U.S. patent application number 13/355205 was filed with the patent office on 2013-01-31 for treatment with anti-vegf antibodies.
This patent application is currently assigned to Genentech, Inc.. The applicant listed for this patent is Gwendolyn Fyfe, Eric Holmgren, Robert D. Mass, William Novotny. Invention is credited to Gwendolyn Fyfe, Eric Holmgren, Robert D. Mass, William Novotny.
Application Number | 20130028862 13/355205 |
Document ID | / |
Family ID | 33551495 |
Filed Date | 2013-01-31 |
United States Patent
Application |
20130028862 |
Kind Code |
A1 |
Fyfe; Gwendolyn ; et
al. |
January 31, 2013 |
TREATMENT WITH ANTI-VEGF ANTIBODIES
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.
Inventors: |
Fyfe; Gwendolyn; (San
Francisco, CA) ; Holmgren; Eric; (Palo Alto, CA)
; Mass; Robert D.; (Mill Valley, CA) ; Novotny;
William; (Foster City, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Fyfe; Gwendolyn
Holmgren; Eric
Mass; Robert D.
Novotny; William |
San Francisco
Palo Alto
Mill Valley
Foster City |
CA
CA
CA
CA |
US
US
US
US |
|
|
Assignee: |
Genentech, Inc.
South San Francisco
CA
|
Family ID: |
33551495 |
Appl. No.: |
13/355205 |
Filed: |
January 20, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13019414 |
Feb 2, 2011 |
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13355205 |
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12576085 |
Oct 8, 2009 |
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13019414 |
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11763263 |
Jun 14, 2007 |
7622115 |
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12576085 |
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10857249 |
May 28, 2004 |
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11763263 |
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60474480 |
May 30, 2003 |
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Current U.S.
Class: |
424/85.4 ;
424/133.1; 424/158.1 |
Current CPC
Class: |
A61P 35/04 20180101;
A61K 31/337 20130101; A61K 31/519 20130101; C07K 16/22 20130101;
A61K 45/06 20130101; A61K 31/4545 20130101; A61P 35/00 20180101;
A61K 39/39541 20130101; A61P 31/00 20180101; C07K 2317/24 20130101;
A61K 31/282 20130101; C07K 2317/567 20130101; A61K 31/555 20130101;
A61K 2039/505 20130101; A61K 31/513 20130101; A61K 31/573 20130101;
A61K 38/09 20130101; A61K 39/3955 20130101; A61P 35/02 20180101;
A61K 31/4745 20130101; A61K 9/0019 20130101; A61K 38/50 20130101;
C07K 2317/565 20130101; C07K 16/3046 20130101; A61K 39/39558
20130101; A61P 43/00 20180101; C07K 2317/76 20130101; A61K 31/7068
20130101; A61K 38/212 20130101; A61K 31/525 20130101; C07K 2317/21
20130101; A61K 31/522 20130101; A61P 9/00 20180101; A61K 31/282
20130101; A61K 2300/00 20130101; A61K 31/522 20130101; A61K 2300/00
20130101; A61K 31/525 20130101; A61K 2300/00 20130101; A61K 31/573
20130101; A61K 2300/00 20130101; A61K 38/09 20130101; A61K 2300/00
20130101; A61K 38/212 20130101; A61K 2300/00 20130101; A61K 38/50
20130101; A61K 2300/00 20130101; A61K 39/39541 20130101; A61K
2300/00 20130101; A61K 39/39558 20130101; A61K 2300/00 20130101;
A61K 39/3955 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/85.4 ;
424/158.1; 424/133.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61P 35/00 20060101 A61P035/00; A61P 35/04 20060101
A61P035/04; A61K 38/21 20060101 A61K038/21 |
Claims
1. A method of treating cancer in a human patient, comprising
administering to the patient effective amounts of an anti-VEGF
antibody and an anti-neoplastic composition, wherein said
anti-neoplastic composition comprises at least one chemotherapeutic
agent.
2. The method of claim 1, wherein the cancer is selected from the
group consisting of breast cancer, colorectal cancer, rectal
cancer, non-small cell lung cancer, non-Hodgkins lymphoma (NHL),
renal cell cancer, prostate cancer, liver cancer, pancreatic
cancer, soft-tissue sarcoma, kaposi's sarcoma, carcinoid carcinoma,
head and neck cancer, melanoma, ovarian cancer, mesothelioma, and
multiple myeloma.
3. The method of claim 1, wherein the cancer is metastatic.
4. The method of claim 1, wherein the patient is previously
untreated.
5. The method of claim 1, wherein 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,
epipodopyyllotoxins, antibiotics, L-Asparaginase, topoisomerase
inhibitor, interferons, platinum cooridnation complexes,
anthracenedione substituted urea, methyl hydrazine derivatives,
adrenocortical suppressant, adrenocorticosteroides, progestins,
estrogens, antiestrogen, androgens, antiandrogen, and
gonadotropin-releasing hormone analog.
6. The method of claim 5, wherein the chemotherapeutic agent is
selected from the group consisting of 5-fluorouracil (5-FU),
leucovorin, irinotecan, oxaliplatin, capecitabine, paclitaxel and
doxetaxel.
7. The method of claim 1, wherein the anti-neoplastic composition
comprises a combination of at least two chemotherapeutic
agents.
8. The method of claim 7, wherein the anti-neoplastic composition
comprises 5-FU and leucovorin.
9. The method of claim 7, wherein the anti-neoplastic composition
comprises 5-FU, leucovorin and irinotecan.
10. The method of claim 1, wherein upon completing treatment with
the anti-VEGF antibody and the anti-neoplastic composition, the
patient receives further chemotherapeutic treatment with at least
one chemotherapeutic agent.
11. The method of claim 10, wherein the chemotherapeutic agent used
in further chemotherapeutic treatment is selected from the group
consisting of 5-FU, leucovorin, irinotecan, oxaliplatin,
capecitabine, paclitaxel and doxetaxel.
12. The method of claim 11, wherein the chemotherapeutic agent is
oxaliplatin.
13. The method of claim 1, wherein said anti-VEGF antibody binds
the same epitope as the monoclonal anti-VEGF antibody A4.6.1
produced by hybridoma ATCC HB 10709.
14. The method of claim 1, wherein the anti-VEGF antibody is a
human antibody.
15. The method of claim 1, wherein the anti-VEGF antibody is a
humanized antibody.
16. The method of claim 15, wherein the anti-VEGF antibody is a
humanized A4.6.1 antibody or fragment thereof.
17. The method of claim 1, wherein the anti-VEGF antibody is
administered intravenously.
18. The method of claim 1, wherein the anti-VEGF antibody is
administered to the patient at about 5 mg/kg every 2 to 3
weeks.
19. The method of claim 1, whereby the co-administration of the
anti-VEGF antibody and the anti-neoplastic composition effectively
increases the duration of survival of the human patient.
20. The method of claim 19, wherein the duration of survival of the
patient is increased by at least about 2 months when compared to
another patient treated with the anti-neoplastic composition
alone.
21. The method of claim 1, whereby the co-administration of the
anti-VEGF antibody and the anti-neoplastic composition effectively
increases the duration of progression free survival of the human
patient.
22. The method of claim 21, wherein the progression free survival
of the patient is increased by at least about 2 months when
compared to another patient treated with the anti-neoplastic
composition alone.
23. The method of claim 1, whereby the co-administration of the
anti-VEGF antibody and the anti-neoplastic composition effectively
increases the response rate in a group of human patients.
24. The method of claim 23, wherein the response rate of the group
of human patients is significantly increased with a Chi-square
p-value of less than 0.005 when compared to another group of
patients treated with the anti-neoplastic composition alone.
25. The method of claim 1, whereby the co-administration of the
anti-VEGF antibody and the anti-neoplastic composition effectively
increases the duration of response of the human patient.
26. The method of claim 25, wherein the duration of response of the
patient is increased by at least about 2 months when compared to
another patient treated with the anti-neoplastic composition
alone.
27. A method of treating a human patient susceptible to or
diagnosed with colorectal cancer, comprising administering to the
patient effective amounts of an anti-VEGF antibody.
28. The method of claim 27, wherein the colorectal cancer is
metastatic.
29. The method of claim 27, wherein said anti-VEGF antibody binds
the same epitope as the monoclonal anti-VEGF antibody A4.6.1
produced by hybridoma ATCC HB 10709.
30. The method of claim 27, wherein the anti-VEGF antibody is a
human antibody.
31. The method of claim 27, wherein the anti-VEGF antibody is a
humanized antibody.
32. The method of claim 31, wherein the anti-VEGF antibody is a
humanized A4.6.1 antibody or fragment thereof.
33. The method of claim 27, wherein the anti-VEGF antibody is
administered by intravenous infusion.
34. The method of claim 27, wherein the anti-VEGF antibody is
administered to the patient at about 5 mg/kg every 2 to 3
weeks.
35. The method of claim 27, further comprising administering to the
patient one or more chemotherapeutic agents.
36. The method of claim 35, wherein 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,
epipodopyyllotoxins, antibiotics, L-Asparaginase, topoisomerase
inhibitor, interferons, platinum cooridnation complexes,
anthracenedione substituted urea, methyl hydrazine derivatives,
adrenocortical suppressant, adrenocorticosteroides, progestins,
estrogens, antiestrogen, androgens, antiandrogen, and
gonadotropin-releasing hormone analog.
37. The method of claim 35, wherein the chemotherapeutic agent is
selected from the group consisting of 5-fluorouracil, leucovorin,
irinotecan, oxaliplatin, capecitabine, paclitaxel and
doxetaxel.
38. A method of treating a human patient or a group of human
patients having metastatic colorectal cancer, comprising
administering to the patient effective amounts of an anti-VEGF
antibody composition and an anti-neoplastic composition, wherein
said anti-neoplastic composition comprises a fluorouracil based
combination of chemotherapeutic agents, whereby the
co-administration of the anti-VEGF antibody and the anti-neoplastic
composition results in statistically significant and clinically
meaningful improvement of the treated patient as measured by the
duration of survival, progression free survival, response rate or
duration of response.
39. The method of claim 38, wherein the anti-neoplastic composition
comprises 5-FU, leucovorin and irinotecan.
40. The method of claim 39, wherein the anti-neoplastic composition
comprises the regimen having 500 mg/m.sup.2 5-FU, 20 mg/m.sup.2
leucovorin and 125 mg/m.sup.2 irinotecan and is administered to the
patient in repeating 6-week cycles consisting of weekly
administrations for 4 weeks followed by 2 weeks of rest, and
wherein the anti-VEGF antibody is administered to the patient at 5
mg/kg every other week.
41. The method of claim 38, wherein the anti-neoplastic composition
comprises 5-FU and leucovorin.
42. The method of claim 41, wherein the 5-FU and leucovorin are
administered to the patient at 500 mg/m.sup.2 each in repeating 8
week cycles consisting of weekly administrations for 4 weeks
followed by 2 weeks of rest, and wherein the anti-VEGF antibody is
administered to the patient at 5 mg/kg every other week.
43. The method of claim 41 for human patients considered
non-optimal candidates for first-line irinotecan therapy.
44. The method of claim 38, wherein the anti-neoplastic composition
comprises 5-FU, leucovorin and oxaliplatin.
45. An article of manufacture comprising a container, a composition
within the container comprising an anti-VEGF antibody and a package
insert instructing the user of the composition to administer to a
cancer patient the anti-VEGF antibody composition and an
anti-neoplastic composition comprising at least one
chemotherapeutic agent.
46. A kit for treating cancer in a human patient comprising a
package comprising an anti-VEGF antibody composition and
instructions for using the anti-VEGF antibody composition and an
anti-neoplastic composition comprising at least one
chemotherapeutic agent for treating cancer in a patient.
Description
[0001] This is a continuation application claiming priority to U.S.
application Ser. No. 13/019,414, filed Feb. 2, 2011, which is a
continuation application claiming priority to U.S. application Ser.
No. 12/576,085, filed Oct. 8, 2009, which is a continuation
application claiming priority to U.S. application Ser. No.
11/763,263, filed Jun. 14, 2007, which is a continuation
application of U.S. application Ser. No. 10/857,249, filed May 28,
2004, which is a non-provisional application claiming priority to
U.S. provisional Application No. 60/474,480, filed May 30, 2003,
the contents of which are incorporated herein by reference.
FIELD OF THE INVENTION
[0002] This invention relates in general to treatment of human
diseases and pathological conditions. More specifically, the
invention relates to anti-angiogenesis therapy of cancer, either
alone or in combination with other anti-cancer therapies.
BACKGROUND OF THE INVENTION
[0003] 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. It is also
predicted that cancer may surpass cardiovascular diseases as the
number one cause of death within 5 years. Solid tumors are
responsible for most of those deaths. Although there have been
significant advances in the medical treatment of certain cancers,
the overall 5-year survival rate for all cancers has improved only
by about 10% in the past 20 years. Cancers, or malignant tumors,
metastasize and grow rapidly in an uncontrolled manner, making
timely detection and treatment extremely difficult. Furthermore,
cancers can arise from almost any tissue in the body through
malignant transformation of one or a few normal cells within the
tissue, and each type of cancer with particular tissue origin
differs from the others.
[0004] Current methods of cancer treatment are relatively
non-selective. Surgery removes the diseased tissue; radiotherapy
shrinks solid tumors; and chemotherapy kills rapidly dividing
cells. Chemotherapy, in particular, results in numerous side
effects, in some cases so severe as to limit the dosage that can be
given and thus preclude the use of potentially effective drugs.
Moreover, cancers often develop resistance to chemotherapeutic
drugs.
[0005] Thus, there is an urgent need for specific and more
effective cancer therapies.
[0006] 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 (1987) Science 235:442-447). 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. Thus, it has been generally
assumed that the vascular compartment is necessary, not only for
organ development and differentiation during embryogenesis, but
also for wound healing and reproductive functions in the adult.
[0007] Angiogenesis is also implicated in the pathogenesis of a
variety of disorders, including but not limited to, tumors,
proliferative retinopathies, age-related macular degeneration,
rheumatoid arthritis (RA), and psoriasis. Angiogenesis is essential
for the growth of most primary tumors and their subsequent
metastasis. Tumors can absorb sufficient nutrients and oxygen by
simple diffusion up to a size of 1-2 mm, at which point their
further growth requires the elaboration of vascular supply. This
process is thought to involve recruitment of the neighboring host
mature vasculature to begin sprouting new blood vessel capillaries,
which grow towards, and subsequently infiltrate, the tumor mass. In
addition, tumor angiogenesis involve the recruitment of circulating
endothelial precursor cells from the bone marrow to promote
neovascularization. Kerbel (2000) Carcinogenesis 21:505-515; Lynden
et al. (2001) Nat. Med. 7:1194-1201.
[0008] 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. (1999) Science
284:1994-1998.
[0009] In view of the remarkable physiological and pathological
importance of angiogenesis, much work has been dedicated to the
elucidation of the factors capable of regulating this process. It
is suggested that the angiogenesis process is regulated by a
balance between pro- and anti-angiogenic molecules, and is derailed
in various diseases, especially cancer. Carmeliet and Jain (2000)
Nature 407:249-257.
[0010] Vascular endothelial cell growth factor (VEGF), which is
also termed VEGF-A or vascular permeability factor (VPF), has been
reported as a pivotal regulator of both normal and abnormal
angiogenesis. Ferrara and Davis-Smyth (1997) Endocrine Rev.
