treat the breast cancer, thereby prolonging progression free survival of the subject.

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

5. A commercial method comprising marketing an anti-VEGF antibody therapy concurrent with chemotherapy, followed by an anti-VEGF maintenance therapy, for treating ovarian cancer in a human subject, thereby prolonging progression-free survival of the subject.

6. A kit for treating previously untreated ovarian cancer in a human patient, comprising a package comprising an anti-VEGF antibody composition and instructions for using the anti-VEGF antibody composition in combination with paclitaxel and carboplatin followed by an anti-VEGF maintenance therapy, wherein the instructions recite that the progression free survival of a patient receiving paclitaxel, carboplatin, and anti-VEGF antibodies is 18.3 months with a hazard ratio of 0.79.