U.S. patent application number 15/930798 was filed with the patent office on 2021-02-25 for anti-angiogenesis therapy for the treatment of ovarian cancer.
This patent application is currently assigned to Genentech, Inc.. The applicant listed for this patent is Genentech, Inc.. Invention is credited to Jakob Dupont, Amreen Husain, Cornelia Irl, Hoa Nguyen, Mika A. Sovak, Jing Yi.
Application Number | 20210052728 15/930798 |
Document ID | / |
Family ID | 1000005197343 |
Filed Date | 2021-02-25 |
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United States Patent
Application |
20210052728 |
Kind Code |
A1 |
Dupont; Jakob ; et
al. |
February 25, 2021 |
ANTI-ANGIOGENESIS THERAPY FOR THE TREATMENT OF OVARIAN CANCER
Abstract
This invention concerns in general treatment of diseases and
pathological conditions with anti-VEGF antibodies. More
specifically, the invention concerns the treatment of human
patients susceptible to or diagnosed with cancer using an anti-VEGF
antibody, preferably in combination with one or more additional
anti-tumor therapeutic agents for the treatment of ovarian
cancer.
Inventors: |
Dupont; Jakob;
(Hillsborough, CA) ; Irl; Cornelia; (San Maateo,
CA) ; Husain; Amreen; (San Mateo, CA) ; Sovak;
Mika A.; (Burlingame, CA) ; Yi; Jing; (San
Francisco, CA) ; Nguyen; Hoa; (South San Francisco,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Genentech, Inc. |
South San Francisco |
CA |
US |
|
|
Assignee: |
Genentech, Inc.
South San Francisco
CA
|
Family ID: |
1000005197343 |
Appl. No.: |
15/930798 |
Filed: |
May 13, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16059964 |
Aug 9, 2018 |
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15930798 |
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15421184 |
Jan 31, 2017 |
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16059964 |
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15184332 |
Jun 16, 2016 |
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15421184 |
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14157351 |
Jan 16, 2014 |
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15184332 |
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13032532 |
Feb 22, 2011 |
8778340 |
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14157351 |
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61439819 |
Feb 4, 2011 |
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61360059 |
Jun 30, 2010 |
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61351231 |
Jun 3, 2010 |
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61307095 |
Feb 23, 2010 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/282 20130101;
A61K 39/39558 20130101; A61K 2039/545 20130101; A61K 31/7068
20130101; A61K 31/337 20130101; C07K 16/22 20130101; A61K 2039/54
20130101; A61K 9/0019 20130101; C07K 2317/56 20130101; G06Q 99/00
20130101; C07K 2317/24 20130101; A61K 39/3955 20130101; C07K
16/3069 20130101 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61K 31/282 20060101 A61K031/282; A61K 31/337 20060101
A61K031/337; G06Q 99/00 20060101 G06Q099/00; C07K 16/22 20060101
C07K016/22; A61K 31/7068 20060101 A61K031/7068; A61K 9/00 20060101
A61K009/00; C07K 16/30 20060101 C07K016/30 |
Claims
1. A method of treating a patient diagnosed with recurrent
platinum-sensitive ovarian, fallopian tube or primary peritoneal
cancer, comprising subjecting the patient to a treatment regimen
combining paclitaxel and carboplatin with the concurrent
administration of an effective amount of an anti-VEGF antibody
followed by anti-VEGF antibody maintenance therapy, wherein the
anti-VEGF antibody has a heavy chain variable region comprising the
following amino acid sequence: TABLE-US-00017 (SEQ ID NO. 1)
EVQLVESGGG LVQPGGSLRL SCAASGYTFT NYGMNWVRQA PGKGLEWVGW INTYTGEPTY
AADFKRRFTF SLDTSKSTAY LQMNSLRAED TAVYYCAKYP HYYGSSHWYF DVWGQGTLVT
VSS
and a light chain variable region comprising the following amino
acid sequence: TABLE-US-00018 (SEQ ID NO. 2) DIQMTQSPSS LSASVGDRVT
ITCSASQDIS NYLNWYQQKP GKAPKVLIYF TSSLHSGVPS RFSGSGSGTD FTLTISSLQP
EDFATYYCQQ YSTVPWTFGQ GTKVEIKR,
and wherein the treatment regimen effectively extends the
progression free survival of the patient.
2. The method of claim 1, wherein the anti-VEGF antibody is
bevacizumab.
3. The method of claim 2, wherein the paclitaxel is administered
intravenously at 175 mg/m.sup.2.
4. The method of claim 2, wherein the carboplatin is administered
intravenously at an area under the concentration-time curve (AUC)
of 4 or an AUC of 6.
5. The method of claim 2, wherein the anti-VEGF antibody
maintenance therapy is administered at 15 mg/kg.
6. The method of claim 1, wherein the progression free survival of
the patient is extended by at least about 2.3 months or more
compared to another patient not treated with anti-VEGF
antibody.
7. The method of claim 2, wherein the anti-VEGF antibody is
administered at 15 mg/kg.
8. The method of claim 2, wherein the patient is diagnosed with
Stage III or Stage IV recurrent platinum-sensitive ovarian
cancer.
9. The method of claim 2, wherein the patient is diagnosed with
recurrent platinum-sensitive primary peritoneal cancer.
10. The method of claim 2, wherein the patient is diagnosed with
recurrent platinum-sensitive fallopian tube cancer.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 16/059,964, filed Aug. 9, 2018 which is a continuation of U.S.
application Ser. No. 15/421,184, filed Jan. 31, 2017, which is a
continuation of U.S. application Ser. No. 15/184,332, filed Jun.
16, 2016 (now abandoned), which is a continuation of U.S.
application Ser. No. 14/157,351, filed Jan. 16, 2014 (now
abandoned), which is a continuation of U.S. application Ser. No.
13/032,532, filed Feb. 22, 2011, now U.S. Pat. No. 8,778,340, which
claims priority to and the benefit of U.S. Provisional Application
Ser. No. 61/439,819, filed Feb. 4, 2011, U.S. Provisional
Application Ser. No. 61/360,059, filed Jun. 30, 2010, U.S.
Provisional Application Ser. No. 61/351,231, filed Jun. 3, 2010,
and U.S. Provisional Application Ser. No. 61/307,095, filed Feb.
23, 2010, the specifications of which are incorporated herein in
their entirety.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted in ASCII format via EFS-Web and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on May 11, 2020, is named Sequence_Listing.txt and is 2,728 bytes
in size.
FIELD OF THE INVENTION
[0003] This invention relates in general to treatment of human
diseases and pathological conditions. More specifically, the
invention relates to anti-angiogenesis therapy, either alone or in
combination with other anti-cancer therapies, for the treatment of
ovarian cancer.
BACKGROUND
[0004] Cancer remains to be one of the most deadly threats to human
health. In the U.S., cancer affects nearly 1.3 million new patients
each year, and is the second leading cause of death after heart
disease, accounting for approximately 1 in 4 deaths. For women with
ovarian and peritoneal cancer, after initial surgical diagnosis,
staging and cytoreduction, the standard primary systemic
chemotherapy for women with advanced epithelial ovarian, and
peritoneal primary cancer consists of chemotherapy with a platinum
and taxane combination, usually carboplatin and paclitaxel. See,
e.g., McGuire W P, et al. Cyclophosphamide and cisplatin compared
with paclitaxel and cisplatin in patients with stage III and stage
IV ovarian cancer. N Eng J Med 334:1-6, 1996; Piccart M J, et al.
Randomized intergroup trial of cisplatin paclitaxel versus
cisplatin-cyclophosphamide in women with advanced epithelial
ovarian cancer: three-year results. J Natl Cancer Inst 92:699-708,
20003; Alberts D S, et al. Improved therapeutic index of
carboplatin plus cyclophosphamide versus cisplatin plus
cyclophosphamide: final report by the Southwest Oncology Group of a
phase III randomized trial in stages III and IV ovarian cancer. J
Clin Oncol 10:706-17, 1992; du Bois A, et al. A randomized clinical
trial of cisplatin/paclitaxel versus carboplatin/paclitaxel as
first-line treatment of ovarian cancer. J Natl Cancer Inst
September 3; 95.(17):1320.-9. 95:1320, 2003; Ozols R F, et al.
Phase III trial of carboplatin and paclitaxel compared with
cisplatin and paclitaxel in patients with optimally resected stage
III ovarian cancer: a Gynecologic Oncology Group study. J Clin
Oncol 21:3194-200, 2003; and, Swenerton K, et al.
Cisplatin-cyclophosphamide versus carboplatin-cyclophosphamide in
advanced ovarian cancer: a randomized phase III study of the
National Cancer Institute of Canada Clinical Trials Group. J Clin
Oncol 10:718-26, 1992. While advances have been made in patient
management, this disease still carries a high fatality to case
ratio for all gynecologic malignancies diagnosed in the United
States. It is estimated that in 2004, 25,580 new cases will have
been diagnosed and 16,090 women will have died of the disease. See,
e.g., Jemal A, et al. Cancer statistics, 2004. CA Cancer J Clin
54:8-29, 2004. Improvements are needed in primary therapeutic
strategies.
[0005] Angiogenesis is an important cellular event in which
vascular endothelial cells proliferate, prune and reorganize to
form new vessels from preexisting vascular network. There are
compelling evidences that the development of a vascular supply is
essential for normal and pathological proliferative processes
(Folkman and Klagsbrun Science 235:442-447 (1987)). Delivery of
oxygen and nutrients, as well as the removal of catabolic products,
represent rate-limiting steps in the majority of growth processes
occurring in multicellular organisms.
[0006] While induction of new blood vessels is considered to be the
predominant mode of tumor angiogenesis, recent data have indicated
that some tumors may grow by co-opting existing host blood vessels.
The co-opted vasculature then regresses, leading to tumor
regression that is eventually reversed by hypoxia-induced
angiogenesis at the tumor margin. Holash et al. Science
284:1994-1998 (1999).
[0007] 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 including VEGF-B, VEGF-C,
VEGF-D, VEGF-E, VEGF-F, and PlGF. VEGF-A primarily binds to two
high affinity receptor tyrosine kinases, VEGFR-1 (Flt-1) and
VEGFR-2 (Flk-1/KDR), the latter being the major transmitter of
vascular endothelial cell mitogenic signals of VEGF-A.
Additionally, neuropilin-1 has been identified as a receptor for
heparin-binding VEGF-A isoforms, and may play a role in vascular
development.
[0008] In addition to being an angiogenic factor in angiogenesis
and vasculogenesis, VEGF, as a pleiotropic growth factor, exhibits
multiple biological effects in other physiological processes, such
as endothelial cell survival, vessel permeability and vasodilation,
monocyte chemotaxis and calcium influx. Ferrara and Davis-Smyth
(1997), supra. Moreover, studies have reported mitogenic effects of
VEGF on a few non-endothelial cell types, such as retinal pigment
epithelial cells, pancreatic duct cells and Schwann cells. Guerrin
et al. J. Cell Physiol. 164:385-394 (1995); Oberg-Welsh et al. Mol.
Cell. Endocrinol. 126:125-132 (1997); Sondell et al. J. Neurosci.
19:5731-5740 (1999). VEGF expression is upregulated in a majority
of malignancies and the overexpression of VEGF often correlates
with a more advanced stage or with a poorer prognosis in many solid
tumors.
[0009] Since ovarian cancer is still one of the most deadly
threats, additional cancer treatments for patients are needed. The
invention addresses these and other needs, as will be apparent upon
review of the following disclosure.
SUMMARY
[0010] Provided is the use of anti-VEGF antagonists for treating
ovarian cancer. For example, uses of anti-VEGF antibodies for
effectively treating women with newly diagnosed, previously
untreated ovarian, fallopian tube or primary peritoneal cancer or
platinum sensitive recurrent (or previously treated) ovary,
primary, peritoneal, or fallopian tube carcinoma are provided. Data
is provided from a randomized phase III clinical trial of
bevacizumab (AVASTIN.RTM.) in combination with chemotherapy regimes
in subjects (e.g., women) with newly diagnosed, previously
untreated stage III (sub optimally and macroscopic optimally
debulked) and IV epithelial ovarian, primary peritoneal or
fallopian tube cancer (Example 1). Data is also provided from a
randomized phase III clinical trial of bevacizumab (AVASTIN.RTM.)
in combination with chemotherapy regimes in subjects (e.g., women)
with newly diagnosed, high risk stage I and IIa (Grade 3 or clear
cell carcinoma only) and stage IIb-IV epithelial ovarian, fallopian
tube or primary peritoneal cancer, who have undergone initial
surgery and who would not be considered for cytoreductive surgery
prior to disease progression (Example 2). Data is also provided
from a placebo-controlled, randomized, multicenter Phase III study
evaluating the efficacy and safety of bevacizumab (15 mg/kg, Day 1,
every 21 days), administered in combination with carboplatin (area
under the curve [AUC] 4, Day 1, every 21 days) with gemcitabine
(1000 mg/m.sup.2, Day 1 and Day 8, every 21 days) in women with
platinum sensitive recurrent epithelial ovarian, primary
peritoneal, or fallopian tube carcinoma (Example 3). Such
chemotherapy regimes include taxane therapy (e.g., paclitaxel or
docetaxel), platinum based chemotherapy (e.g., carboplatin) or
gemcitabine, and combinations thereof. The success of the trials
show that providing anti-VEGF antibody (e.g., bevacizumab) when
combined with chemotherapy and continued as maintenance therapy
provides statistically significant and clinically meaningful
benefits to ovarian cancer patients.
[0011] The results obtained in clinical studies of the use of
bevacizumab in both concurrent and maintenance treatment in human
subjects with previously untreated and recurrent ovarian cancer
show that the efficacy, as evaluated by progression free survival
(PFS) was positive especially when compared to PFS data for
treatment with chemotherapeutic agents alone. Subjects in the
clinical trials who received bevacizumab in concurrent treatment in
combination with taxane therapy (e.g., paclitaxel or docetaxel),
and platinum based chemotherapy (e.g., carboplatin) or platinum
based chemotherapy (e.g., carboplatin) and gemcitabine and
maintenance therapy with bevacizumab had an increase in progression
free survival compared to subjects treated with taxane therapy
(e.g., paclitaxel or docetaxel), and platinum based chemotherapy
(e.g., carboplatin) alone or platinum based chemotherapy (e.g.,
carboplatin) and gemcitabine alone.
[0012] Accordingly, the 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, and then administering
the anti-VEGF antibody for maintenance therapy wherein with said
treatment the progression free survival of the patient is
increased. The treatment regimen combining the chemotherapy with
the administration of the anti-VEGF and then the administration of
anti-VEGF maintenance therapy effectively extends the progression
free survival (PFS) of the patient.
[0013] In certain embodiments, the PFS is extended about 1 month,
1.2 months, 2 months, 2.9 months, 3 months, 3.8 months, 4 months, 6
months, 7 months, 8 months, 9 months, 1 year, about 2 years, about
3 years, etc, compared to a control. In one embodiment, the PFS is
extended about 2.9 months to 3.8 months (e.g., with the treatment
regimen combining the chemotherapy with the administration of the
anti-VEGF and then the administration of anti-VEGF maintenance
therapy) compared to a control. In one embodiment, the PFS is
extended at least about 3.8 months (e.g., with the treatment
regimen combining the chemotherapy with the administration of the
anti-VEGF and then the administration of anti-VEGF maintenance
therapy) compared to a control. In another embodiment, the PFS is
extended about 2.3 months (e.g., with the treatment regimen
combining the chemotherapy with the administration of the anti-VEGF
and then the administration of anti-VEGF maintenance therapy)
compared to a control. In one embodiment, the PFS is extended about
6 months (e.g., with the treatment regimen combining the
chemotherapy with the administration of the anti-VEGF and then the
administration of anti-VEGF maintenance therapy) compared to a
control.
[0014] Any chemotherapeutic agent exhibiting anticancer activity
can be used according to 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 inhibitor, interferons, platinum coordination
complexes, taxanes anthracenedione substituted urea, methyl
hydrazine derivatives, adrenocortical suppressant,
adrenocorticosteroides, progestins, estrogens, antiestrogen,
androgens, antiandrogen, gemcitabine and gonadotropin-releasing
hormone analog. In certain embodiments, the chemotherapeutic agent
is for example, taxane, paclitaxel, docetaxel, paclitaxel
protein-bound particles (e.g., Abraxane.RTM.), gemcitabine,
platinum analogs, carboplatin, or combinations thereof. Two or more
chemotherapeutic agents can be used in a cocktail to be
administered in combination with administration of the anti-VEGF
antibody, e.g., taxane and platinum analogs or gemcitabine and
platinum analogs. In one embodiment, it is carboplatin and
paclitaxel. In one embodiment, it is carboplatin and docetaxel. In
another embodiment, it is gemcitabine and carboplatin.
[0015] Clinical benefits of the treatments according to the
invention can be measured by, for example, duration of progression
free survival (PFS), time to treatment failure, objective response
rate and duration of response.
[0016] Kits are also provided. 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 that
the progression free survival for patients receiving taxane therapy
and carboplatin therapy and bevacizumab is 14.1 months with a
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 package 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 for patients receiving
paclitaxel, carboplatin and anti-VEGF antibody is 18.3 months with
a hazard ratio of 0.79. In certain embodiments, a kit comprises an
anti-VEGF antibody that has a heavy chain variable region
comprising the following amino acid sequence:
TABLE-US-00001 (SEQ ID No. 1) EVQLVESGGG LVQPGGSLRL SCAASGYTFT
NYGMNWVRQA PGKGLEWVGW INTYTGEPTY AADFKRRFTF SLDTSKSTAY LQMNSLRAED
TAVYYCAKYP HYYGSSHWYF DVWGQGTLVT VSS
and a light chain variable region comprising the following amino
acid sequence:
TABLE-US-00002 (SEQ ID No. 2) DIQMTQSPSS LSASVGDRVT ITCSASQDIS
NYLNWYQQKP GKAPKVLIYF TSSLHSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ
YSTVPWTFGQ GTKVEIKR.
In certain embodiments, the anti-VEGF antibody is bevacizumab in
the kit. In certain embodiments, the kit is for a patient that has
stage III or IV ovarian cancer.
[0017] Accordingly, the invention features a method of instructing
a human subject with, e.g., ovarian, cancer by providing
instructions to receive treatment with an anti-VEGF antibody so as
to increase progression free survival of the subject, to decrease
the subject's risk of cancer recurrence or to increase the
subject's likelihood of survival. In some embodiments the method
further comprises providing instructions to receive treatment with
at least one chemotherapeutic agent. In some embodiments, the
method further comprises providing instructions to receive
treatment with at least two chemotherapeutic agents. In certain
embodiments, the treatment with the anti-VEGF antibody is both
concurrent and sequential to the treatment with the
chemotherapeutic agent. In certain embodiments the subject is
treated as instructed by the method of instructing.
[0018] The invention also provides a promotional method, comprising
promoting the administration of an anti-VEGF antibody for treatment
of, e.g., ovarian, cancer in a human subject. In some embodiments
the method further comprises promoting the administration of at
least one chemotherapeutic agent. In certain embodiments of the
invention, the administration of the anti-VEGF antibody is both
concurrent and sequential to administration of the chemotherapeutic
agent(s). Promotion may be conducted by any means available. In
some embodiments the promotion is by a package insert accompanying
a commercial formulation of the anti-VEGF antibody. The promotion
may also be by a package insert accompanying a commercial
formulation of the chemotherapeutic agent(s). Promotion may be by
written or oral communication to a physician or health care
provider. In some embodiments the promotion is by a package insert
where the package inset provides instructions to receive concurrent
therapy with an anti-VEGF antibody and at least one chemotherapy
agent(s) and maintenance therapy with an anti-VEGF antibody. In
some embodiments the promotion is followed by the treatment of the
subject with an anti-VEGF antibody with one or more
chemotherapeutic agent(s) followed by maintenance therapy with an
anti-VEGF antibody.
[0019] The invention provides a business method, comprising
marketing an anti-VEGF antibody for treatment of, e.g., ovarian,
cancer in a human subject in combination with at least one
chemotherapy agent followed by anti-VEGF maintenance therapy so as
to increase progression free survival, or decrease the subject's
likelihood of cancer recurrence or increase the subject's
likelihood of survival. In some embodiments the marketing is
followed by treatment of the subject with the anti-VEGF antibody
with the chemotherapeutic agent(s) followed by anti-VEGF
maintenance therapy. In some embodiments the method further
comprises marketing two or more chemotherapeutic agents for use in
combination with the anti-VEGF antibody followed by anti-VEGF
maintenance therapy. In some embodiments the marketing is followed
by treatment of the subject with the anti-VEGF antibody with the
chemotherapeutic agents followed by anti-VEGF maintenance
therapy.
[0020] Also provided is a business method, comprising marketing a
chemotherapeutic agent in combination with an anti-VEGF antibody
followed by anti-VEGF maintenance therapy for treatment of, e.g.,
ovarian, cancer in a human subject so as to increase progression
free survival, or decrease the subject's likelihood of cancer
recurrence or increase the subject's likelihood of survival. In
some embodiments, the marketing is followed by treatment of the
subject with the combination of the chemotherapeutic agent and the
anti-VEGF antibody followed by the anti-VEGF maintenance therapy.
Also provided is a business method, comprising marketing two or
more chemotherapeutic agents in combination with an anti-VEGF
antibody followed by anti-VEGF maintenance therapy for treatment
of, e.g., ovarian, cancer in a human subject so as to increase
progression free survival, or decrease the subject's likelihood of
cancer recurrence or increase the subject's likelihood of survival.
In some embodiments, the marketing is followed by treatment of the
subject with the combination of the chemotherapeutic agents and the
anti-VEGF antibody followed by anti-VEGF maintenance therapy.
[0021] In each of the methods of the invention the anti-VEGF
antibody may be substituted with a VEGF specific antagonist, e.g.,
a VEGF receptor molecule or 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, can 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 bevacizumab
(AVASTIN.RTM.), a G6 antibody, a B20 antibody, and fragments
thereof. In certain embodiments, the anti-VEGF antibody has a heavy
chain variable region comprising the following amino acid
sequence:
TABLE-US-00003 (SEQ ID No. 1) EVQLVESGGG LVQPGGSLRL SCAASGYTFT
NYGMNWVRQA PGKGLEWVGW INTYTGEPTY AADFKRRFTF SLDTSKSTAY LQMNSLRAED
TAVYYCAKYP HYYGSSHWYF DVWGQGTLVT VSS
and a light chain variable region comprising the following amino
acid sequence:
TABLE-US-00004 (SEQ ID No. 2) DIQMTQSPSS LSASVGDRVT ITCSASQDIS
NYLNWYQQKP GKAPKVLIYF TSSLHSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ
YSTVPWTFGQ GTKVEIKR.
[0022] The antibody, or antigen-binding fragment thereof, can also
be an antibody that lacks an Fc portion, an F(ab').sub.2, an Fab,
or an Fv structure.
[0023] In one embodiment, the treatment is a combination of a
VEGF-specific antagonist, e.g., 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., anti-VEGF antibody, and two or more
chemotherapeutic agents followed by VEGF antagonist maintenance
therapy.
[0024] Each of the methods or uses of the invention may be
practiced in relation to the treatment of cancers 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, liver cancer, bladder cancer, hepatoma, breast cancer,
colon cancer, colorectal cancer, endometrial or uterine carcinoma,
salivary gland carcinoma, kidney cancer, liver cancer, prostate
cancer, renal 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 embodiment, the subject has newly
diagnosed previously untreated ovarian cancer. In some embodiments,
the subject has newly diagnosed, previously untreated, stage III
(sub optimally and macroscopic optimally debulked) and IV
epithelial ovarian primary peritoneal or fallopian tube cancer. In
some embodiments, the subject has platinum sensitive recurrent
epithelial ovarian, primary peritoneal, or fallopian tube
carcinoma.
[0025] Each of the above aspects can further include monitoring the
subject for recurrence of the cancer. Monitoring can be
accomplished, for example, by evaluating progression free survival
(PFS) or overall survival (OS) or objective response rate (ORR). In
one embodiment, the PFS is evaluated after initiation of
treatment.
