U.S. patent application number 10/203399 was filed with the patent office on 2004-12-09 for therapeutic method for reducing angiogenesis.
Invention is credited to Kerbel, Robert.
Application Number | 20040248781 10/203399 |
Document ID | / |
Family ID | 26874652 |
Filed Date | 2004-12-09 |
United States Patent
Application |
20040248781 |
Kind Code |
A1 |
Kerbel, Robert |
December 9, 2004 |
Therapeutic method for reducing angiogenesis
Abstract
A method of controlling or treating an angiogenic dependent
condition in a mammal, preferably in a human by administering an
anti-angiogenic molecule such as an angiogenesis growth factor
antagonist, and a chemotherapeutic agent in amounts and frequencies
effective, in combination, to produce a regression or arrest of
said condition while minimizing or preventing significant toxicity
of the chemotherapeutic agent. Also a kit for controlling or
treating an angiogenic dependent condition in a mammal, preferably
in a human, comprising an anti-angiogenic molecule, such as an
angiogenesis growth factor antagonist, and a chemotherapeutic agent
in amounts effective, in combination, to produce a regression or
arrest of said condition while minimizing or preventing significant
toxicity of the chemotherapeutic agent.
Inventors: |
Kerbel, Robert; (Toronto,
CA) |
Correspondence
Address: |
KENYON & KENYON
ONE BROADWAY
NEW YORK
NY
10004
US
|
Family ID: |
26874652 |
Appl. No.: |
10/203399 |
Filed: |
October 6, 2003 |
PCT Filed: |
January 29, 2001 |
PCT NO: |
PCT/US01/02839 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10203399 |
Oct 6, 2003 |
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09539692 |
Mar 31, 2000 |
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60178791 |
Jan 28, 2000 |
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60178791 |
Jan 28, 2000 |
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Current U.S.
Class: |
514/8.1 ;
514/13.3; 514/17.2; 514/19.3; 514/19.4; 514/19.5; 514/8.2; 514/8.9;
514/9.1; 514/9.6 |
Current CPC
Class: |
A61K 39/3955 20130101;
A61K 2300/00 20130101; C07K 16/2863 20130101; A61K 2039/505
20130101; A61P 43/00 20180101; A61P 37/02 20180101; G01N 33/5011
20130101; A61K 39/3955 20130101; A61P 35/00 20180101 |
Class at
Publication: |
514/012 |
International
Class: |
A61K 038/17 |
Claims
What is claimed is:
1. A method of treating or controlling an angiogenic dependent
condition in a mammal comprising: administering an anti-angiogenic
molecule and a chemotherapeutic agent to the mammal, in amounts and
frequencies effective, in combination, to produce a regression or
arrest of said condition while minimizing or preventing significant
toxicity of the chemotherapeutic agent.
2. The method of claim 1, wherein the anti-angiogenic condition is
selected from the group consisting of a neoplasm, a
collagen-vascular disease or an auto-immune disease.
3. The method of claim 2, wherein the neoplasm is a solid
tumor.
4. The method of claim 3, wherein the solid tumor is selected from
the group consisting of breast carcinoma, lung carcinoma, prostate
carcinoma, colon carcinoma, prostate carcinoma, ovarian carcinoma,
neuroblastoma, central nervous system tumor, neuroblastoma,
glioblastoma multiforme or melanoma.
5. The method of claim 1, wherein the mammal is a human.
6. The method of claim 1, wherein the anti-angiogenic molecule
inhibits or blocks the action of an vascular endothelium survival
factor.
7. The method of claim 6, wherein the vascular endothelial survival
factor is selected from the group consisting of VEGF, VEGF
receptor,.alpha..sub.v.beta..sub.3. .alpha..sub.v.beta..sub.3
receptor, Tie2/tek ligand, Tie2/tek, endoglin ligand, endoglin,
neuropilin ligand, neuropilin, thrombospondin ligand,
thrombospondin, PDGF.alpha., PDGF.alpha. receptor, PDGF.beta.,
PDGF.beta. receptor, aFGF, aFGF receptor, bFGF, bFGF receptor,
TGF.beta., TGF.beta. receptor, EGF, EGF receptor, angiostatin,
angiostatin receptor, angiopoetin, angiopoeitin receptor, PLGF,
PLGF receptor, VPF, or VPF receptor.
8. The method of claim 6, wherein the vascular endothelial survival
factor is a receptor.
9. The method of claim 8, wherein the vascular endothelial survival
factor is an angiogenesis growth factor receptor.
10. The method of claim 9, wherein the angiogenesis growth factor
receptor is a VEGF receptor.
11. The method of claim 10, wherein the VEGF receptor is selected
from the group consisting of flk-1/KDR receptor, or flt-4
receptor.
12. The method of claim 6, wherein the vascular endothelial
survival factor is a ligand to a receptor.
13. The method of claim 10, wherein the ligand is selected from the
group consisting of VEGF, VEGF-B, VEGF-C, or VEGF-D.
14. The method of claim 6, wherein the anti-angiogenic molecule is
selected from the group consisting of an antibody, antibody
fragment, small molecule or peptide.
15. The method of claim 14, wherein the molecule is an antibody or
fragment selected from the group consisting of mouse, rat, rabbit,
chimeric, humanized or human antibody or fragment.
16. The method of claim 6, wherein the anti-angiogenic molecule is
IMC-1C11.
17. The method of claim 16, wherein the IMC-1C11 is administered in
a dose of from about 5 mg/m.sup.2 to about 700 mg/m.sup.2 from
about daily to about every 7 days.
18. The method of claim 17, wherein the IMC-1C11 is administered in
a dose of from about 7.5 mg/m.sup.2 to about 225 mg/m.sup.2, about
twice per week.
19. The method of claim 16, wherein the IMC-1C11 is administered at
a dose and frequency sufficient to substantially saturate the VEGF
receptor.
20. The method of claim 6, wherein the anti-angiogenic molecule is
administered in a dose and frequency sufficient to substantially
saturate the target of the anti-angiogenic molecule.
21. The method of claim 1, wherein the chemotherapeutic agent is
selected from the group consisting of vinca alkaloid, camptothecan,
taxane, or platinum analogue.
22. The method of claim 21, wherein the chemotherapeutic agent is
selected from the group consisting of vincristine, vinblastine,
vinorelbine, vindesine, paclitaxel, docetaxel, 5 FU, cisplatin,
carboplatin, iranotecan, topotecan or cyclophosphamide.
23. The method of claim 22, wherein the chemotherapeutic agent is
administered at less than about 50% of the maximum tolerated
dose.
24. The method of claim 23, wherein the chemotherapeutic agent is
administered at less than about 20% of the maximum tolerated
dose.
25. The method of claim 24, wherein the chemotherapeutic agent is
administered at less than about 10% of the maximum tolerated
dose.
26. The method of claim 22, wherein the chemotherapeutic agent is
vinblastine administered in a dose from about 0.5 mg/m.sup.2 to
about 3 mg/m.sup.2 from about once every 3 days to about once every
7 days.
27. The method of claim 1, wherein the chemotherapeutic agent is
administered in a dosage and frequency that is of substantially
equivalent efficacy to vinblastine in a dose from about 0.5
mg/M.sup.2 to about 3 mg/m.sup.2 from about once every 3 days to
about once every 7 days.
28. The method of claim 1, wherein the chemotherapeutic agent is
administered more frequently than about once every three weeks.
29. The method of claim 28, wherein the chemotherapeutic agent is
administered more frequently than about every seven days.
30. A kit for treating an angiogenic dependent condition in a
mammal comprising: an anti-angiogenic molecule; and, a
chemotherapeutic agent, to be administered in amounts and
frequencies effective, in combination, to produce a regression or
arrest of the condition while minimizing or preventing significant
toxicity of the chemotherapeutic agent, when administered in
combination.