18:4-25; Ferrara (1999) J. Mol. Med. 77:527-543. Compared to other
growth factors that contribute to the processes of vascular
formation, VEGF is unique in its high specificity for endothelial
cells within the vascular system. VEGF is essential for embryonic
vasculogenesis and angiogenesis. Carmeliet et al. (1996) Nature
380:435-439; Ferrara et al. (1996) Nature 380:439-442. Furthermore,
VEGF is required for the cyclical blood vessel proliferation in the
female reproductive tract and for bone growth and cartilage
formation. Ferrara et al. (1998) Nature Med. 4:336-340; Gerber et
al. (1999) Nature Med. 5:623-628.
[0011] 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, recent 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. (1995) J. Cell Physiol. 164:385-394;
Oberg-Welsh et al. (1997) Mol. Cell. Endocrinol. 126:125-132;
Sondell et al. (1999) J. Neurosci. 19:5731-5740.
[0012] Substantial evidence also implicates VEGF's critical role in
the development of conditions or diseases that involve pathological
angiogenesis. The VEGF mRNA is overexpressed by the majority of
human tumors examined (Berkman et al. J Clin Invest 91:153-159
(1993); Brown et al. Human Pathol. 26:86-91 (1995); Brown et al.
Cancer Res. 53:4727-4735 (1993); Mattern et al. Brit. J. Cancer.
73:931-934 (1996); and Dvorak et al. Am J. Pathol. 146:1029-1039
(1995)). Also, the concentration of VEGF in eye fluids are highly
correlated to the presence of active proliferation of blood vessels
in patients with diabetic and other ischemia-related retinopathies
(Aiello et al. N. Engl. J. Med. 331:1480-1487 (1994)). Furthermore,
recent studies have demonstrated the localization of VEGF in
choroidal neovascular membranes in patients affected by AMD (Lopez
et al. Invest. Ophtalmo. Vis. Sci. 37:855-868 (1996)).
[0013] Given its central role in promoting tumor growth, VEGF
provides an attractive target for therapeutic intervention. Indeed,
a variety of therapeutic strategies aimed at blocking VEGF or its
receptor signaling system are currently being developed for the
treatment of neoplastic diseases. Rosen (2000) Oncologist 5:20-27;
Ellis et al. (2000) Oncologist 5:11-15; Kerbel (2001) J. Clin.
Oncol. 19:45 S-51S. So far, VEGF/VEGF receptor blockade by
monoclonal antibodies and inhibition of receptor signaling by
tyrosine kinase inhibitors are the best studied approaches. VEGFR-1
ribozymes, VEGF toxin conjugates, and soluble VEGF receptors are
also being investigated.
[0014] The anti-VEGF antibody "Bevacizumab (BV)", also known as
"rhuMAb VEGF" or "Avastin.TM.", is a recombinant humanized
anti-VEGF monoclonal antibody generated according to Presta et al.
(1997) Cancer Res. 57:4593-4599. 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. Bevacizumab
has a molecular mass of about 149,000 daltons and is glycosylated.
Bevacizumab is being investigated clinically for treating various
cancers, and some early stage trials have shown promising results.
Kerbel (2001) J. Clin. Oncol. 19:45 S-51S; De Vore et al. (2000)
Proc. Am. Soc. Clin. Oncol. 19:485a; Johnson et al. (2001) Proc.
Am. Soc. Clin. Oncol. 20:315a; Kabbinavar et al. (2003) J. Clin.
Oncol. 21:60-65.
SUMMARY OF THE INVENTION
[0015] The present invention concerns methods of using anti-VEGF
antibody for treating diseases and pathological conditions. In
particular, the invention provides an effective approach for
treating cancers, partially based on the unexpected results that
adding anti-VEGF antibody to a standard chemotherapy results in
statistically significant and clinically meaningful improvements
among cancer patients.
[0016] Accordingly, in one aspect, the invention provides a method
of treating cancer in a human patient, comprising administering to
the patient effective amounts of an anti-VEGF antibody and an
anti-neoplastic composition, wherein said anti-neoplastic
composition comprises at least one chemotherapeutic agent.
[0017] The cancer amendable for treatment by the present invention
include, but not limited to, carcinoma, lymphoma, blastoma,
sarcoma, and leukemia or lymphoid malignancies. More particular
examples of such cancers include 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, ovarian 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. Preferably, the cancer is
selected from the group consisting of breast cancer, colorectal
cancer, rectal cancer, non-small cell lung cancer, non-Hodgkins
lymphoma (NHL), renal cell cancer, prostate cancer, liver cancer,
pancreatic cancer, soft-tissue sarcoma, kaposi's sarcoma, carcinoid
carcinoma, head and neck cancer, melanoma, ovarian cancer,
mesothelioma, and multiple myeloma. More preferably, the cancer is
colorectal cancer. The cancerous conditions amendible for treatment
of the invention include metastatic cancers. The method of the
present invention is particularly suitable for the treatment of
vascularized tumors.
[0018] Any chemotherapeutic agent exhibiting anticancer activity
can be used according to the present invention. Preferably, 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,
epipodopyyllotoxins, antibiotics, L-Asparaginase, topoisomerase
inhibitor, interferons, platinum cooridnation complexes,
anthracenedione substituted urea, methyl hydrazine derivatives,
adrenocortical suppressant, adrenocorticosteroides, progestins,
estrogens, antiestrogen, androgens, antiandrogen, and
gonadotropin-releasing hormone analog. More preferably, the
chemotherapeutic agent is selected from the group consisting of
5-fluorouracil (5-FU), leucovorin (LV), irenotecan, oxaliplatin,
capecitabine, paclitaxel and doxetaxel. Two or more
chemotherapeutic agents can be used in a cocktail to be
administered in combination with administration of the anti-VEGF
antibody. One preferred combination chemotherapy is
fluorouracil-based, comprising 5-FU and one or more other
chemotherapeutic agent(s). Suitable dosing regimens of combination
chemotherapies are known in the art and described in, for example,
Saltz et al. (1999) Proc ASCO 18:233a and Douillard et al. (2000)
Lancet 355:1041-7.
[0019] In one aspect, the present invention provides a method for
increasing the duration of survival of a human patient having
cancer, comprising administering to the patient effective amounts
of an anti-VEGF antibody composition and an anti-neoplastic
composition, wherein said anti-neoplastic composition comprises at
least one chemotherapeutic agent, whereby the co-administration of
the anti-VEGF antibody and the anti-neoplastic composition
effectively increases the duration of survival.
[0020] In another aspect, the present invention provides a method
for increasing the progression free survival of a human patient
having cancer, comprising administering to the patient effective
amounts of an anti-VEGF antibody composition and an anti-neoplastic
composition, wherein said anti-neoplastic composition comprises at
least one chemotherapeutic agent, whereby the co-administration of
the anti-VEGF antibody and the anti-neoplastic composition
effectively increases the duration of progression free
survival.
[0021] Furthermore, the present invention provides a method for
treating a group of human patients having cancer, comprising
administering to the patient effective amounts of an anti-VEGF
antibody composition and an anti-neoplastic composition, wherein
said anti-neoplastic composition comprises at least one
chemotherapeutic agent, whereby the co-administration of the
anti-VEGF antibody and the anti-neoplastic composition effectively
increases the response rate in the group of patients.
[0022] In yet another aspect, the present invention provides a
method for increasing the duration of response of a human patient
having cancer, comprising administering to the patient effective
amounts of an anti-VEGF antibody composition and an anti-neoplastic
composition, wherein said anti-neoplastic composition comprises at
least one chemotherapeutic agent, whereby the co-administration of
the anti-VEGF antibody and the anti-neoplastic composition
effectively increases the duration of response.
[0023] The invention also provides a method of treating a human
patient susceptible to or diagnosed with colorectal cancer,
comprising administering to the patient effective amounts of an
anti-VEGF antibody. The colorectal cancer can be metastatic. The
anti-VEGF antibody treatment can be further combined with a
standard chemotherapy for colorectal cancer such as the Saltz
(5-FU/LV/irinotecan) regimen described by Saltz et al. (1999).
[0024] In one preferred embodiment, the invention provides a method
of treating a human patient or a group of human patients having
metastatic colorectal cancer, comprising administering to the
patient effective amounts of an anti-VEGF antibody composition and
an anti-neoplastic composition, wherein said anti-neoplastic
composition comprises at least one chemotherapeutic agent, whereby
the co-administration of the anti-VEGF antibody and the
anti-neoplastic composition results in statistically significant
and clinically meaningful improvement of the treated patient as
measured by the duration of survival, progression free survival,
response rate or duration of response. Preferably, the
anti-neoplastic composition is a fluorouracil based combination
regimen. More preferably the combination regimen comprises
5-FU+leucovorin, 5-FU+leucovorin+irinotecan (IFL), or
5-FU+leucorvin+oxaliplatin (FOLFOX).
[0025] The invention provides an article of manufacture comprising
a container, a composition within the container comprising an
anti-VEGF antibody and a package insert instructing the user of the
composition to administer to a cancer patient the anti-VEGF
antibody composition and an anti-neoplastic composition comprising
at least one chemotherapeutic agent.
[0026] The invention also provides a kit for treating cancer in a
patient comprising a package comprising an anti-VEGF antibody
composition and instructions for using the anti-VEGF antibody
composition and an anti-neoplastic composition comprising at least
one chemotherapeutic agent for treating cancer in a patient.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] FIG. 1 represents Kaplan-Meier estimates of survival. The
median duration of survival (indicated by the dotted lines) was
20.3 months in the group given irinotecan, fluorouracil, and
leucovorin (IFL) plus bevacizumab, as compared with 15.6 months in
the group given IFL plus placebo, corresponding to a hazard ratio
for death of 0.66 (P<0.001).
[0028] FIG. 2 represents Kaplan-Meier estimates of progression-free
survival. The median duration of progression-free survival
(indicated by the dotted lines) was 10.6 months in the group given
irinotecan, fluorouracil, and leucovorin (IFL) plus bevacizumab, as
compared with 6.2 months in the group given IFL plus placebo,
corresponding to a hazard ratio for progression of 0.54
(P<0.001).
[0029] FIGS. 3A-3C provide analysis of duration of survival by
different subgroups of patients divided by baseline
characteristics.
[0030] FIG. 4 represents Kaplan-Meier estimates of survival
comparing the group given 5-FU/LV plus placebo vs. the group given
5-FU/LV plus bevacizumab (BV).
[0031] FIG. 5 represents Kaplan-Meier estimates of progression-free
survival comparing the group given 5-FU/LV plus placebo vs. the
group given 5-FU/LV plus bevacizumab (BV).
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
I. Definitions
[0032] The terms "VEGF" and "VEGF-A" are used interchangeably to
refer to the 165-amino acid vascular endothelial cell growth factor
and related 121-, 189-, and 206-amino acid vascular endothelial
cell growth factors, as described by 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.
The term "VEGF" is also used to refer to truncated forms 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
present application, e.g., by "VEGF (8-109)," "VEGF (1-109)" or
"VEGF.sub.165." 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.
[0033] An "anti-VEGF antibody" is an antibody that binds to VEGF
with sufficient affinity and specificity. Preferably, 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. 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 P1GF, PDGF or bFGF. A preferred anti-VEGF
antibody is a monoclonal antibody that binds to the same epitope as
the monoclonal anti-VEGF antibody A4.6.1 produced by hybridoma ATCC
HB 10709. More preferably the anti-VEGF antibody is a recombinant
humanized anti-VEGF monoclonal antibody generated according to
Presta et al. (1997) Cancer Res. 57:4593-4599, including but not
limited to the antibody known as bevacizumab (BV; Avastin.TM.).
[0034] 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.
[0035] 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.
[0036] 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.
[0037] 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.
[0038] 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.
[0039] Unless indicated otherwise, the expression "multivalent
antibody" is used throughout this specification to denote an
antibody comprising three or more antigen binding sites. The
multivalent antibody is preferably engineered to have the three or
more antigen binding sites and is generally not a native sequence
IgM or IgA antibody.
[0040] "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).
[0041] 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 present
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.
[0042] The monoclonal antibodies herein specifically include
"chimeric" antibodies 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)).
[0043] "Humanized" forms of non-human (e.g., murine) antibodies are
chimeric antibodies that 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,
framework region (FR) residues of the human immunoglobulin are
replaced by corresponding non-human residues. Furthermore,
humanized antibodies may comprise residues that 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 FRs
are those of a human immunoglobulin sequence. The humanized
antibody optionally will also 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).
[0044] 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. PNAS (USA)
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.
[0045] 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).
[0046] An "isolated" polypeptide 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 polypeptide, and may include enzymes, hormones, and other
proteinaceous or nonproteinaceous solutes. In preferred
embodiments, the polypeptide will be purified (1) to greater than
95% by weight of polypeptide 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, preferably, silver stain. Isolated polypeptide
includes the polypeptide in situ within recombinant cells since at
least one component of the polypeptide's natural environment will
not be present. Ordinarily, however, isolated polypeptide will be
prepared by at least one purification step.
[0047] 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 below. 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.
[0048] 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.
[0049] 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.
[0050] An "agonist antibody" is an antibody which binds to and
activates a receptor. Generally, the receptor activation capability
of the agonist antibody will be at least qualitatively similar (and
may be essentially quantitatively similar) to a native agonist
ligand of the receptor. An example of an agonist antibody is one
which binds to a receptor in the TNF receptor superfamily and
induces apoptosis of cells expressing the TNF receptor. Assays for
determining induction of apoptosis are described in WO98/51793 and
WO99/37684, both of which are expressly incorporated herein by
reference.
[0051] 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 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.
[0052] 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, time to disease progression (TTP), the response rates
(RR), duration of response, and/or quality of life.
[0053] "Treatment" refers to both therapeutic treatment and
prophylactic or preventative measures. Those in need of treatment
include those already with the disorder as well as those in which
the disorder is to be prevented.
[0054] The terms "cancer" and "cancerous" refer to or describe the
physiological condition in mammals that is typically characterized
by unregulated cell growth. Examples of cancer include but are not
limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia.
More particular examples of such cancers include 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, ovarian
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.
[0055] The term "mammalian host" as used herein refers to any
compatible transplant recipient. By "compatible" is meant a
mammalian host that will accept the donated graft. Preferably, the
host is human. If both the donor of the graft and the host are
human, they are preferably matched for HLA class II antigens so as
to improve histocompatibility.
[0056] 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.
[0057] The term "anti-neoplastic composition" refers to a
composition useful in treating cancer comprising at least one
active therapeutic agent capable of inhibiting or preventing tumor
growth or function, and/or causing destruction of tumor cells.
Therapeutic agents suitable in an anti-neoplastic composition for
treating cancer include, but not limited to, chemotherapeutic
agents, radioactive isotopes, toxins, cytokines such as
interferons, and antagonistic agents targeting cytokines, cytokine
receptors or antigens associated with tumor cells. For example,
therapeutic agents useful in the present invention can be
antibodies such as anti-HER2 antibody and anti-CD20 antibody, or
small molecule tyrosine kinase inhibitors such as VEGF receptor
inhibitors and EGF receptor inhibitors. Preferably the therapeutic
agent is a chemotherapeutic agent.
[0058] A "chemotherapeutic agent" 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 antiobiotic chromophores), aclacinomysins, actinomycin,
authramycin, azaserine, bleomycins, cactinomycin, carabicin,
caminomycin, 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; elfornithine; 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;
sizofuran; 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.TM. Cremophor-free,
albumin-engineered nanoparticle formulation of paclitaxel (American
Pharmaceutical Partners, Schaumberg, Illinois), and TAXOTERE.RTM.
doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil;
GEMZAR.RTM. gemcitabine; 6-thioguanine; mercaptopurine;
methotrexate; platinum coordination complexes 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 (e.g., CPT-11);
topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO);
retinoids such as retinoic acid; capecitabine; and pharmaceutically
acceptable salts, acids or derivatives of any of the above.