[0026] Depending on the type and severity of the disease, preferred
dosages for the anti-VEGF antibody, e.g., bevacizumab, are
described herein and can range from about 1 .mu.g/kg to about 50
mg/kg, most preferably from about 5 mg/kg to about 15 mg/kg,
including but not limited to 5 mg/kg, 7.5 mg/kg, 10 mg/kg or 15
mg/kg. The frequency of administration will vary depending on the
type and severity of the disease. For repeated administrations over
several days or longer, depending on the condition, the treatment
is sustained until the cancer is treated or the desired therapeutic
effect is achieved, as measured by the methods described herein or
known in the art. In one example, the anti-VEGF antibody of the
invention is administered once every week, every two weeks, or
every three weeks, at a dose range from about 5 mg/kg to about 15
mg/kg, including but not limited to 5 mg/kg, 7.5 mg/kg, 10 mg/kg or
15 mg/kg. However, other dosage regimens may be useful. The
progress of the therapy of the invention is easily monitored by
conventional techniques and assays. In certain embodiments of the
invention, anti-VEGF therapy is provided as maintenance therapy. In
further embodiments, anti-VEGF therapy is provided for at least 14
months (including concurrent anti-VEGF therapy with chemotherapy
and anti-VEGF maintenance therapy). In other embodiments, anti-VEGF
therapy is provided for at least 12 months (including concurrent
anti-VEGF therapy with chemotherapy and anti-VEGF maintenance
therapy).
[0027] In additional embodiments of each of the above aspects, the
VEGF-specific antagonist, e.g., anti-VEGF antibody, is administered
locally or systemically (e.g., orally or intravenously). In other
embodiments, one aspect of the treatment is with the VEGF-specific
antagonist in a monotherapy or a monotherapy for the duration of
the VEGF-specific antagonist treatment period, e.g., in extended
treatment phase or maintenance therapy, as assessed by the
clinician or described herein. In certain embodiments, the
anti-VEGF maintenance therapy is given for at least cycles 7
through 22. In other embodiments, the anti-VEGF maintenance therapy
is given for at least cycles 7 through 18.
[0028] In other embodiments, treatment with the VEGF-specific
antagonist is in combination with an additional anti-cancer
therapy, including but not limited to, surgery, radiation therapy,
chemotherapy, differentiating therapy, biotherapy, immune therapy,
an angiogenesis inhibitor, a cytotoxic agent and an
anti-proliferative compound. Treatment with the VEGF-specific
antagonist can also include any combination of the above types of
therapeutic regimens. In some embodiments, the chemotherapeutic
agent and the VEGF-specific antagonist are administered
concurrently followed by anti-VEGF maintenance therapy. In some
embodiments, two or more chemotherapeutic agents and the
VEGF-specific antagonist are administered concurrently followed by
anti-VEGF maintenance therapy.
[0029] In the embodiments which include an additional anti-cancer
therapy, the subject can be further treated with the additional
anti-cancer therapy before, during (e.g., simultaneously), or after
administration of the VEGF-specific antagonist. In one embodiment,
the VEGF-specific antagonist, administered either alone or with an
anti-cancer therapy, can be administered as maintenance
therapy.
[0030] Other features and advantages of the invention will be
apparent from the following Detailed Description, the drawings, and
the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0031] FIG. 1 depicts the study design for the ovarian cancer trial
described in Example 1.
[0032] FIG. 2 depicts a diagram of the study design for the ovarian
cancer trial using bevacizumab (BEV) or placebo with various
chemotherapies.
[0033] FIG. 3 depicts select adverse events from the trial depicted
in FIG. 2.
[0034] FIG. 4 depicts select adverse events by treatment phase from
the trial depicted in FIG. 2.
[0035] FIG. 5 depicts Investigator-assessed progression free
survival (PFS) of Arm I, Arm II and Arm III of the trial depicted
in FIG. 2.
[0036] FIG. 6 depicts PFS values for Arm I and Arm III of the trial
depicted in FIG. 2 and the ramifications of using CA-125 marker as
determinant of progression.
[0037] FIG. 7 depicts a subgroup analyses of patients in Arm III
verses Arm I of the trial depicted in FIG. 2.
[0038] FIG. 8 depicts the study design for the ovarian cancer trial
described in Example 2.
[0039] FIG. 9 depicts a summary of the progression free survival
(PFS) analysis of the trial depicted in FIG. 8. "CP" corresponds to
Arm A in FIG. 8. "CPB7.5+" corresponds to Arm B in FIG. 8.
[0040] FIG. 10 depicts a graph of the PFS results from the trial
depicted in FIG. 8. "CP" corresponds to Arm A in FIG. 8. "CPB7.5+"
corresponds to Arm B in FIG. 8.
[0041] FIG. 11 depicts the study design for the ovarian cancer
trial described in Example 3.
DETAILED DESCRIPTION
I. Definitions
[0042] The term "VEGF" or "VEGF-A" is used to refer to the
165-amino acid human vascular endothelial cell growth factor and
related 121-, 145-, 189-, and 206-amino acid human vascular
endothelial cell growth factors, as described by, e.g., Leung et
al. Science, 246:1306 (1989), and Houck et al. Mol. Endocrin.,
5:1806 (1991), together with the naturally occurring allelic and
processed forms thereof. VEGF-A is part of a gene family including
VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, and PlGF. VEGF-A primarily
binds to two high affinity receptor tyrosine kinases, VEGFR-1
(Flt-1) and VEGFR-2 (Flk-1/KDR), the latter being the major
transmitter of vascular endothelial cell mitogenic signals of
VEGF-A. Additionally, neuropilin-1 has been identified as a
receptor for heparin-binding VEGF-A isoforms, and may play a role
in vascular development. The term "VEGF" or "VEGF-A" also refers to
VEGFs from non-human species such as mouse, rat, or primate.
Sometimes the VEGF from a specific species is indicated by terms
such as hVEGF for human VEGF or mVEGF for murine VEGF. The term
"VEGF" is also used to refer to truncated forms or fragments of the
polypeptide comprising amino acids 8 to 109 or 1 to 109 of the
165-amino acid human vascular endothelial cell growth factor.
Reference to any such forms of VEGF may be identified in the
application, e.g., by "VEGF (8-109)," "VEGF (1-109)" or "VEGF165."
The amino acid positions for a "truncated" native VEGF are numbered
as indicated in the native VEGF sequence. For example, amino acid
position 17 (methionine) in truncated native VEGF is also position
17 (methionine) in native VEGF. The truncated native VEGF has
binding affinity for the KDR and Flt-1 receptors comparable to
native VEGF.
[0043] An "anti-VEGF antibody" is an antibody that binds to VEGF
with sufficient affinity and specificity. The antibody selected
will normally have a binding affinity for VEGF, for example, the
antibody may bind hVEGF with a Kd value of between 100 nM-1 pM.
Antibody affinities may be determined by a surface plasmon
resonance based assay (such as the BIAcore assay as described in
PCT Application Publication No. WO2005/012359); enzyme-linked
immunoabsorbent assay (ELISA); and competition assays (e.g. RIA's),
for example. In certain embodiments, the anti-VEGF antibody of the
invention can be used as a therapeutic agent in targeting and
interfering with diseases or conditions wherein the VEGF activity
is involved. Also, the antibody may be subjected to other
biological activity assays, e.g., in order to evaluate its
effectiveness as a therapeutic. Such assays are known in the art
and depend on the target antigen and intended use for the antibody.
Examples include the HUVEC inhibition assay; tumor cell growth
inhibition assays (as described in WO 89/06692, for example);
antibody-dependent cellular cytotoxicity (ADCC) and
complement-mediated cytotoxicity (CDC) assays (U.S. Pat. No.
5,500,362); and agonistic activity or hematopoiesis assays (see WO
95/27062). An anti-VEGF antibody will usually not bind to other
VEGF homologues such as VEGF-B or VEGF-C, nor other growth factors
such as PlGF, PDGF or bFGF.
[0044] A "VEGF antagonist" refers to a molecule capable of
neutralizing, blocking, inhibiting, abrogating, reducing or
interfering with VEGF activities including its binding to one or
more VEGF receptors. VEGF antagonists include anti-VEGF antibodies
and antigen-binding fragments thereof, receptor molecules and
derivatives which bind specifically to VEGF thereby sequestering
its binding to one or more receptors, anti-VEGF receptor antibodies
and VEGF receptor antagonists such as small molecule inhibitors of
the VEGFR tyrosine kinases.
[0045] A "native sequence" polypeptide comprises a polypeptide
having the same amino acid sequence as a polypeptide derived from
nature. Thus, a native sequence polypeptide can have the amino acid
sequence of naturally-occurring polypeptide from any mammal. Such
native sequence polypeptide can be isolated from nature or can 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., an
extracellular domain sequence), naturally-occurring variant forms
(e.g., alternatively spliced forms) and naturally-occurring allelic
variants of the polypeptide.
[0046] A polypeptide "variant" means a biologically active
polypeptide having at least about 80% amino acid sequence identity
with the native sequence polypeptide. Such variants include, for
instance, polypeptides wherein one or more amino acid residues are
added, or deleted, at the N- or C-terminus of the polypeptide.
Ordinarily, a variant will have at least about 80% amino acid
sequence identity, more preferably at least about 90% amino acid
sequence identity, and even more preferably at least about 95%
amino acid sequence identity with the native sequence
polypeptide.
[0047] The term "antibody" is used 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.
[0048] Throughout the present specification and claims, the
numbering of the residues in an immunoglobulin heavy chain is that
of the EU index as in Kabat et al., Sequences of Proteins of
Immunological Interest, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda, Md. (1991), expressly incorporated
herein by reference. The "EU index as in Kabat" refers to the
residue numbering of the human IgG1 EU antibody.
[0049] The "Kd" or "Kd value" according to this invention is in one
embodiment measured by a radiolabeled VEGF binding assay (RIA)
performed with the Fab version of the antibody and a VEGF molecule
as described by the following assay that measures solution binding
affinity of Fabs for VEGF by equilibrating Fab with a minimal
concentration of (.sup.125I)-labeled VEGF(109) in the presence of a
titration series of unlabeled VEGF, then capturing bound VEGF with
an anti-Fab antibody-coated plate (Chen, et al., (1999) J. Mol Biol
293:865-881). In one example, to establish conditions for the
assay, microtiter plates (Dynex) are coated overnight with 5 ug/ml
of a capturing anti-Fab antibody (Cappel Labs) in 50 mM sodium
carbonate (pH 9.6), and subsequently blocked with 2% (w/v) bovine
serum albumin in PBS for two to five hours at room temperature
(approximately 23.degree. C.). In a non-adsorbant plate (Nunc
#269620), 100 pM or 26 pM [.sup.125I]VEGF(109) are mixed with
serial dilutions of a Fab of interest, e.g., Fab-12 (Presta et al.,
(1997) Cancer Res. 57:4593-4599). The Fab of interest is then
incubated overnight; however, the incubation may continue for 65
hours to insure that equilibrium is reached. Thereafter, the
mixtures are transferred to the capture plate for incubation at
room temperature for one hour. The solution is then removed and the
plate washed eight times with 0.1% Tween-20 in PBS. When the plates
had dried, 150 ul/well of scintillant (MicroScint-20; Packard) is
added, and the plates are counted on a Topcount gamma counter
(Packard) for ten minutes. Concentrations of each Fab that give
less than or equal to 20% of maximal binding are chosen for use in
competitive binding assays. According to another embodiment the Kd
or Kd value is measured by using surface plasmon resonance assays
using a BIAcore.TM.-2000 or a BIAcore.TM.-3000 (BIAcore, Inc.,
Piscataway, N.J.) at 25.degree. C. with immobilized hVEGF (8-109)
CM5 chips at .about.10 response units (RU). Briefly,
carboxymethylated dextran biosensor chips (CM5, BIAcore Inc.) are
activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide
hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the
supplier's instructions. Human VEGF is diluted with 10 mM sodium
acetate, pH 4.8, into 5 ug/ml (.about.0.2 uM) before injection at a
flow rate of 5 ul/minute to achieve approximately 10 response units
(RU) of coupled protein. Following the injection of human VEGF, 1M
ethanolamine is injected to block unreacted groups. For kinetics
measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM)
are injected in PBS with 0.05% Tween 20 (PBST) at 25.degree. C. at
a flow rate of approximately 25 ul/min. Association rates
(k.sub.on) and dissociation rates (k.sub.off) are calculated using
a simple one-to-one Langmuir binding model (BIAcore Evaluation
Software version 3.2) by simultaneous fitting the association and
dissociation sensorgram. The equilibrium dissociation constant (Kd)
was calculated as the ratio k.sub.off/k.sub.on. See, e.g., Chen,
Y., et al., (1999) J. Mol Biol 293:865-881. If the on-rate exceeds
10.sup.6 M.sup.-1 S.sup.-1 by the surface plasmon resonance assay
above, then the on-rate is can be determined by using a fluorescent
quenching technique that measures the increase or decrease in
fluorescence emission intensity (excitation=295 nm; emission=340
nm, 16 nm band-pass) at 25.degree. C. of a 20 nM 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 as
measured in a spectrometer, such as a stop-flow equipped
spectrophometer (Aviv Instruments) or a 8000-series SLM-Aminco
spectrophotometer (ThermoSpectronic) with a stirred cuvette.
[0050] A "blocking" antibody or an antibody "antagonist" is one
which inhibits or reduces biological activity of the antigen 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 antibodies or antagonist antibodies
completely or substantially inhibit the biological activity of the
antigen.
[0051] Unless indicated otherwise, the expression "multivalent
antibody" is used throughout this specification to denote an
antibody comprising three or more antigen binding sites. For
example, the multivalent antibody is engineered to have the three
or more antigen binding sites and is generally not a native
sequence IgM or IgA antibody.
[0052] "Antibody fragments" comprise only a portion of an intact
antibody, generally including an antigen binding site of the intact
antibody and thus retaining the ability to bind antigen. Examples
of antibody fragments encompassed by the present definition
include: (i) the Fab fragment, having VL, CL, VH and CH1 domains;
(ii) the Fab' fragment, which is a Fab fragment having one or more
cysteine residues at the C-terminus of the CH1 domain; (iii) the Fd
fragment having VH and CH1 domains; (iv) the Fd' fragment having VH
and CH1 domains and one or more cysteine residues at the C-terminus
of the CH1 domain; (v) the Fv fragment having the VL and VH domains
of a single arm of an antibody; (vi) the dAb fragment (Ward et al.,
Nature 341, 544-546 (1989)) which consists of a VH domain; (vii)
isolated CDR regions; (viii) F(ab').sub.2 fragments, a bivalent
fragment including two Fab' fragments linked by a disulphide bridge
at the hinge region; (ix) single chain antibody molecules (e.g.
single chain Fv; scFv) (Bird et al., Science 242:423-426 (1988);
and Huston et al., PNAS (USA) 85:5879-5883 (1988)); (x) "diabodies"
with two antigen binding sites, comprising a heavy chain variable
domain (VH) connected to a light chain variable domain (VL) in the
same polypeptide chain (see, e.g., EP 404,097; WO 93/11161; and
Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993));
(xi) "linear antibodies" comprising a pair of tandem Fd segments
(VH-CH1-VH-CH1) which, together with complementary light chain
polypeptides, form a pair of antigen binding regions (Zapata et al.
Protein Eng. 8(10):1057-1062 (1995); and U.S. Pat. No.
5,641,870).
[0053] 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
mutations that may be present in minor amounts. Monoclonal
antibodies are highly specific, being directed against a single
antigen. Furthermore, in contrast to polyclonal antibody
preparations that typically include 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,
the monoclonal antibodies to be used in accordance with the
invention may be made by the hybridoma method first described by
Kohler et al., Nature 256:495 (1975), or may be made by recombinant
DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The "monoclonal
antibodies" may also be isolated from phage antibody libraries
using the techniques described in Clackson et al., Nature
352:624-628 (1991) or Marks et al., J. Mol. Biol. 222:581-597
(1991), for example.
[0054] An "Fv" fragment is an antibody fragment which contains a
complete antigen recognition and binding site. This region consists
of a dimer of one heavy and one light chain variable domain in
tight association, which can be covalent in nature, for example in
scFv. It is in this configuration that the three CDRs of each
variable domain interact to define an antigen binding site on the
surface of the V.sub.H-V.sub.L dimer. Collectively, the six CDRs or
a subset thereof 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, although usually at a lower affinity
than the entire binding site.
[0055] As used herein, "antibody variable domain" refers to the
portions of the light and heavy chains of antibody molecules that
include amino acid sequences of Complementarity Determining Regions
(CDRs; ie., CDR1, CDR2, and CDR3), and Framework Regions (FRs).
V.sub.H refers to the variable domain of the heavy chain. V.sub.L
refers to the variable domain of the light chain. According to the
methods used in this invention, the amino acid positions assigned
to CDRs and FRs may be defined according to Kabat (Sequences of
Proteins of Immunological Interest (National Institutes of Health,
Bethesda, Md., 1987 and 1991)). Amino acid numbering of antibodies
or antigen binding fragments is also according to that of
Kabat.
[0056] As used herein, the term "Complementarity Determining
Regions" (CDRs; i.e., CDR1, CDR2, and CDR3) refers to the amino
acid residues of an antibody variable domain the presence of which
are necessary for antigen binding. Each variable domain typically
has three CDR regions identified as CDR1, CDR2 and CDR3. Each
complementarity determining region may comprise amino acid residues
from a "complementarity determining region" as defined by Kabat
(i.e. about residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the
light chain variable domain and 31-35 (H1), 50-65 (H2) and 95-102
(H3) in the heavy chain variable domain; Kabat et al., Sequences of
Proteins of Immunological Interest, 5th Ed. Public Health Service,
National Institutes of Health, Bethesda, Md. (1991)) and/or those
residues from a "hypervariable loop" (i.e. about residues 26-32
(L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domain
and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain
variable domain; Chothia and Lesk J. Mol. Biol. 196:901-917
(1987)). In some instances, a complementarity determining region
can include amino acids from both a CDR region defined according to
Kabat and a hypervariable loop. For example, the CDRH1 of the heavy
chain of antibody 4D5 includes amino acids 26 to 35.
[0057] "Framework regions" (hereinafter FR) are those variable
domain residues other than the CDR residues. Each variable domain
typically has four FRs identified as FR1, FR2, FR3 and FR4. If the
CDRs are defined according to Kabat, the light chain FR residues
are positioned at about residues 1-23 (LCFR1), 35-49 (LCFR2), 57-88
(LCFR3), and 98-107 (LCFR4) and the heavy chain FR residues are
positioned about at residues 1-30 (HCFR1), 36-49 (HCFR2), 66-94
(HCFR3), and 103-113 (HCFR4) in the heavy chain residues. If the
CDRs comprise amino acid residues from hypervariable loops, the
light chain FR residues are positioned about at residues 1-25
(LCFR1), 33-49 (LCFR2), 53-90 (LCFR3), and 97-107 (LCFR4) in the
light chain and the heavy chain FR residues are positioned about at
residues 1-25 (HCFR1), 33-52 (HCFR2), 56-95 (HCFR3), and 102-113
(HCFR4) in the heavy chain residues. In some instances, when the
CDR comprises amino acids from both a CDR as defined by Kabat and
those of a hypervariable loop, the FR residues will be adjusted
accordingly. For example, when CDRH1 includes amino acids H26-H35,
the heavy chain FR1 residues are at positions 1-25 and the FR2
residues are at positions 36-49.
[0058] The "Fab" fragment contains a variable and constant domain
of the light chain and a variable domain and the first constant
domain (CH1) of the heavy chain. F(ab').sub.2 antibody fragments
comprise a pair of Fab fragments which are generally covalently
linked near their carboxy termini by hinge cysteines between them.
Other chemical couplings of antibody fragments are also known in
the art.
[0059] "Single-chain Fv" or "scFv" antibody fragments comprise the
V.sub.H and V.sub.L domains of antibody, wherein these domains are
present in a single polypeptide chain. Generally the Fv polypeptide
further comprises a polypeptide linker between the V.sub.H and
V.sub.L domains, which enables the scFv to form the desired
structure for antigen binding. For a review of scFv, see Pluckthun
in The Pharmacology of Monoclonal Antibodies, Vol 113, Rosenburg
and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994).
[0060] The term "diabodies" refers to small antibody fragments with
two antigen-binding sites, which fragments comprise a heavy chain
variable domain (V.sub.H) connected to a light chain variable
domain (V.sub.L) in the same polypeptide chain (V.sub.H and
V.sub.L). By using a linker that is too short to allow pairing
between the two domains on the same chain, the domains are forced
to pair with the complementary domains of another chain and create
two antigen-binding sites. Diabodies are described more fully in,
for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc.
Natl. Acad. Sci. USA, 90:6444-6448 (1993).
[0061] The expression "linear antibodies" refers to the antibodies
described in Zapata et al., Protein Eng., 8(10):1057-1062 (1995).
Briefly, these antibodies comprise a pair of tandem Fd segments
(V.sub.H-C.sub.H1-V.sub.H-C.sub.H1) which, together with
complementary light chain polypeptides, form a pair of antigen
binding regions. Linear antibodies can be bispecific or
monospecific.
[0062] The monoclonal antibodies herein 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,
while the remainder of the chain(s) 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; and Morrison
et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).
[0063] "Humanized" forms of non-human (e.g., murine) antibodies are
chimeric antibodies which contain minimal sequence derived from
non-human immunoglobulin. For the most part, humanized antibodies
are human immunoglobulins (recipient antibody) in which residues
from a hypervariable region of the recipient 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 by corresponding non-human residues. Furthermore,
humanized antibodies may comprise residues which are not found in
the recipient antibody or in the donor antibody. These
modifications are made to further refine antibody performance. In
general, the humanized antibody will comprise substantially all of
at least one, and typically two, variable domains, 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 also will comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin. For further details, see Jones et al., Nature
321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988);
and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).
[0064] A "human antibody" is one which possesses an amino acid
sequence which corresponds to that of an antibody produced by a
human and/or has been made using any of the techniques for making
human antibodies as disclosed herein. This definition of a human
antibody specifically excludes a humanized antibody comprising
non-human antigen-binding residues. Human antibodies can be
produced using various techniques known in the art. In one
embodiment, the human antibody is selected from a phage library,
where that phage library expresses human antibodies (Vaughan et al.
Nature Biotechnology 14:309-314 (1996): Sheets et al. Proc. Natl.
Acad. Sci. 95:6157-6162 (1998)); Hoogenboom and Winter, J. Mol.
Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581
(1991)). Human antibodies can also be made by introducing human
immunoglobulin loci into transgenic animals, e.g., mice in which
the endogenous immunoglobulin genes have been partially or
completely inactivated. Upon challenge, human antibody production
is observed, which closely resembles that seen in humans in all
respects, including gene rearrangement, assembly, and antibody
repertoire. This approach is described, for example, in U.S. Pat.
Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425;
5,661,016, and in the following scientific publications: Marks et
al., Bio/Technology 10: 779-783 (1992); Lonberg et al., Nature 368:
856-859 (1994); Morrison, Nature 368:812-13 (1994); Fishwild et
al., Nature Biotechnology 14: 845-51 (1996); Neuberger, Nature
Biotechnology 14: 826 (1996); Lonberg and Huszar, Intern. Rev.
Immunol. 13:65-93 (1995). Alternatively, the human antibody may be
prepared via immortalization of human B lymphocytes producing an
antibody directed against a target antigen (such B lymphocytes may
be recovered from an individual or may have been 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.
[0065] An "affinity matured" antibody is one with one or more
alterations in one or more CDRs thereof which result an improvement
in the affinity of the antibody for antigen, compared to a parent
antibody which does not possess those alteration(s). Preferred
affinity matured antibodies will have nanomolar or even picomolar
affinities for the target antigen. Affinity matured antibodies are
produced by procedures known in the art. Marks et al.
Bio/Technology 10:779-783 (1992) describes affinity maturation by
VH and VL domain shuffling. Random mutagenesis of CDR and/or
framework residues is described by: Barbas et al. Proc Nat. Acad.
Sci, USA 91:3809-3813 (1994); Schier et al. Gene 169:147-155
(1995); Yelton et al. J. Immunol. 155:1994-2004 (1995); Jackson et
al., J. Immunol. 154(7):3310-9 (1995); and Hawkins et al., J. Mol.
Biol. 226:889-896 (1992).
[0066] A "functional antigen binding site" of an antibody is one
which is capable of binding a target antigen. The antigen binding
affinity of the antigen binding site is not necessarily 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 one of a variety of methods known for evaluating antibody
binding to an antigen. Moreover, the antigen binding affinity of
each of the antigen binding sites of a multivalent antibody herein
need not be quantitatively the same. For the multimeric antibodies
herein, the number of functional antigen binding sites can be
evaluated using ultracentrifugation analysis as described in
Example 2 of U.S. Patent Application Publication No. 20050186208.