31. The kit of claim 30, wherein the angiogenic dependent condition
is selected from the group consisting of neoplasm,
collagen-vascular disease or autoimmune disease.
32. The kit of claim 31, wherein the neoplasm is a solid tumor.
33. The kit claim 32, wherein the solid tumor is selected from the
group consisting of breast carcinoma, lung carcinoma, prostate
carcinoma, colon carcinoma, prostate carcinoma, ovarian carcinoma,
neuroblastoma, central nervous system tumor, neuroblastoma,
glioblastoma multiforme or melanoma.
34. The kit of claim 30, wherein the mammal is a human.
35. The kit of claim 30, wherein the anti-angiogenic molecule
inhibits or blocks the action of a vascular endothelium survival
factor.
36. The kit of claim 35, wherein the vascular endothelial survival
factor is selected from the group consisting of VEGF, VEGF
receptor,.alpha..sub.v.beta..sub.3 . .alpha..sub.v.beta..sub.3
receptor, Tie2/tek ligand, Tie2/tek, endoglin ligand, endoglin,
neuropilin ligand, neuropilin, thrombospondin ligand,
thrombospondin, PDGF.alpha., PDGF.alpha. receptor, PDGF.beta.,
PDGF.beta. receptor, aFGF, aFGF receptor, bFGF, bFGF receptor,
TGF.beta., TGF.beta. receptor, EGF, EGF receptor, angiostatin,
angiostatin receptor, angiopoetin, angiopoeitin receptor, PLGF,
PLGF receptor, VPF, or VPF receptor.
37. The kit of claim 30, wherein the vascular endothelial survival
factor is a receptor.
38. The kit of claim 37, wherein the vascular endothelial survival
factor is a an angiogenesis growth factor receptor.
39. The kit of claim 37, wherein the angiogenesis growth factor
receptor is a VEGF receptor.
40. The kit of claim 39, wherein the VEGF receptor is selected from
the group consisting of flk-1/KDR receptor, or flt-4 receptor.
41. The kit of claim 35, wherein the vascular endothelial survival
factor is a ligand for a receptor.
42. The kit of claim 39, wherein the ligand is selected from the
group consisting of VEGF, VEGF-B, VEGF-C, or VEGF-D.
43. The kit of claim 35, wherein the anti-angiogenic molecule is
selected from the group consisting of an antibody, antibody
fragment, small molecule or peptide.
44. The kit of claim 43, wherein the molecule is an antibody or
fragment selected from the group consisting of mouse, rat, rabbit,
chimeric, humanized or human antibody or fragment.
45. The kit of claim 35, wherein the antibody is IMC-1C11.
46. The kit of claim 45, wherein the IMC-1C11 is provided for
administration in a dose of from about 5 mg/m.sup.2 to about 700
mg/m.sup.2 about every 1 day to about every 7 days.
47. The kit of claim 46, wherein the IMC-1C11 is provided for
administration in a dose of from about 7.5 mg/m.sup.2 to about 225
mg/m.sup.2, about twice per week.
48. The kit of claim 46, wherein the IMC-1C11 is provided for
administration at a dose and frequency sufficient to substantially
saturate the VEGF receptor.
49. The kit of claim 35, wherein the anti-angiogenic molecule is
provided for administration in a dose and frequency sufficient to
substantially saturate the target of the anti-angiogenic
molecule.
50. The kit of claim 30, wherein the chemotherapeutic agent is
selected from the group consisting of a vinca alkaloid, a
camptothecan, a taxane, or a platinum analogue.
51. The kit of claim 50, wherein the chemotherapeutic agent is
selected from the group consisting of vincristine, vinblastine,
vinorelbine, vindesine, paclitaxel, docetaxel, 5 FU, cisplatin,
carboplatin, iranotecan, topotecan or cyclophosphamide.
52. The kit of claim 51, wherein the chemotherapeutic agent is
provided for administration at less than about 50% of the maximum
tolerated dose.
53. The kit of claim 49, wherein the chemotherapeutic agent is
provided for administration at less than about 20% of the maximum
tolerated dose.
54. The kit of claim 52, wherein the chemotherapeutic agent is
provided for administration at less than about 10% of the maximum
tolerated dose.
55. The kit of claim 51, wherein the chemotherapeutic agent is
vinblastine, provided for administration in a dose from about 0.5
mg/m.sup.2 to about 3 mg/m.sup.2 from about once every 3 days to
about once every 7 days.
56. The kit of claim 30, wherein the chemotherapeutic agent is
provided for administration at a dosage and frequency that is of
substantially equivalent efficacy to vinblastine is a dose from
about 0.5 mg/m.sup.2 to about 3 mg/m.sup.2 from about once every 3
days to about once every 7 days.
57. The kit of claim 30, wherein the chemotherapeutic agent is
provided for administration more frequently than about every three
weeks.
58. The kit of claim 57, wherein the chemotherapeutic agent is
provided for administration more frequently than about every seven
days.
Description
[0001] The present application claims the benefit of priority from
U.S. Provisional Application No. 60/178791, filed on Jan. 28, 2000,
which is hereby incorporated in its entirety by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to the inhibition or
prevention of angiogenesis as a means to control or treat an
angiogenic dependent condition, a condition characterized by, or
dependent upon, blood vessel proliferation. The invention further
relates to the use of an anti-angiogenic molecule in combination
with a chemotherapeutic agent.
BACKGROUND OF THE INVENTION
[0003] Angiogenesis is a highly complex process of developing new
blood vessels that involves the proliferation and migration of, and
tissue infiltration by capillary endothelial cells from
pre-existing blood vessels, cell assembly into tubular structures,
joining of newly forming tubular assemblies to closed-circuit
vascular systems, and maturation of newly formed capillary vessels.
The molecular bases of many of these aspects are still not
understood.
[0004] Angiogenesis is important in normal physiological processes
including embryonic development, follicular growth, and wound
healing, as well as in pathological conditions such as tumor growth
and in non-neoplastic diseases involving abnormal
neovascularization, including neovascular glaucoma (Folkman, J. and
Klagsbrun, M. Science 235:442-447 (1987). Other disease states
include but are not limited to, neoplastic diseases, including but
not limited to solid tumors, autoimmune diseases and collagen
vascular diseases such as, for example, rheumatoid arthritis, and
ophthalmalogical conditions such as diabetic retinopathy,
retrolental fibroplasia and neovascular glaucoma Conditions or
diseases to which persistent or uncontrolled angiogenesis
contribute have been termed angiogenic dependent or angiogenic
associated diseases.
[0005] One means of controlling such diseases and pathological
conditions comprises restricting the blood supply to those cells
involved in mediating or causing the disease or condition. For
example, in the case of neoplastic disease, solid tumors develop to
a size of about a few millimeters, and further growth is not
possible, absent angiogenesis within the tumor. In the past,
strategies to limit the blood supply to tumors have included
occluding blood vessels supplying portions of organs in which
tumors are present. Such approaches require the site of the tumor
to be identified and are generally limited to treatment to a single
site, or small number of sites. An additional disadvantage of
direct mechanical restriction of a blood supply is that collateral
blood vessels develop, often quite rapidly, restoring the blood
supply to the tumor.
[0006] Other approaches have focused on the modulation of factors
that are involved in the regulation of angiogenesis. While usually
quiescent, vascular endothelial proliferation is highly regulated,
even during angiogenesis. Examples of factors that have been
implicated as possible regulators of angiogenesis in vivo include,
but are not limited to, transforming growth factor beta
(TGF.beta.), acidic and basic fibroblast growth factor (aFGF and
bFGF), platelet derived growth factor (PDGF), and vascular
endothelial growth factor (VEGF) (Klagsbrun, M. and D'Amore, P. (1
991) Annual Rev. Physiol. 53: 217-239).