[0059] 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.cndot.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.
[0060] 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.
[0061] 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.
[0062] 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.
[0063] 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.
[0064] 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.
[0065] 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.
[0066] 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.
[0067] 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.
[0068] An "angiogenic factor" is a growth factor which stimulates
the development of blood vessels. The preferred angiogenic factor
herein is Vascular Endothelial Growth Factor (VEGF).
[0069] 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.
[0070] An "isolated" nucleic acid molecule is a nucleic acid
molecule that is identified and separated from at least one
contaminant nucleic acid molecule with which it is ordinarily
associated in the natural source of the polypeptide nucleic acid.
An isolated nucleic acid molecule is other than in the form or
setting in which it is found in nature. Isolated nucleic acid
molecules therefore are distinguished from the nucleic acid
molecule as it exists in natural cells. However, an isolated
nucleic acid molecule includes a nucleic acid molecule contained in
cells that ordinarily express the polypeptide where, for example,
the nucleic acid molecule is in a chromosomal location different
from that of natural cells.
[0071] The expression "control sequences" refers to DNA sequences
necessary for the expression of an operably linked coding sequence
in a particular host organism. The control sequences that are
suitable for prokaryotes, for example, include a promoter,
optionally an operator sequence, and a ribosome binding site.
Eukaryotic cells are known to utilize promoters, polyadenylation
signals, and enhancers.
[0072] Nucleic acid is "operably linked" when it is placed into a
functional relationship with another nucleic acid sequence. For
example, DNA for a presequence or secretory leader is operably
linked to DNA for a polypeptide if it is expressed as a preprotein
that participates in the secretion of the polypeptide; a promoter
or enhancer is operably linked to a coding sequence if it affects
the transcription of the sequence; or a ribosome binding site is
operably linked to a coding sequence if it is positioned so as to
facilitate translation. Generally, "operably linked" means that the
DNA sequences being linked are contiguous, and, in the case of a
secretory leader, contiguous and in reading phase. However,
enhancers do not have to be contiguous. Linking is accomplished by
ligation at convenient restriction sites. If such sites do not
exist, the synthetic oligonucleotide adaptors or linkers are used
in accordance with conventional practice.
[0073] As used herein, the expressions "cell," "cell line," and
"cell culture" are used interchangeably and all such designations
include progeny. Thus, the words "transformants" and "transformed
cells" include the primary subject cell and cultures derived
therefrom without regard for the number of transfers. It is also
understood that all progeny may not be precisely identical in DNA
content, due to deliberate or inadvertent mutations. Mutant progeny
that have the same function or biological activity as screened for
in the originally transformed cell are included. Where distinct
designations are intended, it will be clear from the context.
Ii. Production of Anti-VEGF Antibodies
A. Antibody Preparation
(i) VEGF Antigen
[0074] Means for preparing and characterizing antibodies are well
known in the art. A description follows as to exemplary techniques
for the production of anti-VEGF antibodies used in accordance with
the present invention. 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.
[0075] 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.
[0076] 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 (1995)
Laboratory Investigation 72:615-618, and the references cited
therein).
[0077] 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.
[0078] Several molecules structurally related to VEGF have also
been identified recently, 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. (1998) J. Biological Chem.
273:31273-31281; Meyer et al. (1999) EMBO J., 18:363-374. A
receptor tyrosine kinase, Flt-4 (VEGFR-3), has been identified as
the receptor for VEGF-C and VEGF-D. Joukov et al. (1996) EMBO. J.
15:1751; Lee et al. (1996) Proc. Natl. Acad. Sci. USA 93:1988-1992;
Achen et al. (1998) Proc. Natl. Acad. Sci. USA 95:548-553. VEGF-C
has recently been shown to be involved in the regulation of
lymphatic angiogenesis. Jeltsch et al. (1997) Science
276:1423-1425.
[0079] 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.
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.
(ii) Polyclonal Antibodies
[0080] 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=NR, where R and
R.sup.1 are different alkyl groups.
[0081] 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.
(iii) Monoclonal Antibodies
[0082] Monoclonal antibodies may be made using the hybridoma method
first described by Kohler et al., Nature, 256:495 (1975), or may be
made by recombinant DNA methods (U.S. Pat. No. 4,816,567).
[0083] 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)).
[0084] 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.
[0085] 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)).
[0086] 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).
[0087] 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.
[0088] 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.
[0089] 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.
[0090] 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.
[0091] 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.
[0092] 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.
(iv) Humanized and Human Antibodies
[0093] 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.
[0094] 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. Immunol., 151:2623 (1993)).
[0095] 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.
[0096] Alternatively, 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 (J.sub.H) 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). Human
antibodies can also be derived from phage-display libraries
(Hoogenboom et al., J. Mol. Biol., 227:381 (1991); Marks et al., J.
Mol. Biol., 222:581-597 (1991); Vaughan et al. Nature Biotech
14:309 (1996)).
(v) Antibody Fragments
[0097] 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.
(vi) Multispecific Antibodies
[0098] Multispecific antibodies have binding specificities for at
least two different antigens. While such molecules normally will
only bind two antigens (i.e. bispecific antibodies, BsAbs),
antibodies with additional specificities such as trispecific
antibodies are encompassed by this expression when used herein.
Examples of BsAbs include those with one arm directed against a
tumor cell antigen and the other arm directed against a cytotoxic
trigger molecule such as anti-Fc.gamma.RI/anti-CD15,
anti-p185.sup.HER2/Fc.gamma.RIII (CD16), anti-CD3/anti-malignant
B-cell (1D10), anti-CD3/anti-p185.sup.HER2, anti-CD3/anti-p97,
anti-CD3/anti-renal cell carcinoma, anti-CD3/anti-OVCAR-3,
anti-CD3/L-D1 (anti-colon carcinoma), anti-CD3/anti-melanocyte
stimulating hormone analog, anti-EGF receptor/anti-CD3,
anti-CD3/anti-CAMA1, anti-CD3/anti-CD19, anti-CD3/MoV18,
anti-neural cell ahesion molecule (NCAM)/anti-CD3, anti-folate
binding protein (FBP)/anti-CD3, anti-pan carcinoma associated
antigen (AMOC-31)/anti-CD3; BsAbs with one arm which binds
specifically to a tumor antigen and one arm which binds to a toxin
such as anti-saporin/anti-Id-1, anti-CD22/anti-saporin,
anti-CD7/anti-saporin, anti-CD38/anti-saporin, anti-CEA/anti-ricin
A chain, anti-interferon-.alpha.(IFN-.alpha.)/anti-hybridoma
idiotype, anti-CEA/anti-vinca alkaloid; BsAbs for converting enzyme
activated prodrugs such as anti-CD30/anti-alkaline phosphatase
(which catalyzes conversion of mitomycin phosphate prodrug to
mitomycin alcohol); BsAbs which can be used as fibrinolytic agents
such as anti-fibrin/anti-tissue plasminogen activator (tPA),
anti-fibrin/anti-urokinase-type plasminogen activator (uPA); BsAbs
for targeting immune complexes to cell surface receptors such as
anti-low density lipoprotein (LDL)/anti-Fc receptor (e.g.
Fc.gamma.RI, Fc.gamma.RII or Fc.gamma.RIII); BsAbs for use in
therapy of infectious diseases such as anti-CD3/anti-herpes simplex
virus (HSV), anti-T-cell receptor:CD3 complex/anti-influenza,
anti-Fc.gamma.R/anti-HIV; BsAbs for tumor detection in vitro or in
vivo such as anti-CEA/anti-EOTUBE, anti-CEA/anti-DPTA,
anti-p185.sup.HER2/anti-hapten; BsAbs as vaccine adjuvants; and
BsAbs as diagnostic tools such as anti-rabbit IgG/anti-ferritin,
anti-horse radish peroxidase (HRP)/anti-hormone,
anti-somatostatin/anti-substance P, anti-HRP/anti-FITC,
anti-CEA/anti-.beta.-galactosidase. Examples of trispecific
antibodies include anti-CD3/anti-CD4/anti-CD37,
anti-CD3/anti-CD5/anti-CD37 and anti-CD3/anti-CD8/anti-CD37.
Bispecific antibodies can be prepared as full length antibodies or
antibody fragments (e.g. F(ab').sub.2 bispecific antibodies).
[0099] Methods for making bispecific antibodies are known in the
art. Traditional production of full length bispecific antibodies is
based on the coexpression of two immunoglobulin heavy chain-light
chain pairs, where the two chains have different specificities
(Millstein et al., Nature, 305:537-539 (1983)). Because of the
random assortment of immunoglobulin heavy and light chains, these
hybridomas (quadromas) produce a potential mixture of 10 different
antibody molecules, of which only one has the correct bispecific
structure. Purification of the correct molecule, which is usually
done by affinity chromatography steps, is rather cumbersome, and
the product yields are low. Similar procedures are disclosed in WO
93/08829, and in Traunecker et al., EMBO J., 10:3655-3659
(1991).
[0100] According to a different approach, antibody variable domains
with the desired binding specificities (antibody-antigen combining
sites) are fused to immunoglobulin constant domain sequences. The
fusion preferably is with an immunoglobulin heavy chain constant
domain, comprising at least part of the hinge, CH2, and CH3
regions. It is preferred to have the first heavy-chain constant
region (CH1) containing the site necessary for light chain binding,
present in at least one of the fusions. DNAs encoding the
immunoglobulin heavy chain fusions and, if desired, the
immunoglobulin light chain, are inserted into separate expression
vectors, and are co-transfected into a suitable host organism. This
provides for great flexibility in adjusting the mutual proportions
of the three polypeptide fragments in embodiments when unequal
ratios of the three polypeptide chains used in the construction
provide the optimum yields. It is, however, possible to insert the
coding sequences for two or all three polypeptide chains in one
expression vector when the expression of at least two polypeptide
chains in equal ratios results in high yields or when the ratios
are of no particular significance.
[0101] In a preferred embodiment of this approach, the bispecific
antibodies are composed of a hybrid immunoglobulin heavy chain with
a first binding specificity in one arm, and a hybrid immunoglobulin
heavy chain-light chain pair (providing a second binding
specificity) in the other arm. It was found that this asymmetric
structure facilitates the separation of the desired bispecific
compound from unwanted immunoglobulin chain combinations, as the
presence of an immunoglobulin light chain in only one half of the
bispecific molecule provides for a facile way of separation. This
approach is disclosed in WO 94/04690. For further details of
generating bispecific antibodies see, for example, Suresh et al.,
Methods in Enzymology, 121:210 (1986). According to another
approach described in WO96/27011, the interface between a pair of
antibody molecules can be engineered to maximize the percentage of
heterodimers which are recovered from recombinant cell culture. The
preferred interface comprises at least a part of the C.sub.H3
domain of an antibody constant domain. In this method, one or more
small amino acid side chains from the interface of the first
antibody molecule are replaced with larger side chains (e.g.
tyrosine or tryptophan). Compensatory "cavities" of identical or
similar size to the large side chain(s) are created on the
interface of the second antibody molecule by replacing large amino
acid side chains with smaller ones (e.g. alanine or threonine).
This provides a mechanism for increasing the yield of the
heterodimer over other unwanted end-products such as
homodimers.
[0102] Bispecific antibodies include cross-linked or
"heteroconjugate" antibodies. For example, one of the antibodies in
the heteroconjugate can be coupled to avidin, the other to biotin.
Such antibodies have, for example, been proposed to target immune
system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for
treatment of HIV infection (WO 91/00360, WO 92/200373, and EP
03089). Heteroconjugate antibodies may be made using any convenient
cross-linking methods. Suitable cross-linking agents are well known
in the art, and are disclosed in U.S. Pat. No. 4,676,980, along
with a number of cross-linking techniques.
[0103] Techniques for generating bispecific antibodies from
antibody fragments have also been described in the literature. For
example, bispecific antibodies can be prepared using chemical
linkage. Brennan et al., Science, 229: 81 (1985) describe a
procedure wherein intact antibodies are proteolytically cleaved to
generate F(ab').sub.2 fragments. These fragments are reduced in the
presence of the dithiol complexing agent sodium arsenite to
stabilize vicinal dithiols and prevent intermolecular disulfide
formation. The Fab' fragments generated are then converted to
thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB
derivatives is then reconverted to the Fab'-thiol by reduction with
mercaptoethylamine and is mixed with an equimolar amount of the
other Fab'-TNB derivative to form the bispecific antibody. The
bispecific antibodies produced can be used as agents for the
selective immobilization of enzymes.
[0104] Recent progress has facilitated the direct recovery of
Fab'-SH fragments from E. coli, which can be chemically coupled to
form bispecific antibodies. Shalaby et al., J. Exp. Med., 175:
217-225 (1992) describe the production of a fully humanized
bispecific antibody F(ab').sub.2 molecule. Each Fab' fragment was
separately secreted from E. coli and subjected to directed chemical
coupling in vitro to form the bispecific antibody. The bispecific
antibody thus formed was able to bind to cells overexpressing the
VEGF receptor and normal human T cells, as well as trigger the
lytic activity of human cytotoxic lymphocytes against human breast
tumor targets.
[0105] Various techniques for making and isolating bispecific
antibody fragments directly from recombinant cell culture have also
been described. For example, bispecific antibodies have been
produced using leucine zippers. Kostelny et al., J. Immunol.,
148(5):1547-1553 (1992). The leucine zipper peptides from the Fos
and Jun proteins were linked to the Fab' portions of two different
antibodies by gene fusion. The antibody homodimers were reduced at
the hinge region to form monomers and then re-oxidized to form the
antibody heterodimers. This method can also be utilized for the
production of antibody homodimers. The "diabody" technology
described by Hollinger et al., Proc. Natl. Acad. Sci. USA,
90:6444-6448 (1993) has provided an alternative mechanism for
making bispecific antibody fragments. The fragments comprise a
heavy-chain variable domain (V.sub.H) connected to a light-chain
variable domain (V.sub.L) by a linker which is too short to allow
pairing between the two domains on the same chain. Accordingly, the
V.sub.H and V.sub.L domains of one fragment are forced to pair with
the complementary V.sub.L and V.sub.H domains of another fragment,
thereby forming two antigen-binding sites. Another strategy for
making bispecific antibody fragments by the use of single-chain Fv
(sFv) dimers has also been reported. See Gruber et al., J.
Immunol., 152:5368 (1994).
[0106] Antibodies with more than two valencies are contemplated.
For example, trispecific antibodies can be prepared. Tutt et al. J.
Immunol. 147: 60 (1991).
(vii) Effector Function Engineering
[0107] It may be desirable to modify the antibody of the invention
with respect to effector function, so as to enhance the
effectiveness of the antibody in treating cancer, for example. For
example 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).
(viii) Immunoconjugates
[0108] 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).
[0109] 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.
[0110] 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.
[0111] 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).
(ix) Immunoliposomes
[0112] 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.
[0113] 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 present 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)
(x) Antibody Dependent Enzyme Mediated Prodrug Therapy (ADEPT)
[0114] The antibody of the present invention may also be used in
ADEPT by conjugating the antibody to a prodrug-activating enzyme
which converts a prodrug (e.g. a peptidyl chemotherapeutic agent,
see WO81/01145) to an active anti-cancer drug. See, for example, WO
88/07378 and U.S. Pat. No. 4,975,278.