According to this method of analysis, different ratios of target
antigen to multimeric antibody are combined and the average
molecular weight of the complexes is calculated assuming differing
numbers of functional binding sites. These theoretical values are
compared to the actual experimental values obtained in order to
evaluate the number of functional binding sites.
[0067] An antibody having a "biological characteristic" of a
designated antibody is one which possesses one or more of the
biological characteristics of that antibody which distinguish it
from other antibodies that bind to the same antigen.
[0068] In order to screen for antibodies which bind to an epitope
on an antigen bound by an antibody of interest, a routine
cross-blocking assay such as that described in Antibodies, A
Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and
David Lane (1988), can be performed.
[0069] A "species-dependent antibody" is one which has a stronger
binding affinity for an antigen from a first mammalian species than
it has for a homologue of that antigen from a second mammalian
species. Normally, the species-dependent antibody "binds
specifically" to a human antigen (i.e. has a binding affinity
(K.sub.d) value of no more than about 1.times.10.sup.-7 M,
preferably no more than about 1.times.10.sup.-8 M and most
preferably no more than about 1.times.10.sup.-9 M) but has a
binding affinity for a homologue of the antigen from a second
nonhuman mammalian species which is at least about 50 fold, or at
least about 500 fold, or at least about 1000 fold, weaker than its
binding affinity for the human antigen. The species-dependent
antibody can be any of the various types of antibodies as defined
above, but typically is a humanized or human antibody.
[0070] As used herein, "antibody mutant" or "antibody variant"
refers to an amino acid sequence variant of the species-dependent
antibody wherein one or more of the amino acid residues of the
species-dependent antibody have been modified. Such mutants
necessarily have less than 100% sequence identity or similarity
with the species-dependent antibody. In one embodiment, the
antibody mutant will have an amino acid sequence having at least
75% amino acid sequence identity or similarity with the amino acid
sequence of either the heavy or light chain variable domain of the
species-dependent antibody, more preferably at least 80%, more
preferably at least 85%, more preferably at least 90%, and most
preferably at least 95%. 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 same
residue) or similar (i.e. amino acid residue from the same group
based on common side-chain properties, see below) with the
species-dependent antibody residues, after aligning the sequences
and introducing gaps, if necessary, to achieve the maximum percent
sequence identity. None of N-terminal, C-terminal, or internal
extensions, deletions, or insertions into the antibody sequence
outside of the variable domain shall be construed as affecting
sequence identity or similarity.
[0071] To increase the half-life of the antibodies or polypeptide
containing the amino acid sequences of this invention, one can
attach a salvage receptor binding epitope to the antibody
(especially an antibody fragment), as described, e.g., in U.S. Pat.
No. 5,739,277. For example, a nucleic acid molecule encoding the
salvage receptor binding epitope can be linked in frame to a
nucleic acid encoding a polypeptide sequence of this invention so
that the fusion protein expressed by the engineered nucleic acid
molecule comprises the salvage receptor binding epitope and a
polypeptide sequence of this invention. As used herein, the term
"salvage receptor binding epitope" refers to an epitope of the Fc
region of an IgG molecule (e.g., IgG.sub.1, IgG.sub.2, IgG.sub.3,
or IgG.sub.4) that is responsible for increasing the in vivo serum
half-life of the IgG molecule (e.g., Ghetie et al., Ann. Rev.
Immunol. 18:739-766 (2000), Table 1). Antibodies with substitutions
in an Fc region thereof and increased serum half-lives are also
described in WO00/42072, WO 02/060919; Shields et al., J. Biol.
Chem. 276:6591-6604 (2001); Hinton, J. Biol. Chem. 279:6213-6216
(2004)). In another embodiment, the serum half-life can also be
increased, 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 a portion of
serum albumin that binds to the FcRn receptor or a serum albumin
binding peptide so that serum albumin binds to the antibody or
polypeptide, e.g., such polypeptide sequences are disclosed in
WO01/45746. In one embodiment, the serum albumin peptide to be
attached comprises an amino acid sequence of DICLPRWGCLW. In
another embodiment, the half-life of a Fab is increased by these
methods. See also, Dennis et al. J. Biol. Chem. 277:35035-35043
(2002) for serum albumin binding peptide sequences.
[0072] A "chimeric VEGF receptor protein" is a VEGF receptor
molecule having amino acid sequences derived from at least two
different proteins, at least one of which is as VEGF receptor
protein. In certain embodiments, the chimeric VEGF receptor protein
is capable of binding to and inhibiting the biological activity of
VEGF.
[0073] An "isolated" antibody is one that has been identified and
separated and/or recovered from a component of its natural
environment. Contaminant components of its natural environment are
materials that would interfere with diagnostic or therapeutic uses
for the antibody, and may include enzymes, hormones, and other
proteinaceous or nonproteinaceous solutes. In certain embodiments,
the antibody will be purified (1) to greater than 95% by weight of
antibody as determined by the Lowry method, and most preferably
more than 99% by weight, (2) to a degree sufficient to obtain at
least 15 residues of N-terminal or internal amino acid sequence by
use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE
under reducing or nonreducing conditions using Coomassie blue or,
silver stain. Isolated antibody includes the antibody in situ
within recombinant cells since at least one component of the
antibody's natural environment will not be present. Ordinarily,
however, isolated antibody will be prepared by at least one
purification step.
[0074] By "fragment" is meant a portion of a polypeptide or nucleic
acid molecule that contains, preferably, at least 10%, 20%, 30%,
40%, 50%, 60%, 70%, 80%, 90%, 95%, or more of the entire length of
the reference nucleic acid molecule or polypeptide. A 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 amino acids or more.
[0075] An "anti-angiogenesis agent" or "angiogenesis inhibitor"
refers to a small molecular weight substance, a polynucleotide, a
polypeptide, an isolated protein, a recombinant protein, an
antibody, or conjugates or fusion proteins thereof, that inhibits
angiogenesis, vasculogenesis, or undesirable vascular permeability,
either directly or indirectly. It should be understood that the
anti-angiogenesis agent includes those agents that bind and block
the angiogenic activity of the angiogenic factor or its receptor.
For example, an anti-angiogenesis agent is an antibody or other
antagonist to an angiogenic agent as defined throughout the
specification or known in the art, e.g., but are not limited to,
antibodies to VEGF-A or to the VEGF-A receptor (e.g., KDR receptor
or Flt-1 receptor), VEGF-trap, anti-PDGFR inhibitors such as
Gleevec.TM. (Imatinib Mesylate). Anti-angiogensis agents also
include native angiogenesis inhibitors, e.g., angiostatin,
endostatin, etc. See, e.g., Klagsbrun and D'Amore, Annu. Rev.
Physiol., 53:217-39 (1991); Streit and Detmar, Oncogene,
22:3172-3179 (2003) (e.g., Table 3 listing anti-angiogenic therapy
in malignant melanoma); Ferrara & Alitalo, Nature Medicine
5:1359-1364 (1999); Tonini et al., Oncogene, 22:6549-6556 (2003)
(e.g., Table 2 listing known antiangiogenic factors); and Sato.
Int. J. Clin. Oncol 8:200-206 (2003) (e.g., Table 1 lists
anti-angiogenic agents used in clinical trials).
[0076] A "maintenance" dose herein refers to one or more doses of a
therapeutic agent administered to the patient over or after a
treatment period. Usually, the maintenance doses are administered
at spaced treatment intervals, such as approximately every week,
approximately every 2 weeks, approximately every 3 weeks, or
approximately every 4 weeks. In one embodiment, the maintenance
doses are as depicted in FIG. 1 (extended therapy), FIG. 2 or FIG.
8 or FIG. 11 herein.
[0077] "Survival" refers to the patient remaining alive, and
includes progression free survival (PFS) and overall survival (OS).
Survival can be estimated by the Kaplan-Meier method, and any
differences in survival are computed using the stratified log-rank
test.
[0078] "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 Response
Evaluation Criteria in Solid Tumors (RECIST). In one aspect of the
invention, PFS can be assessed by CA-125 levels as a determinant of
progression.
[0079] "Overall survival" refers to the patient remaining alive for
a defined 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., from initiation of treatment or from initial
diagnosis. In the studies underlying the invention the event used
for survival analysis was death from any cause.
[0080] By "extending survival" or "increasing the likelihood of
survival" is meant increasing PFS and/or OS in a treated patient
relative to an untreated patient (i.e. relative to a patient not
treated with a VEGF-specific antagonist, e.g., a VEGF antibody), or
relative to a control treatment protocol, such as treatment only
with the chemotherapeutic agent, such as those use in the standard
of care for ovarian cancer. For example extended PFS is the time
that the patient remains alive, without return of the cancer, e.g.,
for a defined period of time such as about 1 month, 2 months, 2.3
months, 2.9 months, 3 months, 3.8 months, 4 months, 6 months, 7
months, 8 months, 9 months, 1 year, about 2 years, about 3 years,
etc., from initiation of treatment or from initial diagnosis,
compared to a control (e.g., patient not treated with the same VEGF
specific antagonist). In one embodiment, the PFS is extended about
2.9 months to 3.8 months compared to a control. In one embodiment,
the PFS is extended at least about 3.8 months compared to a
control. In another embodiment, the PFS is extended by about 2.3
months. In one embodiment, the PFS is extended about 6 months
compared to a control. In certain embodiment, survival is monitored
for at least about one month, two months, four months, six months,
nine 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 the
initiation of treatment or following the initial diagnosis.
[0081] Hazard ratio (HR) is a statistical definition for rates of
events. For the purpose of the invention, hazard ratio is defined
as representing the probability of an event in the experimental arm
divided by the probability of an event in the control arm at any
specific point in time. "Hazard ratio" in progression free survival
analysis is a summary of the difference between two progression
free survival curves, representing the reduction in the risk of
death on treatment compared to control, over a period of
follow-up.
[0082] The term "concurrently" is used herein to refer to
administration of two or more therapeutic agents, where at least
part of the administration overlaps in time. Accordingly,
concurrent administration includes a dosing regimen when the
administration of one or more agent(s) continues after
discontinuing the administration of one or more other agent(s).
[0083] By "monotherapy" is meant a therapeutic regimen that
includes only a single therapeutic agent for the treatment of the
cancer or tumor during the course of the treatment period.
Monotherapy using a VEGF-specific antagonist means that the
VEGF-specific antagonist is administered in the absence of an
additional anti-cancer therapy during treatment period.
[0084] By "maintenance therapy" is meant a therapeutic regimen that
is given to reduce the likelihood of disease recurrence or
progression. Maintenance therapy can be provided for any length of
time, including extended time periods up to the life-span of the
subject. Maintenance therapy can be provided after initial therapy
or in conjunction with initial or additional therapies. Dosages
used for maintenance therapy can vary and can include diminished
dosages as compared to dosages used for other types of therapy. In
certain embodiments of the invention, maintenance therapy is
provided for at least 16 cycles after completion of the
chemotherapy concurrently with 5 cycles of anti-VEGF therapy. In
other embodiments of the invention, maintenance therapy is provided
for at least 12 cycles after completion of the chemotherapy
concurrently with 6 cycles of anti-VEGF therapy. In one embodiment,
maintenance therapy is as depicted in FIG. 1, FIG. 2, FIG. 8 or
FIG. 11.
[0085] The terms "cancer" and "cancerous" refer to or describe the
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
micrometastatses. Examples of cancer include but are 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, platinum
sensitive recurrent epithelial ovarian, primary peritoneal, or
fallopian tube carcinoma, 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),
cancer of the peritoneum, hepatocellular cancer, gastric or stomach
cancer (including gastrointestinal cancer), pancreatic cancer,
glioblastoma, cervical cancer, liver cancer, bladder cancer,
hepatoma, breast cancer, colon cancer, colorectal cancer,
endometrial or uterine carcinoma, salivary gland carcinoma, kidney
or renal cancer, liver cancer, prostate cancer, vulval cancer,
thyroid cancer, hepatic carcinoma and various types of head and
neck cancer, as well as 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 immunoblastic NHL; high grade lymphoblastic
NHL; high grade small non-cleaved cell NHL; bulky disease NHL;
mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's
Macroglobulinemia); chronic lymphocytic leukemia (CLL); acute
lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic
myeloblastic leukemia; and post-transplant lymphoproliferative
disorder (PTLD), as well as abnormal vascular proliferation
associated with phakomatoses, edema (such as that associated with
brain tumors), and Meigs' syndrome.
[0086] By "metastasis" is meant the spread of
http://en.wikipedia.org/wiki/Cancer cancer from its primary site to
other places in the body. Cancer cells can break away from a
primary tumor, penetrate into lymphatic and blood vessels,
circulate through the bloodstream, and grow in a distant focus
(metastasize) in normal tissues elsewhere in the body. Metastasis
can be local or distant. Metastasis is a sequential process,
contingent on tumor cells breaking off from the primary tumor,
traveling through the bloodstream, and stopping at a distant site.
At the new site, the cells establish a blood supply and can grow to
form a life-threatening mass. Both stimulatory and inhibitory
molecular pathways within the tumor cell regulate this behavior,
and interactions between the tumor cell and host cells in the
distant site are also significant.
[0087] By "subject" is meant 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. Patients are
also subjects herein. Typically, the subject is female.
[0088] For the methods of the present invention, the term
"instructing" a subject means providing directions for applicable
therapy, medication, treatment, treatment regimens, and the like,
by any means, but preferably in writing, such as in the form of
package inserts or other written promotional material.
[0089] For the methods of the present invention, the term
"promoting" means offering, advertising, selling, or describing a
particular drug, combination of drugs, or treatment modality, by
any means, including writing, such as in the form of package
inserts. Promoting herein refers to promotion of a therapeutic
agent, such as a VEGF antagonist, e.g., anti-VEGF antibody or
chemotherapeutic agent, for an indication, such as ovarian cancer
treatment, where such promoting is authorized 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
[0090] The term "marketing" is used herein to describe the
promotion, selling or distribution of a product (e.g., drug).
Marketing specifically includes packaging, advertising, and any
business activity with the purpose of commercializing a
product.
[0091] A "population" of subjects refers to a group of subjects
with cancer, such as in a clinical trial, or as seen by oncologists
following FDA approval for a particular indication, such as ovarian
cancer therapy.
[0092] The term "anti-cancer therapy" refers to a therapy useful in
treating cancer. Examples of anti-cancer therapeutic agents
include, but are limited to, e.g., surgery, chemotherapeutic
agents, growth inhibitory agents, cytotoxic agents, agents used in
radiation therapy, anti-angiogenesis agents, apoptotic agents,
anti-tubulin agents, and other agents to treat cancer, such as
anti-HER-2 antibodies, anti-CD20 antibodies, an epidermal growth
factor receptor (EGFR) antagonist (e.g., a tyrosine kinase
inhibitor), HER1/EGFR inhibitor (e.g., erlotinib (Tarceva.RTM.),
platelet derived growth factor inhibitors (e.g., Gleevec.TM.
(Imatinib Mesylate)), a COX-2 inhibitor (e.g., celecoxib),
interferons, cytokines, antagonists (e.g., neutralizing antibodies)
that bind to one or more of the following targets ErbB2, ErbB3,
ErbB4, PDGFR-beta, BlyS, APRIL, BCMA or VEGF receptor(s),
TRAIL/Apo2, and other bioactive and organic chemical agents, etc.
Combinations thereof are also included in the invention.
[0093] The term "cytotoxic agent" as used herein refers to a
substance that inhibits or prevents the function of cells and/or
causes destruction of cells. The term is intended to include
radioactive isotopes (e.g. At.sup.211, I.sup.131, I.sup.125,
Y.sup.90, Re.sup.186, Re.sup.188, Sm.sup.153, Bi.sup.212, P.sup.32
and 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.
[0094] A "chemotherapeutic agent" is a chemical compound useful in
the treatment of cancer. Examples of chemotherapeutic agents
include is a chemical compound useful in the treatment of cancer.
Examples of chemotherapeutic agents include alkylating agents such
as thiotepa and CYTOXAN.RTM. cyclosphosphamide; alkyl sulfonates
such as busulfan, improsulfan and piposulfan; aziridines such as
benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and
methylamelamines including altretamine, triethylenemelamine,
trietylenephosphoramide, triethiylenethiophosphoramide and
trimethylolomelamine; acetogenins (especially bullatacin and
bullatacinone); a camptothecin (including the synthetic analogue
topotecan); bryostatin; callystatin; CC-1065 (including its
adozelesin, carzelesin and bizelesin synthetic analogues);
cryptophycins (particularly cryptophycin 1 and cryptophycin 8);
dolastatin; duocarmycin (including the synthetic analogues, KW-2189
and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin;
spongistatin; nitrogen mustards such as chlorambucil,
chlornaphazine, cholophosphamide, estramustine, ifosfamide,
mechlorethamine, mechlorethamine oxide hydrochloride, melphalan,
novembichin, phenesterine, prednimustine, trofosfamide, uracil
mustard; nitrosureas such as carmustine, chlorozotocin,
fotemustine, lomustine, nimustine, and ranimnustine; antibiotics
such as the enediyne antibiotics (e. g., calicheamicin, especially
calicheamicin gamma1I and calicheamicin omegaI1 (see, e.g., Agnew,
Chem Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, including
dynemicin A; bisphosphonates, such as clodronate; an esperamicin;
as well as neocarzinostatin chromophore and related chromoprotein
enediyne antibiotic chromophores), aclacinomysins, actinomycin,
authramycin, azaserine, bleomycins, cactinomycin, carabicin,
carminomycin, carzinophilin, chromomycinis, dactinomycin,
daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine,
ADRIAMYCIN.RTM. doxorubicin (including morpholino-doxorubicin,
cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and
deoxydoxorubicin), epirubicin, esorubicin, idarubicin,
marcellomycin, mitomycins such as mitomycin C, mycophenolic acid,
nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin,
quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,
ubenimex, zinostatin, zorubicin; anti-metabolites such as
methotrexate and 5-fluorouracil (5-FU); folic acid analogues such
as denopterin, methotrexate, pteropterin, trimetrexate; purine
analogs such as fludarabine, 6-mercaptopurine, thiamiprine,
thioguanine; pyrimidine analogs such as ancitabine, azacitidine,
6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine,
enocitabine, floxuridine; androgens such as calusterone,
dromostanolone propionate, epitiostanol, mepitiostane,
testolactone; anti-adrenals such as aminoglutethimide, mitotane,
trilostane; folic acid replenisher such as frolinic acid;
aceglatone; aldophosphamide glycoside; aminolevulinic acid;
eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate;
defofamine; demecolcine; diaziquone; eflornithine; elliptinium
acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea;
lentinan; lonidainine; maytansinoids such as maytansine and
ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine;
pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic
acid; 2-ethylhydrazide; procarbazine; PSK.RTM. polysaccharide
complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;
sizofiran; spirogermanium; tenuazonic acid; triaziquone;
2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2
toxin, verracurin A, roridin A and anguidine); urethan; vindesine;
dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman;
gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa;
taxoids, e.g., TAXOL.RTM. paclitaxel (Bristol-Myers Squibb
Oncology, Princeton, N.J.), ABRAXANE.RTM. Cremophor-free,
albumin-engineered nanoparticle formulation of paclitaxel (American
Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE.RTM.
doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil;
GEMZAR.RTM. gemcitabine; 6-thioguanine; mercaptopurine;
methotrexate; platinum analogs such as cisplatin, oxaliplatin and
carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide;
mitoxantrone; vincristine; NAVELBINE.RTM. vinorelbine; novantrone;
teniposide; edatrexate; daunomycin; aminopterin; xeloda;
ibandronate; irinotecan (Camptosar, CPT-11) (including the
treatment regimen of irinotecan with 5-FU and leucovorin);
topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO);
retinoids such as retinoic acid; capecitabine; combretastatin;
leucovorin (LV); oxaliplatin, including the oxaliplatin treatment
regimen (FOLFOX); lapatinib (Tykerb.RTM.); inhibitors of PKC-alpha,
Raf, H-Ras, EGFR (e.g., erlotinib))(Tarceva.RTM. and VEGF-A that
reduce cell proliferation and pharmaceutically acceptable salts,
acids or derivatives of any of the above.
[0095] Also included in this definition are anti-hormonal agents
that act to regulate or inhibit hormone action on tumors such as
anti-estrogens and selective estrogen receptor modulators (SERMs),
including, for example, tamoxifen (including NOLVADEX.RTM.
tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen,
trioxifene, keoxifene, LY117018, onapristone, and FARESTON
toremifene; aromatase inhibitors that inhibit the enzyme aromatase,
which regulates estrogen production in the adrenal glands, such as,
for example, 4(5)-imidazoles, aminoglutethimide, MEGASE.RTM.
megestrol acetate, AROMASIN.RTM. exemestane, formestanie,
fadrozole, RIVISOR.RTM. vorozole, FEMARA.RTM. letrozole, and
ARIMIDEX.RTM. anastrozole; and anti-androgens such as flutamide,
nilutamide, bicalutamide, leuprolide, and goserelin; as well as
troxacitabine (a 1,3-dioxolane nucleoside cytosine analog);
antisense oligonucleotides, particularly those which inhibit
expression of genes in signaling pathways implicated in abherant
cell proliferation, such as, for example, PKC-alpha, Ralf and
H-Ras; ribozymes such as a VEGF expression inhibitor (e.g.,
ANGIOZYME.RTM. ribozyme) and a HER2 expression inhibitor; vaccines
such as gene therapy vaccines, for example, ALLOVECTIN.RTM.
vaccine, LEUVECTIN.RTM. vaccine, and VAXID.RTM. vaccine;
PROLEUKIN.RTM. rIL-2; LURTOTECAN.RTM. topoisomerase 1 inhibitor;
ABARELIX.RTM. rmRH; and pharmaceutically acceptable salts, acids or
derivatives of any of the above.
[0096] The term "cytokine" is a generic term for proteins released
by one cell population which act on another cell as intercellular
mediators. Examples of such cytokines are lymphokines, monokines,
and traditional polypeptide hormones. Included among the cytokines
are growth hormone such as human growth hormone, N-methionyl human
growth hormone, and bovine growth hormone; parathyroid hormone;
thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein
hormones such as follicle stimulating hormone (FSH), thyroid
stimulating hormone (TSH), and luteinizing hormone (LH); epidermal
growth factor; hepatic growth factor; fibroblast growth factor;
prolactin; placental lactogen; tumor necrosis factor-alpha and
-beta; mullerian-inhibiting substance; mouse
gonadotropin-associated peptide; inhibin; activin; vascular
endothelial growth factor; integrin; thrombopoietin (TPO); nerve
growth factors such as NGF-alpha; platelet-growth factor;
transforming growth factors (TGFs) such as TGF-alpha and TGF-beta;
insulin-like growth factor-I and -II; erythropoietin (EPO);
osteoinductive factors; interferons such as interferon-alpha, -beta
and -gamma colony stimulating factors (CSFs) such as macrophage-CSF
(M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF
(G-CSF); interleukins (ILs) such as IL-1, IL-1alpha, IL-2, IL-3,
IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; a tumor
necrosis factor such as TNF-alpha or TNF-beta; and other
polypeptide factors including LIF and kit ligand (KL). As used
herein, the term cytokine includes proteins from natural sources or
from recombinant cell culture and biologically active equivalents
of the native sequence cytokines.
[0097] A "growth inhibitory agent" when used herein refers to a
compound or composition which inhibits growth of a cell in vitro
and/or in vivo. Thus, the growth inhibitory agent may be one which
significantly reduces the percentage of cells in S phase. Examples
of growth inhibitory agents include agents that block cell cycle
progression (at a place other than S phase), such as agents that
induce G1 arrest and M-phase arrest. Classical M-phase blockers
include the vincas (vincristine and vinblastine), TAXOL.RTM., and
topo II inhibitors such as doxorubicin, epirubicin, daunorubicin,
etoposide, and bleomycin. Those agents that arrest G1 also spill
over into S-phase arrest, for example, DNA alkylating agents such
as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin,
methotrexate, 5-fluorouracil, and ara-C. Further information can be
found in The Molecular Basis of Cancer, Mendelsohn and Israel,
eds., Chapter 1, entitled "Cell cycle regulation, oncogenes, and
antineoplastic drugs" by Murakami et al. (WB Saunders:
Philadelphia, 1995), especially p. 13.