[0007] One growth factor of particular interest is VEGF. An
endothelial-cell specific mitogen, VEGF acts as an angiogenesis
inducer by specifically promoting the proliferation of endothelial
cells. It is a homodimeric glycoprotein consisting of two 23 kD
subunits with structural similarity to PDGF. Four different
monomeric isoforms of VEGF resulting from alternative splicing of
mRNA have been identified. These include two membrane bound forms
(VEGF.sub.206 and VEGF.sub.189) and two soluble forms (VEGF.sub.165
and VEGF121). VEGF.sub.165 is the most abundant isoform in all
human tissues except placenta.
[0008] VEGF is expressed in embryonic tissues (Breier et al.,
Development (Camb.) 114:521 (1992)), macrophages, and proliferating
epidermal keratinocytes during wound healing (Brown et al., J. Exp.
Med., 176:1375 (1992)), and maybe responsible for tissue edema
associated with inflammation (Fenrara et al., Endocr. Rev. 13:18
(1992)). In situ hybridization studies have demonstrated high
levels of VEGF expression in a number of human tumor lines
including glioblastoma multiforme, hemangioblastoma, other central
nervous system neoplasms and AIDS-associated Kaposi's sarcoma
(Plate, K. et al. (1992) Nature 359: 845-848; Plate, K. et al.
(1993) Cancer Res. 53: 5822-5827; Berkman, R. et al. (1993) J.
Clin. Invest. 91: 153-159; Nakamura, S. et al. (1992) AIDS Weekly,
13 (1)). High levels of VEGF also have been reported in hypoxia
induced angiogenesis (Shweiki, D. et al. (1992) Nature 359:
843-845).
[0009] VEGF mediates its biological effect through high affinity
VEGF receptors which are selectively expressed on endothelial cells
during, for example, embryogenesis (Millauer, B., et al. (1993)
Cell 72: 835-846) and tumor formation. VEGF receptors typically are
class III receptor-type tyrosine kinases characterized by having
several, typically 5 or 7, immunoglobulin-like loops in their
amino-terminal extracellular receptor ligand-binding domains
(Kaipainen et al., J. Exp. Med. 178:2077-2088 (1993)). The other
two regions include a transmembrane region and a carboxy-terminal
intracellular catalytic domain interrupted by an insertion of
hydrophilic interkinase sequences of variable lengths, called the
kinase insert domain (Terman et al., Oncogene 6:1677-1683 (1991)).
VEGF receptors include flt-1, sequenced by Shibuya M. et al.,
Oncogene 5, 519-524 (1990); flk-1, sequenced by Matthews W. et al.
Proc. Natl. Acad. Sci. USA, 88:9026-9030 (1991) and KDR, the human
homologue of flk-1, described in PCT/US92/01300, filed Feb. 20,
1992, and in Terman et al., Oncogene 6:1677-1683 (1991).
[0010] High levels of flk-1 are expressed by endothelial cells that
infiltrate gliomas (Plate, K. et al., (1992) Nature 359: 845-848),
and are specifically upregulated by VEGF produced by human
glioblastomas (Plate, K et al. (1993) Cancer Res. 53: 5822-5827).
The finding of high levels of flk-1 expression in glioblastoma
associated endothelial cells (GAEC) suggests that receptor activity
is induced during tumor formation, since flk-1 transcripts are
barely detectable in normal brain endothelial cells. This
upregulation is confined to the vascular endothelial cells in close
proximity to the tumor. Blocking VEGF activity with neutralizing
anti-VEGF monoclonal antibodies (mAbs) results in inhibition of the
growth of human tumor xenografts in nude mice (Kim, K. et al.
(1993) Nature 362: 841-844), suggesting a direct role for VEGF in
tumor-related angiogenesis.
[0011] Various chemotherapeutic drugs also have been shown to block
functions of activated, dividing endothelial cells critical to
angiogenesis, or to kill such cells. Such collateral damaging
effects on a genetically stable normal host cell, in addition to
the chemotherapeutic agent's effect upon the tumor cells,
contribute significantly to the in vivo anti-tumor efficacy of
chemotherapy. However, the standard use of chemotherapeutic agents
has obvious undesirable side-effects upon the normal cells of
patients, limiting its use. Administration of chemotherapeutic
agents in their usual doses and at usual dosage frequencies are
commonly associated with side-effects, including, but not limited
to, myelosuppression, neurotoxicity, cardiotoxicity, alopecia,
nausea and vomiting, nephrotoxicity, and gastrointestinal toxicity.
Further, patients' tumors often also develop resistance to the
chemotherapeutic agents after initial exposure to the drugs.
[0012] A desirable method and composition for controlling
angiogenesis should be well tolerated, have few or no side-effects,
and prevent new vessel formation at sites of disease without
interfering with required physiologic angiogenesis in normal sites.
It should be effective and, in the case of neoplastic disease,
overcome the problem of the development of drug resistance by tumor
cells. In so doing, it should permit targeted therapy without the
accurate identification of all disease sites. The present invention
addresses many of the problems with existing materials and
methods.
SUMMARY OF THE INVENTION
[0013] The present invention provides a method of treating an
angiogenic dependent condition in a mammal comprising administering
an anti-angiogenic molecule and a chemotherapeutic agent to the
mammal, in an amount and frequency effective, in combination, to
produce a regression or arrest of the condition without significant
toxicity from the chemotherapeutic agent. The angiogenic dependent
condition may be selected from the group consisting of neoplasm,
collagen-vascular disease or auto-immune disease, including a solid
tumor neoplasm, including breast carcinoma, lung carcinoma,
prostate carcinoma, colon carcinoma, prostate carcinoma, ovarian
carcinoma, neuroblastoma, central nervous system tumor,
neuroblastoma, glioblastoma multiforme or melanoma. The mammal
receiving the treatment is preferably a human.
[0014] The anti-angiogenic molecules inhibit the action of a
vascular endothelium survival factor, which include receptors and
their ligands. Vascular endothelium survival factors include
receptors, including angiogenic growth factors such as VEGF
receptor, including flk-1/KDR receptor, or flt-4 receptor and VEGF.
Examples of other vascular endothelial survival factors are
integrin .alpha..sub.v.beta..sub.3. .alpha..sub.v.beta..sub.3
ligand, Tie2/tek ligand, Tie2/tek, endoglin ligand, endoglin,
neuropilin ligand, neuropilin, thrombospondin ligand,
thrombospondin, PDGF.alpha., PDGF.alpha. receptor, PDGF.beta.,
PDGF.beta. receptor, aFGF, aFGF receptor, bFGF, bFGF receptor,
TGF.beta., TGF.beta. receptor, EGF, EGF receptor, angiostatin,
angiostatin receptor, angiopoetin, angiopoeitin receptor, PLGF,
PLGF receptor, VPF, or VPF receptor. Optionally, the ligand is
selected from the group consisting of VEGF (VEGF-A), VEGF-B,
VEGF-C, or VEGF-D. The anti-angiogenic molecule may be selected
from the group consisting of antibody, antibody fragment, small
molecule or peptide.
[0015] Preferred embodiments of the present invention include
antibodies selected from the group consisting of mouse antibody,
rat antibody, chimeric antibody, humanized antibody or human
antibody. A preferred antibody is IMC-1C11.
[0016] Preferably, IMC-1C11 is administered in a dose of from about
5 mg/m.sup.2 to about 700 mg/m.sup.2 about daily to about every 7
days, more preferably a dose of from about 7.5 mg/m.sup.2 to about
225 mg/m.sup.2, about twice per week. Optionally, the IMC-1C11 is
administered at a dose and frequency sufficient to substantially
saturate the VEGF receptor. Optionally, the anti-angiogenic
molecule is administered in a dose and frequency sufficient to
substantially saturate the target of the anti-angiogenic molecule.
In another embodiment, the anti-angiogenic molecule is administered
in a dose equivalent to that of IMC-1C11, administered in a dose of
from about 5 mg/m.sup.2 to about 700 mg/m.sup.2 about daily to
about every 7 days, more preferably a dose of from about 7.5
mg/m.sup.2 to about 225 mg/m.sup.2, about twice per week.