[0115] The enzyme component of the immunoconjugate useful for ADEPT
includes any enzyme capable of acting on a prodrug in such a way so
as to convert it into its more active, cytotoxic form.
[0116] Enzymes that are useful in the method of this invention
include, but are not limited to, alkaline phosphatase useful for
converting phosphate-containing prodrugs into free drugs;
arylsulfatase useful for converting sulfate-containing prodrugs
into free drugs; cytosine deaminase useful for converting non-toxic
5-fluorocytosine into the anti-cancer drug, 5-fluorouracil;
proteases, such as serratia protease, thermolysin, subtilisin,
carboxypeptidases and cathepsins (such as cathepsins B and L), that
are useful for converting peptide-containing prodrugs into free
drugs; D-alanylcarboxypeptidases, useful for converting prodrugs
that contain D-amino acid substituents; carbohydrate-cleaving
enzymes such as .beta.-galactosidase and neuraminidase useful for
converting glycosylated prodrugs into free drugs; .beta.-lactamase
useful for converting drugs derivatized with .beta.-lactams into
free drugs; and penicillin amidases, such as penicillin V amidase
or penicillin G amidase, useful for converting drugs derivatized at
their amine nitrogens with phenoxyacetyl or phenylacetyl groups,
respectively, into free drugs. Alternatively, antibodies with
enzymatic activity, also known in the art as "abzymes", can be used
to convert the prodrugs of the invention into free active drugs
(see, e.g., Massey, Nature 328: 457-458 (1987)). Antibody-abzyme
conjugates can be prepared as described herein for delivery of the
abzyme to a tumor cell population.
[0117] The enzymes of this invention can be covalently bound to the
antibody by techniques well known in the art such as the use of the
heterobifunctional crosslinking reagents discussed above.
Alternatively, fusion proteins comprising at least the antigen
binding region of an antibody of the invention linked to at least a
functionally active portion of an enzyme of the invention can be
constructed using recombinant DNA techniques well known in the art
(see, e.g., Neuberger et al., Nature, 312: 604-608 (1984)).
(xi) Antibody-Salvage Receptor Binding Epitope Fusions.
[0118] In certain embodiments of the invention, it may be desirable
to use an antibody fragment, rather than an intact antibody, to
increase tumor penetration, for example. In this case, it may be
desirable to modify the antibody fragment in order to increase its
serum half life. This may be achieved, for example, by
incorporation of a salvage receptor binding epitope into the
antibody fragment (e.g. by mutation of the appropriate region in
the antibody fragment or by incorporating the epitope into a
peptide tag that is then fused to the antibody fragment at either
end or in the middle, e.g., by DNA or peptide synthesis).
[0119] The salvage receptor binding epitope preferably constitutes
a region wherein any one or more amino acid residues from one or
two loops of a Fc domain are transferred to an analogous position
of the antibody fragment. Even more preferably, three or more
residues from one or two loops of the Fc domain are transferred.
Still more preferred, the epitope is taken from the CH2 domain of
the Fc region (e.g., of an IgG) and transferred to the CH1, CH3, or
V.sub.H region, or more than one such region, of the antibody.
Alternatively, the epitope is taken from the CH2 domain of the Fc
region and transferred to the C.sub.L region or V.sub.L region, or
both, of the antibody fragment.
(xii) Other Covalent Modifications of the Antibody
[0120] Covalent modifications of the antibody are included within
the scope of this invention. They may be made by chemical synthesis
or by enzymatic or chemical cleavage of the antibody, if
applicable. Other types of covalent modifications of the antibody
are introduced into the molecule by reacting targeted amino acid
residues of the antibody with an organic derivatizing agent that is
capable of reacting with selected side chains or the N- or
C-terminal residues.
[0121] Cysteinyl residues most commonly are reacted with
.alpha.-haloacetates (and corresponding amines), such as
chloroacetic acid or chloroacetamide, to give carboxymethyl or
carboxyamidomethyl derivatives. Cysteinyl residues also are
derivatized by reaction with bromotrifluoroacetone,
.alpha.-bromo-.beta.-(5-imidozoyl)propionic acid, chloroacetyl
phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl
2-pyridyl disulfide, p-chloromercuribenzoate,
2-chloromercuri-4-nitrophenol, or
chloro-7-nitrobenzo-2-oxa-1,3-diazole.
[0122] Histidyl residues are derivatized by reaction with
diethylpyrocarbonate at pH 5.5-7.0 because this agent is relatively
specific for the histidyl side chain. Para-bromophenacyl bromide
also is useful; the reaction is preferably performed in 0.1 M
sodium cacodylate at pH 6.0.
[0123] Lysinyl and amino-terminal residues are reacted with
succinic or other carboxylic acid anhydrides. Derivatization with
these agents has the effect of reversing the charge of the lysinyl
residues. Other suitable reagents for derivatizing
.alpha.-amino-containing residues include imidoesters such as
methyl picolinimidate, pyridoxal phosphate, pyridoxal,
chloroborohydride, trinitrobenzenesulfonic acid, O-methylisourea,
2,4-pentanedione, and transaminase-catalyzed reaction with
glyoxylate.
[0124] Arginyl residues are modified by reaction with one or
several conventional reagents, among them phenylglyoxal,
2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin.
Derivatization of arginine residues requires that the reaction be
performed in alkaline conditions because of the high pK.sub.a of
the guanidine functional group. Furthermore, these reagents may
react with the groups of lysine as well as the arginine
epsilon-amino group.
[0125] The specific modification of tyrosyl residues may be made,
with particular interest in introducing spectral labels into
tyrosyl residues by reaction with aromatic diazonium compounds or
tetranitromethane. Most commonly, N-acetylimidizole and
tetranitromethane are used to form O-acetyl tyrosyl species and
3-nitro derivatives, respectively. Tyrosyl residues are iodinated
using .sup.125I or .sup.131I to prepare labeled proteins for use in
radioimmunoassay.
[0126] Carboxyl side groups (aspartyl or glutamyl) are selectively
modified by reaction with carbodiimides (R--N.dbd.C.dbd.N--R'),
where R and R' are different alkyl groups, such as
1-cyclohexyl-3-(2-morpholinyl-4-ethyl) carbodiimide or
1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermore,
aspartyl and glutamyl residues are converted to asparaginyl and
glutaminyl residues by reaction with ammonium ions.
[0127] Glutaminyl and asparaginyl residues are frequently
deamidated to the corresponding glutamyl and aspartyl residues,
respectively. These residues are deamidated under neutral or basic
conditions. The deamidated form of these residues falls within the
scope of this invention.
[0128] Other modifications include hydroxylation of proline and
lysine, phosphorylation of hydroxyl groups of seryl or threonyl
residues, methylation of the .alpha.-amino groups of lysine,
arginine, and histidine side chains (T. E. Creighton, Proteins:
Structure and Molecular Properties, W.H. Freeman & Co., San
Francisco, pp. 79-86 (1983)), acetylation of the N-terminal amine,
and amidation of any C-terminal carboxyl group.
[0129] Another type of covalent modification involves chemically or
enzymatically coupling glycosides to the antibody. These procedures
are advantageous in that they do not require production of the
antibody in a host cell that has glycosylation capabilities for N-
or O-linked glycosylation. Depending on the coupling mode used, the
sugar(s) may be attached to (a) arginine and histidine, (b) free
carboxyl groups, (c) free sulfhydryl groups such as those of
cysteine, (d) free hydroxyl groups such as those of serine,
threonine, or hydroxyproline, (e) aromatic residues such as those
of phenylalanine, tyrosine, or tryptophan, or (f) the amide group
of glutamine. These methods are described in WO 87/05330 published
11 Sep. 1987, and in Aplin and Wriston, CRC Crit. Rev. Biochem.,
pp. 259-306 (1981).
[0130] Removal of any carbohydrate moieties present on the antibody
may be accomplished chemically or enzymatically. Chemical
deglycosylation requires exposure of the antibody to the compound
trifluoromethanesulfonic acid, or an equivalent compound. This
treatment results in the cleavage of most or all sugars except the
linking sugar (N-acetylglucosamine or N-acetylgalactosamine), while
leaving the antibody intact. Chemical deglycosylation is described
by Hakimuddin, et al. Arch. Biochem. Biophys. 259:52 (1987) and by
Edge et al. Anal. Biochem., 118:131 (1981). Enzymatic cleavage of
carbohydrate moieties on antibodies can be achieved by the use of a
variety of endo- and exo-glycosidases as described by Thotakura et
al. Meth. Enzymol. 138:350 (1987).
[0131] Another type of covalent modification of the antibody
comprises linking the antibody to one of a variety of
nonproteinaceous polymers, e.g., polyethylene glycol, polypropylene
glycol, or polyoxyalkylenes, in the manner set forth in U.S. Pat.
No. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or
4,179,337.
B. Vectors, Host Cells and Recombinant Methods
[0132] The anti-VEGF antibody of the invention can be produced
recombinantly, using techniques and materials readily
obtainable.
[0133] For recombinant production of an anti-VEGF antibody, the
nucleic acid encoding it is isolated and inserted into a replicable
vector for further cloning (amplification of the DNA) or for
expression. DNA encoding the monoclonal antibody 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
antibody). Many vectors are available. The vector components
generally include, but are not limited to, one or more of the
following: a signal sequence, an origin of replication, one or more
marker genes, an enhancer element, a promoter, and a transcription
termination sequence.
(i) Signal Sequence Component
[0134] The antibody of this invention may be produced recombinantly
not only directly, but also as a fusion polypeptide with a
heterologous polypeptide, which is preferably a signal sequence or
other polypeptide having a specific cleavage site at the N-terminus
of the mature protein or polypeptide. The heterologous signal
sequence selected preferably is one that is recognized and
processed (i.e., cleaved by a signal peptidase) by the host cell.
For prokaryotic host cells that do not recognize and process the
native antibody signal sequence, the signal sequence is substituted
by a prokaryotic signal sequence selected, for example, from the
group of the alkaline phosphatase, penicillinase, 1 pp, or
heat-stable enterotoxin II leaders. For yeast secretion the native
signal sequence may be substituted by, e.g., the yeast invertase
leader, a factor leader (including Saccharomyces and Kluyveromyces
.alpha.-factor leaders), or acid phosphatase leader, the C.
albicans glucoamylase leader, or the signal described in WO
90/13646. In mammalian cell expression, mammalian signal sequences
as well as viral secretory leaders, for example, the herpes simplex
gD signal, are available.
[0135] The DNA for such precursor region is ligated in reading
frame to DNA encoding the antibody.
(ii) Origin of Replication Component
[0136] Both expression and cloning vectors contain a nucleic acid
sequence that enables the vector to replicate in one or more
selected host cells. Generally, in cloning vectors this sequence is
one that enables the vector to replicate independently of the host
chromosomal DNA, and includes origins of replication or
autonomously replicating sequences. Such sequences are well known
for a variety of bacteria, yeast, and viruses. The origin of
replication from the plasmid pBR322 is suitable for most
Gram-negative bacteria, the 2.mu. plasmid origin is suitable for
yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or
BPV) are useful for cloning vectors in mammalian cells. Generally,
the origin of replication component is not needed for mammalian
expression vectors (the SV40 origin may typically be used only
because it contains the early promoter).
(iii) Selection Gene Component
[0137] Expression and cloning vectors may contain a selection gene,
also termed a selectable marker. Typical selection genes encode
proteins that (a) confer resistance to antibiotics or other toxins,
e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b)
complement auxotrophic deficiencies, or (c) supply critical
nutrients not available from complex media, e.g., the gene encoding
D-alanine racemase for Bacilli.
[0138] One example of a selection scheme utilizes a drug to arrest
growth of a host cell. Those cells that are successfully
transformed with a heterologous gene produce a protein conferring
drug resistance and thus survive the selection regimen. Examples of
such dominant selection use the drugs neomycin, mycophenolic acid
and hygromycin.
[0139] Another example of suitable selectable markers for mammalian
cells are those that enable the identification of cells competent
to take up the antibody nucleic acid, such as DHFR, thymidine
kinase, metallothionein-I and -II, preferably primate
metallothionein genes, adenosine deaminase, ornithine
decarboxylase, etc.
[0140] For example, cells transformed with the DHFR selection gene
are first identified by culturing all of the transformants in a
culture medium that contains methotrexate (Mtx), a competitive
antagonist of DHFR. An appropriate host cell when wild-type DHFR is
employed is the Chinese hamster ovary (CHO) cell line deficient in
DHFR activity.
[0141] Alternatively, host cells (particularly wild-type hosts that
contain endogenous DHFR) transformed or co-transformed with DNA
sequences encoding antibody, wild-type DHFR protein, and another
selectable marker such as aminoglycoside 3'-phosphotransferase
(APH) can be selected by cell growth in medium containing a
selection agent for the selectable marker such as an
aminoglycosidic antibiotic, e.g., kanamycin, neomycin, or G418. See
U.S. Pat. No. 4,965,199.
[0142] A suitable selection gene for use in yeast is the trp1 gene
present in the yeast plasmid YRp7 (Stinchcomb et al., Nature,
282:39 (1979)). The trp1 gene provides a selection marker for a
mutant strain of yeast lacking the ability to grow in tryptophan,
for example, ATCC No. 44076 or PEP4-1. Jones, Genetics, 85:12
(1977). The presence of the trp1 lesion in the yeast host cell
genome then provides an effective environment for detecting
transformation by growth in the absence of tryptophan. Similarly,
Leu2-deficient yeast strains (ATCC 20,622 or 38,626) are
complemented by known plasmids bearing the Leu2 gene.
[0143] In addition, vectors derived from the 1.6 .mu.m circular
plasmid pKD1 can be used for transformation of Kluyveromyces
yeasts. Alternatively, an expression system for large-scale
production of recombinant calf chymosin was reported for K. lactis.
Van den Berg, Bio/Technology, 8:135 (1990). Stable multi-copy
expression vectors for secretion of mature recombinant human serum
albumin by industrial strains of Kluyveromyces have also been
disclosed. Fleer et al., Bio/Technology, 9:968-975 (1991).
(iv) Promoter Component
[0144] Expression and cloning vectors usually contain a promoter
that is recognized by the host organism and is operably linked to
the antibody nucleic acid. Promoters suitable for use with
prokaryotic hosts include the phoA promoter, .beta.-lactamase and
lactose promoter systems, alkaline phosphatase, a tryptophan (trp)
promoter system, and hybrid promoters such as the tac promoter.
However, other known bacterial promoters are suitable. Promoters
for use in bacterial systems also will contain a Shine-Dalgarno
(S.D.) sequence operably linked to the DNA encoding the
antibody.
[0145] Promoter sequences are known for eukaryotes. Virtually all
eukaryotic genes have an AT-rich region located approximately 25 to
30 bases upstream from the site where transcription is initiated.
Another sequence found 70 to 80 bases upstream from the start of
transcription of many genes is a CNCAAT region where N may be any
nucleotide. At the 3' end of most eukaryotic genes is an AATAAA
sequence that may be the signal for addition of the poly A tail to
the 3' end of the coding sequence. All of these sequences are
suitably inserted into eukaryotic expression vectors.
[0146] Examples of suitable promoting sequences for use with yeast
hosts include the promoters for 3-phosphoglycerate kinase or other
glycolytic enzymes, such as enolase, glyceraldehyde-3-phosphate
dehydrogenase, hexokinase, pyruvate decarboxylase,
phosphofructokinase, glucose-6-phosphate isomerase,
3-phosphoglycerate mutase, pyruvate kinase, triosephosphate
isomerase, phosphoglucose isomerase, and glucokinase.