[0098] The term "prodrug" as used in this application refers to a
precursor or derivative form of a pharmaceutically active substance
that is less cytotoxic to tumor cells compared to the parent drug
and is capable of being enzymatically activated or converted into
the more active parent form. See, e.g., Wilman, "Prodrugs in Cancer
Chemotherapy" Biochemical Society Transactions, 14, pp. 375-382,
615th Meeting Belfast (1986) and Stella et al., "Prodrugs: A
Chemical Approach to Targeted Drug Delivery," Directed Drug
Delivery, Borchardt et al., (ed.), pp. 247-267, Humana Press
(1985). The prodrugs of this invention include, but are not limited
to, phosphate-containing prodrugs, thiophosphate-containing
prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs,
D-amino acid-modified prodrugs, glycosylated prodrugs,
.beta.-lactam-containing prodrugs, optionally substituted
phenoxyacetamide-containing prodrugs or optionally substituted
phenylacetamide-containing prodrugs, 5-fluorocytosine and other
5-fluorouridine prodrugs which can be converted into the more
active cytotoxic free drug. Examples of cytotoxic drugs that can be
derivatized into a prodrug form for use in this invention include,
but are not limited to, those chemotherapeutic agents described
above.
[0099] By "radiation therapy" is meant the use of directed gamma
rays or beta rays to induce sufficient damage to a cell so as to
limit its ability to function normally or to destroy the cell
altogether. It will be appreciated that there will be many ways
known in the art to determine the dosage and duration of treatment.
Typical treatments are given as a one time administration and
typical dosages range from 10 to 200 units (Grays) per day.
[0100] By "reduce or inhibit" is meant the ability to cause an
overall decrease preferably of 20% or greater, more preferably of
50% or greater, and most preferably of 75%, 85%, 90%, 95%, or
greater. Reduce or inhibit can refer to the symptoms of the
disorder being treated, the presence or size of metastases or
micrometastases, the size of the primary tumor, the presence or the
size of the dormant tumor, or the size or number of the blood
vessels in angiogenic disorders.
[0101] The term "intravenous infusion" refers to introduction of a
drug into the vein of an animal or human patient over a period of
time greater than approximately 5 minutes, preferably between
approximately 30 to 90 minutes, although, according to the
invention, intravenous infusion is alternatively administered for
10 hours or less.
[0102] The term "intravenous bolus" or "intravenous push" refers to
drug administration into a vein of an animal or human such that the
body receives the drug in approximately 15 minutes or less,
preferably 5 minutes or less.
[0103] The term "subcutaneous administration" refers to
introduction of a drug under the skin of an animal or human
patient, preferable within a pocket between the skin and underlying
tissue, by relatively slow, sustained delivery from a drug
receptacle. The pocket may be created by pinching or drawing the
skin up and away from underlying tissue.
[0104] The term "subcutaneous infusion" refers to introduction of a
drug under the skin of an animal or human patient, preferably
within a pocket between the skin and underlying tissue, by
relatively slow, sustained delivery from a drug receptacle for a
period of time including, but not limited to, 30 minutes or less,
or 90 minutes or less. Optionally, the infusion may be made by
subcutaneous implantation of a drug delivery pump implanted under
the skin of the 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 time period spanning the
length of the treatment regimen.
[0105] The term "subcutaneous bolus" refers to drug administration
beneath the skin of an animal or human patient, where bolus drug
delivery is preferably less than approximately 15 minutes, more
preferably less than 5 minutes, and most preferably less than 60
seconds. Administration is preferably within a pocket between the
skin and underlying tissue, where the pocket is created, for
example, by pinching or drawing the skin up and away from
underlying tissue.
[0106] A "disorder" is any condition that would benefit from
treatment with the antibody. This includes chronic and acute
disorders or diseases including those pathological conditions which
predispose the mammal to the disorder in question. Non-limiting
examples of disorders to be treated herein include cancer; benign
and malignant tumors; leukemias and lymphoid malignancies;
neuronal, glial, astrocytal, hypothalamic and other glandular,
macrophagal, epithelial, stromal and blastocoelic disorders; and
inflammatory, angiogenic and immunologic disorders.
[0107] 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, the therapeutically effective amount
of the drug may reduce the number of cancer cells; reduce the tumor
size; inhibit (i.e., slow to some extent and preferably stop)
cancer cell infiltration into peripheral organs; inhibit (i.e.,
slow to some extent and preferably stop) tumor metastasis; inhibit,
to some extent, tumor growth; and/or relieve to some extent one or
more of the symptoms associated with the disorder. To the extent
the drug may prevent growth and/or kill existing cancer cells, it
may be cytostatic and/or cytotoxic. For cancer therapy, efficacy in
vivo can, for example, be measured by assessing the duration of
survival, duration of progression free survival (PFS), extension of
progression free survival (PFS), the response rates (RR), duration
of response, and/or quality of life.
[0108] "Treatment" refers to therapeutic treatment for those in
need of treatment include those already with the disorder.
[0109] "Prophylactic or preventative treatment" refers to those in
which the disorder is to be prevented.
[0110] The word "label" when used herein refers to a detectable
compound or composition which is conjugated directly or indirectly
to the polypeptide. The label may be itself be detectable (e.g.,
radioisotope labels or fluorescent labels) or, in the case of an
enzymatic label, may catalyze chemical alteration of a substrate
compound or composition which is detectable.
II. Anti-VEGF Antibodies and Antagonists
[0111] Uses of anti-VEGF antagonists for treating ovarian cancer
are provided herein. Angiogenesis is one of the cardinal processes
leading to invasion and metastasis of solid tumors. The
angiogenic-signaling pathway may be triggered by the release of
angiogenic promoters such as vascular endothelial growth factor
(VEGF) from tumor cells into the local microenvironment. There is
accumulating evidence that angiogenesis plays a role in ovarian
cancer disease prognosis and possibly progression and prognosis.
See, e.g., Yoneda J, et al., Expression of angiogenesis-related
genes and progression of human ovarian carcinomas in nude mice. J
Natl Cancer Inst 90:447-54, 1998; Nakanishi Y, et al. The
expression of vascular endothelial growth factor and transforming
growth factor-beta associates with angiogenesis in epithelial
ovarian cancer. Int J Gynecol Pathol 16:256-62, 1997; Gasparini G,
et al. Prognostic and predictive value of tumour angiogenesis in
ovarian carcinomas. Int J Cancer 69:205-11, 1996; Hollingsworth H
C, et al., Tumor angiogenesis in advanced stage ovarian carcinoma.
Am J Pathol 147:33-41, 1995; Paley P J, et al. Vascular endothelial
growth factor expression in early stage ovarian carcinoma. Cancer
80:98-106, 1997; Alvarez A A, et al., The prognostic significance
of angiogenesis in epithelial ovarian carcinoma. Clin Cancer Res
5:587-91, 1999; Gasparini G. The rationale and future potential of
angiogenesis inhibitors in neoplasia. Drugs 58:17-38, 1999; van
Hinsbergh V W, et al., Angiogenesis and anti-angiogenesis:
perspectives for the treatment of solid tumors. Ann Oncol 10 Suppl
4:60-3, 1999; Malonne H, et al., Mechanisms of tumor angiogenesis
and therapeutic implications: angiogenesis inhibitors. Clin Exp
Metastasis 17:1-14, 1999; Folkman J. Tumor angiogenesis:
therapeutic implications. N Eng J Med 285:1182-6, 1971; Kim K J, et
al. Inhibition of vascular endothelial growth factor-induced
angiogenesis suppresses tumour growth in vivo. Nature 362:841-4,
1993; and, Luo J C, et al., Differential inhibition of fluid
accumulation and tumor growth in two mouse ascites tumors by an
anti vascular endothelial growth factor/permeability factor
neutralizing antibody. Cancer Res 58:2594-600, 1998.
(i) VEGF Antigen
[0112] The VEGF antigen to be used for production of antibodies may
be, e.g., the VEGF.sub.165 molecule as well as other isoforms of
VEGF or a fragment thereof containing the desired epitope. Other
forms of VEGF useful for generating anti-VEGF antibodies of the
invention will be apparent to those skilled in the art.
[0113] Human VEGF was obtained by first screening a cDNA library
prepared from human cells, using bovine VEGF cDNA as a
hybridization probe. Leung et al. (1989) Science, 246:1306. One
cDNA identified thereby encodes a 165-amino acid protein having
greater than 95% homology to bovine VEGF; this 165-amino acid
protein is typically referred to as human VEGF (hVEGF) or
VEGF.sub.165. The mitogenic activity of human VEGF was confirmed by
expressing the human VEGF cDNA in mammalian host cells. Media
conditioned by cells transfected with the human VEGF cDNA promoted
the proliferation of capillary endothelial cells, whereas control
cells did not. Leung et al. (1989) Science, supra.
[0114] Although a vascular endothelial cell growth factor could be
isolated and purified from natural sources for subsequent
therapeutic use, the relatively low concentrations of the protein
in follicular cells and the high cost, both in terms of effort and
expense, of recovering VEGF proved commercially unavailing.
Accordingly, further efforts were undertaken to clone and express
VEGF via recombinant DNA techniques. (See, e.g., Ferrara,
Laboratory Investigation 72:615-618 (1995), and the references
cited therein).
[0115] VEGF is expressed in a variety of tissues as multiple
homodimeric forms (121, 145, 165, 189, and 206 amino acids per
monomer) resulting from alternative RNA splicing. VEGF.sub.121 is a
soluble mitogen that does not bind heparin; the longer forms of
VEGF bind heparin with progressively higher affinity. The
heparin-binding forms of VEGF can be cleaved in the carboxy
terminus by plasmin to release a diffusible form(s) of VEGF. Amino
acid sequencing of the carboxy terminal peptide identified after
plasmin cleavage is Arg.sub.110-Ala.sub.111. Amino terminal "core"
protein, VEGF (1-110) isolated as a homodimer, binds neutralizing
monoclonal antibodies (such as the antibodies referred to as 4.6.1
and 3.2E3.1.1) and soluble forms of VEGF receptors with similar
affinity compared to the intact VEGF.sub.165 homodimer.
[0116] Several molecules structurally related to VEGF have also
been identified including placenta 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). A receptor
tyrosine kinase, Flt-4 (VEGFR-3), has been identified as the
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 lymphatic
angiogenesis. Jeltsch et al. Science 276:1423-1425 (1997).
(ii) Anti-VEGF Antibodies
[0117] Anti-VEGF antibodies that are useful in the methods of the
invention to treat ovarian cancer include any antibody, or antigen
binding fragment thereof, that bind with sufficient affinity and
specificity to VEGF and can reduce or inhibit the biological
activity of VEGF. An anti-VEGF antibody will usually not bind to
other VEGF homologues such as VEGF-B or VEGF-C, nor other growth
factors such as PlGF, PDGF, or bFGF.
[0118] In certain embodiments of the invention, the anti-VEGF
antibodies include, but are not limited to, a monoclonal antibody
that binds to the same epitope as the monoclonal anti-VEGF antibody
A4.6.1 produced by hybridoma ATCC HB 10709; a recombinant humanized
anti-VEGF monoclonal antibody generated according to Presta et al.
(1997) Cancer Res. 57:4593-4599. In one embodiment, the anti-VEGF
antibody is "Bevacizumab (BV)", also known as "rhuMAb VEGF" or
"AVASTIN.RTM.". AVASTIN.RTM. is commercially available in certain
countries. It comprises mutated human IgG1 framework regions and
antigen-binding complementarity-determining regions from the murine
anti-hVEGF monoclonal antibody A.4.6.1 that blocks binding of human
VEGF to its receptors. Approximately 93% of the amino acid sequence
of bevacizumab, including most of the framework regions, is derived
from human IgG1, and about 7% of the sequence is derived from the
murine antibody A4.6.1.
[0119] Bevacizumab and other humanized anti-VEGF antibodies are
further described in U.S. Pat. No. 6,884,879 issued Feb. 26, 2005.
Additional antibodies include the G6 or B20 series antibodies
(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 content of these patent applications
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; WO98/45332; WO 96/30046; WO94/10202; EP 0666868B1; U.S.
Patent Application Publication Nos. 2006009360, 20050186208,
20030206899, 20030190317, 20030203409, and 20050112126; and Popkov
et al., Journal of Immunological Methods 288:149-164 (2004). Other
antibodies include those that bind to a functional epitope on human
VEGF comprising of residues F17, M18, D19, Y21, Y25, Q89, I91,
K101, E103, and C104 or, alternatively, comprising residues F17,
Y21, Q22, Y25, D63, I83 and Q89.
[0120] In one embodiment of the invention, the anti-VEGF antibody
has a heavy chain variable region comprising the following amino
acid sequence:
TABLE-US-00005 (SEQ ID No. 1) EVQLVESGGG LVQPGGSLRL SCAASGYTFT
NYGMNWVRQA PGKGLEWVGW INTYTGEPTY AADFKRRFTF SLDTSKSTAY LQMNSLRAED
TAVYYCAKYP HYYGSSHWYF DVWGQGTLVT VSS
and a light chain variable region comprising the following amino
acid sequence:
TABLE-US-00006 (SEQ ID No. 2) DIQMTQSPSS LSASVGDRVT ITCSASQDIS
NYLNWYQQKP GKAPKVLIYF TSSLHSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ
YSTVPWTFGQ GTKVEIKR.
[0121] A "G6 series antibody" according to this invention, is an
anti-VEGF antibody that is derived from a sequence of a G6 antibody
or G6-derived antibody according to any one of FIGS. 7, 24-26, and
34-35 of PCT Publication No. WO2005/012359, the entire disclosure
of which is expressly incorporated herein by reference. See also
PCT Publication No. WO2005/044853, the entire disclosure of which
is expressly incorporated herein by reference. In one embodiment,
the G6 series antibody binds to a functional epitope on human VEGF
comprising residues F17, Y21, Q22, Y25, D63, I83 and Q89.
[0122] A "B20 series antibody" according to this invention is an
anti-VEGF antibody that is derived from a sequence of the B20
antibody or a B20-derived antibody according to any one of FIGS.
27-29 of PCT Publication No. WO2005/012359, the entire disclosure
of which is expressly incorporated herein by reference. See also
PCT Publication No. WO2005/044853, and U.S. Patent Application
60/991,302, the content of these patent applications are expressly
incorporated herein by reference. In one embodiment, the B20 series
antibody binds to a functional epitope on human VEGF comprising
residues F17, M18, D19, Y21, Y25, Q89, I91, K101, E103, and
C104.
[0123] A "functional epitope" according to this invention refers to
amino acid residues of an antigen that contribute energetically to
the binding of an antibody. Mutation of any one of the
energetically contributing residues of the antigen (for example,
mutation of wild-type VEGF by alanine or homolog mutation) will
disrupt the binding of the antibody such that the relative affinity
ratio (IC50mutant VEGF/IC50wild-type VEGF) of the antibody will be
greater than 5 (see Example 2 of WO2005/012359). In one embodiment,
the relative affinity ratio is determined by a solution binding
phage displaying ELISA. Briefly, 96-well Maxisorp immunoplates
(NUNC) are coated overnight at 4.degree. C. with an Fab form of the
antibody to be tested at a concentration of 2 ug/ml in PBS, and
blocked with PBS, 0.5% BSA, and 0.05% Tween20 (PBT) for 2 h at room
temperature. Serial dilutions of phage displaying hVEGF alanine
point mutants (residues 8-109 form) or wild type hVEGF (8-109) in
PBT are first incubated on the Fab-coated plates for 15 min at room
temperature, and the plates are washed with PBS, 0.05% Tween20
(PBST). The bound phage is detected with an anti-M13 monoclonal
antibody horseradish peroxidase (Amersham Pharmacia) conjugate
diluted 1:5000 in PBT, developed with 3,3',
5,5'-tetramethylbenzidine (TMB, Kirkegaard & Perry Labs,
Gaithersburg, Md.) substrate for approximately 5 min, quenched with
1.0 M H3PO4, and read spectrophotometrically at 450 nm. The ratio
of IC50 values (IC50,ala/IC50,wt) represents the fold of reduction
in binding affinity (the relative binding affinity).
(iii) VEGF Receptor Molecules
[0124] Two VEGF receptors have been identified, Flt-1 (also called
VEGFR-1) and KDR (also called VEGFR-2). Shibuya et al. (1990)
Oncogene 8:519-527; de Vries et al. (1992) Science 255:989-991;
Terman et al. (1992) Biochem. Biophys. Res. Commun. 187:1579-1586.
The specificity of each receptor for each VEGF family member varies
but VEGF-A binds to both Flt-1 and KDR. Neuropilin-1 has been shown
to be a selective VEGF receptor, able to bind the heparin-binding
VEGF isoforms (Soker et al. (1998) Cell 92:735-45). Both Flt-I and
KDR belong to the family of receptor tyrosine kinases (RTKs). The
RTKs comprise a large family of transmembrane receptors with
diverse biological activities. At present, at least nineteen (19)
distinct RTK subfamilies have been identified. The receptor
tyrosine kinase (RTK) family includes receptors that are crucial
for the growth and differentiation of a variety of cell types
(Yarden and Ullrich (1988) Ann. Rev. Biochem. 57:433-478; Ullrich
and Schlessinger (1990) Cell 61:243-254). The intrinsic function of
RTKs is activated upon ligand binding, which results in
phosphorylation of the receptor and multiple cellular substrates,
and subsequently in 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 a specific growth factor (ligand), typically
followed by receptor dimerization, stimulation of the intrinsic
protein tyrosine kinase activity and receptor
trans-phosphorylation. Binding sites are thereby created for
intracellular signal transduction molecules and lead to the
formation of complexes with a spectrum of cytoplasmic signaling
molecules that facilitate the appropriate cellular response. (e.g.,
cell division, differentiation, metabolic effects, changes in the
extracellular microenvironment) see, Schlessinger and Ullrich
(1992) Neuron 9:1-20. Structurally, both Flt-1 and KDR have seven
immunoglobulin-like domains in the extracellular domain, a single
transmembrane region, and a consensus tyrosine kinase sequence
which is interrupted by a kinase-insert domain. Matthews et al.
(1991) Proc. Natl. Acad. Sci. USA 88:9026-9030; Terman et al.
(1991) Oncogene 6:1677-1683.
[0125] VEGF receptor molecules, or fragments thereof, that
specifically bind to VEGF can be used in the methods of the
invention to bind to and sequester the VEGF protein, thereby
preventing it from signaling. In certain embodiments, the VEGF
receptor molecule, or VEGF binding fragment thereof, is a soluble
form, such as sFlt-1. A 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 natural
receptors present on the surface of target cells. Also included are
VEGF receptor fusion proteins, examples of which are described
below.
[0126] A chimeric VEGF receptor protein is a receptor molecule
having amino acid sequences derived from at least two different
proteins, at least one of which is a VEGF receptor protein (e.g.,
the flt-1 or KDR receptor), that is 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 can be linked to amino acid sequences from other unrelated
proteins, for example, immunoglobulin sequences. Other amino acid
sequences to which Ig-like domains are combined will be readily
apparent to those of ordinary skill in the art. Examples of
chimeric VEGF receptor proteins include, e.g., soluble Flt-1/Fc,
KDR/Fc, or FLt-1/KDR/Fc (also known as VEGF Trap). (See for example
PCT Application Publication No. WO97/44453)
[0127] A soluble VEGF receptor protein or chimeric VEGF receptor
proteins of the invention includes VEGF receptor proteins which are
not fixed to the surface of cells via a transmembrane domain. As
such, soluble forms of the VEGF receptor, including chimeric
receptor proteins, while capable of binding to and inactivating
VEGF, do not comprise a transmembrane domain and thus generally do
not become associated with the cell membrane of cells in which the
molecule is expressed.
III. Therapeutic Uses of Anti-VEGF Antibodies
[0128] The invention encompasses antiangiogenic therapy, a novel
cancer treatment strategy aimed at inhibiting the development of
tumor blood vessels required for providing nutrients to support
tumor growth. Because angiogenesis is involved in both primary
tumor growth and metastasis, the antiangiogenic treatment provided
by the invention is capable of inhibiting the neoplastic growth of
tumor at the primary site as well as preventing metastasis of
tumors at the secondary sites, therefore allowing attack of the
tumors by other therapeutics. In addition, ovarian cancer is
associated with a high level of circulating vascular endothelial
growth factor (VEGF), a protein associated with tumor growth and
spread. Studies of women with ovarian cancer have shown a
correlation between a high level of VEGF and a poorer prognosis
(Alvarez A et al. 1999 Clin Cancer Res.; 5:587-591; Yamamoto S et
al. 1997 Br J Cancer; 76:1221-1227).
[0129] Specifically, 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 combining at least
chemotherapy concurrent with the 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 (sub optimally and macroscopic optimally debulked) or
stage IV epithelial ovarian primary peritoneal or fallopian tube
cancer. In other embodiments, the patient has stage I and IIa
(Grade 3 or clear cell carcinoma only) or stage IIb-IV epithelial
ovarian, fallopian tube or primary peritoneal cancer. In another
embodiment, the invention provides a method of treating a patient
diagnosed with recurrent or previously treated ovarian cancer,
comprising subjecting the patient to a treatment regimen combining
at least chemotherapy concurrent with the administration of an
effective amount of an anti-VEGF antibody followed by anti-VEGF
maintenance therapy.
Combination Therapies
[0130] The invention features the use of a combination of at least
one VEGF-specific antagonist with one or more additional
anti-cancer therapies followed by anti-VEGF maintenance therapy.
Examples of anti-cancer therapies include, without limitation,
surgery, radiation therapy (radiotherapy), biotherapy,
immunotherapy, chemotherapy, or a combination of these therapies.
In addition, cytotoxic agents, anti-angiogenic and
anti-proliferative agents can be used in combination with the
VEGF-specific antagonist.
[0131] In certain aspects, the invention provides a method of
treating ovarian cancer, by administering effective amounts 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 combined treatment
methods of the invention. An exemplary and non-limiting list of
chemotherapeutic agents contemplated is provided herein under
"Definition", or described herein.
[0132] In one example, the invention features the use of a
VEGF-specific antagonist with one or more chemotherapeutic agents
(e.g., a cocktail) or any combination thereof. In certain
embodiments, the chemotherapeutic agent is for example, taxane,
paclitaxel, docetaxel, paclitaxel protein-bound particles (e.g.,
Abraxane.RTM.), platinum analogs, carboplatin, gemcitabine, or
combinations thereof therapy. In one embodiment, the
chemotherapeutic agents are carboplatin and pacilataxel or
docetaxel. In another embodiment, the chemotherapeutic agents are
carboplatin and gemcitabine. The combined administration includes
simultaneous administration, using separate formulations or a
single pharmaceutical formulation, and consecutive administration
in either order, wherein preferably there is a time period while
both (or all) active agents simultaneously exert their biological
activities followed by maintenance therapy with a VEGF specific
antagonist, e.g., as outlined in FIG. 1, FIG. 2, or FIG. 8 or FIG.
11. Preparation and dosing schedules for such chemotherapeutic
agents may be used according to manufacturers' instructions or as
determined empirically by the skilled practitioner. Preparation and
dosing schedules for chemotherapy are also described in
Chemotherapy Service Ed., M. C. Perry, Williams & Wilkins,
Baltimore, Md. (1992). The chemotherapeutic agent may precede, or
follow administration of the VEGF-specific antagonist or may be
given simultaneously therewith. In certain embodiments of the
invention, the dosing schedules and amounts are as set forth in
FIG. 1, FIG. 2 or FIG. 8 or FIG. 11.
[0133] In some other aspects, other therapeutic agents useful for
combination tumor therapy with the antibody of the invention
include antagonist of other factors that are 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 a one embodiment, the VEGF antibody is
co-administered with a growth inhibitory agent. For example, the
growth inhibitory agent may be administered first, followed by the
VEGF antibody. However, simultaneous administration or
administration of the VEGF antibody first is also contemplated.
Suitable dosages for the growth inhibitory agent are those
presently used and may be lowered due to the combined action
(synergy) of the growth inhibitory agent and anti-VEGF
antibody.