[0017] The chemotherapeutic agent may be selected from the group
consisting of vinca alkaloid, camptothecan, taxane, or platinum
analogue, including vincristine, vinblastine, vinorelbine,
vindesine, paclitaxel, docetaxel, 5 FU, cisplatin, carboplatin,
iranotecan, topotecan or cyclophosphamide. The chemotherapeutic
agent is administered in a low-dose regimen. Preferably the
chemotherapeutic agent is administered at less than about 50% of
the maximum tolerated dose, more preferably at less than about 20%
of the maximum tolerated dose, most preferably at less than about
10% of the maximum tolerated dose. In one preferred embodiment the
vinblastine is administered in a dose from about 0.5 mg/m.sup.2 to
about 3 mg/m.sup.2 from about once every 3 days to about once every
7 days. In another embodiment, the chemotherapeutic agent is
administered in a dosage and frequency that is of substantially
equivalent efficacy to vinblastine in a dose from about 0.5
mg/m.sup.2 to about 3 mg/m.sup.2 from about once every 3 days to
about once every 7 days.
[0018] Optionally the chemotherapeutic agent is administered more
frequently than about every three weeks, or more frequently than
about every seven days.
[0019] The present invention also includes a kit for treating an
angiogenic dependent condition in a mammal comprising the
anti-angiogenic molecule and the chemotherapeutic agent, which are
provided to be administered in an amount and frequency effective,
in combination, to produce a regression or arrest of the condition
while minimizing or preventing significant toxicity of the
chemotherapeutic agent.
BRIEF DESCRIPTION OF FIGURES
[0020] FIG. 1 is the encoding nucleotide sequence and deduced amino
acid sequence of V.sub.H and V.sub.L domains of IMC-1C11
(c-p1C11).
DETAILED DESCRIPTION OF THE INVENTION
[0021] Throughout this application, various articles and patents
are referenced. Disclosures of these publications in their
entireties are hereby incorporated by reference into this
application.
[0022] The present invention comprises a method of treating or
controlling an angiogenic dependent condition in a mammal,
comprising administering an anti-angiogenic molecule and a
chemotherapeutic agent in combined amounts effective to produce a
regression or arrest to the angiogenic dependent condition, while
minimizing or preventing significant toxicity.
[0023] The benefits of the combination of an anti-angiogenic
molecule and a chemotherapeutic agent of the present invention
include an improvement in the treatment and control of an
angiogenic dependant condition with reduced doses of a
chemotherapeutic agent administered at increased frequency, without
significant toxicity, together with a anti-angiogenic molecule. The
combination can be administered for a prolonged period of time, or
optionally a shorter duration of treatment may be administered due
to the increased effectiveness of the combination. Toxicity is
reduced or eliminated without a loss of effectiveness. The
administration of the treatment of the invention can overcome the
problems of drug resistance that develops with standard
chemotherapeutic regimens.
[0024] The anti-angiogenic molecule functions to inhibit or prevent
angiogenesis and thereby treating the angiogenic dependent
condition by inhibiting, blocking, or antagonizing the effect of
vascular endothelial survival factors. These survival factors are
receptors and their ligands, upon which vascular endothelium
depends, either directly or indirectly, for growth and/or survival.
They play a role in allowing vascular endothelial cells to recovery
from injury or insult, which, absent the effect of the survival
factor would result in cell death or apoptosis. Survival factors
include vascular endothelial cell growth factors or mitogens, as
well as those factors which do not appear to have a direct
growth-stimulatory effect but allow the cells to recover from
injury.
[0025] Examples of survival factors include VEGF receptors,
including but not limited to flt-1 (VEGFR1), flk-1/KDR (VEGFR2),
flt-4 (VEGFR3), their ligands VEGF, VEGF-B, VEGF-C, and VEGF-D,
integrin .alpha.V.beta.3, Tie2/tek, endoglin (CD105), neuropilin,
thrombospondin and their ligands, and PDGF.alpha., PDGF.beta.,
aFGF, bFGF, and TGF.beta., as well as EGF, angiostatin, and
angiopoeitin, vascular permiability factor (VPF), and placenta-like
growth factor (PLGF) and their receptors.
[0026] The survival factors that are receptors are located on
vascular endothelial cells or may be located on other cell types
including, but not limited to, tumor cells. The anti-angiogenic
molecule inhibit binding to ,and/or activation of, receptors,
inhibit their expression, or inhibit the binding of or expression
of ligands. Suitable anti-angiogenic molecules include, but are not
limited to antibody, antibody fragment, small molecule or peptide.
An antibody can be derived from any mammalian species. Optionally,
the antibody is of mouse, rat, rabbit, or human origin. Preferably
the antibody is chimeric, more preferably the antibody is
humanized, and even more preferably the antibody is human. Suitable
antibody fragments include, for example, Fab fragment, Fab'
fragment, F(ab').sub.2 fragment, monovalent single chain antibody
(scFv), and diabodies (DAB).
[0027] Examples of suitable anti-angiogenic molecules that are
antagonists to vascular endothelium survival factors include, but
are not limited to, VEGF receptor antagonist or VEGF antagonist, as
disclosed in U.S. Pat. Nos. 5,840,301, 5,861,499, 5,874,542,
5,955,311, and 5,730,977, which are incorporated in their entirety
by reference, aFGF receptor antagonist, aFGF antagonist, bFGF
receptor antagonist, bFGF antagonist, PDGF receptor antagonist,
PDGF antagonist, TGF.beta. antagonist, Tie2/tek antagonist (P. Lin
et al., Inhibition of Tumor Angiogenesis Using a Soluble Receptor
Establishes a Role for Tie2 in Pathologic Vascular Growth. J. Clin.
Invest. 100(8) 2072 (1997)), endoglin (CD105) antagonist, as
disclosed in U.S. Pat. Nos. 5,855,866, and 5,660,827, neuropilin
antagonist, thrombospondin antagonist, and antagonists to the
receptors for PDGF.alpha., PDGF.beta., aFGF, bFGF, or TGF.beta., as
well as antagonists to the receptors for EGF, angiostatin,
angiopoeitin, or VPF (Vascular Permeability Factor) as disclosed in
U.S. Pat. Nos. 5,036,003 and 5,659,013. Also encompassed within the
scope of the present invention are integrin receptor antagonists as
disclosed in U.S. Pat. Nos. 6,017,926, 6,017,925, 5,981,546,
5,952,341, and 5,919,792, integrin .alpha.V.beta..sub.3
antagonists, as disclosed in U.S. Pat. Nos. 5,780,426, 5,773,412,
5,767,071, 5,759,996, 5,753,230, 5,652,110, and 5,652,109,
antagonists to placenta-like growth factor (PLGF) as disclosed in
European Patent Application EP506477A1, thrombospondin antagonists
as disclosed in U.S. Pat. Nos. 5,840,692, 5,770,563, 5,654,277,
5,648,461, 5,506,208, 5,399,667, 5,200,397, 5,192,744, and
5,190,918, as well as those disclosed in U.S. Pat. Nos. 5,965,132,
6,004,555 and 5,877,289, and PCT Applications Nos. WO 99/16465, WO
97/05250, WO 98/33917. Also included, are molecules such as
thalidomide, TNP-470, interferon-a (INF-.alpha.), and
interleukin-12 (IL-12).
[0028] In many cases, the expression of a receptor and/or ligand is
upregulated in an region of angiogenesis. However, although located
in an area of abnormal cells responsible for the specific disease,
exposed to high levels of ligand, and having upregulated receptors,
the cells of the vascular endothelium are largely normal and
responsive to normal regulatory mechanisms. An advantage to
blocking a receptor, rather than the ligand, is that are that fewer
anti-angiogenic molecules may be needed to achieve such inhibition,
as levels of receptor expression may be more constant than those of
the environmentally induced ligand. Because the receptors exist on
essentially normal endothelial cells, their behavior is less likely
to escape normal regulatory control. Although there are advantages
to targeting receptors, it is also possible, and within the scope
of the present invention, to inhibit angiogenesis by targeting the
ligand for the receptor, either alone or in combination with
blockade of the receptor. Optionally, antagonism of the receptor is
combined with antagonism of the ligand in order to achieve even
more efficient inhibition of angiogenesis.