[0147] Other yeast promoters, which are inducible promoters having
the additional advantage of transcription controlled by growth
conditions, are the promoter regions for alcohol dehydrogenase 2,
isocytochrome C, acid phosphatase, degradative enzymes associated
with nitrogen metabolism, metallothionein,
glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible
for maltose and galactose utilization. Suitable vectors and
promoters for use in yeast expression are further described in EP
73,657. Yeast enhancers also are advantageously used with yeast
promoters.
[0148] Antibody transcription from vectors in mammalian host cells
is controlled, for example, by promoters obtained from the genomes
of viruses such as polyoma virus, fowlpox virus, adenovirus (such
as Adenovirus 2), bovine papilloma virus, avian sarcoma virus,
cytomegalovirus, a retrovirus, hepatitis-B virus and most
preferably Simian Virus 40 (SV40), from heterologous mammalian
promoters, e.g., the actin promoter or an immunoglobulin promoter,
from heat-shock promoters, provided such promoters are compatible
with the host cell systems.
[0149] The early and late promoters of the SV40 virus are
conveniently obtained as an SV40 restriction fragment that also
contains the SV40 viral origin of replication. The immediate early
promoter of the human cytomegalovirus is conveniently obtained as a
HindIII E restriction fragment. A system for expressing DNA in
mammalian hosts using the bovine papilloma virus as a vector is
disclosed in U.S. Pat. No. 4,419,446. A modification of this system
is described in U.S. Pat. No. 4,601,978. See also Reyes et al.,
Nature 297:598-601 (1982) on expression of human .beta.-interferon
cDNA in mouse cells under the control of a thymidine kinase
promoter from herpes simplex virus. Alternatively, the rous sarcoma
virus long terminal repeat can be used as the promoter.
(v) Enhancer Element Component
[0150] Transcription of a DNA encoding the antibody of this
invention by higher eukaryotes is often increased by inserting an
enhancer sequence into the vector. Many enhancer sequences are now
known from mammalian genes (globin, elastase, albumin,
.alpha.-fetoprotein, and insulin). Typically, however, one will use
an enhancer from a eukaryotic cell virus. Examples include the SV40
enhancer on the late side of the replication origin (bp 100-270),
the cytomegalovirus early promoter enhancer, the polyoma enhancer
on the late side of the replication origin, and adenovirus
enhancers. See also Yaniv (1982) Nature 297:17-18 on enhancing
elements for activation of eukaryotic promoters. The enhancer may
be spliced into the vector at a position 5' or 3' to the
antibody-encoding sequence, but is preferably located at a site 5'
from the promoter.
(vi) Transcription Termination Component
[0151] Expression vectors used in eukaryotic host cells (yeast,
fungi, insect, plant, animal, human, or nucleated cells from other
multicellular organisms) will also contain sequences necessary for
the termination of transcription and for stabilizing the mRNA. Such
sequences are commonly available from the 5' and, occasionally 3',
untranslated regions of eukaryotic or viral DNAs or cDNAs. These
regions contain nucleotide segments transcribed as polyadenylated
fragments in the untranslated portion of the mRNA encoding the
antibody. One useful transcription termination component is the
bovine growth hormone polyadenylation region. See WO94/11026 and
the expression vector disclosed therein.
(vii) Selection and Transformation of Host Cells
[0152] Suitable host cells for cloning or expressing the DNA in the
vectors herein are the prokaryote, yeast, or higher eukaryote cells
described above. Suitable prokaryotes for this purpose include
eubacteria, such as Gram-negative or Gram-positive organisms, for
example, Enterobacteriaceae such as Escherichia, e.g., E. coli,
Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g.,
Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and
Shigella, as well as Bacilli such as B. subtilis and B.
licheniformis (e.g., B. licheniformis 41P disclosed in DD 266,710
published 12 Apr. 1989), Pseudomonas such as P. aeruginosa, and
Streptomyces. One preferred E. coli cloning host is E. coli 294
(ATCC 31,446), although other strains such as E. coli B, E. coli
X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable.
These examples are illustrative rather than limiting.
[0153] In addition to prokaryotes, eukaryotic microbes such as
filamentous fungi or yeast are suitable cloning or expression hosts
for antibody-encoding vectors. Saccharomyces cerevisiae, or common
baker's yeast, is the most commonly used among lower eukaryotic
host microorganisms. However, a number of other genera, species,
and strains are commonly available and useful herein, such as
Schizosaccharomyces pombe; Kluyveromyces hosts such as, e.g., K.
lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K.
wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum
(ATCC 36,906), K. thermotolerans, and K. marxianus; yarrowia (EP
402,226); Pichia pastoris (EP 183,070); Candida; Trichoderma reesia
(EP 244,234); Neurospora crassa; Schwanniomyces such as
Schwanniomyces occidentalis; and filamentous fungi such as, e.g.,
Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such
as A. nidulans and A. niger.
[0154] Suitable host cells for the expression of glycosylated
antibody are derived from multicellular organisms. Examples of
invertebrate cells include plant and insect cells. Numerous
baculoviral strains and variants and corresponding permissive
insect host cells from hosts such as Spodoptera frugiperda
(caterpillar), Aedes aegypti (mosquito), Aedes albopictus
(mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori
have been identified. A variety of viral strains for transfection
are publicly available, e.g., the L-1 variant of Autographa
californica NPV and the Bm-5 strain of Bombyx mori NPV, and such
viruses may be used as the virus herein according to the present
invention, particularly for transfection of Spodoptera frugiperda
cells. Plant cell cultures of cotton, corn, potato, soybean,
petunia, tomato, and tobacco can also be utilized as hosts.
[0155] However, interest has been greatest in vertebrate cells, and
propagation of vertebrate cells in culture (tissue culture) has
become a routine procedure. Examples of useful mammalian host cell
lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC
CRL 1651); human embryonic kidney line (293 or 293 cells subcloned
for growth in suspension culture, Graham et al., J. Gen Virol.
36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10);
Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al., Proc. Natl.
Acad. Sci. USA 77:4216 (1980)); mouse sertoli cells (TM4, Mather,
Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL
70); African green monkey kidney cells (VERO-76, ATCC CRL-1587);
human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney
cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC
CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells
(Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51);
TR1 cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982));
MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).
[0156] Host cells are transformed with the above-described
expression or cloning vectors for antibody production and cultured
in conventional nutrient media modified as appropriate for inducing
promoters, selecting transformants, or amplifying the genes
encoding the desired sequences.
(viii) Culturing the Host Cells
[0157] The host cells used to produce the antibody of this
invention may be cultured in a variety of media. Commercially
available media such as Ham's F10 (Sigma), Minimal Essential Medium
((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's
Medium ((DMEM), Sigma) are suitable for culturing the host cells.
In addition, any of the media described in Ham et al., Meth. Enz.
58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S.
Pat. No. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469;
WO 90/03430; WO 87/00195; or U.S. Pat. No. Re. 30,985 may be used
as culture media for the host cells. Any of these media may be
supplemented as necessary with hormones and/or other growth factors
(such as insulin, transferrin, or epidermal growth factor), salts
(such as sodium chloride, calcium, magnesium, and phosphate),
buffers (such as HEPES), nucleotides (such as adenosine and
thymidine), antibiotics (such as GENTAMYCIN.TM. drug), trace
elements (defined as inorganic compounds usually present at final
concentrations in the micromolar range), and glucose or an
equivalent energy source. Any other necessary supplements may also
be included at appropriate concentrations that would be known to
those skilled in the art. The culture conditions, such as
temperature, pH, and the like, are those previously used with the
host cell selected for expression, and will be apparent to the
ordinarily skilled artisan.
(ix) Antibody Purification
[0158] When using recombinant techniques, the antibody can be
produced intracellularly, in the periplasmic space, or directly
secreted into the medium. If the antibody is produced
intracellularly, as a first step, the particulate debris, either
host cells or lysed fragments, is removed, for example, by
centrifugation or ultrafiltration. Carter et al., Bio/Technology
10:163-167 (1992) describe a procedure for isolating antibodies
which are secreted to the periplasmic space of E. coli. Briefly,
cell paste is thawed in the presence of sodium acetate (pH 3.5),
EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min.
Cell debris can be removed by centrifugation. Where the antibody is
secreted into the medium, supernatants from such expression systems
are generally first concentrated using a commercially available
protein concentration filter, for example, an Amicon or Millipore
Pellicon ultrafiltration unit. A protease inhibitor such as PMSF
may be included in any of the foregoing steps to inhibit
proteolysis and antibiotics may be included to prevent the growth
of adventitious contaminants.
[0159] The antibody composition prepared from the cells can be
purified using, for example, hydroxylapatite chromatography, gel
electrophoresis, dialysis, and affinity chromatography, with
affinity chromatography being the preferred purification technique.
The suitability of protein A as an affinity ligand depends on the
species and isotype of any immunoglobulin Fc domain that is present
in the antibody. Protein A can be used to purify antibodies that
are based on human .gamma.1, .gamma.2, or .gamma.4 heavy chains
(Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is
recommended for all mouse isotypes and for human .gamma.3 (Guss et
al., EMBO J. 5:15671575 (1986)). The matrix to which the affinity
ligand is attached is most often agarose, but other matrices are
available. Mechanically stable matrices such as controlled pore
glass or poly(styrenedivinyl)benzene allow for faster flow rates
and shorter processing times than can be achieved with agarose.
Where the antibody comprises a C.sub.H3 domain, the Bakerbond
ABX.TM. resin (J. T. Baker, Phillipsburg, N.J.) is useful for
purification. Other techniques for protein purification such as
fractionation on an ion-exchange column, ethanol precipitation,
Reverse Phase HPLC, chromatography on silica, chromatography on
heparin SEPHAROSE.TM. chromatography on an anion or cation exchange
resin (such as a polyaspartic acid column), chromatofocusing,
SDS-PAGE, and ammonium sulfate precipitation are also available
depending on the antibody to be recovered.
[0160] Following any preliminary purification step(s), the mixture
comprising the anti-VEGF antibody and contaminants may be subjected
to low pH hydrophobic interaction chromatography using an elution
buffer at a pH between about 2.5-4.5, preferably performed at low
salt concentrations (e.g., from about 0-0.25M salt).
III. Pharmaceutical Formulations
[0161] Therapeutic formulations of the antibodies used in
accordance with the present 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.
[0162] 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.
[0163] 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).
[0164] The formulations to be used for in vivo administration must
be sterile. This is readily accomplished by filtration through
sterile filtration membranes.
[0165] 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
microcapsules. 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 .gamma. 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.
IV. Therapeutic Uses of Anti-VEGF Antibodies
[0166] It is contemplated that, according to the present invention,
the anti-VEGF antibodies may be used to treat various neoplasms or
non-neoplastic conditions characterized by pathological
angiogenesis. Non-neoplastic conditions that are amenable to
treatment include rheumatoid arthritis, psoriasis, atherosclerosis,
diabetic and other proliferative retinopathies including
retinopathy of prematurity, retrolental fibroplasia, neovascular
glaucoma, age-related macular degeneration, thyroid hyperplasias
(including Grave's disease), corneal and other tissue
transplantation, chronic inflammation, lung inflammation, nephrotic
syndrome, preeclampsia, ascites, pericardial effusion (such as that
associated with pericarditis), and pleural effusion.
[0167] The antibodies of the invention are preferably used in the
treatment of tumors in which angiogenesis plays an important role
in tumor growth, including cancers and benign tumors. Examples of
cancer to be treated herein include, but are not limited to,
carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More
particular examples of such cancers include 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, ovarian 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. More particularly, cancers that
are amenable to treatment by the antibodies of the invention
include breast cancer, colorectal cancer, rectal cancer, non-small
cell lung cancer, non-Hodgkins lymphoma (NHL), renal cell cancer,
prostate cancer, liver cancer, pancreatic cancer, soft-tissue
sarcoma, kaposi's sarcoma, carcinoid carcinoma, head and neck
cancer, melanoma, ovarian cancer, mesothelioma, and multiple
myeloma. More preferably, the methods of the invention are used to
treat colorectal cancer in a human patient.
[0168] The present 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.
Combination Therapies
[0169] It is contemplated that when used to treat various diseases
such as tumors, the antibodies of the invention can be combined
with other therapeutic agents suitable for the same or similar
diseases. When used for treating cancer, antibodies of the present
invention may be used in combination with conventional cancer
therapies, such as surgery, radiotherapy, chemotherapy or
combinations thereof.
[0170] 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). Preferably, 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.
[0171] 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 preferred 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.
Chemotherapeutic Agents
[0172] In certain aspects, the present invention provides a method
of treating cancer, by administering effective amounts of an
anti-VEGF antibody and one or more chemotherapeutic agents to a
patient susceptible to, or diagnosed with, 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".
[0173] As will be understood by those of ordinary skill in the art,
the appropriate doses of chemotherapeutic agents will be generally
around those already employed in clinical therapies wherein 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.
[0174] By way of example only, standard chemotherapy treatments for
metastatic colorectal cancer are described herein below.
[0175] In one preferred embodiment, the methods of the invention
are used to treat colorectal cancer including metastatic colorectal
cancer. Colorectal cancer is the third most common cause of cancer
mortality in the United States. It was estimated that approximately
129,000 new cases of colorectal cancer would be diagnosed and
56,000 deaths would occur due to colorectal cancer in the United
States in 1999, Landis et al., Cancer J. Clin. 49:8-31 (1999).
Approximately 70% of colorectal cancer patients present with
disease that is potentially curable by surgical resection, August
et al., Cancer Metastasis Rev 3:303-24 (1984). However, the
prognosis for the 30% who present with advanced or metastatic
disease and for the 20% who relapse following resection is poor.
The median survival for those with metastatic disease is 12-14
months, Advanced Colorectal Cancer Meta-Analysis Project, J Clin
Oncol 10:896-903 (1992).
[0176] The standard treatment for metastatic colorectal cancer in
the United States has been until recently chemotherapy with
5-fluorouracil (5-FU) plus a biochemical modulator of 5-FU,
leucovorin, Advanced Colorectal Cancer Meta-Analysis Project, J
Clin Oncol 10:896-903 (1992); Moertel N Engl J Med 330:1136-42
(1994). The combination of 5-FU/leucovorin provides infrequent,
transient shrinkage of colorectal tumors but has not been
demonstrated to prolong survival compared with 5-FU alone (Advanced
Colorectal Cancer Meta-Analysis Project, J Clin Oncol 10:896-903
(1992)), and 5-FU has not been demonstrated to prolong survival
compared with an ineffective therapy plus best supportive care,
Ansfield et al. Cancer 39:34-40 (1977). The lack of a demonstrated
survival benefit for 5-FU/leucovorin may be due in part to
inadequately sized clinical trials. In a large randomized trial of
patients receiving adjuvant chemotherapy for resectable colorectal
cancer, 5-FU/leucovorin demonstrated prolonged survival compared
with lomustine (MeCCNU), vincristine, and 5-FU (MOF; Wolmark et al.
J Clin Oncol 11:1879-87 (1993).