[0134] The formulation herein may also contain more than one active
compound as necessary for the particular indication being treated,
preferably those with complementary activities that do not
adversely affect each other. For example, it may be desirable to
further provide antibodies which bind to EGFR, VEGF (e.g. an
antibody which binds a different epitope on VEGF), VEGFR, ErbB2
(e.g., Herceptin.RTM.) or another antibody used in oncology
indications in the one formulation. Alternatively, or in addition,
the composition may comprise a cytotoxic agent, cytokine, growth
inhibitory agent and/or small molecule VEGFR antagonist. Such
molecules are suitably present in combination in amounts that are
effective for the purpose intended. In certain embodiments, VEGF
antagonist (e.g., anti-VEGF antibody) is the treatment for ovarian
cancer. In certain embodiments, VEGF antagonist (e.g., anti-VEGF
antibody) is combined with carboplatin and paclitaxel followed by
anti-VEGF maintenance therapy. In certain embodiments, VEGF
antagonist (e.g., anti-VEGF antibody) is combined with cisplatin
and paclitaxel followed by anti-VEGF maintenance therapy. In
certain embodiments, VEGF antagonist (e.g., anti-VEGF antibody) is
combined with carboplatin and docetaxel followed by anti-VEGF
maintenance therapy. In certain embodiments, VEGF antagonist (e.g.,
anti-VEGF antibody) is combined with carboplatin and gemcitabine
followed by anti-VEGF maintenance therapy.
[0135] In certain aspects, other therapeutic agents useful for
combination cancer therapy with the antibody of the invention
include other anti-angiogenic agents. Many anti-angiogenic agents
have been identified and are known in the arts, 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 a VEGF receptor antagonist such as VEGF
variants, soluble VEGF receptor fragments, aptamers capable of
blocking VEGF or VEGFR, neutralizing anti-VEGFR antibodies, low
molecule weight inhibitors of VEGFR tyrosine kinases and any
combinations thereof. Alternatively, or in addition, two or more
anti-VEGF antibodies may be co-administered to the patient.
[0136] For the prevention or treatment of disease, the appropriate
dosage of VEGF-specific antagonist will depend on the type of
disease to be treated, as defined above, the severity and course of
the disease, whether the VEGF-specific antagonist is administered
for preventive or therapeutic purposes, previous therapy, the
patient's clinical history and response to the VEGF-specific
antagonist, and the discretion of the attending physician. The
VEGF-specific antagonist is suitably administered to the patient at
one time or over a series of treatments. In a combination therapy
regimen, the VEGF-specific antagonist and the one or more
anti-cancer therapeutic agent of the invention are administered in
a therapeutically effective or synergistic amount. As used herein,
a therapeutically effective amount is such that co-administration
of a VEGF-specific antagonist and one or more other therapeutic
agents, or administration of a composition of the invention,
results in reduction or inhibition of the cancer as described
above. A therapeutically synergistic amount is that amount of a
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
increase progression free survival.
[0137] The VEGF-specific antagonist and the one or more other
therapeutic agents can be administered simultaneously or
sequentially in an amount and for a time sufficient to reduce or
eliminate the occurrence or recurrence of a tumor, a dormant tumor,
or a micrometastases. The VEGF-specific antagonist can be
administered as maintenance therapy to prevent or reduce the
likelihood of recurrence of the tumor or increase progression free
survival of the patient.
[0138] As will be understood by those of ordinary skill in the art,
the appropriate doses of chemotherapeutic agents or other
anti-cancer agents will be generally around those already employed
in clinical therapies, e.g., where the chemotherapeutics are
administered alone or in combination with other chemotherapeutics.
Variation in dosage will likely occur depending on the condition
being treated. The physician administering treatment will be able
to determine the appropriate dose for the individual subject.
[0139] In addition to the above therapeutic regimes, the patient
may be subjected to radiation therapy.
[0140] In certain embodiments, the administered VEGF antibody is an
intact, naked antibody. However, the VEGF antibody may be
conjugated with a cytotoxic agent. In certain embodiments, the
conjugated antibody and/or antigen to which it is bound is/are
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, calicheamicins, ribonucleases and DNA
endonucleases.
[0141] The invention also features a method of instructing a human
subject with ovarian cancer by providing instructions to receive
treatment with an anti-VEGF antibody so as to increase the time for
progression free survival, to decrease the subject's risk of cancer
recurrence or to increase the subject's likelihood of survival. In
some embodiments the method further comprises providing
instructions to receive treatment with at least one
chemotherapeutic agent followed by anti-VEGF maintenance therapy.
In some embodiments the method further comprises providing
instructions to receive treatment with two or more chemotherapeutic
agents followed by anti-VEGF maintenance therapy. The treatment
with the anti-VEGF antibody may be concurrent with the treatment
with the chemotherapeutic agent(s). In certain embodiments the
subject is treated as instructed by the method of instructing.
Treatment of ovarian cancer by administration of an anti-VEGF
antibody with or without chemotherapy may be continued until cancer
recurrence or death. In certain embodiments of the invention, the
patient is treated with at least 16 cycles of anti-VEGF therapy
after concurrent therapy with chemotherapeutic agent(s). In other
embodiments of the invention, the patient is treated with at least
12 cycles of anti-VEGF therapy after concurrent therapy with
chemotherapeutic agent(s).
[0142] The invention further provides a promotional method,
comprising promoting the administration of an anti-VEGF antibody
for treatment of ovarian cancer in a human subject. In some
embodiments the method further comprises promoting the
administration of at least one chemotherapeutic agent followed by
anti-VEGF maintenance therapy. In some embodiments the method
further comprises promoting the administration of two or more
chemotherapeutic agent followed by anti-VEGF maintenance therapy.
Administration of the anti-VEGF antibody may be concurrent with
administration of the chemotherapeutic agent(s). Promotion may be
conducted by any means available. In some embodiments the promotion
is by a package insert accompanying a commercial formulation of the
anti-VEGF antibody. The promotion may also be by a package insert
accompanying a commercial formulation of the chemotherapeutic
agent(s). Promotion may be by written or oral communication to a
physician or health care provider. In some embodiments the
promotion is by a package insert where the package inset provides
instructions to receive ovarian cancer therapy with anti-VEGF
antibody. In a further embodiment, the package insert include some
or all of the results under Example 1 or Example 2 or Example 3. In
some embodiments the promotion is followed by the treatment of the
subject with the anti-VEGF antibody with or without the
chemotherapeutic agent(s).
[0143] The invention provides a business method, comprising
marketing an anti-VEGF antibody for treatment of ovarian cancer in
a human subject so as to increase the subject's time for
progression free survival, to decrease the subject's likelihood of
cancer recurrence or increase the subject's likelihood of survival.
In some embodiments the method further comprises marketing a
chemotherapeutic agent for use in combination with the anti-VEGF
antibody followed by anti-VEGF maintenance therapy. In some
embodiments the marketing is followed by treatment of the subject
with the anti-VEGF antibody with or without the chemotherapeutic
agent followed by anti-VEGF maintenance therapy. In some
embodiments the method further comprises marketing two or more
chemotherapeutic agents for use in combination with the anti-VEGF
antibody followed by anti-VEGF maintenance therapy. In some
embodiments the marketing is followed by treatment of the subject
with the anti-VEGF antibody with or without the chemotherapeutic
agents followed by anti-VEGF maintenance therapy.
[0144] Also provided is a business method, comprising marketing a
chemotherapeutic agent in combination with an anti-VEGF antibody
for treatment of ovarian cancer in a human subject so as to
increase the subject's time for progression free survival, to
decrease the subject's likelihood of cancer recurrence or increase
the subject's likelihood of survival. In some embodiments the
marketing is followed by treatment of the subject with the
combination of the chemotherapeutic agent and the anti-VEGF
antibody followed by anti-VEGF maintenance therapy. Also provided
is a business method, comprising marketing two or more
chemotherapeutic agents in combination with an anti-VEGF antibody
followed by anti-VEGF maintenance therapy for treatment of ovarian
cancer in a human subject so as to increase the subject's time for
progression free survival, to decrease the subject's likelihood of
cancer recurrence or increase the subject's likelihood of survival.
In some embodiments the marketing is followed by treatment of the
subject with the combination of the chemotherapeutic agents and the
anti-VEGF antibody followed by anti-VEGF maintenance therapy.
IV Dosages, and Duration
[0145] The VEGF-specific antagonist composition will be formulated,
dosed, and administered in a fashion consistent with good medical
practice. Factors for consideration in this context include the
particular disorder being treated, the particular subject being
treated, the clinical condition of the individual patient, the
cause of the disorder, the site of delivery of the agent, the
method of administration, the scheduling of administration, and
other factors known to medical practitioners. The "therapeutically
effective amount" of the VEGF-specific antagonist to be
administered will be governed by such considerations, and is the
minimum amount necessary to prevent, ameliorate, or treat, or
stabilize, the cancer; to increase the time until progression
(duration of progression free survival) or to treat or prevent the
occurrence or recurrence of a tumor, a dormant tumor, or a
micrometastases. The VEGF-specific antagonist need not be, but is
optionally, formulated with one or more agents currently used to
prevent or treat cancer or a risk of developing a 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 with administration routes
as used hereinbefore or about from 1 to 99% of the heretofore
employed dosages.
[0146] Depending on the type and severity of the disease, about 1
.mu.g/kg to 100 mg/kg (e.g., 0.1-20 mg/kg) of VEGF-specific
antagonist is an initial candidate dosage for administration to the
patient, whether, for example, by one or more separate
administrations, or by continuous infusion. A typical daily dosage
might range from about 1 .mu.g/kg to about 100 mg/kg or more,
depending on the factors mentioned above. Particularly desirable
dosages include, for example, 5 mg/kg, 7.5 mg/kg, 10 mg/kg, and 15
mg/kg. For repeated administrations over several days or longer,
depending on the condition, the treatment is sustained 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 once every week, every
two weeks, or every three weeks, at a dose range from about 5 mg/kg
to about 15 mg/kg, including but not limited to 5 mg/kg, 7.5 mg/kg,
10 mg/kg or 15 mg/kg. The progress of the therapy of the invention
is easily monitored by conventional techniques and assays. In other
embodiments, such dosing regimen is used in combination with a
chemotherapy regimen (including but not limited to one or more
chemotherapeutic agent(s)) as the first line therapy for treating
previously untreated ovarian cancer followed by maintenance
therapy. In other embodiments, such dosing regimen is used in
combination with a chemotherapy regimen (including but not limited
to one or more chemotherapeutic agent(s)) as the second line
therapy for treating recurrent ovarian cancer followed by
maintenance therapy. Further information about suitable dosages is
provided in the Examples below.
[0147] The duration of therapy will continue for as long as
medically indicated or until a desired therapeutic effect (e.g.,
those described herein) is achieved. In certain embodiments, the
VEGF-specific antagonist therapy is continued for 1 month, 2
months, 4 months, 6 months, 8 months, 10 months, 1 year, 2 years, 3
years, 4 years, 5 years, or for a period of years up to the
lifetime of the subject. In certain embodiments, the anti-VEGF
therapy is continued for at least 16 cycles after the concurrent
anti-VEGF treatment with chemotherapeutic agents. In other
embodiments, the anti-VEGF therapy is continued for at least 12
cycles after the concurrent anti-VEGF treatment with
chemotherapeutic agents.
[0148] The VEGF-specific antagonists of the invention are
administered to a subject, e.g., a human patient, in accord with
known methods, such as intravenous administration as a bolus or by
continuous infusion over a period of time, by intramuscular,
intraperitoneal, intracerobrospinal, subcutaneous, intra-articular,
intrasynovial, intrathecal, oral, topical, or inhalation routes.
Local administration is particularly desired if extensive side
effects or toxicity is associated with VEGF antagonism. An ex vivo
strategy can also be used for therapeutic applications. Ex vivo
strategies involve transfecting or transducing cells obtained from
the 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 range of types including, without
limitation, 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.
[0149] 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 desired for local immunosuppressive treatment,
intralesional administration. Parenteral infusions include
intramuscular, intravenous, intraarterial, intraperitoneal, or
subcutaneous administration. In addition, the antibody is suitably
administered by pulse infusion, particularly with declining doses
of the antibody. Preferably the dosing is given by injections, most
preferably intravenous or subcutaneous injections, depending in
part on whether the administration is brief or chronic.
[0150] In another example, the VEGF-specific antagonist compound is
administered locally, e.g., by direct injections, when the disorder
or location of the tumor permits, and the injections can be
repeated periodically. The VEGF-specific antagonist can also be
delivered systemically to the subject or directly to the tumor
cells, e.g., to a tumor or a tumor bed following surgical excision
of the tumor, in order to prevent or reduce local recurrence or
metastasis, for example of a dormant tumor or micrometastases.
[0151] Alternatively, an inhibitory nucleic acid molecule or
polynucleotide containing a nucleic acid sequence encoding a
VEGF-specific antagonist can be delivered to the appropriate cells
in the subject. In certain embodiments, the nucleic acid can be
directed to the tumor itself.
[0152] The nucleic acid can be introduced into the cells by any
means appropriate 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 methods of gene delivery include liposome
mediated transfection, electroporation, calcium phosphate/DEAE
dextran methods, gene gun, and microinjection.
V. Pharmaceutical Formulations
[0153] Therapeutic formulations of the antibodies used in
accordance with the invention are prepared for storage by mixing an
antibody having the desired degree of purity with optional
pharmaceutically acceptable carriers, excipients or stabilizers
(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed.
(1980)), in the form of lyophilized formulations or aqueous
solutions. 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 octadecyldimethylbenzyl ammonium chloride;
hexamethonium chloride; benzalkonium chloride, benzethonium
chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as
methyl or propyl paraben; catechol; resorcinol; cyclohexanol;
3-pentanol; and m-cresol); low molecular weight (less than about 10
residues) 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 counter-ions such as
sodium; metal complexes (e.g. Zn-protein complexes); and/or
non-ionic surfactants such as TWEEN.TM., PLURONICS.TM. or
polyethylene glycol (PEG). Preferred lyophilized anti-VEGF antibody
formulations are described in WO 97/04801, expressly incorporated
herein be reference.
[0154] Optionally, the formulation contains a pharmaceutically
acceptable salt, typically, e.g., sodium chloride, and preferably
at about physiological concentrations. Optionally, the formulations
of the invention can contain a pharmaceutically acceptable
preservative. In some embodiments the preservative concentration
ranges from 0.1 to 2.0%, typically v/v. Suitable preservatives
include those known in the pharmaceutical arts. Benzyl alcohol,
phenol, m-cresol, methylparaben, and propylparaben are examples of
preservatives. Optionally, the formulations of the invention can
include a pharmaceutically acceptable surfactant at a concentration
of 0.005 to 0.02%.
[0155] Typically, bevacizumab is supplied for therapeutic uses in
100 mg and 400 mg preservative-free, single-use vials to deliver 4
ml or 16 ml of bevacizumab (25 mg/ml). The 100 mg product is
formulated in 240 mg .alpha.,.alpha.-trehalose dehydrate, 23.2 mg
sodium phosphate (monobasic, monohydrate), 4.8 mg sodium phosphate
(dibasic, anhydrous), 1.6 mg polysorbate 20, and Water for
Injection, USP. The 400 mg product is formulated in 960 mg
.alpha.,.alpha.-trehalose dehydrate, 92.8 mg sodium phosphate
(monobasic, monohydrate), 19.2 mg sodium phosphate (dibasic,
anhydrous), 6.4 mg polysorbate 20, and Water for Injection, USP.
See also the label for bevacizumab.
[0156] The formulation herein may also contain more than one active
compound as necessary for the particular indication being treated,
preferably those with complementary activities that do not
adversely affect each other. For example, it may be desirable to
further provide antibodies which bind to EGFR, VEGF (e.g. an
antibody which binds a different epitope on VEGF), VEGFR, or ErbB2
(e.g., Herceptin.RTM.) in the one formulation. Alternatively, or in
addition, the composition may comprise a cytotoxic agent, cytokine,
growth inhibitory agent and/or small molecule VEGFR antagonist.
Such molecules are suitably present in combination in amounts that
are effective for the purpose intended.
[0157] The active ingredients may also be entrapped in
microcapsules prepared, for example, by coacervation techniques or
by interfacial polymerization, for example, hydroxymethylcellulose
or gelatin-microcapsules and poly-(methylmethacylate)
microcapsules, respectively, in colloidal drug delivery systems
(for example, liposomes, albumin microspheres, microemulsions,
nano-particles and nanocapsules) or in macroemulsions. Such
techniques are disclosed in Remington's Pharmaceutical Sciences
16th edition, Osol, A. Ed. (1980).
[0158] Sustained-release preparations may 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
microcapsule. Examples of sustained-release matrices include
polyesters, hydrogels (for example,
poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),
polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic
acid and y ethyl-L-glutamate, non-degradable ethylene-vinyl
acetate, degradable lactic acid-glycolic acid copolymers such as
the 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 enable release
of molecules for over 100 days, certain hydrogels release proteins
for shorter time periods. When encapsulated antibodies remain in
the body for a long time, they may denature or aggregate as a
result of exposure to moisture at 37.degree. C., resulting in a
loss of biological activity and possible changes in immunogenicity.
Rational strategies can be devised for stabilization depending on
the mechanism involved. For example, if the aggregation mechanism
is discovered to be intermolecular S--S bond formation through
thio-disulfide interchange, stabilization may be achieved by
modifying sulfhydryl residues, lyophilizing from acidic solutions,
controlling moisture content, using appropriate additives, and
developing specific polymer matrix compositions.
[0159] The formulations to be used for in vivo administration
should be sterile. This is readily accomplished by filtration
through sterile filtration membranes.
VI Efficacy of the Treatment
[0160] The main advantage of the treatment of the invention is the
ability of producing marked anti-cancer effects in a human patient
without causing significant toxicities or adverse effects, so that
the patient benefited from the treatment overall. The efficacy of
the treatment of the invention can be measured by various endpoints
commonly used in evaluating cancer treatments, including but not
limited to, tumor regression, tumor weight or size shrinkage, 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 invention target the
tumor vasculature and not necessarily the neoplastic cells
themselves, they represent a unique class of anticancer drugs, and
therefore unique measures and definitions of clinical responses to
drugs may be employed. For example, tumor shrinkage of greater than
50% in a 2-dimensional analysis is the standard cut-off for
declaring a response. However, the anti-VEGF antibody of the
invention may cause inhibition of metastatic spread without
shrinkage of the primary tumor, or may simply exert a tumouristatic
effect. Accordingly, optionally other approaches to determining
efficacy of an anti-angiogenic therapy are employed, including for
example, measurement of plasma or urinary markers of angiogenesis
and measurement of response through radiological imaging.
[0161] In another embodiment, the invention provides methods for
increasing progression free survival of a human patient susceptible
to or diagnosed with a cancer. Time to disease progression is
defined as the time from administration of the drug until disease
progression or death. In a preferred embodiment, the combination
treatment of the invention using anti-VEGF antibody and one or more
chemotherapeutic agents followed by anti-VEGF maintenance therapy
significantly increases progression free survival by at least about
1 month, 2 months, 2.3 months, 2.9 months, 3.0 months, 3.8 months,
preferably by about 1 to about 6.1 months, when compared to a
treatment without anti-VEGF antibody maintenance therapy. In one
embodiment, the PFS median in months (95% CI) is increased 3.8
months (0.717 (0.625, 0.824) with one-sided p-value (log rank) of
<0.001)) in the patients treated with bevacizumab and taxane
therapy (e.g., docetaxel or paclitaxel) and carboplatin followed by
anti-VEGF maintenance therapy compared to control. In another
embodiment, the difference in median PFS in months (95% CI) between
patients receiving paclitaxel and carboplatin alone versus
paclitaxel, carboplatin and anti-VEGF antibody followed by
anti-VEGF maintenance therapy is 2.3 months with HR=0.79 and
p-value (Log-Rank Test) of 0.0010.
VII Antibody Production
(i) Polyclonal Antibodies
[0162] Polyclonal antibodies are preferably raised in animals by
multiple subcutaneous (sc) or intraperitoneal (ip) injections of
the relevant antigen and an adjuvant. It may be useful to conjugate
the relevant antigen to a protein that is immunogenic in the
species to be immunized, e.g., keyhole limpet hemocyanin, serum
albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a
bifunctional or derivatizing agent, for example, maleimidobenzoyl
sulfosuccinimide ester (conjugation through cysteine residues),
N-hydroxysuccinimide (through lysine residues), glutaraldehyde,
succinic anhydride, SOCl.sub.2, or R.sup.1N.dbd.C.dbd.NR, where R
and R.sup.1 are different alkyl groups.
[0163] Animals are immunized against the antigen, immunogenic
conjugates, or derivatives by combining, e.g., 100 .mu.g or 5 .mu.g
of the protein or conjugate (for rabbits or mice, respectively)
with 3 volumes of Freund's complete adjuvant and injecting the
solution intradermally at multiple sites. One month later the
animals are boosted with 1/5 to 1/10 the original amount of peptide
or conjugate in Freund's complete adjuvant by subcutaneous
injection at multiple sites. Seven to 14 days later the animals are
bled and the serum is assayed for antibody titer. Animals are
boosted until the titer plateaus. Preferably, the animal is boosted
with the conjugate of the same antigen, but conjugated to a
different protein and/or through a different cross-linking reagent.
Conjugates also can be made in recombinant cell culture as protein
fusions. Also, aggregating agents such as alum are suitably used to
enhance the immune response.
(ii) Monoclonal Antibodies
[0164] Various methods for making monoclonal antibodies herein are
available in the art. For example, the monoclonal antibodies may be
made using the hybridoma method first described by Kohler et al.,
Nature, 256:495 (1975), or by recombinant DNA methods (U.S. Pat.
No. 4,816,567).
[0165] In the hybridoma method, a mouse or other appropriate host
animal, such as a hamster or macaque monkey, is immunized as
hereinabove described 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. Lymphocytes then are fused with myeloma cells
using a suitable fusing agent, such as polyethylene glycol, to form
a hybridoma cell (Goding, Monoclonal Antibodies: Principles and
Practice, pp. 59-103 (Academic Press, 1986)).
[0166] The hybridoma cells thus prepared are seeded and grown in a
suitable culture medium that preferably contains one or more
substances that inhibit the growth or survival of the unfused,
parental myeloma cells. For example, if the parental myeloma cells
lack the enzyme hypoxanthine guanine phosphoribosyl transferase
(HGPRT or HPRT), the culture medium for the hybridomas typically
will include hypoxanthine, aminopterin, and thymidine (HAT medium),
which substances prevent the growth of HGPRT-deficient cells.
[0167] Preferred myeloma cells are those that fuse efficiently,
support stable high-level production of antibody by the selected
antibody-producing cells, and are sensitive to a medium such as HAT
medium. Among these, 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, Rockville, Md. USA. Human
myeloma and mouse-human heteromyeloma cell lines also have been
described for the production of 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)).
[0168] Culture medium in which hybridoma cells are growing is
assayed for production of monoclonal antibodies directed against
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 immunoabsorbent assay
(ELISA).
[0169] After hybridoma cells are identified that produce antibodies
of the desired specificity, affinity, and/or activity, the clones
may be subcloned by limiting dilution procedures and grown by
standard methods (Goding, Monoclonal Antibodies: Principles and
Practice, pp. 59-103 (Academic Press, 1986)). Suitable culture
media for this purpose include, for example, D-MEM or RPMI-1640
medium. In addition, the hybridoma cells may be grown in vivo as
ascites tumors in an animal.
[0170] The monoclonal antibodies secreted by the subclones are
suitably separated from the culture medium, ascites fluid, or serum
by conventional immunoglobulin purification procedures such as, for
example, protein A-Sepharose, hydroxylapatite chromatography, gel
electrophoresis, dialysis, or affinity chromatography.
[0171] DNA encoding the monoclonal antibodies is readily isolated
and sequenced using conventional procedures (e.g., by using
oligonucleotide probes that are capable of binding specifically to
genes encoding the heavy and light chains of the monoclonal
antibodies). The hybridoma cells serve as a preferred source of
such DNA. Once isolated, the DNA may be placed into expression
vectors, which are then transfected into host cells such as E. coli
cells, simian COS cells, Chinese hamster ovary (CHO) cells, or
myeloma cells that do not otherwise produce immunoglobulin protein,
to obtain the synthesis of monoclonal antibodies in the recombinant
host cells. Recombinant production of antibodies will be described
in more detail below.
[0172] In a further embodiment, antibodies or antibody fragments
can be isolated from antibody phage libraries generated using the
techniques described in McCafferty et al., Nature, 348:552-554
(1990). Clackson et al., Nature, 352:624-628 (1991) and Marks et
al., J. Mol. Biol., 222:581-597 (1991) describe the isolation of
murine and human antibodies, respectively, using phage libraries.
Subsequent publications describe the production of high affinity
(nM range) human antibodies by chain shuffling (Marks et al.,
Bio/Technology, 10:779-783 (1992)), as well as combinatorial
infection and in vivo recombination as a strategy for constructing
very large phage libraries (Waterhouse et al., Nuc. Acids. Res.,
21:2265-2266 (1993)). Thus, these techniques are viable
alternatives to traditional monoclonal antibody hybridoma
techniques for isolation of monoclonal antibodies.