[0029] In a preferred embodiment of the invention, the
anti-angiogenic molecule is an antagonist to VEGF or the VEGF
receptor. The expression of the VEGF receptor and ligand is low in
normal endothelial cells that are not in or near a region of
angiogenesis. In contrast, VEGF receptors present on tumor
infiltrating vascular endothelial cells are upregulated, as is the
expression of the VEGF ligand by tumor cells. VEGF (or VEGF-A) is
the ligand for VEGFR1 and VEGFR2, VEGF-B is the ligand for VEGFR2,
VEGF-C is the ligand for VEGFR3, VEGFR4, and possibly VEGFR2, and
VEGF-D is the ligand for VEGFR2 and VEGFR3. Blocking the
interaction between VEGF and its receptors can inhibit
angiogenesis, and thereby tumor growth, while not significantly
effecting normal endothelial cells at other sites, where vascular
endothelial cell receptors have not been upregulated. In one
embodiment of the present invention, antagonism of the VEGF
receptor is combined with antagonism of the VEGF ligand in order to
achieve even more efficient inhibition of angiogenesis. In other
embodiments of the invention, VEGF (VEGF-A), VEGF-B, VEGF-C or
VEGF-D is the ligand which is inhibited by the anti-angiogenesis
molecule. Optionally, the effect of more than one form of VEGF is
inhibited.
[0030] Examples of an antagonist to a receptor are the antibodies
DC101, described in the Examples, an antagonist of flk-1, and
A.4.6.1 and its chimeric and humanized form as disclosed in L. G.
Presta, Humanization of an Anti-vascular Endothelial Growth Factor
Monoclonal Antibody for the Therapy of Solid Tumors and Other
Disorders. Cancer Research, 57, 4593-4599 (1997), which is hereby
incorporated by reference. A preferred antibody is the mouse-human
chimeric antibody IMC-1C11 which is a KDR antagonist, and is
disclosed in U.S. application Ser. No. 09/240,736, which is hereby
incorporated by reference. The encoding nucleotide sequences and
deduced amino acid sequences of the V.sub.H and V.sub.L domains are
shown in FIG. 1.
[0031] The present invention provides a low dose application of a
chemotherapeutic agent that provides effective therapy without
significant side-effects. An effective amount of a chemotherapeutic
agent is an amount sufficient to inhibit or contribute to the
inhibition of angiogenesis, when administered with an
anti-angiogenic molecule, without resulting in significant
toxicity. The meaning of significant toxicity is well known to one
of ordinary skill in the art, and includes toxicities that
cumulatively or acutely effect a patient's quality of life and/or
limit the amount of chemotherapeutic agent than can be
administered. Examples of chemotherapy induced toxicity that can be
minimized or prevented by the present invention include, but are
not limited to, myelosuppression, neurotoxicity, cardiotoxicity,
alopecia, nausea and vomiting, nephrotoxicity, and gastrointestinal
toxicity. The low dose administration of a chemotherapeutic agent
without significant toxicity permits prolonged treatment if
desired. Additionally, the low dose manner of chemotherapy
administration in the present invention can overcome the problem of
the development of chemotherapeutic drug resistance by the
patient's tumor cells that occurs with current chemotherapeutic
regimens. The present invention delays, reduces, or even
circumvents the problem of acquired drug resistance by targeting
the genetically stable endothelial cells of newly formed tumor
blood vessels, rather than genetically unstable tumor cells which
are prone to mutate and develop resistance. Encompassed within the
scope of the present invention is the administration of amounts of
chemotherapy that are insufficient to have a cytotoxic effect on
tumor cells yet have anti-angiogenic properties as a result of the
drug's effect on vascular endothelial cells.
[0032] The chemotherapeutic agent of the present invention
functions, in combination with the anti-angiogenic molecule, to
cause a cytotoxic effect on the vascular endothelial cells involved
in angiogenesis. A number of chemotherapeutic agents have been
identified as having anti-angiogenic activity and are suitable for
use in the practice of the present invention. Examples include, but
are not limited to, taxanes, including but not limited to
paclitaxel and docetaxel, camptothecin analogues, including but not
limited to iranotecan and topotecan, platinum analogues including
but not limited to cisplatin and carboplatin, 5FU, and vinca
alkaloid, including but not limited to vinblastine, vincristine
vindesine and vinorelbine.
[0033] The low-dose administration of chemotherapeutic agents, to
achieve therapeutic effects without significant toxicity (side
effects) is routinely possible by the practice of the present
invention. Applying standard methods of defining optimal dosage
levels and schedules to the teachings of the present invention, one
of ordinary skill in the art readily can determine a more or most
desirable low-dose regimen for a selected chemotherapeutic agent
when used in combination with an anti-angiogenic molecule, as
detailed in the present application. A low-dose regimen will
administer the chemotherapy at frequent intervals or continually,
at less than about 50% [Should this be lower?] of the maximum
tolerated dose (MTD), more preferably less than about 20% of the
MTD, and most preferably, less than about 10% of the MTD, although
the preferred dose depends on the particular chemotherapy. The
preferred dose will be a dose effective to inhibit or prevent
progression of the angiogenic dependent condition, when
administered in combination with the anti-angiogenic molecule of
the present invention, while minimizing or preventing the
development of significant chemotherapy related toxicity.
Optionally the dose of chemotherapy will be effective to inhibit or
prevent progression of the angiogenic dependent condition even when
administered alone. Optionally the dose of chemotherapy will be one
which does not exert a direct cytotoxic effect on tumor cells, yet
has an antitumor effect mediated by its anti-angiogenic properties.
Optionally, the low-dose regimen of the present invention will
administer chemotherapy at a dose intensity of less than 50% of
that when administered alone to treat a particular neoplasm. More
preferably the low-dose regimen of the present invention will
administer chemotherapy at a dose intensity of less than 20% of
that when administered alone to treat a particular neoplasm.
[0034] In the prior art, chemotherapy is usually given
intermittently, commonly in the form of a bolus infusion or an
infusion lasting from about 20 minutes to about three hours, at
about the maximum tolerated dose (MTD) with long rest periods
(e.g., 3 weeks) between successive drug exposures. It has been
suggested that these rest periods provide the endothelial cell
compartment of a tumor an opportunity to repair some of the damage
inflicted by the chemotherapy (T. Browder, et al., Antiangiogenic
Scheduling of Chemotherapy Improves Efficacy Against Experimental
Drug-Resistant Cancer. Cancer Res. (In press) 1999). Administering
lower doses of a chemotherapeutic drug, such as cyclophosphamide,
more frequently such as weekly, enables circumvention of many
problems associated with standard chemotherapeutic doses. This
anti-angiogenic scheduling of chemotherapy optimizes
antitumor/anti-vascular effects. For example, a sub-line of the
Lewis Lung Carcinoma, previously selected in vivo for acquired
resistance to the MTD of cyclophosphamide, is rendered sensitive
again to the drug in vivo by employing continuous low dose therapy
of the same drug.
[0035] The invention provides low-dose administration of
chemotherapy administered at short intervals, for example, from
about every 4 to about every 6 hours, to about daily to weekly, or
optionally administered continuously. The preferred time interval
between administration of successive doses of chemotherapeutic
agent is that amount of time that is of sufficiently short duration
that the blood levels of the chemotherapeutic agent (or its active
metabolite) will remain at about a concentration sufficient to
exert an anti-angiogenic effect for substantially the duration of
treatment. Preferably, such a blood level will be maintained for at
least about 30% [Should this be lower] of the time between doses,
more preferably for at least 50% of the time between doses, most
preferably for at least about 70% of the time between doses.