[0177] In the United States, 5-FU/leucovorin chemotherapy is
commonly administered according to one of two schedules: the Mayo
Clinic and Roswell Park regimens. The Mayo Clinic regimen consists
of an intensive course of 5-FU plus low-dose leucovorin (425 mg/m2
5-FU plus 20 mg/m2 leucovorin administered daily by intravenous
[IV] push for 5 days, with courses repeated at 4- to 5-week
intervals), Buroker et al. J Clin Oncol 12:14-20 (1994). The
Roswell Park regimen consists of weekly 5-FU plus high-dose
leucovorin (500-600 mg/m2 5-FU administered by IV push plus 500
mg/m2 leucovorin administered as a 2-hour infusion weekly for 6
weeks, with courses repeated every 8 weeks), Petrelli et al., J
Clin Oncol 7:1419-26 (1989). Clinical trials comparing the Mayo
Clinic and Roswell Park regimens have not demonstrated a difference
in efficacy but have been underpowered to do so, Buroker et al., J
Clin Oncol 12:14-20 (1994); Poon et al., J Clin Oncol 7:1407-18
(1989). The toxicity profiles of the two regimens are different,
with the Mayo Clinic regimen resulting in more leukopenia and
stomatitis and the Roswell Park regimen resulting in more frequent
diarrhea. Patients with newly diagnosed metastatic colorectal
cancer receiving either regimen can expect a median time to disease
progression of 4-5 months and a median survival of 12-14 months,
Petrelli et al., J Clin Oncol 7:1419-26 (1989); Advanced Colorectal
Cancer Meta-Analysis Project, J Clin Oncol 10:896-903 (1992);
Buroker et al., J Clin Oncol 12:14-20 (1994); Cocconi et al., J
Clin Oncol 16:2943-52 (1998).
[0178] Recently, a new first-line therapy for metastatic colorectal
cancer has emerged. Two randomized clinical trials, each with
approximately 400 patients, evaluated irinotecan in combination
with 5-FU/leucovorin, Saltz et al., Proc ASCO 18:233a (1999);
Douillard et al., Lancet 355:1041-7 (2000). In both studies, the
combination of irinotecan/5-FU/leucovorin demonstrated
statistically significant increases in survival (of 2.2 and 3.3
months), time to disease progression and response rates as compared
with 5-FU/leucovorin alone. The benefits of irinotecan came at a
price of increased toxicity: addition of irinotecan to
5-FU/leucovorin was associated with an increased incidence of
National Cancer Institute Common Toxicity Criteria (NCI-CTC) Grade
3/4 diarrhea, Grade 3/4 vomiting, Grade 4 neutropenia, and asthenia
compared with 5-FU/leucovorin alone. There is also evidence showing
that single-agent irinotecan prolongs survival in the second-line
setting, Cunningham et al., Lancet 352:1413-18 (1998); Rougier et
al., Lancet 352:1407-12 (1998). Two randomized studies have
demonstrated that irinotecan prolongs survival in patients who have
progressed following 5-FU therapy. One study compared irinotecan to
best supportive care and showed a 2.8-month prolongation of
survival; the other study compared irinotecan with infusional 5-FU
and showed a 2.2-month prolongation of survival. The question of
whether irinotecan has more effect on survival in the first- or
second-line setting has not been studied in a well-controlled
fashion.
Dosage and Administration
[0179] The antibodies and chemotherapeutic agents of the invention
are administered to 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. Intravenous or
subcutaneous administration of the antibody is preferred.
[0180] In one embodiment, the treatment of the present invention
involves the combined administration of an anti-VEGF antibody and
one or more chemotherapeutic agents. The present invention
contemplates administration of cocktails of different
chemotherapeutic agents. The combined administration includes
coadministration, 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. 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 antibody or may be
given simultaneously therewith.
[0181] For the prevention or treatment of disease, the appropriate
dosage of antibody will depend on the type of disease to be
treated, as defined above, the severity and course of the disease,
whether the antibody is administered for preventive or therapeutic
purposes, previous therapy, the patient's clinical history and
response to the antibody, and the discretion of the attending
physician. The antibody is suitably administered to the patient at
one time or over a series of treatments. In a combination therapy
regimen, the compositions of the present invention are administered
in a therapeutically effective or synergistic amount. As used
herein, a therapeutically effective amount is such that
co-administration of anti-VEGF antibody and one or more other
therapeutic agents, or administration of a composition of the
present invention, results in reduction or inhibition of the
targeting disease or condition. A therapeutically synergistic
amount is that amount of anti-VEGF antibody and one or more other
therapeutic agents necessary to synergistically or significantly
reduce or eliminate conditions or symptoms associated with a
particular disease.
[0182] Depending on the type and severity of the disease, about 1
.mu.g/kg to 50 mg/kg (e.g. 0.1-20 mg/kg) of antibody 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. For repeated administrations over several days or longer,
depending on the condition, the treatment is sustained until a
desired suppression of disease symptoms occurs. However, other
dosage regimens may be useful. In a preferred aspect, the antibody
of the invention is administered every two to three weeks, at a
dose ranged from about 5 mg/kg to about 15 mg/kg. More preferably,
such dosing regimen is used in combination with a chemotherapy
regimen as the first line therapy for treating metastatic
colorectal cancer. In some aspects, the chemotherapy regimen
involves the traditional high-dose intermittent administration. In
some other aspects, the chemotherapeutic agents are administered
using smaller and more frequent doses without scheduled breaks
("metronomic chemotherapy"). The progress of the therapy of the
invention is easily monitored by conventional techniques and
assays.
[0183] Further information about suitable dosages is provided in
the Example below.
Efficacy of the Treatment
[0184] The main advantage of the treatment of the present 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 may require unique measures and
definitions of clinical responses to drugs. 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, novel approaches to determining
efficacy of an anti-angiogenic therapy should be employed,
including for example, measurement of plasma or urinary markers of
angiogenesis and measurement of response through radiological
imaging.
[0185] In one embodiment, the present invention can be used for
increasing the duration of survival of a human patient susceptible
to or diagnosed with a cancer. Duration of survival is defined as
the time from first administration of the drug to death. In a
preferred aspect, the anti-VEGF antibody of the invention is
administered to the human patient in combination with one or more
chemotherapeutic agents, thereby the duration of survival of the
patient is effectively increased as compared to a chemotherapy
alone. For example, patient group treated with the anti-VEGF
antibody combined with a chemotherapeutic cocktail of at least two,
preferably three, chemotherapeutic agents may have a median
duration of survival that is at least about 2 months, preferably
between about 2 and about 5 months, longer than that of the patient
group treated with the same chemotherapeutic cocktail alone, said
increase being statistically significant. Duration of survival can
also be measured by stratified hazard ratio (HR) of the treatment
group versus control group, which represents the risk of death for
a patient during the treatment. Preferably, a combination treatment
of anti-VEGF antibody and one or more chemotherapeutic agents
significantly reduces the risk of death by at least about 30%
(i.e., a stratified HR of about 0.70), preferably by at least about
35% (i.e., a stratified HR of about 0.65), when compared to a
chemotherapy alone.
[0186] In another embodiment, the present 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. In a preferred embodiment, the
combination treatment of the invention using anti-VEGF antibody and
one or more chemotherapeutic agents significantly increases
progression free survival by at least about 2 months, preferably by
about 2 to about 5 months, when compared to a treatment with
chemotherapy alone.
[0187] In yet another embodiment, the treatment of the present
invention significantly increases response rate in a group of human
patients susceptible to or diagnosed with a cancer who are treated
with various therapeutics. Response rate is defined as the
percentage of treated patients who responded to the treatment. In
one aspect, the combination treatment of the invention using
anti-VEGF antibody and one or more chemotherapeutic agents
significantly increases response rate in the treated patient group
compared to the group treated with chemotherapy alone, said
increase having a Chi-square p-value of less than 0.005.
[0188] In one aspect, the present invention provides methods for
increasing duration of response in a human patient or a group of
human patients susceptible to or diagnosed with a cancer. Duration
of response is defined as the time from the initial response to
disease progression. In a combination treatment of the invention
using anti-VEGF antibody and one or more chemotherapeutic agents, a
statistically significant increase of at least 2 months in duration
of response is obtainable and preferred.
Safety of the Treatment
[0189] The present invention provides methods of effectively
treating cancers without significant adverse effects to the human
patient subject to treatment. The clinical outcomes of the
treatment according to the invention are somewhat unexpected, in
that several adverse events thought to be associated with
anti-angiogenic therapies are not observed during the course of
treatments according to the present invention. For example,
previous clinical studies suggested that treatment with anti-VEGF
antibodies may cause thrombosis (fatal in certain case),
hypertension, proteinuria and epistaxis (bleeding). However,
combination therapy of the invention using anti-VEGF antibody
combined with a chemotherapy cocktail comprising at least two,
preferably three, chemotherapeutic agents does not significantly
increase incident occurrences of these adverse events, when
compared with the chemotherapy alone. Thus, the treatment of the
present invention unexpectedly contains side effects at acceptable
level, at the same time significantly improve anticancer
efficacy.
V. Articles of Manufacture
[0190] 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 a warning that the composition is not to be used in
combination with anthacycline-type chemotherapeutic agent, e.g.
doxorubicin, or epirubicin, or instructing the user of the
composition to administer the anti-VEGF antibody composition and an
antineoplastic composition to a patient.
Deposit of Materials
[0191] 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-00001 Antibody Designation ATCC No. Deposit Date A4.6.1
ATCC HB-10709 Mar. 29, 1991
[0192] The following examples are intended merely to illustrate the
practice of the present 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.
VI. Examples
Example 1
Addition of an Anti-VEGF Antibody to Bolus
Irinotecan/Fluorouracil/Leucovorin (IFL) in First Line Metastatic
Colorectal Cancer
[0193] A multicenter, Phase III, randomized, active-controlled
trial was conducted to evaluate the efficacy and safety of
bevacizumab when added to standard first-line chemotherapy used to
treat metastatic colorectal cancer. The trial enrolled over 900
patients with histologically confirmed, previously untreated,
bi-dimensionally measurable metastatic colorectal cancer.
Methods and Materials
Anti-VEGF Antibody Bevacizumab
[0194] The anti-VEGF antibody "Bevacizumab (BV)", also known as
"rhuMAb VEGF" or "Avastin.TM.", is a recombinant humanized
anti-VEGF monoclonal antibody generated according to Presta et al.
(1997) Cancer Res. 57:4593-4599. 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. U.S. Pat. No.
6,582,959; WO 98/45331. 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. Bevacizumab has a molecular mass
of about 149,000 daltons and is glycosylated.
[0195] Identities of the polypeptide and sites of glycosylation
were deduced from the amino acid composition and peptide map. The
size and charge characteristics of the molecule and the purity of
the clinical lots were demonstrated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis or capillary
electrophoresis non-gel sieving, isoelectric focusing, as well as
ion-exchange and size-exclusion chromatography. The activity of
bevacizumab was quantified by a binding enzyme-linked immunosorbent
assay or a kinase receptor assay for recombinant human VEGF.
[0196] bevacizumab was produced by recombinant DNA technology,
using a genetically engineered Chinese hamster ovary cell line. The
protein was purified from the cell culture medium by routine
methods of column chromatography and filtration. The final product
was tested for quality, identity, safety, purity, potency,
strength, and excipient/chemical composition according to U.S. Food
and Drug Administration guidelines. The purity of bevacizumab is
>95%. bevacizumab is supplied as a clear to slightly opalescent,
sterile liquid ready for parenteral administration.
Patient Selection
[0197] Eligible patients had histologically confirmed metastatic
colorectal carcinoma, with bidimensionally measurable disease.
Other inclusion criteria included an age of at least 18 years, an
Eastern Cooperative Oncology Group (ECOG) performance status of 0
or 1 (Oken et al. (1982) Am. J. Clin. Oncol. 5:649-55), a life
expectancy of more than three months, and written informed consent.
Adequate hematologic, hepatic, and renal function (including
urinary excretion of no more than 500 mg of protein per day) was
also required.
[0198] Exclusion criteria included prior chemotherapy or biologic
therapy for metastatic disease (adjuvant or radiosensitizing use of
fluoropyrimidines with or without leucovorin or levamisole more
than 12 months before study entry was permitted), receipt of
radiotherapy within 14 days before the initiation of study
treatment, major surgery within 28 days before the initiation
ofstudytreatment, clinically significant cardiovascular disease,
clinically detectable ascites, pregnancy or lactation, regular use
of aspirin (more than 325 mg per day) or other nonsteroidal
andinflammatory agents, preexisting bleeding diatheses or
coagulopathy or the need for full-dose anticoagulation, and known
central nervous system metastases.
Study Design
[0199] Eligible patients were assigned to treatment with the use of
a dynamic randomization algorithm that was designed to achieve
overall balance between groups; randomization was stratified
according to study center, baseline ECOG performance status (0 vs.
1), site of primary disease (colon vs. rectum), and number of
metastatic sites (one vs. more than one). Initially, patients were
randomly assigned in a 1:1:1 ratio to receive IFL plus placebo, IFL
plus bevacizumab, or fluorouracil and leucovorin plus bevacizumab
(Table 1), each of which was to continue until disease progression
or unacceptable adverse effects occurred or for a maximum of 96
weeks.
TABLE-US-00002 TABLE 1 First-Line Treatment Regimens* Treatment
Starting Dose Schedule Irinotecan 125 mg/m.sup.2 of body- Once
weekly for 4 wk; cycle Fluorouracil surface area repeated every 6
wk Leucovorin 500 mg/m.sup.2 Every 2 wk Placebo 20 mg/m.sup.2
Irinotecan 125 mg/m.sup.2 Once weekly for 4 wk; cycle Fluorouracil
500 mg/m.sup.2 repeated every 6 wk Leucovorin 20 mg/m.sup.2 Every 2
wk Bevacizumab 5 mg/kg of body weight Fluorouracil 500 mg/m.sup.2
Once weekly for 4 wk; cycle Leucovorin 500 mg/m.sup.2 repeated
every 8 wk Bevacizumab 5 mg/kg Every 2 wk *Treatment with
fluorouracil, leucovorin, and bevacizumab was discontinued after
the safety of adding bevacizumab to the regimen of irinotecan,
fluorouracil, and leucovorin was confirmed. Confirmation occurred
after the randomization of 313 patients. All drugs were given
intravenously.
[0200] An interim analysis was scheduled to be performed after 300
patients underwent randomization, at which time an unblinded,
independent data-monitoring committee was to assess the safety of
IFL plus bevacizumab, on the basis of all the available safety
information, including the number of deaths in each group, but in
the absence of information related to tumor response. If the
data-monitoring committee found no untoward adverse events
attributable to the addition of bevacizumab to IFL, the enrollment
of patients in the group assigned to receive fluorouracil and
leucovorin plus bevacizumab was to be discontinued, and additional
patients would be randomly assigned in a 1:1 ratio to receive
either IFL plus placebo or IFL plus bevacizumab. However, if the
data-monitoring committee concluded that the safety profile of IFL
plus bevacizumab was unacceptable, assignment to that treatment was
to be discontinued, and patients would instead be randomly assigned
in a 1:1 ratio to receive either the combination of fluorouracil
and leucovorin plus bevacizumab or IFL plus placebo.
[0201] Tumor responses and progression were determined with the use
of the Response Evaluation Criteria in Solid Tumors. Therasse et
al. (2000) J. Natl. Cancer Inst. 92:205-16. At the time of disease
progression, the treatment assignment was revealed and patients
could be offered second-line treatment. Such patients in the group
assigned to bevacizumab-containing treatment had the option to
continue bevacizumab during this second-line treat-ment. No
crossovers were allowed in the group given IFL plus placebo.