[0173] The DNA also may be modified, for example, by substituting
the coding sequence for human heavy- and light-chain constant
domains in place of the homologous murine sequences (U.S. Pat. No.
4,816,567; Morrison, et al., Proc. Natl Acad. Sci. USA, 81:6851
(1984)), or by covalently joining to the immunoglobulin coding
sequence all or part of the coding sequence for a
non-immunoglobulin polypeptide.
[0174] 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-combining site
of an antibody to create a chimeric bivalent antibody comprising
one antigen-combining site having specificity for an antigen and
another antigen-combining site having specificity for a different
antigen.
(iii) Humanized and Human Antibodies
[0175] A humanized antibody has one or more amino acid residues
introduced into it from a source which is non-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 essentially performed 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)), by substituting rodent CDRs or
CDR sequences for the corresponding sequences of a human antibody.
Accordingly, such "humanized" antibodies are chimeric antibodies
(U.S. Pat. No. 4,816,567) wherein substantially less than an intact
human variable domain has been substituted by 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 substituted by residues
from analogous sites in rodent antibodies.
[0176] The choice of human variable domains, both light and heavy,
to be used in making the humanized antibodies is very important to
reduce antigenicity. According to the so-called "best-fit" method,
the sequence of the variable domain of a rodent antibody is
screened against the entire library of known human variable-domain
sequences. The human sequence which is closest to that of the
rodent is then accepted 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 method uses
a particular framework derived from the consensus sequence of all
human antibodies of a particular subgroup of light or heavy chains.
The same framework may be used for several different humanized
antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285
(1992); Presta et al., J. Immnol., 151:2623 (1993)).
[0177] It is further important that antibodies be humanized with
retention of high affinity for the antigen and other favorable
biological properties. To achieve this goal, according to a
preferred method, humanized antibodies are prepared by a process of
analysis of the parental sequences and various conceptual humanized
products using three-dimensional models of the parental and
humanized sequences. Three-dimensional immunoglobulin models are
commonly available and are familiar to those skilled in the art.
Computer programs are available which illustrate and display
probable three-dimensional conformational structures of selected
candidate immunoglobulin sequences. Inspection of these displays
permits analysis of the likely role of the residues in the
functioning of the candidate immunoglobulin sequence, i.e., the
analysis of residues that influence the ability of the candidate
immunoglobulin to bind its antigen. In this way, FR residues can be
selected and combined from the recipient and import sequences so
that the desired antibody characteristic, such as increased
affinity for the target antigen(s), is achieved. In general, the
CDR residues are directly and most substantially involved in
influencing antigen binding.
[0178] Humanized anti-VEGF antibodies and affinity matured variants
thereof are described in, for example, U.S. Pat. No. 6,884,879
issued Feb. 26, 2005.
[0179] It is now possible to produce transgenic animals (e.g.,
mice) that are capable, upon immunization, of producing a full
repertoire of human antibodies in the absence of endogenous
immunoglobulin production. For example, it has been described that
the homozygous deletion of the antibody heavy-chain joining region
(JO gene in chimeric and germ-line mutant mice results in complete
inhibition of endogenous antibody production. Transfer of the human
germ-line immunoglobulin gene array in such germ-line 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., Year in Immuno., 7:33 (1993); and Duchosal et
al. Nature 355:258 (1992).
[0180] Alternatively, phage display technology (McCafferty et al.,
Nature 348:552-553 (1990)) can be used to produce human antibodies
and antibody fragments in vitro, from immunoglobulin variable (V)
domain gene repertoires from unimmunized donors. According to this
technique, antibody V domain genes are cloned in-frame into either
a major or minor coat protein gene of a filamentous bacteriophage,
such as M13 or fd, and displayed as functional antibody fragments
on the surface of the phage particle. Because the filamentous
particle contains a single-stranded DNA copy of the phage genome,
selections based on the functional properties of the antibody also
result in selection of the gene encoding the antibody exhibiting
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 their 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 can be used for phage display. Clackson
et al., Nature, 352:624-628 (1991) isolated a diverse array of
anti-oxazolone antibodies from a small random combinatorial library
of V genes derived from the spleens of immunized mice. A repertoire
of V genes from unimmunized human donors can be constructed and
antibodies to a diverse array of antigens (including self-antigens)
can be isolated 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.
[0181] As discussed above, human antibodies may also be generated
by in vitro activated B cells (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, issued Mar. 24, 1998.
(iv) Antibody Fragments
[0182] Various techniques have been developed for the production of
antibody fragments. Traditionally, these fragments were derived via
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, these
fragments can now be produced directly by recombinant host cells.
For example, the antibody fragments can be isolated from the
antibody phage libraries discussed above. Alternatively, Fab'-SH
fragments can be directly recovered from E. coli and chemically
coupled to form F(ab').sub.2 fragments (Carter et al.,
Bio/Technology 10:163-167 (1992)). According to another approach,
F(ab').sub.2 fragments can be isolated directly from recombinant
host cell culture. Other techniques for the production of 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.
(v) Other Amino Acid Sequence Modifications
[0183] Amino acid sequence modification(s) of the antibodies
described herein are contemplated. For example, it may be desirable
to improve the binding affinity and/or other biological properties
of the antibody. Amino acid sequence variants of the antibody 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 sequences of the
antibody. Any combination of deletion, insertion, and substitution
is made to arrive at the final construct, provided that the final
construct possesses the desired characteristics. The amino acid
changes also may alter post-translational processes of the
antibody, such as changing the number or position of glycosylation
sites.
[0184] A useful method for identification of certain residues or
regions of the antibody that are preferred locations for
mutagenesis is called "alanine scanning mutagenesis" as described
by Cunningham and Wells Science, 244:1081-1085 (1989). Here, a
residue or group of target residues are identified (e.g., charged
residues such as arg, asp, his, lys, and glu) and replaced by a
neutral or negatively charged amino acid (most preferably alanine
or polyalanine) to affect the interaction of the amino acids with
antigen. Those amino acid locations demonstrating functional
sensitivity to the substitutions then are refined by introducing
further or other variants at, or for, the sites of substitution.
Thus, while the site for introducing an amino acid sequence
variation is predetermined, the nature of the mutation per se need
not be predetermined. For example, to analyze the performance of a
mutation at a given site, ala scanning or random mutagenesis is
conducted at the target codon or region and the expressed antibody
variants are screened for the desired activity.
[0185] Amino acid sequence insertions include amino- and/or
carboxyl-terminal fusions ranging in length from one residue to
polypeptides containing a hundred or more residues, as well as
intrasequence insertions of single or multiple amino acid residues.
Examples of terminal insertions include antibody with an N-terminal
methionyl residue or the antibody fused to a cytotoxic polypeptide.
Other insertional variants of the antibody molecule include the
fusion to the N- or C-terminus of the antibody to an enzyme (e.g.
for ADEPT) or a polypeptide which increases the serum half-life of
the antibody.
[0186] Another type of variant is an amino acid substitution
variant. These variants have at least one amino acid residue in the
antibody molecule replaced by a different residue. The sites of
greatest interest for substitutional mutagenesis include the
hypervariable regions, but FR alterations are also
contemplated.
[0187] Substantial modifications in the biological properties of
the antibody are accomplished by selecting substitutions that
differ significantly in their effect on maintaining (a) the
structure of the polypeptide backbone in the area of the
substitution, for example, as a sheet or helical conformation, (b)
the charge or hydrophobicity of the molecule at the target site, or
(c) the bulk of the side chain. Amino acids may be grouped
according to similarities in the properties of their side chains
(in A. L. Lehninger, in Biochemistry, second ed., pp. 73-75, Worth
Publishers, New York (1975)):
[0188] (1) non-polar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P),
Phe (F), Trp (W), Met (M)
[0189] (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr
(Y), Asn (N), Gln (Q)
[0190] (3) acidic: Asp (D), Glu (E)
[0191] (4) basic: Lys (K), Arg (R), His(H)
[0192] Alternatively, naturally occurring residues may be divided
into groups based on common side-chain properties:
[0193] (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;
[0194] (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;
[0195] (3) acidic: Asp, Glu;
[0196] (4) basic: His, Lys, Arg;
[0197] (5) residues that influence chain orientation: Gly, Pro;
[0198] (6) aromatic: Trp, Tyr, Phe.
[0199] Non-conservative substitutions will entail exchanging a
member of one of these classes for another class.
[0200] Any cysteine residue not involved in maintaining the proper
conformation of the antibody also may be substituted, generally
with serine, to improve the oxidative stability of the molecule and
prevent aberrant crosslinking. Conversely, cysteine bond(s) may be
added to the antibody to improve its stability (particularly where
the antibody is an antibody fragment such as an Fv fragment).
[0201] A particularly preferred type of substitutional variant
involves substituting one or more hypervariable region residues of
a parent antibody (e.g. a humanized or human antibody). Generally,
the resulting variant(s) selected for further development will have
improved biological properties relative to the parent antibody from
which they are generated. A convenient way for generating such
substitutional variants involves affinity maturation using phage
display. Briefly, several hypervariable region sites (e.g. 6-7
sites) are mutated to generate all possible amino substitutions at
each site. The antibody variants thus generated are displayed in a
monovalent fashion from filamentous phage particles as fusions to
the gene III product of M13 packaged within each particle. The
phage-displayed variants are then screened for their biological
activity (e.g. binding affinity) as herein disclosed. In order to
identify candidate hypervariable region sites for modification,
alanine scanning mutagenesis can be performed to identify
hypervariable region residues contributing significantly to antigen
binding. Alternatively, or additionally, it may be beneficial to
analyze a crystal structure of the antigen-antibody complex to
identify contact points between the antibody and human VEGF. Such
contact residues and neighboring residues are candidates for
substitution according to the techniques elaborated herein. Once
such variants are generated, the panel of variants is subjected to
screening as described herein and antibodies with superior
properties in one or more relevant assays may be selected for
further development.
[0202] Another type of amino acid variant of the antibody alters
the original glycosylation pattern of the antibody. By altering is
meant deleting one or more carbohydrate moieties found in the
antibody, and/or adding one or more glycosylation sites that are
not present in the antibody.
[0203] 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 the recognition sequences for
enzymatic attachment of the carbohydrate moiety to the asparagine
side chain. Thus, the presence of either of these tripeptide
sequences in a polypeptide creates a potential glycosylation site.
O-linked glycosylation refers to the attachment of one of the
sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino
acid, most commonly serine or threonine, although 5-hydroxyproline
or 5-hydroxylysine may also be used.
[0204] Addition of glycosylation sites to the antibody is
conveniently accomplished by altering the amino acid sequence such
that it contains one or more of the above-described tripeptide
sequences (for N-linked glycosylation sites). The alteration may
also be made by the addition of, or substitution by, one or more
serine or threonine residues to the sequence of the original
antibody (for O-linked glycosylation sites).
[0205] Where the antibody comprises an Fc region, the carbohydrate
attached thereto may be altered. For example, antibodies with a
mature carbohydrate structure that lacks fucose attached to an Fc
region of the antibody are described in US Pat Appl No US
2003/0157108 A1, Presta, L. See also US 2004/0093621 A1 (Kyowa
Hakko Kogyo Co., Ltd). Antibodies with a bisecting
N-acetylglucosamine (GlcNAc) in the carbohydrate attached to an Fc
region of the antibody are referenced in WO03/011878, Jean-Mairet
et al. and U.S. Pat. No. 6,602,684, Umana et al. Antibodies with at
least one galactose residue in the oligosaccharide attached to an
Fc region of the antibody are reported in WO97/30087, Patel et al.
See, also, WO98/58964 (Raju, S.) and WO99/22764 (Raju, S.)
concerning antibodies with altered carbohydrate attached to the Fc
region thereof.
[0206] It may be desirable to modify the antibody of the invention
with respect to effector function, e.g. so as to enhance
antigen-dependent cell-mediated cyotoxicity (ADCC) and/or
complement dependent cytotoxicity (CDC) of the antibody. This may
be achieved by introducing one or more amino acid substitutions in
an Fc region of the antibody. Alternatively or additionally,
cysteine residue(s) may be introduced in the Fc region, thereby
allowing interchain disulfide bond formation in this region. The
homodimeric antibody thus generated may have improved
internalization capability and/or increased complement-mediated
cell killing and antibody-dependent cellular cytotoxicity (ADCC).
See Caron et al., J. Exp Med. 176:1191-1195 (1992) and Shopes, B.
J. Immunol. 148:2918-2922 (1992). Homodimeric antibodies with
enhanced anti-tumor activity may also be prepared using
heterobifunctional cross-linkers as described in Wolff et al.
Cancer Research 53:2560-2565 (1993). Alternatively, an antibody can
be engineered which has dual Fc regions and may thereby have
enhanced complement lysis and ADCC capabilities. See Stevenson et
al. Anti-Cancer Drug Design 3:219-230 (1989).
[0207] WO00/42072 (Presta, L.) describes antibodies with improved
ADCC function in the presence of human effector cells, where the
antibodies comprise amino acid substitutions in the Fc region
thereof. Preferably, the antibody with improved ADCC comprises
substitutions at positions 298, 333, and/or 334 of the Fc region
(Eu numbering of residues). Preferably the altered Fc region is a
human IgG1 Fc region comprising or consisting of substitutions at
one, two or three of these positions. Such substitutions are
optionally combined with substitution(s) which increase C1q binding
and/or CDC.
[0208] Antibodies with altered C1q binding and/or complement
dependent cytotoxicity (CDC) are described in WO99/51642, U.S. Pat.
No. 6,194,551B1, U.S. Pat. No. 6,242,195B1, U.S. Pat. No.
6,528,624B1 and U.S. Pat. No. 6,538,124 (Idusogie et al.). The
antibodies comprise an amino acid substitution at one or more of
amino acid positions 270, 322, 326, 327, 329, 313, 333 and/or 334
of the Fc region thereof (Eu numbering of residues).
[0209] To increase the serum half life of the antibody, one may
incorporate a salvage receptor binding epitope into the antibody
(especially an antibody fragment) as described in U.S. Pat. No.
5,739,277, for example. As used herein, the term "salvage receptor
binding epitope" refers to an epitope of the Fc region of an IgG
molecule (e.g., IgG.sub.1, IgG.sub.2, IgG.sub.3, or IgG.sub.4) that
is responsible for increasing the in vivo serum half-life of the
IgG molecule.
[0210] Antibodies with improved binding to the neonatal Fc receptor
(FcRn), and increased half-lives, are described in WO00/42072
(Presta, L.) and US2005/0014934A1 (Hinton et al.). These antibodies
comprise an Fc region with one or more substitutions therein which
improve 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 numbering of
residues). The preferred Fc region-comprising antibody variant with
improved FcRn binding comprises amino acid substitutions at one,
two or three of positions 307, 380 and 434 of the Fc region thereof
(Eu numbering of residues). In one embodiment, the antibody has
307/434 mutations.
[0211] Engineered antibodies with three or more (preferably four)
functional antigen binding sites are also contemplated (US Appln
No. US2002/0004587 A1, Miller et al.).
[0212] Nucleic acid molecules encoding amino acid sequence variants
of the antibody are 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 variant or a non-variant version
of the antibody.
(vi) Immunoconjugates
[0213] The invention also pertains to immunoconjugates comprising
the antibody described herein conjugated to a cytotoxic agent such
as a chemotherapeutic agent, toxin (e.g. an enzymatically active
toxin of bacterial, fungal, plant or animal origin, or fragments
thereof), or a radioactive isotope (i.e., a radioconjugate).
[0214] Chemotherapeutic agents useful in the generation of such
immunoconjugates have been described above. Enzymatically active
toxins and fragments thereof which can be used include diphtheria A
chain, nonbinding active fragments of diphtheria toxin, exotoxin A
chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain,
modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin
proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S),
Momordica charantia inhibitor, curcin, crotin, Sapaonaria
officinalis inhibitor, gelonin, mitogellin, restrictocin,
phenomycin, enomycin and the tricothecenes. A variety of
radionuclides are available for the production of radioconjugate
antibodies. Examples include .sup.212Bi, .sup.131I, .sup.131In,
.sup.90Y and .sup.186Re.
[0215] Conjugates of the antibody and cytotoxic agent are made
using a variety of bifunctional protein coupling agents such as
N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP),
iminothiolane (IT), bifunctional derivatives of imidoesters (such
as dimethyl adipimidate HCL), active esters (such as disuccinimidyl
suberate), aldehydes (such as glutareldehyde), bis-azido compounds
(such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium
derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active
fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For
example, a ricin immunotoxin can 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 chelating agent for conjugation of
radionucleotide to the antibody. See WO94/11026.
[0216] In another embodiment, the antibody may be conjugated to a
"receptor" (such streptavidin) for utilization in tumor
pretargeting wherein the antibody-receptor conjugate is
administered to the patient, followed by removal of unbound
conjugate from the circulation using a clearing agent and then
administration of a "ligand" (e.g. avidin) which is conjugated to a
cytotoxic agent (e.g. a radionucleotide).
(vii) Immunoliposomes
[0217] The antibody disclosed herein may also be formulated as
immunoliposomes. Liposomes containing the antibody are prepared by
methods known in the art, such as described in 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. Pat. Nos. 4,485,045
and 4,544,545. Liposomes with enhanced circulation time are
disclosed in U.S. Pat. No. 5,013,556.
[0218] Particularly useful liposomes can be generated by the
reverse phase evaporation method with a lipid composition
comprising phosphatidylcholine, cholesterol and PEG-derivatized
phosphatidylethanolamine (PEG-PE). Liposomes are extruded through
filters of defined pore size to yield liposomes with the desired
diameter. Fab' fragments of the antibody of the invention can be
conjugated to the liposomes as described in Martin et al. J. Biol.
Chem. 257: 286-288 (1982) via a disulfide interchange reaction. A
chemotherapeutic agent (such as Doxorubicin) is optionally
contained within the liposome. See Gabizon et al. J. National
Cancer Inst. 81(19)1484 (1989)
VIII. Articles of Manufacture and Kits
[0219] In another embodiment of the invention, an article of
manufacture containing materials useful for the treatment of the
disorders described above is provided. The article of manufacture
comprises a container, a label and a package insert. Suitable
containers include, for example, bottles, vials, syringes, etc. The
containers may be formed from a variety of materials such as glass
or plastic. The container holds a composition which is effective
for treating the condition and may have a sterile access port (for
example the container may be an intravenous solution bag or a vial
having a stopper pierceable by a hypodermic injection needle). At
least one active agent in the composition is an anti-VEGF antibody.
The label on, or associated with, the container indicates that the
composition is used for treating the condition of choice. The
article of manufacture may further comprise a second container
comprising a pharmaceutically-acceptable buffer, such as
phosphate-buffered saline, Ringer's solution and dextrose solution.
It may further 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 inserts with instructions for use, including
for example instructing the user of the composition to administer
the anti-VEGF antibody composition and a chemotherapeutic agent to
the patient, e.g., taxane, paclitaxel, docetaxel, paclitaxel
protein-bound particles (e.g., Abraxane.RTM.), platinum analogue,
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.
[0220] The VEGF-specific antagonist can be packaged alone or in
combination with other anti-cancer therapeutic compounds as a kit.
The kit can include optional components that aid in the
administration of the unit dose to patients, such as vials for
reconstituting powder forms, syringes for injection, customized IV
delivery systems, inhalers, etc. Additionally, the unit dose kit
can contain instructions for preparation and administration of the
compositions. In certain embodiments, the instructions comprises
instructions for use, including for example instructing the user of
the composition to administer the anti-VEGF antibody composition
and a chemotherapeutic agent to the patient, e.g., taxane,
paclitaxel, docetaxel, paclitaxel protein-bound particles (e.g.,
Abraxane.RTM.), platinum analogue, carboplatinm, gemcitabine, or
combinations thereof 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. The kit may be
manufactured as a single use unit dose for one patient, multiple
uses for a particular patient (at a constant dose or in which the
individual compounds may vary in potency as therapy progresses); or
the kit may contain multiple doses suitable for administration to
multiple patients ("bulk packaging"). The kit components may be
assembled in cartons, blister packs, bottles, tubes, and the
like.
Deposit of Materials
[0221] The following hybridoma cell line has been deposited under
the provisions of the Budapest Treaty with the American Type
Culture Collection (ATCC), Manassas, Va., USA:
TABLE-US-00007 Antibody Designation ATCC No. Deposit Date A4.6.1
ATCC HB-10709 Mar. 29, 1991
[0222] The following examples are intended merely to illustrate the
practice of the invention and are not provided by way of
limitation. The disclosures of all patent and scientific
literatures cited herein are expressly incorporated in their
entirety by reference.
EXAMPLES
Example 1. A Phase III Trial of Carboplatin and Paclitaxel Plus
Placebo Versus Carboplatin and Paclitaxel Plus Concurrent
Bevacizumab Followed by Placebo, Versus Carboplatin and Paclitaxel
Plus Concurrent and Extended Bevacizumab, in Women with Newly
Diagnosed, Previously Untreated, Stage III (Sub Optimally and
Macroscopic Optimally Debulked) or IV, Epithelial Ovarian, Primary
Peritoneal or Fallopian Tube Cancer
[0223] Results are presented from a phase III randomized study to
evaluate new treatment programs for patients with International
Federation of Gynecologic Oncology (FIGO) stages III and IV,
epithelial ovarian, peritoneal primary or fallopian tube cancer.
Primary Objectives include to determine if the addition of 5
concurrent cycles of bevacizumab to 6 cycles of standard therapy
(carboplatin and paclitaxel) [Arm II] increases the duration of
progression-free survival (PFS) when compared to 6 cycles of
standard therapy alone [Arm I] in women with newly diagnosed stage
III (with any gross residual disease) and stage IV, epithelial
ovarian, peritoneal primary or fallopian tube cancer; and, to
determine if the addition of 5 concurrent cycles of bevacizumab
plus extended bevacizumab for 16 cycles beyond the 6 cycles of
standard therapy (carboplatin and paclitaxel) [Arm III] increases
progression-free survival when compared to 6 cycles of standard
therapy [Arm I] in women with newly diagnosed stage III (with any
gross residual disease) and stage IV, epithelial ovarian,
peritoneal primary or fallopian tube cancer.
[0224] GOG-0182-ICON5 was a 5-arm randomized clinical trial
comparing standard therapy (carboplatin and paclitaxel) with four
investigational arms incorporating gemcitabine, topotecan and
liposomal doxorubicin, either in combination or in sequence with
paclitaxel and carboplatin. Major ovarian cancer clinical trials
groups throughout the world participated in this study. This
international collaboration provided a unique opportunity to accrue
large numbers of patients in a timely manner which facilitated the
simultaneous evaluation of multiple agents in a prospective
randomized trial. With international participation, accrual
exceeded 1,200 patients per year, and the trial reached its
targeted accrual goal within four years of activation.
[0225] While the results of GOG-0182-ICON5 helped establish optimum
chemotherapy for previously untreated patients with advanced
ovarian and peritoneal primary cancer, the next generation of
clinical trials will explore the impact of molecular targeted
therapies in conjunction with chemotherapy. In particular, growth
factor signal transduction inhibitors and anti-angiogenic agents as
single agents and in combination with chemotherapy drugs are
currently undergoing trials in women with these tumors. Many of
these agents have been shown to have cytostatic effects and have
shown synergy with chemotherapy in experimental models of human
cancer. In this phase III trial, the impact on outcome of active
biologic agents in combination with standard chemotherapeutic
therapy plus or minus extended single agent administration,
compared with standard chemotherapeutic therapy alone, in patients
with advanced disease was evaluated.