Therapy is continued for a period of time from about 10 days to
about 6 months, or as determined by one of skill in the art.
Optionally, treatment will continue chronically for a period longer
than six months for as long as is needed. The present invention
reduces host toxicity, allows for longer term administration of the
chemotherapeutic agent in diseases or pathological conditions
requiring it, and does not sacrifice, and perhaps even improves,
antitumor efficacy. Optionally, increased efficacy will permit the
use of shorter durations of therapy for selected angiogenic
dependent conditions.
[0036] The anti-angiogenic molecule is administered in dosages and
dose frequencies sufficient to substantially saturate the selected
target receptor or ligand. Substantial saturation is saturation of
at least about 50% of targeted receptors. A more preferred level of
saturation is at least about 80%, and a most preferred level of
saturation is about 100%. Optionally, the anti-angiogenic molecule
is administered at a dose and frequency sufficiently short to
maintain a blood level sufficient to saturate the targeted survival
factor for at least about 50% of the time between doses, more
preferably at least about 70% of the time and most preferably at
least about 90% of the time interval between doses. Using the
concentrations required to achieve receptor saturation or ligand
neutralization in vitro, and by analysis of serum concentrations of
anti-angiogenic molecule in vivo, both the appropriate dose and
schedule can be determined readily by one of skill in the art.
[0037] A preferred embodiment of the invention is the combination
of a chemotherapeutic agent and a VEGF receptor antagonist. It has
been shown that a major function of VEGF is to promote the survival
of endothelial cells comprising newly formed vessels (L. E.
Benjamin, et Al., Selective Ablation of Immature Blood Vessels in
Established Human Tumors Follows Vascular Endothelial Growth Factor
Withdrawal. J.Clin.Invest. 103:159-165 (1999), T. Alon, et al.,
Vascular Endothelial Growth Factor Acts as a Survival Factor for
Newly Formed Retinal Vessels and Has Implications for Retinopathy
of Prematurity. Nature Med. 1:1024-1028 (1995), R. K. Jain, et al.,
Endothelial Cell Death, Angiogenesis, and Microvascular Function
after Castration in an Androgen-Dependent Tumor: Role of Vascular
Endothelial Growth Factor. Proc.Natl.Acad.Sci. U.S.A.
95:10820-10825 (1998)) Hence, the ability of such cells to cope
with the damage inflicted by continuous or frequent exposure to a
chemotherapeutic drug is selectively and significantly impaired,
(M. J. Prewett, et al., Antivascular Endothelial Growth Factor
Receptor (Fetal Liver Kinase 1) Monoclonal Antibody Inhibits Tumor
Angiogenesis and Growth of Several Mouse and Human Tumors. Cancer
Res 59:5209-5218. (1999); T. A. Fong, et al., SU5416 Is a Potent
and Selective Inhibitor of the Vascular Endothelial Growth Factor
Receptor (Flk-1/kdr) That Inhibits Tyrosine Kinase Catalysis, Tumor
Vascularization, and Growth of Multiple Tumor Types. Cancer Res
59:99-106 (1999); N. Ferrara, et al., Clinical Applications of
Angiogenic Growth Factors and Their Inhibitors. Nat.Med.
5:1359-1364. (1999)). It is believed that the combination of
continuous chemotherapy with, for example, interruption of the cell
rescue mechanisms provided by activation of the VEGF receptor plays
a role in inducing vascular endothelial cell apoptosis.
[0038] A preferred embodiment of the present invention comprises
the administration of the antibody IMC-1C11, a KDR receptor
antagonist with a chemotherapeutic agent. A preferred dose of
IMC-1C11, is an amount that is sufficient to adequately saturate
the targeted receptors or ligand. In in vitro experiments, 50%
saturation of VEGF receptors was obtained as an IMC-1C11
concentration of 0.2 .mu.g/ml, and 100% at a concentration of 3
.mu.g/ml. A preferred level of saturation is about at least 50%, a
more preferred level is about at least 80%, and a most preferred
level is about 100% saturation. For therapy, a preferred dose
regimen of IMC-1C11 is from about 5 mg/m.sup.2 to about 700
mg/M.sup.2, more preferably from about 7.5 mg/M.sup.2 to about 225
mg/m.sup.2, administered about twice per week.
[0039] Another preferred embodiment of the invention combines
IMC-1C11 in the doses described above with, vinblastine,
administered in a low dose regimen, at a dose from about 0.5 mg
/m.sup.2 to about 3 mg/m.sup.2 from about every 3 days to about
every 7 days. Optionally, a suitable chemotherapeutic agent other
than vinblastine is administered in a dosage and frequency that is
of substantially equivalent efficacy to vinblastine (in the
combination) at a dose from about 0.5 mg /m.sup.2 to about 3
mg/m.sup.2 from about every 3 days to about every 7 days.
[0040] In other embodiments of the present invention, doses of an
anti-angiogenic molecule in amounts and dosing frequencies
sufficient to provide levels of receptor or ligand saturation
equivalent to that of IMC-1C11 in the doses about are combined with
a chemotherapeutic agent in a dose and frequency equivalent to that
of vinblastine above, and therapy is carried out for as long as is
needed. An equivalent dose is one that, in the combination, is
substantially as effective in arresting or inhibiting the
angiogenic dependent condition, while being substantially as
effective in minimizing or preventing significant chemotherapy
induced toxicity. In one preferred embodiment of the present
invention, an equivalent dose of another chemotherapeutic agent is
determined based on data derived from an animal model, an example
of which is included herein, utilizing a chemotherapy-resistant
cell line so that any observed antitumor effect is due to an effect
on the vascular endothelium. A preferred dose of vinblastine in a
mouse is from about 1 mg/m.sup.2 to about 2 mg/m.sup.2 more
preferably about 1.5 mg/m.sup.2 administered every three days. The
MTD of this drug in mice is approximately 4-5 times that of a
human, and a preferred dose is {fraction (1/16)}-{fraction (1/20)}
of the MTD in mice. A preferred dose of DC011 in a mouse is about
800 .mu.g administered intraperitoneally every three days. The use
of DC101 and vinblastine showed a therapeutic effect upon
neuroblastoma cell lines grown as xenografts in SCID mice. (L.
Witte, L, et al., Monoclonal antibodies targeting the VEGF
receptor-2 (flk1/KDR) as an anti-angiogenic therapeutic strategy.
Cancer Metastasis Rev. 17:155-161. (1998); Prewitt, 1999). In yet
another preferred embodiment of the present invention, low-dose
vinblastine is administered every 3 days in combination with
IMC-1C11 (p1C11).
[0041] In one aspect of the present invention, there is provided a
kit comprising an anti-angiogenic molecule and a chemotherapeutic
agent to be administered to a mammal in an amount effective to
produce a regression or arrest of an angiogenic dependent condition
while minimizing or preventing significant toxicity of the
chemotherapeutic agent. Such a kit optionally comprises an
anti-angiogenic molecule and a chemotherapeutic agent in one or
more than one containers for administration at about the same time
points or at different times. Optionally, the anti-angiogenic
molecule is administered intermittently and the chemotherapeutic
agent is administered continuously or in a manner that permits the
maintenance of a suitable blood concentration. It is an aspect of
the present invention that such treatment optionally is
administered for a prolonged period or chronically, without
substantial chemotherapy induced toxicity. Routes of administration
include but are not limited to oral and parenteral, including but
not limited to intravenous, subcutaneous, percutaneous, intrathecal
and intraperitoneal. Patients that may be treated with the methods
and compositions of the present invention include any patients with
an angiogenic dependent disease.