Patients assigned to a treatment containing bevacizumab who had no
signs of progressive disease at the end of the 96-week study period
could continue to receive bevacizumab in a separate extension
study. Patients in a group receiving bevacizumab who had a
confirmed complete response or unacceptable adverse effects from
chemotherapy could discontinue chemotherapy and receive bevacizumab
alone.
[0202] Bevacizumab (or placebo) was administered concomitantly with
chemotherapy. Doses of bevacizumab and chemotherapy were
recalculated if a patient's weight changed by at least 10 percent
during the study. Standard intracycle and intercycle dose
modifications of irinotecan and fluorouracil (according to the
package insert)' were permitted in patients with treatment-related
adverse events. The doses of leucovorin and bevacizumab were not
altered.
[0203] In the analysis of survival and subsequent treatment, all
patients were followed until death, loss to follow-up, or
termination of the study.
Assessments
[0204] After the baseline evaluation, tumor status was assessed
every 6 weeks for the first 24 weeks of the study and then every 12
weeks for the remainder of therapy. All complete and partial
responses required confirmation at least four weeks after they were
first noted.
[0205] Safety was assessed on the basis of reports of adverse
events, laboratory-test results, and vital sign measurements.
Adverse events were categorized according to the Common Toxicity
Criteria of the National Cancer Institute, version 2, in which a
grade of 1 indicates mild adverse events, a grade of 2 moderate
adverse events, a grade of 3 serious adverse events, and a grade of
4 life-threatening adverse events. Prespecified safety measures
included the incidence of all adverse events, all serious adverse
events, and adverse events that have been associated with
bevacizumab--hypertension, thrombosis, bleeding of grade 3 or 4,
and proteinuria--as well as diarrhea of grade 3 or 4, and changes
from baseline in various laboratory values and vital signs.
[0206] To monitor the safety of the regimen of IFL plus placebo and
of IFL plus bevacizumab, the incidence of death, serious adverse
events, diarrhea of grade 3 or 4, bleeding of grade 3 or 4 from any
source, and thrombosis was monitored during the study in an
un-blinded fashion by the data-safety monitoring committee until
the completion of recruitment or the time of the interim analysis
of efficacy, whichever came first.
Statistical Analysis
[0207] The primary outcome measure was the duration of overall
survival; survival was measured without regard to subsequent
treatments. There was no crossover between groups, however.
Survival analysis techniques such as the Kaplan-Meier method,
log-rank test, and Cox proportional hazards model were used.
Secondary outcome measures were progression-free survival,
objective response rates (complete and partial responses), the
duration of responses, and the quality of life.
[0208] For patients who were alive at the time of analysis, data on
survival were censored at the time of the last contact.
Progression-free survival was defined as the time from
randomization to progression or death during the study, with death
during the study defined as any death that occurred within 30 days
after the last dose of bevacizumab or chemotherapy. For patients
without disease progression at the time of the final analysis, data
on progression-free survival were censored at the last assessment
of tumor status or on day 0 if no further assessment was performed
after baseline. Patients without adequate follow-up data were
categorized as having no response.
[0209] To detect a hazard ratio of 0.75 for death in the group
given IFL plus bevacizumab as compared with the control group,
approximately 385 deaths were required. All calculations were
performed with the log-rank test and involved two-sided P values,
with an alpha value of 0.05, a statistical power of 80 percent, and
one interim analysis of efficacy.
[0210] Interim analyses were conducted in an un-blinded fashion. An
interim analysis of safety was conducted after the random
assignment of approximately 100 patients to each group. A second
interim analysis of safety and efficacy was performed after 193
deaths had occurred (half the number of required events).
[0211] Efficacy analyses were performed according to the
intention-to-treat principle. Safety analyses included all patients
who received at least one dose of study medication.
Results
Characteristics of the Patients
[0212] During a period of about twenty months, 923 patients
underwent randomization at 164 sites in the United States,
Australia, and New Zealand. After 313 patients had been randomly
assigned to one of the three groups--100 to IFL plus placebo, 103
to IFL plus bevacizumab, and 110 to fluorouracil, leucovorin, and
bevacizumab--assignment to the group given fluorouracil,
leucovorin, and bevacizumab was halted (the results in this group
are not reported). This step was required by the protocol after the
first formal interim analysis of safety concluded that the regimen
of IFL plus bevacizumab had an acceptable safety profile and that
assignment to this group could continue.
[0213] The intention-to-treat analysis of the primary end point of
overall survival included 411 patients in the group given IFL plus
placebo and 402 patients in the group given IFL plus bevacizumab.
Table 2 shows selected demographic and baseline characteristics,
which were well balanced between the groups. Similar numbers of
patients in each group had previously undergone surgery or received
radiation therapy or adjuvant chemotherapy for colorectal
cancer.
Treatment
[0214] The median duration of therapy was 27.6 weeks in the group
given IFL plus placebo and 40.4 weeks in the group given IFL plus
bevacizumab. The percentage of the planned dose of irinotecan that
was given was similar in the two groups (78 percent in the group
given IFL plus placebo and 73 percent in the group given IFL plus
bevacizumab).
[0215] As of the date of data cutoff, 33 patients in the group
given IFL plus placebo and 71 in the group given IFL plus
bevacizumab were still taking their assigned initial therapy. The
rates of use of second-line therapies that may have affected
survival, such as oxaliplatin or metastasectomy, were well balanced
between the two groups. In both groups, approximately 50 percent of
patients received some form of second line therapy; 25 percent of
all patients received oxaliplatin, and less than 2 percent of
patients underwent metastasectomy.
TABLE-US-00003 TABLE 2 Selected Demographic and Baseline
Characteristics.* IFL IFL plus Placebo plus Bevacizumab
Characteristic (N = 411) (N = 402) Sex (%) MALE 60 59 FEMALE 40 41
MEAN AGE (YR) 59.2 59.5 Race (%) White 80 79 Black 11 12 Other 9 9
Location of center (%) United States 99 99 Australia or New Zealand
<1 <1 ECOG performance status (%) 0 55 58 1 44 41 2 <1
<1 Type of cancer (%) Colon 81 77 Rectal 19 23 Number of
metastatic sites (%) 1 39 37 >1 61 63 Prior cancer therapy (%)
Adjuvant chemotherapy 28 24 Radiation therapy 14 15 Median duration
of 4 4 metastatic disease (mo) *There were no significant
differences between groups. IFL denotes irinotecan, fluorouracil,
and leucovorin, and ECOG Eastern Cooperative Oncology Group.
Efficacy
[0216] The median duration of overall survival, the primary end
point, was significantly longer in the group given IFL plus
bevacizumab than in the group given IFL plus placebo (20.3 months
vs. 15.6 months), which corresponds to a hazard ratio for death of
0.66 (P<0.001) (Table 3 and FIG. 1), or a reduction of 34
percent in the risk of death in the bevacizumab group. The one-year
survival rate was 74.3 percent in the group given IFL plus
bevacizumab and 63.4 percent in the group given IFL plus placebo
(P<0.001). In the subgroup of patients who received second-line
treatment with oxaliplatin, the median duration of overall survival
was 25.1 months in the group given IFL plus bevacizumab and 22.2
months in the group given IFL plus placebo.
[0217] The addition of bevacizumab to IFL was associated with
increases in the median duration of progression-free survival (10.6
months vs. 6.2 months; hazard ratio for progression, 0.54, for the
comparison with the group given IFL plus placebo; P<0.001);
response rate (44.8 percent vs. 34.8 percent; P=0.004); and the
median duration of response (10.4 months vs. 7.1 months; hazard
ratio for progression, 0.62; P=0.001) (Table 3). FIG. 2 shows the
Kaplan-Meier estimates of progression free survival. Treatment
effects were consistent across prespecified subgroups, including
those defined according to age, sex, race, ECOG performance status,
location of the primary tumor, presence or absence of prior
adjuvant therapy, duration of metastatic disease, number of
metastatic sites, years since the diagnosis of colorectal cancer,
presence or absence of prior radiotherapy, baseline tumor burden,
and serum concentrations of albumin, alkaline phosphatase, and
lactate dehydrogenase.
TABLE-US-00004 TABLE 3 Analysis of Efficacy* IFL plus IFL plus End
Point Placebo Bevacizumab P Value Median survival (mo) 15.6 20.3
<0.001 Hazard ratio for death 0.66 One-year survival rate (%)
63.4 74.3 <0.001 Progression-free survival (mo) 6.2 10.6
<0.001 Hazard ratio for progression 0.54 Overall response rate
(%) 34.8 44.8 0.004 Complete response 2.2 3.7 Partial response 32.6
41.0 Median duration of response (mo) 7.1 10.4 0.001 Hazard ratio
for relapse 0.62 *IFL denotes irinotecan, fluorouracil, and
leucovorin.
Safety
[0218] Table 4 presents the incidence of selected grade 3 or 4
adverse events during the assigned treatment, without adjustment
for the median duration of therapy (27.6 weeks in the group given
IFL plus placebo and 40.4 weeks in the group given IFL plus
bevacizumab). The incidence of any grade 3 or 4 adverse events was
approximately 10 percentage points higher among patients receiving
IFL plus bevacizumab than among patients receiving IFL plus
placebo, largely because of an increase in the incidence of grade 3
hypertension (requiring treatment) and small increases in the
incidence of grade 4 diarrhea and leukopenia. However, there was no
significant difference in the incidence of adverse events leading
to hospitalization or to the discontinuation of study treatment or
in the 60-day rate of death from any cause.
TABLE-US-00005 TABLE 4 Selected Adverse Events.* IFL plus IFL plus
Placebo Bevacizumab (N = 397) (N = 393) Adverse Event percent Any
grade 3 or 4 adverse event 74.0 84.9** Adverse event leading to
hospitalization 39.6 44.9 Adverse event leading to discontinuation
of 7.1 8.4 treatment Adverse event leading to death 2.8 2.6 Death
within 60 days 4.9 3.0 Grade 3 or 4 leukopenia 31.1 37.0
Hypertension Any 8.3 22.4** Grade 3 2.3 11.0** Any thrombotic event
16.2 19.4 Deep thrombophletitis 6.3 8.9 Pulmonary embolus 5.1 3.6
Grade 3 or 4 bleeding 2.5 3.1 Proteinuria Any 21.7 26.5 Grade 2 5.8
3.1 Grade 3 0.8 0.8 Gastrointestinal perforation 0.0 1.5 *Data were
not adjusted for differences in the median duration of therapy
between the group given irinotecan, fluorouracil, and leucovorin
(IFL) plus placebo and the group given IFL plus bevacizumab (27.6
weeks vs. 40.4 weeks). **P < 0.01. Only patients who received at
least one study-drug treatment are included.
[0219] Phase 1 and 2 trials had identified hemorrhage,
thromboembolism, proteinuria, and hypertension as possible
bevacizumab-associated adverse effects. However, in the present
study, only the incidence of hypertension was clearly increased in
the group given IFL plus bevacizumab, as compared with the group
given IFL plus placebo. All episodes of hyperten-sion were
manageable with standard oral antihypertensive agents (e.g.,
calcium-channel blockers, angiotensin-converting-enzyme inhibitors,
and diuretics). There were no discontinuations of bevacizumab
therapy, hypertensive crises, or deaths related to hypertension in
the bevacizumab group.
[0220] Rates of grade 2 or 3 proteinuria (there were no episodes of
grade 4 proteinuria or nephrotic syndrome) and grade 3 or 4
bleeding from any cause were similar in the two groups, although
all three cases of grade 4 bleeding were in the group given IFL
plus bevacizumab. The incidence of all venous and arterial
thrombotic events was 19.4 percent in the group given IFL plus
bevacizumab and 16.2 percent in the group given IFL plus placebo
(P=0.26).
[0221] Gastrointestinal perforation occurred in six patients (1.5
percent) receiving IFL plus bevacizumab. One patient died as a
direct result of this event, whereas the other five recovered
(three of them were able to restart treatment without subsequent
complications). Of the six patients with a perforation, three had a
confirmed complete or partial response to IFL plus bevacizumab.
Factors other than the study treatment that may have been
associated with gastrointestinal perforation were colon surgery
within the previous two months in two patients and peptic-ulcer
disease in one patient.
[0222] The results of this phase III study provide direct support
for a broadly applicable use of antiangiogenic agents in the
treatment of cancer. The addition of bevacizumab, an anti-VEGF
antibody, to IFL chemotherapy conferred a clinically meaningful and
statistically significant improvement in cancer patients as
measured by, for example, overall survival, progression-free
survival, response rate and duration of response. The increase of
4.7 months in the median duration of survival attributable to
bevacizumab is as large as or larger than that observed in any
other phase 3 trial for the treatment of colorectal cancer.
Goldberg et al. (2004) J. Clin. Oncol. 22:23-30. The median
survival of 20.3 months in the bevacizumab-treated population
occurred in spite of the limited availability of oxaliplatin for
second-line therapy during this trial.
[0223] As compared with IFL alone, the regimen of IFL plus
bevacizumab increased progression-free survival from a median of
6.2 months to 10.6 months, the overall response rate from 34.8
percent to 44.8 percent, and the median duration of response from
7.1 months to 10.4 months. These improvements are clinically
meaningful. It was not predicted that the absolute improvement in
the response rate of 10 percent with IFL plus bevacizumab would
have been associated with an increase in survival of this
magnitude. This observation suggests that the primary mechanism of
bevacizumab is the inhibition of tumor growth, rather than
cytoreduction.
[0224] This clinical benefit was accompanied by a relatively modest
increase in side effects of treatment, which were easily managed.
There was an absolute increase of approximately 10 percent in the
overall incidence of grade 3 and 4 adverse effects, attributable
largely to hypertension requiring treatment, diarrhea, and
leukopenia. The 60-day rates of death from any cause,
hospitalization, and discontinuation of treatment were not
significantly increased by the addition of bevacizumab to IFL.
[0225] Previous phase 1 and 2 clinical trials suggested that
treatment with bevacizumab alone or with chemotherapy resulted in
an increased incidence of thrombosis, bleeding, proteinuria, and
hypertension. Kabbinavar et al. (2003) J. Clin. Oncol. 21:60-65;
Yang et al. (2003) New Engl. J. Med. 349:427-34. With the exception
of hypertension, an excess of these side effects was not found as
compared with their incidence in the group given IFL plus
placebo--thus highlighting the importance of randomized,
placebo-controlled studies for the evaluation of safety as well as
efficacy. One new potential adverse effect that occurred was
gastrointestinal perforation. This complication was uncommon and
had variable clinical presentations. Severe bowel complications,
particularly in patients with neutropenia, have been reported with
IFL and other chemotherapy regimens for colorectal cancer and in
one series, fistulas were re-ported in over 2 percent of patients
treated with fluorouracil-based regimens. Saltz et al. (2000) New
Engl. J. Med. 343:905-914; Rothenberg et al. (2001) J. Clin. Oncol.
19:3801-7; Tebbutt et al. (2003) Gut 52:568-73. No such events
occurred in the group given IFL plus placebo, whereas six cases
were observed in the group given IFL plus bevacizumab (1.5
percent), sometimes in the setting of overall tumor responses.
Although three of these six patients were able to restart treatment
without subsequent complications, one patient died and two
discontinued therapy permanently as a result of this
complication.
[0226] While previous animal studies and early phase clinical
trials have suggested uses of anti-angiogenic therapy for treating
cancer, the present study showed for the first time that using an
angiogenic inhibitor, such as an anti-VEGF antibody, indeed results
in statistically significant and clinically meaningful benefits for
cancer patients.