[0226] Bevacizumab is a recombinant humanized version of a murine
anti-human VEGF monoclonal antibody, named rhuMAb VEGF. Bevacizumab
has been advanced into clinical development for use as a single
agent to induce tumor growth inhibition in patients with solid
tumors and for use in combination with cytotoxic chemotherapy to
delay the time to disease progression in patients with metastatic
solid tumors. See, e.g., Presta L G, et al. Humanization of an
anti-vascular endothelial growth factor monoclonal antibody for the
therapy of solid tumors and other disorders. Cancer Res 57:4593-9,
1997. The results of two single agent trials of bevacizumab for
patients with recurrent epithelial ovarian and peritoneal primary
cancer have been published. See, e.g., Burger R A, et al., Phase II
trial of bevacizumab in persistent or recurrent epithelial ovarian
cancer or primary peritoneal cancer: a Gynecologic Oncology Group
study. J Clin Oncol 25(33):5165-5171, 2007; and, Cannistra S A, et
al., Phase II Study of Bevacizumab in Patients with Platinum
Resistant Ovarian Cancer or Primary Peritoneal Serous Cancer. J
Clin Oncol 25(33):5180-86, 2007. GOG (GOG-0170-D) utilized two
co-primary efficacy endpoints: clinical response by NCI RECIST
criteria and proportion surviving progression-free for at least 6
months. 62 participants received bevacizumab at 15 mg/kg every 21
days until clinical or radiographic evidence of disease progression
or development of unacceptable toxicity. The primary disease
characteristics were typical of patients with recurrent ovarian
cancer, and approximately 43% of patients were considered primarily
platinum resistant. A 21% response rate was observed, and 40% were
progression-free for at least 6 months, with a median PFS 4.7
months, compared with 1.8 months for a historical control based on
previous negative phase II trials of cytotoxic agents in
populations with similar clinical characteristics. Genentech AVF
2949 examined patients with a higher risk profile in terms of the
potential for disease progression and adverse events, allowing only
patients considered either primarily or secondarily platinum
resistant and having received 2 or 3 previous cytotoxic regimens.
These differences in eligibility ultimately translated into a
higher level of platinum resistance, a greater number of prior
regimens and a slightly worse performance status profile in the AVF
population. Forty four patients were treated at the same dose and
schedule for bevacizumab as used in GOG 170-D. Seven (16%)
responses were documented, and 12 (27%) were progression-free for
at least 6 months.
[0227] In this study two experimental arms were selected to compare
with standard cytotoxic chemotherapy with paclitaxel and
carboplatin: one incorporating 5 cycles of bevacizumab (concurrent
bevacizumab) and the other with bevacizumab for an additional 16
cycles after completion of chemotherapy with paclitaxel and
carboplatin (extended bevacizumab). Administration and doses are
indicated in FIGS. 1 and 2. Calvert Formula for Carboplatin (AUC)
Dosing:
Total dose (mg)=target AUC (in mg/mL/minute)*[GFR (in
mL/minute)+25].
[0228] The statistical design for the primary endpoint the study
was based on 90% power to detect PFS hazard ratio (HR).ltoreq.0.77
(median PFS shift: 14.0 months (historical).fwdarw.18.2 months. The
primary analysis compared investigator-assessed PFS for each
bevacizumab arm verses control ((analysis 1.fwdarw.by RECIST (see,
e.g, Therasse et al., J Natl. Cancer Inst., 92:205-16, 2000),
global clinical deterioration, or CA-125; or by analysis
2.fwdarw.RECIST or global clinical deterioration, censoring
CA-125). The baseline clinical characteristics of the patients are
found in Table 1. The baseline surgical-pathologic characteristics
of the patients are found in Table 2.
[0229] Eligible Patients: Patients with a histologic diagnosis of
epithelial ovarian cancer, peritoneal primary carcinoma or
fallopian tube cancer; FIGO stage III with any gross (macroscopic
or palpable) residual disease or FIGO stage IV, defined surgically
at the completion of initial abdominal surgery and with appropriate
tissue available for histologic evaluation. The minimum surgery
required was an abdominal surgery providing tissue for histologic
evaluation and establishing and documenting the primary site and
stage, as well as a maximal effort at tumor debulking. If
additional surgery was performed, it should have been in accordance
with appropriate surgery for ovarian or peritoneal carcinoma
described in the GOG Surgical Procedures Manual
(https://www.gog.fccc.edu/manuals/pdf/surgman.pdf). However, the
surgeon is not required to have performed all of the items
contained in this section of the GOG Surgical Procedures Manual.
Those patients with stage III cancer in which the largest maximal
diameter of any residual tumor implant at the completion of this
initial surgery is no greater than 1 cm will be defined as
"optimal;" all others will be defined as "suboptimal." Measurable
disease on post-operative imaging studies is not required for
eligibility.
[0230] Patients with the following histologic epithelial cell types
are eligible: Serous adenocarcinoma, endometrioid adenocarcinoma,
mucinous adenocarcinoma, undifferentiated carcinoma, clear cell
adenocarcinoma, mixed epithelial carcinoma, transitional cell
carcinoma, malignant Brenner's Tumor, or adenocarcinoma not
otherwise specified (N.O.S.). However, the histologic features of
the tumor must be compatible with a primary Mullerian epithelial
adenocarcinoma. Patients may have co-existing fallopian tube
carcinoma in-situ so long as the primary origin of invasive tumor
is ovarian, peritoneal or fallopian tube.
[0231] Patients must have adequate:
(1) Bone marrow function: Absolute neutrophil count (ANC) greater
than or equal to 1,500/.mu.l, equivalent to Common Toxicity
Criteria for Adverse Events v3.0 (CTCAE) Grade1. This ANC cannot
have been induced or supported by granulocyte colony stimulating
factors. (2) Platelets greater than or equal to 100,000/.mu.l.
(CTCAE Grade 0-1). (3) Renal function: Creatinine
.ltoreq.1.5.times. institutional upper limit normal (ULN), CTCAE
Grade 1. (4) Hepatic function: (a) Bilirubin less than or equal to
1.5.times.ULN (CTCAE Grade 1). (b) SGOT and alkaline phosphatase
less than or equal to 2.5.times.ULN (CTCAE Grade 1). (c) Neurologic
function: Neuropathy (sensory and motor) less than or equal to
CTCAE Grade 1. (5) Blood coagulation parameters: PT such that
international normalized ratio (INR) is .ltoreq.1.5 (or an in-range
INR, usually between 2 and 3, if a patient is on a stable dose of
therapeutic warfarin for management of venous thrombosis including
pulmonary thrombo-embolus) and a PTT .ltoreq.1.2 times the upper
limit of normal. (6) Patients with a GOG Performance Status of 0,
1, or 2. (7) Patients must be entered between 1 and 12 weeks after
initial surgery performed for the combined purpose of diagnosis,
staging and cytoreduction. (8) Patients with measurable and
non-measurable disease are eligible. Patients may or may not have
cancer-related symptoms. (9) Patients who have met the pre-entry
requirements specified in Section 7.0. (10) An approved informed
consent and authorization permitting release of personal health
information must be signed by the patient or guardian. (11)
Patients in this trial may receive ovarian estrogen +/- progestin
replacement therapy as indicated at the lowest effective dose(s)
for control of menopausal symptoms at any time, but not progestins
for management of anorexia while on protocol directed therapy or
prior to disease progression.
[0232] Ineligible Patients: Patients with a current diagnosis of
borderline epithelial ovarian tumor (formerly "tumors of low
malignant potential") or recurrent invasive epithelial ovarian,
primary peritoneal or fallopian tube cancer treated with surgery
only (such as patients with stage Ia or Ib low grade epithelial
ovarian or fallopian tube cancers) are not eligible. Patients with
a prior diagnosis of a borderline tumor that was surgically
resected and who subsequently develop an unrelated, new invasive
epithelial ovarian, peritoneal primary or fallopian tube cancer are
eligible, provided that they have not received prior chemotherapy
for any ovarian tumor.
[0233] Patients who have received prior radiotherapy to any portion
of the abdominal cavity or pelvis are excluded. Prior radiation for
localized cancer of the breast, head and neck, or skin is
permitted, provided that it was completed more than three years
prior to registration, and the patient remains free of recurrent or
metastatic disease.
[0234] Patients who have received prior chemotherapy for any
abdominal or pelvic tumor including neo-adjuvant chemotherapy for
their ovarian, primary peritoneal or fallopian tube cancer are
excluded. Patients may have received prior adjuvant chemotherapy
for localized breast cancer, provided that it was completed more
than three years prior to registration, and that the patient
remains free of recurrent or metastatic disease.
[0235] Patients who have received any targeted therapy (including
but not limited to vaccines, antibodies, tyrosine kinase
inhibitors) or hormonal therapy for management of their epithelial
ovarian or peritoneal primary cancer.
[0236] Patients with synchronous primary endometrial cancer, or a
past history of primary endometrial cancer, are excluded, unless
all of the following conditions are met: Stage not greater than
I-B; no more than superficial myometrial invasion, without vascular
or lymphatic invasion; no poorly differentiated subtypes, including
papillary serous, clear cell or other FIGO Grade 3 lesions.
[0237] With the exception of non-melanoma skin cancer and other
specific malignancies as noted above, patients with other invasive
malignancies who had (or have) any evidence of the other cancer
present within the last five years or whose previous cancer
treatment contraindicates this protocol therapy are excluded.
[0238] Patients with acute hepatitis or active infection that
requires parenteral antibiotics.
[0239] Patients with serious non-healing wound, ulcer, or bone
fracture. This includes history of abdominal fistula,
gastrointestinal perforation or intra-abdominal abscess within 28
days. Patients with granulating incisions healing by secondary
intention with no evidence of fascial dehiscence or infection are
eligible but require weekly wound examinations.
[0240] Patients with active bleeding or pathologic conditions that
carry high risk of bleeding, such as known bleeding disorder,
coagulopathy, or tumor involving major vessels.
[0241] Patients with history or evidence upon physical examination
of CNS disease, including primary brain tumor, seizures not
controlled with standard medical therapy, any brain metastases, or
history of cerebrovascular accident (CVA, stroke), transient
ischemic attack (TIA) or subarachnoid hemorrhage within six months
of the first date of treatment on this study.
[0242] Patients with clinically significant cardiovascular disease.
This includes: Uncontrolled hypertension, defined as systolic
>150 mm Hg or diastolic >90 mm Hg.; Myocardial infarction or
unstable angina <6 months prior to registration; New York Heart
Association (NYHA) Grade II or greater congestive heart failure;
Serious cardiac arrhythmia requiring medication. This does not
include asymptomatic, atrial fibrillation with controlled
ventricular rate; CTCAE Grade 2 or greater peripheral vascular
disease (at least brief (<24 hrs) episodes of ischemia managed
non-surgically and without permanent deficit); History of CVA
within six months.
[0243] Patients with known hypersensitivity to Chinese hamster
ovary cell products or other recombinant human or humanized
antibodies.
[0244] Patients with clinically significant proteinuria. Urine
protein should be screened by urine protein-creatinine ratio
(UPCR). The UPCR has been found to correlate directly with the
amount of protein excreted in a 24 hour urine collection. See,
e.g., Ginsberg J M, et al., Use of single voided urine samples to
estimate quantitative proteinuria. N Engl J Med 309:1543-6, 1983;
Rodby R A, et al., The urine protein to creatinine ratio as a
predictor of 24-hour urine protein excretion in type 1 diabetic
patients with nephropathy. The Collaborative Study Group. Am J
Kidney Dis 26:904-9, 1995; Schwab S J, et al., Quantitation of
proteinuria by the use of protein-to-creatinine ratios in single
urine samples. Arch Intern Med 147:943-4, 1987; Steinhauslin F,
& Wauters J P. Quantitation of proteinuria in kidney transplant
patients: accuracy of the urinary protein/creatinine ratio. Clin
Nephrol 43:110-5, 1995; Wilson D M, & Anderson R L.
Protein-osmolality ratio for the quantitative assessment of
proteinuria from a random urinalysis sample. Am J Clin Pathol
100:419-24, 1993; and, Zelmanovitz T, et al., Proteinuria is still
useful for the screening and diagnosis of overt diabetic
nephropathy. Diabetes Care 21:1076-9, 1998. Specifically, a UPCR of
1.0 is equivalent to 1.0 gram of protein in a 24 hour urine
collection. Patients must have a UPCR <1.0 to allow
participation in the study.
[0245] Patients with or with anticipation of invasive procedures as
defined below: Major surgical procedure, open biopsy or significant
traumatic injury within 28 days prior to the first date of
bevacizumab/placebo therapy (cycle 2). Major surgical procedure
anticipated 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,
interval or secondary cytoreductive surgery, or second look
surgery. Core biopsy, within 7 days prior to the first date of
bevacizumab/placebo therapy (cycle 2).
[0246] Patients with GOG Performance Grade of 3 or 4.
[0247] Patients who are pregnant or nursing.
[0248] Patients under the age of 18.
[0249] Patients who have received prior therapy with any anti-VEGF
drug, including bevacizumab.
[0250] Patients with clinical symptoms or signs of gastrointestinal
obstruction and who require parenteral hydration and/or
nutrition.
[0251] Patients with other medical history or condition that in the
opinion of the doctor, would preclude study participation.
[0252] Response and progression will be evaluated in this study
using the international criteria proposed by the Response
Evaluation Criteria in Solid Tumors (RECIST) Committee. See, e.g.,
Therasse P, et al. New guidelines to evaluate the response to
treatment in solid tumors. European Organization for Research and
Treatment of Cancer, National Cancer Institute of the United
States, National Cancer Institute of Canada. J Natl Cancer Inst
92:205-16, 2000. Changes in only the largest diameter
(unidimensional measurement) of the tumor lesions are used in the
RECIST criteria.
[0253] CA-125 as a Biologic Marker of Progressive Disease: Serum
levels of CA-125, a tumor-associated glycoprotein antigen, are
elevated in 80% of patients with epithelial ovarian cancer. See,
e.g., Bast et al., N. Engl. J. Med. 309:88307, 1983. CA-125 has
been monitored, often on a frequent basis, to verify response to
therapy, presence of residual disease, and as early evidence of
recurrence. However, CA-125, is not entirely tumor specific, and
can be elevated in a variety of benign conditions, such as
endometriosis, uterine fibroids, and pelvic inflammation; this is
particularly true in pre-menopausal women. In addition, levels of
CA-125 can be discordant with tumor response, both as
false-positive and false-negative trends; the influence of biologic
agents on these inaccuracies is unclear. Nonetheless, it has been
standard practice for patients and physicians interpret a
progressive rise in CA-125 post-therapy as evidence of recurrent or
progressive disease, and will make therapeutic decisions based on
CA-125. The current randomized trial will employ a conservative
formula to define progressive disease based on serial elevation of
CA-125, (in addition to other standard definitions in the
management of solid tumors), but only following 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, progress based upon serum
CA-125 can be determined only during the period following
completion of cytotoxic chemotherapy, if one of the three
conditions are met: 1) patients with elevated CA-125 pretreatment
and normalization of CA-125 must show evidence of CA-125 greater
than or equal to two times the upper normal limited on two
occasions at least one week apart; or 2) patients with elevated
CA-125 pretreatment, which never normalizes must show evidence of
CA-125 greater than or equal to two times the nadir value on two
occasions at least one week apart; or 3) patients with CA-125 in
the normal range pretreatment must show evidence of CA-125 greater
than or equal to two times the upper normal limit on two occasions
at least one week apart.
[0254] Results
[0255] The results of the study demonstrate that bevacizumab is
effective for first line ovarian cancer when combined with
chemotherapy and continued as maintenance therapy. This combination
was effective at increasing PFS. Preliminary assessment of safety
identified bevacizumab related adverse events (AEs) noted in
previous studies. The primary analysis of PFS demonstrated a
progression-free survival (months) median of 10.3 (in arm one of
FIG. 2) compared to 14.1 months in arm three of FIG. 2. The HR (95%
CI) was 0.908 (0.795, 1.04) with a one-sided p-value (log-rank of
0.08) in arm I of FIG. 2 compared to 0.717 (0.625, 0.824) with
one-sided p-value (log-rank) of <0.001 in arm III of FIG. 2. See
FIG. 5. The difference was statistically significant. The treatment
regimen was generally well tolerated and adverse events (including
GI perforation) were similar to previous bevacizumab studies. See
FIG. 3 and FIG. 4. This is the first anti-angiogenic therapy to
demonstrate benefit in this population. FIG. 6 illustrates the
ramification of using CA-125 as a determinant of progression.
CA-125 is an antigenic determinant on a high-molecular weight
glycoprotein recognized by a monoclonal antibody (OC-125), which is
produced using an ovarian cancer cell line as an immunogen. CA 125
has been evaluated as a serum marker for monitoring patients with
epithelial ovarian carcinoma and other cancers. See, e.g.,
references Gyn Oncol 38:373, 1990; Gyn Oncol 38:181, 1990; Amer J
Ob Gyn 160:667, 1989; Amer J Ob Gyn 159:873, 1988; Amer J Ob Gyn
159:341, 1988; Ob Gyn 72:159, 1988; and, Gyn Oncol 36:299, 1990 and
descriptions herein. FIG. 7 illustrates subgroup analyses of Arm I
verses Arm III.
TABLE-US-00008 Median age, years (range) 60 (25-86) 60 (24-88) 60
(22-89) Race, n (%) Non-Hispanic white 526 (84) 519 (83) 521 (84)
Asian 41 (7) 37 (6) 39 (6) Non-Hispanic black 25 (4) 28 (5) 27 (4)
Hispanic 21 (3) 28 (5) 25 (4) Other, specified 8 (1) 5 (<1) 4
(<1) GOG PS, n (%) 0 311 (50) 315 (50) 305 (49) 1 272 (44) 270
(43) 267 (43) 2 42 (7) 40 (6) 51 (8)
TABLE-US-00009 TABLE 2 Baseline Surgical-Pathologic Characteristics
Arm I Arm II Arm III Characteristic, n CP CP + BEV CP + BEV
.fwdarw. BEV (%) (n = 625) (n = 625) (n = 623) Stage/residual size
III optimal 218 (35) 205 (33) 216 (35) (macroscopic) III suboptimal
254 (41) 256 (41) 242 (39) IV 153 (25) 164 (26) 165 (27) Histology
Serous 543 (87) 523 (84) 525 (84) Endometrioid 20 (3) 15 (2) 25 (4)
Clear cell 11 (2) 23 (4) 18 (3) Mucinous 8 (1) 5 (<1) 8 (1)
Tumor grade 3a 412 (66) 435 (70) 430 (69) 2 94 (15) 77 (12) 92 (15)
1 33 (5) 28 (4) 16 (3) Not 86 (14) 85 (14) 85 (14)
specified/pending
Example 2. A Randomized, Two-Arm, Multi-Center Gynaecologic Cancer
Intergroup Trial of Adding Bevacizumab to Standard Chemotherapy
(Carboplatin and Paclitaxel) in Patients with Epithelial Ovarian
Cancer
[0256] Results are presented from a phase III randomized study
(ICON7) to evaluate the safety and efficacy of adding bevacizumab
to standard chemotherapy with carboplatin and paclitaxel. The
primary endpoint was to determine whether the addition of
bevacizumab to standard chemotherapy improves progression free
survival (PFS) when compared to standard chemotherapy alone in
women with newly diagnosed, histologically confirmed, high risk
International Federation of Gynaecology and Obstetrics (FIGO) stage
I and IIa (Grade 3 or clear cell carcinoma only) and FIGO stage
IIb-IV (all grades and all histological types) epithelial ovarian,
fallopian tube or primary peritoneal cancer, who have undergone
initial surgery (either debulking cytoreductive surgery or a biopsy
if the patient has FIGO stage IV disease) and who would not be
considered for cytoreductive surgery prior to disease progression.
Secondary endpoints included overall survival (OS), response rate,
duration of response, biological progression free interval (defined
by increasing CA 125 or PFI.sub.BIO), safety and quality of life.
ICON7 was a 2-arm randomized clinical trial comparing standard
therapy (carboplatin and paclitaxel) with one investigational arm
incorporating bevacizumab in combination with paclitaxel and
carboplatin (see FIG. 8). A total of 1528 eligible women
participated in the trial.
[0257] Bevacizumab is a recombinant humanized version of a murine
anti-human VEGF monoclonal antibody, named rhuMAb VEGF. Bevacizumab
has been advanced into clinical development for use as a single
agent to induce tumor growth inhibition in patients with solid
tumors and for use in combination with cytotoxic chemotherapy to
delay the time to disease progression in patients with metastatic
solid tumors. See, e.g., Presta L G, et al. Humanization of an
anti-vascular endothelial growth factor monoclonal antibody for the
therapy of solid tumors and other disorders. Cancer Res 57:4593-9,
1997.
Patient Selection
[0258] ICON7 included patients with newly diagnosed, histologically
confirmed, high risk FIGO stage I and IIa (Grade 3 or clear cell
carcinoma only) and FIGO stage IIb-IV (all grades and all
histological types) epithelial ovarian, fallopian tube or primary
peritoneal cancer, who have undergone initial surgery (either
debulking cytoreductive surgery or a biopsy if the patient has FIGO
stage IV disease) and who will not be considered for cytoreductive
surgery prior to disease progression. Patients with measurable and
non-measurable disease are eligible. Patients were considered
eligible for enrollment in this trial if they fulfilled all the
inclusion criteria and none of the exclusion criteria as described
below:
Patient Inclusion Criteria:
[0259] Females aged .gtoreq.18 years [0260] Histologically
confirmed, with core biopsy from a disease site as minimum
requirement, (cytology alone was insufficient for diagnosis) [0261]
Epithelial Ovarian cancer [0262] Primary peritoneal carcinoma (must
be of the papillary-serous histological type) or [0263] Fallopian
tube carcinoma [0264] AND meeting the criteria in Table 3 Patients
with clear cell carcinoma of any stage were eligible due to the
poorer prognosis associated with this subtype. Patients with
previous early stage epithelial ovarian or fallopian tube carcinoma
treated with surgery alone were eligible at the time of diagnosis
of abdomino-pelvic recurrence as long as no further interval
cytoreductive therapy was planned prior to disease progression.
[0265] For the purposes of this trial, clear cell carcinoma was
defined as either .gtoreq.50% clear cell elements present or
reported as clear cell carcinoma by the local pathologist.