[0042] The angiogenic dependent diseases encompassed by the scope
of the present invention include, but are not limited to neoplasms,
collegen vascular diseases or autoimmune diseases. All neoplasms
are suitable for treatment with the present invention, however
preferred neoplasms are solid tumors. More preferred are breast
carcinoma, lung carcinoma, prostate carcinoma, colon carcinoma,
prostate carcinoma, ovarian carcinoma, neuroblastoma, central
nervous system tumor, neuroblastoma, glioblastoma multiforme or
melanoma, and a preferred mammal to receive treatment is a
human.
[0043] Antibodies used in this invention may be produced in a
eukaryotic cell. Techniques for the creation of and production of
such antibodies, or portions thereof are well know in the field and
are within the knowledge of one of ordinary skill in the art.
Techniques used for preparation of monoclonal antibodies, include
but are not limited to, the hybridoma technique (Kohler &
Milstein, Nature, 256:495-497 (1975)), the trioma technique, the
human B-cell hybridoma technique (Kozbor et al., Immunology Today
4:72, (1983)), and the EBV-hybridoma technique to produce human
monoclonal antibodies (Cole, et al., 1985, In Monoclonal Antibodies
and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).
[0044] DNA encoding chimerized antibodies may be prepared by
recombining DNA substantially or exclusively encoding human
constant regions and DNA encoding variable regions derived
substantially or exclusively from the sequence of the variable
region of a mammal other than a human. DNA encoding humanized
antibodies may be prepared by recombining DNA encoding constant
regions and variable regions, other than the CDRs, derived
substantially or exclusively from the corresponding human antibody
regions and DNA encoding CDRs derived substantially or exclusively
from a mammal other than a human.
[0045] Each domain of the antibodies of this invention may be a
complete immunoglobulin heavy or light chain variable domain, or it
may be a functional equivalent or a mutant or derivative of a
naturally occurring domain, or a synthetic domain constructed, for
example, in vitro using a technique such as one described in WO
93/11236 (Medical Research Council et al./Griffiths et al.). For
instance, it is possible to join together domains corresponding to
antibody variable domains which are missing at least one amino
acid. The important characterizing feature is the ability of each
domain to associate with a complementary domain to form an antigen
binding site. Accordingly, the terms "variable heavy/light chain
fragment" should not be construed to exclude variants which do not
have a material effect on how the invention works. The DNA
deletions and recombinations of the present invention may be
carried out by known methods, such as those described in PCT
applications WO 93/21319, WO 89/09622, European Patent applications
239,400, 338,745 and 332,424 and/or other standard recombinant DNA
techniques. Conventional methods, such as those employed in the
construction of vectors and plasmids, the insertion of genes
encoding polypeptides into such vectors and plasmids, or the
introduction of plasmids into host cells, are well known to those
of ordinary skill in the art and are described in numerous
publications including Sambrook, J., Fritsch, E. F. and Maniatis,
T. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition, Cold
Spring Harbor Laboratory Press, and in Ausubel et al. (Eds) Current
Protocols in Molecular Biology, Green Publishing Associates/
Wiley-Interscience, New York (1990).
[0046] The invention also includes functional equivalents of the
antibodies described in this specification. Functional equivalents
have binding characteristics comparable to those of the antibodies,
and include, for example, chimerized, humanized and single chain
antibodies as well as fragments thereof. Methods of producing such
functional equivalents are disclosed in PCT Application No. WO
93/21319, European Patent Application No. EPO 239,400; PCT
Application WO 89/09622; European Patent Application No. EP338,745;
and European Patent Application EPO 332,424.
[0047] Functional equivalents include polypeptides with amino acid
sequences substantially the same as the amino acid sequence of the
variable or hypervariable regions of the antibodies of the
invention. "Substantially the same" amino acid sequence is defined
herein as a sequence with at least 70% percent homology to an amino
acid sequence of an antibody of the invention, as determined by the
FASTA search method in accordance with Pearson and Lipman, Proc.
Natl. Acad. Sci. USA 85, 2444-2448 (1988).
[0048] The examples which follow are set forth to aid in
understanding the invention, but are not intended to, and should
not be construed as, limiting the scope of the invention in any
manner.
EXAMPLES
[0049] Cells and culture conditions: Neuroblastoma cell lines
SK-N-MC, SK-N-AS were obtained from American Type Culture
Collection (ATCC) and expanded as a monolayer culture by serial
passage on tissue culture plates (Nunc, Denmark) in DMEM, 5% fetal
bovine serum (Gibco, Grand Island, N.Y., USA). Human umbilical vein
endothelial cells (HUVEC) (Clonetics, San Diego, Calif.) were
expanded on 1% gelatin-coated tissue culture plates in MCDB131
culture medium (JRH Biosciences, Lenexa, Kans., USA) supplemented
with 5 ng/ml bFGF (R&D, Minneapolis, Minn.), 10 units/ml
heparin (Wyeth-Ayerst Canada), 10 ng/ml EGF (UBI, Lake Placid,
N.Y.) and 10% fetal bovine serum.
[0050] In vitro determination of drug sensitivity: Three thousand
cells in 200 .mu.l growth media per well were plated in 96-well
flat bottom tissue culture plates (Nunc, Denmark) and incubated at
37.degree. C., 5% CO.sub.2 for 24 hours prior to initiation of
treatment. The cells were then washed with PBS and treated with
1-500 ng/ml vinblastine sulphate (Calbiochem, La Jolla, Calif.) for
24 hours, in groups of eight wells per dose. The cells were then
pulsed for 6 hrs with 2 .mu.Ci/well of methyl-.sup.3H-thymidine
(Amersham Life Science, Buckinghamshire, England). The plates were
frozen, thawed and the DNA harvested onto a filtermat using a
Titertek Cell Harvester. Incorporated radioactivity was measured on
Wallac 1205 BetaPlate Scintillation Counter (Wallac Oy, Finland)
and proliferation was expressed as a percentage of
.sup.3H-thymidine in treated cells vs. that in controls.
[0051] In vivo tumor growth assessment: SK-N-MC, cells were
harvested using 1% Trypsin EDTA (GibcoBRL, Gaithesburg, Md.), and
single cell suspension of 2.times.106 cells in 0.2 ml of growth
media was injected subcutaneously into the flanks of 4-6 week old
CB-17 SCID mice (Charles River, St-Constant, Quebec). Approximately
3 weeks later, most tumors had grown to .about.0.75 cm.sup.3, and
mice were randomized into groups of 5 animals. Two independent
experiments were performed, each totaling 20 animals in 4 groups.
The treatment was as follows:
[0052] Group I (Control)--0.4 ml of PBS (DC101 vehicle) i.p. every
three days and 0.15 ml injectable saline (vinblastine vehicle) i.p.
every three days.
[0053] Group II--0.4 ml of 2 mg/ml DC101 antibody (800 .mu.g/mouse)
(24) every three days and 0.15 ml of injectable saline i.p. every
three days
[0054] Group III--vinblastine sulfate 0.75 mg/m.sup.2 i.p. bolus at
the start of therapy, followed by 1 mg/m.sup.2/day via subcutaneous
Alzet osmotic pumps (Alza Corp,Palo Alto, Calif.) for 3 weeks,
followed by maintenance therapy with 0.15 ml of 0.067 mg/ml
vinblastine sulfate (1.5 mg/m.sup.2) i.p. every three days, and 0.4
ml of PBS i.p. every three days
[0055] Group IV--combination of DC101 and vinblastine at doses
identical to the single agent groups.
[0056] The body weight, tumor size and general clinical status of
the animals were recorded every 2-3 days. Perpendicular tumor
diameters were measured using a vernier scale caliper and tumor
volume was estimated using the formula for ellipsoid:
(width.sup.2.times.length)/2. Growth curves were statistically
analyzed using repeated measures ANOVA. All animal care was in
accordance with institutional guidelines. As required by
institutional guidelines, the mice were sacrificed when tumor size
reached 1.5 cm.sup.3 or 7.5-10% of their body weight.