Example 2
Addition of Bevacizumab to Bolus 5-FU/Leucovorin in First-Line
Metastatic Colorectal Cancer
[0227] This randomized, phase II trial compared bevacizumab plus
5-fluorouracil and leucovorin (5-FU/LV) versus placebo plus 5-FU/LV
as first-line therapy in patients considered non-optimal candidates
for first-line irinotecan.
Patients and Methods
Patient Eligibility
[0228] Patients with histologically confirmed, previously
untreated, measurable metastatic colorectal cancer were eligible
if, in the judgment of the investigator, they were not optimal
candidates for first-line irinotecan-containing therapy and had at
least one of the following characteristics: age above 65 years,
ECOG PS of 1 or 2, serum albumin equal or less than 3.5 g/dL, or
prior radiotherapy to abdomen or pelvis. Patients were excluded if
they had undergone major surgical procedures or open biopsy, or had
experienced significant traumatic injury, within 28 days prior to
study entry; anticipated need for major surgery during the course
of the study; were currently using or had recently used therapeutic
anticoagulants (except as required for catheter patency),
thrombolytic therapy or chronic, daily treatment with aspirin 325
mg/day) or nonsteroidal anti-inflammatory medications; had a
serious, non-healing wound, ulcer, or bone fracture; had a history
or evidence of CNS metastases; were pregnant or lactating; or had
proteinuria or clinically significant impairment of renal function
at baseline. All patients provided written informed consent for
their participation.
Study Design and Treatments
[0229] An interactive voice response system was used to randomly
assign eligible patients to one of two treatment groups: 5-FU/LV
plus placebo or 5-FU/LV plus bevacizumab. A dynamic randomization
algorithm was utilized to achieve balance overall and within each
of the following categories: study center, baseline ECOG
performance status (0 vs. 1), site of primary disease (colon vs.
rectum), and number of metastatic sites (1 vs. >1). The 5-FU/LV
treatment, comprising LV 500 mg/m.sup.2 over 2 hours and 5-FU 500
mg/m.sup.2 as a bolus midway through the LV infusion (Roswell Park
regimen; Petrelli et al. (1989) J. Clin. Oncol. 7:1419-1426), was
administered weekly for the first 6 weeks of each 8-week cycle.
Chemotherapy was continued until study completion (96 weeks) or
disease progression. Bevacizumab 5 mg/kg or placebo was
administered every 2 weeks. Patients in the bevacizumab arm who had
a confirmed complete response or experienced unacceptable toxicity
as a result of chemotherapy treatment were allowed to discontinue
5-FU/LV and continue receiving bevacizumab alone as first-line
treatment. At the time of disease progression, patients were
unblinded to their treatment assignment and could receive any
second-line treatment at the discretion of the investigator. Only
patients who had been randomized to the bevacizumab group could
receive bevacizumab as a component of second-line treatment. After
completing the study, patients were followed for any subsequent
treatment and survival every 4 months until death, loss to
follow-up, or termination of the study.
Study Assessments
[0230] Patients underwent an assessment of tumor status at baseline
and at completion of every 8-week cycle using appropriate
radiographic techniques, typically spiral CT scanning Tumor
response, or progression, was determined by both the investigator
and an independent radiology facility (IRF) utilizing the Response
Evaluation Criteria in Solid Tumors. Therasse et al. (2000). The
IRF assessment was performed without knowledge of the treatment
assignment or investigator assessment. In addition, patients
completed the Functional Assessment of Cancer Therapy--Colorectal
(FACT-C), Version 4, a validated instrument for assessing quality
of life (QOL) in colorectal cancer patients, at baseline and prior
to each treatment cycle until disease progression. Ward et al.
(1999) Qual. Life Res. 8:181-195.
[0231] Safety was assessed from reports of adverse events,
laboratory test results, and vital sign measurements. Adverse
events and abnormal laboratory results were categorized using the
National Cancer Institute Common Toxicity Criteria (NCI-CTC),
Version 2. Prespecified safety measures included four adverse
events of special interest (hypertension, proteinuria, thrombosis,
and bleeding) based on findings of previous clinical trials of
bevacizumab.
Statistical Analysis
[0232] The primary outcome measure was duration of overall
survival. Secondary outcome measures included progression-free
survival, objective response rate (complete and partial), response
duration, and change in the FACT-C QOL score. Survival duration was
defined as the time from randomization to death. For patients alive
at the time of analysis, duration of survival was censored at the
date of last contact. Progression-free survival was defined as the
time from randomization to the earlier of disease progression or
death on study, defined as death from any cause within 30 days of
the last dose of study drug or chemotherapy. For patients alive
without disease progression at the time of analysis,
progression-free survival was censored at their last tumor
assessment, or day 1 (the first day of study treatment) if no
postbaseline assessment was performed. In the analysis of objective
response, patients without tumor assessments were categorized as
nonresponders. Disease progression and response analyses were based
on the IRF assessments. Change in quality of life was analyzed as
time to deterioration in QOL (TDQ), defined as the length of time
from randomization to a the earliest of a .gtoreq.3-point decrease
from baseline in colon-cancer specific FACT-C subscale score (CCS),
disease progression, or death on study. TDQ was also determined for
the TOI-C (sum of CCS, physical and functional well-being) and
total FACT-C for changes from baseline of 7 and 9 points,
respectively.
[0233] To detect a hazard ratio of 0.61 for death in the
5-FU/LV/bevacizumab group relative to the 5-FU/LV/placebo group,
approximately 133 deaths were required. A two-tailed, log-rank test
at the 0.05 level of significance with 80% power and two interim
analyses were assumed in the calculations. Interim analyses were
conducted by an unblinded, independent Data Monitoring Committee
(DMC). A safety interim analysis was conducted after 44 deaths and
a second safety and efficacy interim analysis was conducted after
89 deaths. The interim efficacy analysis was governed by a formal
group sequential stopping rule based on an O'Brien-Fleming spending
function. Kaplan-Meier methodology was applied to estimate the
median survival, progression free survival, and duration of
response time for each treatment group. Hazard ratios for the
bevacizumab group relative to the placebo group were determined
using the stratified Cox proportional hazards model. A two-sided
stratified log rank test was used to compare the two groups.
Stratified analyses included baseline ECOG performance status, site
of primary disease, and the number of metastatic sites. Objective
response rates were compared by the Chi-squared test. As
exploratory analyses, the Cox proportional hazards model was used
to estimate the effect of risk factors on modifications of
treatment effect for duration of survival and progression-free
survival. Efficacy analyses were performed on the intent-to-treat
population, defined as all randomized patients. Safety analyses
included all patients who received at least one dose of study
drug.
Results
Patient Characteristics
[0234] In a period of twenty three months, 209 patients were
randomized at 60 sites in the United States and Australia/New
Zealand. For the intent-to-treat analysis of the primary endpoint
(overall survival), there were 105 patients in the 5-FU/LV/placebo
group and 104 in the 5-FU/LV/bevacizumab group. Selected
demographic and baseline characteristics similar to those described
in Example 1 were reasonably balanced between treatment groups. Low
serum albumin 3.5 g/dL) at baseline was less common in the
bevacizumab group than in the placebo group.
Treatment
[0235] The median duration of therapy was 23 weeks in the
5-FU/LV/placebo group and 31 weeks in the 5-FU/LV/bevacizumab
group, and the 5-FU dose intensity (percentage of planned 5-FU
doses actually received) in the two groups was similar (92% vs.
84%) during the treatment course. As of the date of date cut-off, 1
patient in the 5-FU/LV/placebo group and 7 in the
5-FU/LV/bevacizumab group remained on the assigned initial therapy.
Subsequent therapies, which may have influenced survival, were used
in approximately 50% of patients in both groups, although more
patients in the 5-FU/LV/placebo group were treated with the active
agents irinotecan and oxaliplatin.
Efficacy
[0236] Overall survival, the primary endpoint, was longer in the
5-FU/LV/bevacizumab group (median, 16.6 months) than in the
5-FU/LV/placebo group (median, 12.9 months), demonstrating a trend
toward significance. The hazard ratio of death was estimated to be
0.79 (95% CI, 0.56 to 1.10; P=0.16; Table 5 and FIG. 4). The
addition of bevacizumab to 5-FU/LV was associated with increases in
median progression-free survival (9.2 vs. 5.5 months; hazard
ratio=0.50; 95% CI, 0.34 to 0.73; P=0.0002, Table 5 and FIG. 4),
response rate (26.0% vs. 15.2%, P=0.055), and median duration of
response (9.2 months vs. 6.8 months; hazard ratio=0.42; 95% CI,
0.15 to 1.17; P=0.088). A further analysis of treatment effect on
overall survival by baseline characteristics showed that patients
with low serum albumin 3.5 g/dL) at baseline appeared to derive a
significant survival benefit (hazard ratio=0.46; 95% CI, 0.29 to
0.74; P=0.001).
TABLE-US-00006 TABLE 5 Summary of Efficacy Analysis 5-FU/LV/
5-FU/LV/ Placebo Bevacizumab Efficacy Parameter (N = 105) (N = 104)
P-value Median survival (months) 12.9 16.6 Hazard ratio 0.79 0.160
95% CI 0.56 to 1.10 Progression-free survival (months) 5.5 9.2
Hazard ratio 0.50 0.0002 95% CI 0.34 to 0.73 Overall response rate
(%) 15.2 26.0 0.055 Complete response 0 0 Partial response 15.2
26.0 Duration of response (months) 6.8 9.2 Hazard ratio 0.42 0.088
95% CI 0.15 to 1.17 5-FU/LV = 5 fluorouracil/leucovorin
[0237] Bevacizumab treatment had no detrimental effect on quality
of life, and the TDQ results suggest a possible beneficial effect.
The median TDQ as measured by the CCS score was 3.0 months in the
5-FU/LV/placebo group and 3.1 months in the 5-FU/LV/bevacizumab
group (hazard ratio=0.79, P=0.188). The median TDQ for
placebo-treated and bevacizumab-treated patients as measured by
secondary TDQ measures was 2.3 and 3.2 months (TOI-C; hazard
ratio=0.71, P=0.048) and 2.6 and 3.6 months (total FACT-C; hazard
ratio=0.66, P=0.016).
Safety
[0238] A total of 204 patients (104 5-FU/LV/placebo and 100
5-FU/LV/bevacizumab) who received at least one dose of study drug
comprised the safety population. A 16% increase (71% versus 87%) in
total grade 3 and 4 toxicities was observed for patients receiving
bevacizumab. Adverse events leading to death or study
discontinuation were similar in the two groups, as were adverse
events known to be associated with 5-FU/LV (specifically, diarrhea
and leukopenia). Two patients, both in the 5-FU/LV/bevacizumab
group, experienced a bowel perforation event. These events occurred
at day 110 and day 338 of treatment, and both were determined to be
associated with a colonic diverticulum at surgical exploration. One
patient died as a result of this complication. Previous clinical
trials had suggested hemorrhage, thromboembolism, proteinuria, and
hypertension as possible bevacizumab-associated toxicities;
however, in this study, no increases were seen in venous
thrombosis, .gtoreq.grade 3 bleeding, or clinically significant
grade 3) proteinuria. Arterial thrombotic events (myocardial
infarction, stroke, or peripheral arterial thrombotic event)
occurred in 10 patients in the 5-FU/LV/bevacizumab group, compared
to 5 patients in the 5-FU/LV/placebo group.
[0239] The 5-FU/LV/placebo group had a higher 60-day all-cause
mortality compared to the 5-FU/LV/bevacizumab group (13.5% vs.
5.0%). Death due to disease progression in the first 60 days was
similar (5.8% vs. 4.0%) in the two groups. In the 5-FU/LV/placebo
group, deaths within the first 60 days not due to disease
progression were attributed to the following: heart failure (1),
sepsis (3), diarrhea (2), respiratory failure (1), and pulmonary
embolus (1). In the 5-FU/LV/bevacizumab group, the single early
death not due to disease progression was attributed to a myocardial
infarction.
[0240] The results of this clinical trial further demonstrate that
bevacizumab, a humanized monoclonal antibody against VEGF, provides
important clinical benefit when added to first-line chemotherapy
for the treatment of metastatic colorectal cancer. When compared
with 5-FU/LV alone, the addition of bevacizumab prolonged median
survival by 3.7 months, progression-free survival by 3.7 months,
and response duration by 2.4 months, and increased the response
rate by 11%.
[0241] These results should be viewed in the context of the study
population. Specifically selected were patients who were poor
candidates for first-line irinotecan-containing therapy, either
because of a low likelihood of benefit or a high likelihood of
treatment-associated toxicities. A careful analysis of the pivotal
irinotecan trials showed that clinical benefit from this agent was
confined to patients with a normal ECOG performance status
(PS=0).21, 22 Advanced age, prior pelvic radiation therapy,
impaired performance status, and low serum albumin have all been
reported to increase irinotecan-associated toxicities. 23-27
Patients with these characteristics are in need of alternative
therapeutic options. A retrospective subset analysis from a smaller
randomized phase II trial was previously conducted evaluating
bevacizumab and 5-FU/LV in CRC and noted bevacizumab provided a
substantial treatment effect in the subset of patients with
baseline PS1 or 2 (median survival, 6.3 months vs. 15.2 months), in
the subset aged 65 years (11.2 months vs. 17.7 months), and in the
subset with serum albumin <3.5 (8.1 months versus 14.1 months).
These results encouraged us to design the current trial,
specifically including a poor-prognosis study population and
powering the trial to detect a large treatment effect on survival.
We were largely successful in enrolling a population different from
that in the concurrently conducted pivotal trial of IFL/placebo
versus IFL/bevacizumab. Compared with the pivotal trial, patients
in the present trial had a higher median age (72 vs. 61 years) and
substantially more patients had a performance status >0 (72% vs.
43%) and albumin 3.5 mg/dL (46% vs. 33%).
[0242] Despite this high-risk study population, the regimen of
5-FU/LV/bevacizumab appeared to be well tolerated. The
well-described bevacizumab-associated adverse event of grade 3
hypertension was seen in 16% of the 5-FU/LV/bevacizumab group
versus 3% in the 5-FU/LV/placebo group. No cases of grade 4
hypertension occurred. Proteinuria of any grade was seen in 38% of
the 5-FU/LV/bevacizumab group versus 19% of the 5-FU/LV/placeb
group; however, only a single patient in the bevacizumab group
developed grade 3 proteinuria, and there were no cases of grade 4
proteinuria. No increases in grade 3 or 4 bleeding or venous
thrombotic events were seen in bevacizumab-treated patients. There
was an imbalance in the incidence of arterial thrombotic events:
10% in the 5-FU/LV/bevacizumab group compared with 4.8% in the
5-FU-/LV placebo group. A similar imbalance was noted in the
pivotal bevacizumab trial (1.0% in the IFL/placebo group and 3.3%
in the IFL/bevacizumab group). The more advanced age of the
population included in the present study may have contributed to a
higher overall incidence of this adverse event, however the
imbalance in both studies is noteworthy. Large, observational
safety trials may be required to further define the incidence and
potential risk factors for these, and other, uncommon adverse
events associated with bevacizumab therapy.
[0243] In summary, these data demonstrate that bevacizumab, when
combined with bolus 5-FU/LV, provides substantial clinical benefit
for patients with previously untreated metastatic colorectal cancer
who are deemed to be poor candidates for irinotecan-containing
therapy. Together with the pivotal trial results, these data
strengthen the evidence that bevacizumab-based, 5-FU/LV-containing
therapy should be considered a standard option for the initial
treatment of metastatic colorectal cancer.
* * * * *