TABLE-US-00010 TABLE 3 Histological Eligibility Criteria FIGO
Eligible Stage Grade 1 Grade 2 Grade 3 Ia No .sup.E No .sup.E Yes
Ib No .sup.E No .sup.E Yes Ic No .sup.E No .sup.E Yes IIa No .sup.E
No .sup.E Yes IIb Yes Yes Yes IIc Yes Yes Yes III Yes Yes Yes IV
Yes Yes Yes Grade refers to 1 (well differentiated), 2 (moderately
differentiated) and 3 (poorly differentiated) E = Except patients
with clear cell carcinoma who are eligible regardless of FIGO
stage
[0266] Patients should have already undergone surgical debulking,
by a surgeon experienced in the management of ovarian cancer, with
the aim of maximal surgical cytoreduction according to the GCIG
Conference Consensus Statement. There must be no planned surgical
debulking prior to disease progression. [0267] Patients with stage
III and IV disease in whom initial surgical debulking was not
appropriate were still be eligible providing [0268] the patient had
a histological diagnosis and [0269] debulking surgery prior to
disease progression was not foreseen [0270] Patients should have
been able to commence systemic therapy within eight weeks of
cytoreductive surgery. If the patient was randomised to the
research arm then the first dose of bevacizumab must be omitted if
the investigator decides to start chemotherapy within 4 weeks of
surgery. [0271] If a patient had two operations, for example an
initial operation to remove what was thought to be a benign cyst
and then a second gynae-oncological operation to formally stage and
maximally debulk the ovarian tumour, then the second operation date
was documented as the date of surgery; the first systemic treatment
started within eight weeks of this date. The date of diagnosis was
recorded as the date of the initial operation where ovarian cancer
was diagnosed. [0272] ECOG performance status (PS) 0-2 [0273] Life
expectancy >12 weeks [0274] Adequate bone marrow function (all
parameters were checked/calculated on post-operative bloods)
(within 28 days prior to randomisation) [0275] Absolute Neutrophil
Count (ANC) 1.5.times.10.sup.9/l [0276] Platelets (PLT)
.gtoreq.100.times.10.sup.9/l [0277] Haemoglobin (Hb) .gtoreq.9 g/dl
(can be post-transfusion) [0278] Adequate coagulation parameters
(all parameters were checked/calculated on post-operative bloods)
(within 28 days prior to randomisation) [0279] Activated
ProThrombin Time (APTT) .ltoreq.1.5.times.ULN; or, [0280]
International Normalised Ratio (INR) .ltoreq.1.5 (measurement of
INR was mandatory if patient was receiving warfarin treatment)
[0281] Adequate liver function (all parameters were
checked/calculated on post-operative bloods) (within 28 days prior
to randomisation) [0282] Serum bilirubin (BR) 1.5.times.ULN [0283]
Serum transaminases 2.5.times.ULN [0284] Urine dipstick for
proteinuria <2+. If urine dipstick is .gtoreq.2+, 24 hour urine
must demonstrate .ltoreq.1 g of protein in 24 hours [0285] Adequate
renal function defined as a serum creatinine .ltoreq.2.0 mg/dl or
.ltoreq.177 .mu.mol/l
Patient Exclusion Criteria:
[0285] [0286] Non-epithelial ovarian cancer, including malignant
mixed Mullerian tumours [0287] Borderline tumours (tumours of low
malignant potential) [0288] Planned intraperitoneal cytotoxic
chemotherapy [0289] Prior systemic anti-cancer therapy for ovarian
cancer (for example chemotherapy, monoclonal antibody therapy,
tyrosine kinase inhibitor therapy or hormonal therapy) [0290]
Surgery (including open biopsy) within 4 weeks prior to anticipated
first dose of bevacizumab (allowing for the fact that bevacizumab
can be omitted from the first cycle of chemotherapy) [0291] Any
planned surgery during the 58 week period from the start of study
treatment (54 weeks of treatment plus 4 additional weeks to allow
for bevacizumab clearance) [0292] Uncontrolled hypertension (blood
pressure measurements were recorded in patients after 5 minutes of
rest, and in the sitting position) (Sustained elevation of BP
>150/100 mmHg despite anti-hypertensive therapy) [0293] Any
previous radiotherapy to the abdomen or pelvis [0294] Significant
traumatic injury during 4 weeks preceding the potential first dose
of bevacizumab [0295] History or clinical suspicion of brain
metastases or spinal cord compression. CT/MRI of the brain is
mandatory (within 4 weeks prior to randomisation) in case of
suspected brain metastases. Spinal MRI is mandatory (within 4 weeks
prior to randomisation) in case of suspected spinal cord
compression [0296] History or evidence upon neurological
examination of central nervous system (CNS) disease, unless
adequately treated with standard medical therapy e.g. uncontrolled
seizures [0297] Previous Cerebro-Vascular Accident (CVA), Transient
Ischaemic Attack (TIA) or Sub-Arachnoid Haemorrhage (SAH) within
six months prior to randomisation [0298] Fertile woman of
childbearing potential not willing to use adequate contraception
(oral contraceptives, intrauterine device or barrier method of
contraception in conjunction with spermicidal jelly or surgically
sterile) for the study duration and at least six months afterwards
[0299] Pregnant or lactating women [0300] Previous exposure to
mouse CA 125 antibody [0301] Treatment with any other
investigational agent, or participation in another clinical trial
within 30 days prior to entering this trial [0302] Malignancies
other than ovarian cancer within 5 years prior to randomisation,
except for adequately treated carcinoma in situ of the cervix
and/or basal cell skin cancer and/or early endometrial carcinoma as
specified below. Patients may have received previous adjuvant
chemotherapy for other malignancies e.g. breast or colorectal
carcinoma if diagnosed over 5 years ago with no evidence of
subsequent recurrence [0303] Patients with synchronous primary
endometrial carcinoma, or a past history of primary endometrial
carcinoma, were excluded unless ALL of the following criteria for
describing the endometrial carcinoma were met [0304] Stage
.ltoreq.Ib [0305] No more than superficial myometrial invasion
[0306] No lymphovascular invasion [0307] Not poorly differentiated
(i.e. not Grade 3 or papillary serous or clear cell) [0308] Known
hypersensitivity to bevacizumab and its excipients or chemotherapy
(including cremophor) [0309] Non healing wound, ulcer or bone
fracture. Patients with granulating incisions healing by secondary
intention with no evidence of facial dehiscence or infection were
eligible but required three weekly wound examinations [0310]
History or evidence of thrombotic or hemorrhagic disorders [0311]
Clinically significant cardiovascular disease, including [0312]
Myocardial infarction or unstable angina within 6 months of
randomisation [0313] New York Heart Association (NYHA) Grade 2
Congestive Heart Failure (CHF) [0314] Poorly controlled cardiac
arrhythmia despite medication (patients with rate-controlled atrial
fibrillation were eligible) [0315] Grade .gtoreq.3 peripheral
vascular disease (i.e. symptomatic and interfering with activities
of daily living [ADL] requiring repair or revision) [0316] Current
or recent (within 10 days prior to cycle 1 treatment) chronic use
of aspirin >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, therefore the use of
prophylactic low-dose aspirin in patients who are at risk of an
arterial thromboembolic event was not prohibited in this trial
protocol) [0317] Current or recent (within 10 days prior to cycle 1
treatment) use of full-dose oral or parenteral anticoagulants or
thrombolytic agent for therapeutic purposes (except for line
patency, in which case INR must be maintained below 1.5) [0318]
Pre-existing sensory or motor neuropathy .gtoreq.Grade 2 [0319]
Evidence of any other disease, metabolic dysfunction, physical
examination finding or laboratory finding giving reasonable
suspicion of a disease or condition that contra-indicates the use
of an investigational drug or puts the patient at high risk for
treatment-related complications
[0320] Tumor assessments, either by CT or MRI scan, with
measurements using the RECIST criteria, were performed after three
and six cycles of chemotherapy, and at around nine months and 12
months in the first year, or after cycle 12 and cycle 18 of
treatment for patients on the research arm. In the second and third
year of the trial tumour assessments were repeated every six months
and thereafter as clinically indicated. These scans were performed
irrespective of whether the patient has been optimally or
sub-optimally debulked, and irrespective of whether there is
measurable disease, or not, on the baseline scan.
[0321] Patients were clinically assessed and CA 125 measured at the
start of every chemotherapy cycle and then six weekly during the
first year of the trial. In the second and third year of the trial
patients were assessed and CA 125 measured every three months. In
the fourth and fifth year patients were clinically assessed and CA
125 measured every six months. Thereafter assessments were yearly.
Progression based on CA 125 criteria alone were verified with a CT
scan. If this was negative then it was repeated at the time of
suspected clinical progression.
[0322] After evidence of protocol defined disease progression,
patients were followed for survival and subsequent treatment for
ovarian cancer every six months during the first five years of
their follow up in the trial and yearly thereafter.
[0323] Regular physical examination and routine blood tests were
performed during treatment to monitor patient safety. Quality of
life (QoL) were assessed using the EORTC QLQ C-30+OV-28 and EQ-5D
questionnaires at the start of every chemotherapy cycle, every six
weeks until the end of the first year and then every three months
until treatment for progression commenced, or to the end of year
two. An additional QoL form was completed by all patients still
alive three years after randomisation. Adverse events and medical
resource use were documented during the study treatment and
follow-up period.
[0324] Results
[0325] The results of the study demonstrate that bevacizumab is
effective for first line ovarian cancer when combined with
chemotherapy and continued as maintenance therapy for a total
duration of 12 months. This combination was effective at increasing
progression-free survival (PFS). The primary analysis of PFS
demonstrated a PFS median of 16.0 months in the chemotherapy arm
(CP) compared to 18.3 months in the chemotherapy plus bevacizumab
arm (CPB7.5+) with a p-value (Log-Rank Test) of 0.0010. The hazard
ratio (HR) (95% CI) was 0.79 (0.68; 0.91). The difference was
significant. The PFS analysis is summarized in FIGS. 9 and 10.
[0326] The baseline characteristics were as follows:
TABLE-US-00011 TABLE 4 Baseline Characteristics-Demographics CP
CPB7.5 + (N = 764) (N = 764) Age in years: Mean (SD) 56.7 (10.6)
56.5 (10.4) Race: White (%) 737 ( 96%) 730 ( 96%) Performance
status (ECOG) 0 (%) 333 ( 44%) 307 ( 41%) 1 (%) 375 ( 49%) 391 (
52%) 2 (%) 54 ( 7%) 55 ( 7%)
TABLE-US-00012 TABLE 5 Baseline Characteristics-History of Ovarian
Cancer CP CPB7.5 + (N = 764) (N = 764) Origin of Cancer Ovary
(Epithelial) (%) 667 ( 87%) 673 ( 88%) Fallopian Tube (%) 29 ( 4%)
27 ( 4%) Primary Peritoneal (%) 56 ( 7%) 50 ( 7%) Multiple
Locations (%) 12 ( 2%) 14 ( 2%) FIGO staging I (%) 65 ( 8%) 54 (
7%) II (%) 80 ( 11%) 83 ( 11%) III (%) 522 ( 68%) 523 ( 68%) IV (%)
97 ( 13%) 104 ( 14%)
TABLE-US-00013 TABLE 6 Baseline Characteristics-History of Ovarian
Cancer CP CPB7.5 + (N = 764) (N = 764) Degree of Differentiation
Grade 1 (%) 56 ( 7%) 41 ( 5%) Grade 2 (%) 142 ( 19%) 175 ( 23%)
Grade 3 (%) 556 ( 74%) 538 ( 71%) Histological Subtype Serous (%)
529 ( 69%) 525 ( 69%) Mucinous (%) 15 ( 2%) 19 ( 2%) Endometroid
(%) 57 ( 7%) 60 ( 8%) Clear cell (%) 60 ( 8%) 67 ( 9%) Other (%) 55
( 7%) 53 ( 7%) Mixed (%) 48 ( 6%) 40 ( 5%)
TABLE-US-00014 TABLE 7 Baseline Characteristics-Surgery of Ovarian
Cancer CP CPB7.5 + (N = 764) (N = 764) Debulking surgery performed:
Yes (%) 747 ( 98%) 751 ( 98%) Debulking surgery outcome: Optimal
(%) 552 ( 74%) 559 ( 74%) Time between surgery and first trial
treatment 35.6 ( 10.2) 35.9 ( 9.9) [days]: Mean (SD)
[0327] Preliminary assessment of adverse events for bevacizumab
were consistent with previous studies.
TABLE-US-00015 TABLE 8 Overview of Adverse Events (AEs) CP CPB7.5 +
(N = 763) (N = 746) Pts w. Serious AE 154 ( 20.2%) 279 ( 37.4%) Pts
w. Grade 3/4/5 AE 385 ( 50.5%) 479 ( 64.2%) Pts who Disc. Any
Treatment 98 ( 12.8%) 293 ( 39.3%) Pts who Disc. Any Treatment due
to AE 68 ( 8.9%) 162 ( 21.7%) All Deaths 131 ( 17.2%) 107 ( 14.3%)
All Related Deaths 1 ( 0.1%) 5 ( 0.7%) Deaths not due to
Progression 16 ( 2.1%) 19 ( 2.5%)
Example 3. A Phase III, Multicenter, Randomized, Blinded,
Placebo-Controlled Trial of Carboplatin and Gemcitabine Plus
Bevacizumab in Patients with Platinum-Sensitive Recurrent Ovary,
Primary Peritoneal, or Fallopian Tube Carcinoma
[0328] Epithelial ovarian carcinoma (EOC) and its histological and
clinical equivalents, primary peritoneal carcinoma (PPC) and
fallopian tube carcinoma, occur at an incidence of approximately
25,000 cases per year in the United States and result in
approximately 14,000 deaths per annum. Because the disease tends to
be asymptomatic in early stages, the majority of patients will
present initially with advanced (Stage III or IV) disease. Despite
the sensitivity of EOC, PPC, and fallopian tube carcinoma to a
number of chemotherapeutic agents, particularly the taxanes and
platinum compounds, only 20%-30% of patients who present with Stage
III or IV disease will be alive at 5 years. Patients with
platinum-sensitive recurrent cancer (defined as recurrence of
disease more than 6 months from the completion of a platinum based
chemotherapy regimen) have higher initial response rates 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
relapsed platinum sensitive disease. Carboplatin and gemcitabine
resulted in a statistically significant progression-free survival
(PFS) compared with carboplatin alone in patients with platinum
sensitive disease. See, e.g., Pfisterer, Plante M, Vergote I, et
al. Gemcitabine plus Carboplatin compared with carboplatin in
patients with platinum-sensitive recurrent ovarian cancer: an
intergroup trial of the AGO-OVAR, the NCIC CTG, and the EORTC GCG.
J. Clin Oncol, 2006; 24:4699-707.
[0329] Angiogenesis appears to be an important factor in both the
development and subsequent progression of EOC. Yoneda and
colleagues (1998) demonstrated in a xenograft model of EOC that
tumor growth rates were directly proportional to vascular density
and that the development of malignant ascites, a feature associated
with poor outcome in EOC, was associated with the expression of
vascular endothelial growth factor (VEGF). See, e.g., Yoneda J,
Kuniyasu H, Crispens M A, et al. Expression of angiogenesis-related
genes and progression of human ovarian carcinomas in nude mice. J
Natl Cancer Inst. 1998 Mar. 18; 90:447-54. Other studies have
demonstrated the association of VEGF expression in EOC with
microvascular density. Moreover, studies have shown that the
expression density of CD31 (a marker of vascular endothelium) by
immunohistochemistry in EOC inversely correlates with survival.
[0330] This example describes a placebo-controlled, randomized,
multicenter Phase III study evaluating the efficacy and safety of
bevacizumab (15 mg/kg, Day 1, every 21 days), administered in
combination with carboplatin (area under the curve [AUC] 4, Day 1,
every 21 days) with gemcitabine (1000 mg/m.sup.2, Day 1 and Day 8,
every 21 days) in women with platinum sensitive recurrent
epithelial ovarian, primary peritoneal, or fallopian tube
carcinoma. About 480 patients were enrolled over a period of
approximately 2.5 years. Patients were randomized in either
carboplatin and gemcitabine with placebo versus carboplatin and
gemcitabine with bevacizumab. In addition, at randomization,
patients were stratified by platinum-sensitive disease (recurrence
6-12 months from last platinum based treatment versus recurrence
>12 months from last platinum-based treatment) and cytoreductive
surgery for recurrent epithelial ovarian, primary peritoneal, or
fallopian tube carcinomas (surgery was performed vs. was not
performed).
TABLE-US-00016 484 women randomized 1:1 to one of two treatment
arms: Phase B (until disease Phase A (cycles 1-6; progression =
three Patients cycle = three weeks) weeks) Arm 1 Chemotherapy +
placebo Placebo Arm 2 Avastin + chemotherapy Avastin
[0331] The study consisted of the two arms shown below. See also
FIG. 11.
[0332] Arm 1: Carboplatin (AUC 4 IV) and gemcitabine (1000
mg/m.sup.2) chemotherapies (6 cycles up to 20 cycles) followed by
placebo
[0333] Arm 2: Avastin (15 mg/kg for 6 cycles up p 10 cycles) in
combination with carboplatin and gemcitabine chemotherapies (6
cycles up to 10 cycles) followed by the continued use of Avastin
(15 mg/kg) alone until disease progression
[0334] The carboplatin dose was calculated to reach a target AUC of
concentration.times.time according to the Calvert formula with use
of an estimated glomerular filtration rate (GFR); e.g., for the
purposes here, the GFR is considered to be equivalent to the
creatinine clearance. Calvert Formula for Carboplatin (AUC)
Dosing
total dose (mg)=target AUC (in mg/mL/minute).times.[GFR (in
mL/minute) 25]
Creatinine clearance can be calculated according to institutional
guidelines.
Patient Selection
[0335] Patients with epithelial carcinoma of the ovary, PPC, or
fallopian tube carcinoma that has recurred >6 months since
platinum-based chemotherapy (first recurrence) will be eligible for
this study. Additional specific inclusion and exclusion criteria
are listed below.
Patient Inclusion Criteria:
[0336] Patients must meet the following criteria to be eligible for
study entry:
[0337] Signed Informed Consent Form
[0338] Age .gtoreq.18 years
[0339] Histologically documented ovarian, primary peritoneal, or
fallopian tube carcinoma that has recurred >6 months after
platinum based chemotherapy
[0340] The patient must have recurrent epithelial ovarian, primary
peritoneal, or fallopian tube carcinoma. This must be the first
recurrence of epithelial ovarian, primary peritoneal, or fallopian
tube carcinoma. [0341] Examples of eligible histological cell types
include: serous adenocarcinoma, endometrioid adenocarcinoma,
mucinous adenocarcinoma, undifferentiated carcinoma, clear cell
adenocarcinoma, transitional cell carcinoma, malignant Brenner's
Tumor, or adenocarcinoma not otherwise specified
[0342] No prior chemotherapy in the recurrent setting
[0343] Measurable disease according to modified RECIST with at
least one lesion that can be accurately measured in at least one
dimension (longest dimension recorded)
[0344] Each measurable lesion must be 20 mm when measured by
conventional techniques, CT and magnetic resonance imaging (MRI),
or 10 mm when measured by spiral CT.
[0345] Greater than 28 days from and recovered from prior radiation
therapy or surgery
[0346] ECOG performance status 0 or 1
[0347] Use of an effective means of contraception (for women of
childbearing potential)
[0348] Ability to comply with study and follow up procedures
Patient Exclusion Criteria
[0349] Patients who meet any of the following criteria will be
excluded from study entry. [0350] Disease-Specific Exclusions
[0351] Prior chemotherapy treatment for recurrent ovarian, primary
peritoneal, or fallopian tube carcinoma: Hormonal therapy (i.e.,
progesterones, estrogens, anti estrogens, aromatase inhibitors)
will not be considered a prior chemotherapy regimen. Concomitant
anti-neoplastic anti-hormonal therapy (including tamoxifen,
aromatase inhibitors, etc.) is not allowed for patients
participating in study treatment. Low-dose (physiologic) estrogen
hormone-replacement therapy (HRT) may be given. [0352] History of
abdominal fistula, gastrointestinal perforation, or intra abdominal
abscess [0353] Patients with clinical symptoms or signs of GI
obstruction or who require parenteral hydration, parenteral
nutrition, or tube feeding [0354] Patients with evidence of
abdominal free air not explained by paracentesis or recent surgical
procedure [0355] General Medical Exclusions [0356] Life expectancy
of <12 weeks [0357] Current, recent (within 4 weeks of Day 1,
Cycle 1), or planned participation in an experimental drug study
[0358] Screening clinical laboratory values [0359] Granulocyte
count <1500/.mu.L, [0360] Platelet count <100,000/.mu.L,
[0361] Hemoglobin <8.5 g/dL (hemoglobin may be supported by
transfusion or erythropoietin or other approved hematopoietic
growth factors) [0362] Serum bilirubin >2.0.times. upper limits
of normal (ULN) [0363] Alkaline phosphatase, aspartate transaminase
(AST), and/or alanine transaminase (ALT) >2.5.times.ULN (AST,
ALT >5.times.ULN for patients with liver metastasis) [0364]
Serum creatinine .gtoreq.1.6 [0365] International normalized ratio
(INR)>1.5 and/or activated partial thromboplastin time (aPTT)
>1.5.times.ULN (except for patients receiving anticoagulation
therapy) [0366] For patients on full-dose warfarin, in-range INR
(usually between 2 and 3) and a PTT <1.2 times the ULN [0367]
History of other malignancies within 5 years of Day 1, Cycle 1,
except for tumors with a negligible risk for metastasis or death,
such as adequately controlled basal-cell carcinoma or squamous cell
carcinoma of the skin or carcinoma in situ of the cervix [0368] Any
other diseases, metabolic dysfunction, physical examination
finding, or clinical laboratory finding giving reasonable suspicion
of a disease or condition that contraindicates the use of an
investigational drug or that may affect the interpretation of the
results or render the patient at high risk for treatment
complications [0369] Bevacizumab-Specific Exclusions [0370] History
of systemic bevacizumab (Avastin.RTM.) or other VEGF or VEGF
receptor targeted agent use [0371] Inadequately controlled
hypertension (defined as systolic blood pressure >150 mmHg
and/or diastolic blood pressure >100 mmHg on antihypertensive
medications) [0372] Prior history of hypertensive crisis or
hypertensive encephalopathy [0373] New York Heart Association Class
II or greater CHF [0374] History of myocardial infarction or
unstable angina within 6 months prior to Day 1, Cycle 1 (day of the
first bevacizumab/placebo infusion) [0375] History of stroke or TIA
within 6 months prior to study enrollment [0376] Known CNS disease
except for treated brain metastasis [0377] Treated brain metastases
are defined as having no evidence of progression or hemorrhage
after treatment and no ongoing requirement for dexamethasone, as
ascertained by clinical examination and brain imaging (MRI or CT)
during the screening period. These metastases must not be located
in the brainstem, midbrain, pons, medulla, or leptomeninges.
Treatment for brain metastases may include whole brain radiotherapy
(WBRT), radiosurgery (Gamma Knife, LINAC, or equivalent) or a
combination as deemed appropriate by the treating physician.
Patients with CNS metastases treated by neurosurgical resection or
brain biopsy performed within 3 months prior to Day 1 will be
excluded. [0378] History of significant vascular disease (e.g.,
aortic aneurysm, aortic dissection) [0379] Recent peripheral
arterial thrombosis within 6 months prior to Day 1, Cycle 1 [0380]
History of hemoptysis (.gtoreq.1/2 teaspoon of bright red blood per
episode) within 1 month prior to Day 1, Cycle 1 [0381] Evidence of
bleeding diathesis or significant coagulopathy (in the absence of
therapeutic anticoagulation) [0382] Major surgical procedure, open
biopsy, or significant traumatic injury within 28 days prior to Day
1, Cycle 1 or anticipation of need for major surgical procedure
during the course of the study [0383] Core biopsy or other minor
surgical procedure, excluding placement of a vascular access
device, within 7 days prior to Day 1, Cycle 1 [0384] Serious,
non-healing wound; active ulcer; or untreated bone fracture [0385]
Proteinuria at screening, as demonstrated by a UPCR of .gtoreq.1.0
at screening [0386] Known hypersensitivity to any component of
bevacizumab [0387] Pregnancy (positive pregnancy test) or lactation
[0388] Patients of childbearing potential must use an effective
means of contraception.
[0389] This study, OCEANS, enrolled a different patient population
from example 1 (GOG 0218) and example 2 (ICON7); women with
previously treated, platinum-sensitive ovarian cancer were eligible
for this trial. Women with ovarian cancer may have receive a
platinum-based chemotherapy as the first line of treatment. The
time between receiving the last dose of platinum-based chemotherapy
and disease coming back (recurrence) is used to help determine the
choice of chemotherapy used in the next line of treatment. Women
have "platinum-sensitive" ovarian cancer if the disease comes back
more than six months after completing initial platinum-based
chemotherapy. Ovarian cancer is considered "platinum-resistant" if
it comes back within six months of completing initial
platinum-based chemotherapy.
[0390] Results
[0391] This phase III study of bevacizumab plus chemotherapy in
women with ovarian cancer met its primary endpoint. The object of
the study was to evaluate the efficacy and safety of adding
bevacizumab in to standard chemotherapy followed by extended use of
bevacizumab alone until disease progression, compared to
chemotherapy alone, in previously treated women with ovarian
cancer. The study showed that bevacizumab plus chemotherapy,
followed by continued use of bevacizumab alone until disease
progression, increased the time women with previously treated
(recurrent), platinum-sensitive ovarian cancer lived without the
disease worsening (progression-free survival or PFS), compared to
chemotherapy alone. PFS is defined as the time from randomization
to disease progression as determined by the investigator or death
due to any cause, whichever occurs first. The primary endpoint of
PFS was assessed by the study investigators. Measurable disease was
investigator assessed using modified RECIST (Therasse et al. 2000),
e.g., every 9 weeks throughout the course of the study. See, e.g.,
Therasse P, Arbuck S G, Eisenhauser E A, et al. New guidelines to
evaluate the response to treatment in solid tumors. J Natl Cancer
Inst 2000; 92:205-1. Secondary endpoints included overall survival
(OS), response rate, duration of response and safety. No new safety
findings were observed and adverse events were consistent with
those observed in previous pivotal trials of bevacizumab.
Sequence CWU 1
1
21123PRTArtificial sequenceSequence is Synthesized 1Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly1 5 10 15Gly Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr 20 25 30Asn Tyr Gly Met
Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 35 40 45Glu Trp Val Gly
Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr 50 55 60Ala Ala Asp Phe
Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser65 70 75Lys Ser Thr Ala
Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 80 85 90Thr Ala Val Tyr
Tyr Cys Ala Lys Tyr Pro His Tyr Tyr Gly Ser 95 100 105Ser His Trp
Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu Val Thr 110 115 120Val Ser
Ser2108PRTArtificial sequenceSequence is Synthesized 2Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val1 5 10 15Gly Asp Arg
Val Thr Ile Thr Cys Ser Ala Ser Gln Asp Ile Ser 20 25 30Asn Tyr Leu
Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 35 40 45Val Leu Ile
Tyr Phe Thr Ser Ser Leu His Ser Gly Val Pro Ser 50 55 60Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile65 70 75Ser Ser Leu
Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 80 85 90Tyr Ser Thr
Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu 95 100 105Ile Lys
Arg
* * * * *
References