[0057] Histology: All tumors were excised, fixed in 10% (v/v)
formalin and processed for immunohistochemical analysis. To obtain
adequate tissue for the combination treatment group, two mice were
sacrificed at 7.5 weeks of treatment. Paraffin blocks were cut to 5
.mu.m sections and stained with haematoxylin/eosin for morphology
evaluation and with Apoptosis Detection System (Promega, Madison,
Wis.) for assessment of programmed cell death.
[0058] Relative tumor vascularity assessed by an FITC-Dextran
perfusion assay: The method was designed to assess the relative
functionality of the tumor vasculature. 2.times.10.sup.6 SK-N-AS
neuroblastoma cells were injected into the flanks of CB-17 SCID
mice. Tumors were allowed to grow to approximately 0.75 cm.sup.3 at
which point tumor bearing mice were then treated with 1 mg/m.sup.2
vinblastine i.p. every three days, 800 .mu.g DC101 i.p. every three
days, combination of the two agents or saline as a control. At 14
days, when divergence in tumor growth between the treatment groups
was clearly evident, 0.2 ml of 25 mg/ml FITC-Dextran in PBS (Sigma,
St. Louis, Mo.) was injected systemically into the lateral tail
vein of each mouse and allowed to circulate for 20-30 minutes. Mice
were then sacrificed by cervical dislocation and blood samples were
collected into heparinized tubes by cardiac puncture for assessment
of systemic fluorescein levels. Tumors were resected from the
surrounding connective tissue being careful to avoid spillage of
intra-vascular contents, weighed and placed into tubes containing
1:10 dispase (Collaborative Research, Two Oaks, Bedford, Mass.). To
normalize for dilution caused by the difference in tumor sizes, 1
ml of 1:10 dispase was added per 0.5 g of tissue. Tumors were
incubated in a dark 37.degree. C. shaker overnight. The tissue was
homogenized, centrifuged at 5000 rpm for 10 minutes, and the
supernatant was collected and stored in the dark until further
analysis. Blood samples were centrifuged immediately following
collection, plasma separated and protected from light at 4.degree.
C. until analysis. Fluorescence readings were obtained on a FL600
Fluorescence Plate Reader (Bio-tek Instruments Inc., Winooski, Vt.,
USA), from a standard curve created by serial dilution of the
FITC-dextran used for injection. The ratio of tumor fluorescence:
plasma fluorescence was assumed to be reflective of the degree of
tumor perfusion.
[0059] In vivo angiogenesis assessment by the Matrigel plug assay
(5,25): Matrigel (Collaborative Biomedical Products, Bedford,
Mass.) stored at -20.degree. C., was thawed at 4.degree. C.
overnight and mixed with 500 ng/ml bFGF. 0.5 ml of this mixture was
then injected subcutaneously into the shaved flanks of twenty 6-8
week old female Balb/cJ mice (Jackson Labs, Bar Harbor, Me.). Five
mice, used as negative controls, were injected with Matrigel alone.
Three days later, treatment mice were randomized into four groups
as follows:
[0060] Group I--saline i.p.,
[0061] Group II--800 .mu.g DC101 i.p.
[0062] Group III--1 mg/m.sup.2 vinblastine i.p.
[0063] Group IV--combination therapy.
[0064] All 25 mice were treated on day 4 and 7 and sacrificed on
day 10. Blood samples were collected into heparinized tubes by
cardiac puncture, centrifuged immediately following their
collection, plasma separated and protected from light at 4.degree.
C. The Matrigel plugs were resected from surrounding connective
tissues, placed into tubes containing 1 ml of 1:10 dispase and
incubated in the dark in a 37.degree. C. shaker overnight. The
following day, the plugs were homogenized, centrifuged at 5000 rpm
for 10 minutes and supematant saved in the dark for analysis of
fluorescence. Fluorescence readings were obtained on FL600
Fluorescence Plate Reader using a standard curve created by serial
dilution of FITC-dextran used for injection. Angiogenic response
was expressed as a ratio of Matrigel plug fluorescence: plasma
fluorescence.
[0065] In vitro determination of differential drug sensitivity:
Prior to undertaking our in vivo experiments we established a dose
of vinblastine, at which significant toxicity of endothelial, but
not tumor, cells was observed. To do so, we optimized growth
conditions to achieve comparable levels of mitotic activity in two
human neuroblastoma cell lines (SK-NM-C and SK-N-AS) and HUVEC. All
three cell lines were grown in DMEM with 10% bovine serum, but the
HUVEC were grown on gelatinized plates and in the presence of
additional growth factors (bFGF and EGF). The untreated controls
show similar levels of .sup.3H-Thymidine incorporation for all
three cell lines thus eliminating the concern that the differences
in proliferation may be inherent. At the higher concentrations of
vinblastine used (e.g.100-400 ng/ml) all three cell populations
were strongly inhibited, especially HUVEC. In striking contrast, at
the lowest concentrations (e.g. 0.78 ng/ml) vinblastine retained
almost the same degree of inhibitory activity against HUVEC,
whereas anti-proliferative activity against two tumor cell lines
was not. The source of this differential sensitivity is not clear,
but it should be noted that at least one of the tumor cell lines,
SK-N-MC, is positive for multidrug resistance-associated protein
(MRP) (26). These in vitro findings suggest that the lowering of
the usual maximum tolerated dose (MTD) used in the clinic may allow
retention of good vinblastine activity against dividing endothelial
cells present in tumors.
[0066] In vivo tumor growth assessment: Building on this in vitro
difference in sensitivity to vinblastine, we went on to evaluate
lower doses of vinblastine in an in vivo model, using an increased
dose frequency to maximize the endothelial injury. Xenografts of
either SK-N-MC neuroepithelioma or SK-N-AS neuroblastoma cell lines
were implanted subcutaneously in the flanks of 4-6 week old. CB-17
SCID mice and grown to .about.0.75 cm.sup.3 before initiation of
treatment. The first treatment group, treated with DC101, an
anti-Flk1 receptor antibody shown previously to inhibit growth of
different kinds of human xenografts in mice and in mouse tumor
models (5), showed an anticipated effectiveness in inhibiting tumor
growth, but, its effect was not sustained. The findings in the
second treatment group (vinblastine alone), were even more
surprising. This agent, traditionally thought to act by inhibiting
tumor cell proliferation through inhibition of tubulin assembly,
produced significant, albeit not sustained, regression of tumor
growth even though used at subclinical low-dose,. This growth delay
in the vinblastine group was further potentiated with the
simultaneous treatment with the anti-flk-1 antibody, DC101. The
combination treatment induced an initial response comparable to the
other treatment groups but then caused further, long term, tumor
regression. To date, the mice in combination therapy group have not
manifested any resistance to the treatment or recurrence of
disease, despite almost seven months of continuous treatment. The
mice remain healthy, with almost no evidence of tumor, except for a
small, barely palpable remnant in one of the mice. [What were the
doses used?]
[0067] Toxicity evaluation: Anti-vascular therapy would be expected
to show minimal toxicity in the post-natal stage of development. To
evaluate this aspect of DC101/vinblastine combination therapy, the
health status of the mice was monitored. Weight was plotted at
regular intervals and considered a surrogate for evaluation of
systemic well being, anorexia, or failure to thrive. No significant
differences in weights were seen between the four groups. The
weight curve of the DC101 group parallels very closely that of the
control group. The vinblastine group showed some weight gain
retardation, but the differences never became significantly
different from controls. Similarly, the toxicity profile in the
combination treatment group was very similar to those in the single
agent groups, with the exception of a transient episode of weight
loss associated with diarrhea. The episode lasted approximately 2-3
weeks and was unlikely to be due to the therapy as the mice
recovered without interruption of treatment. Other usual signs of
drug toxicity in mice such as ruffled fur, anorexia, cachexia, skin
tenting (due to dehydration), skin ulcerations or toxic deaths,
were not seen at the doses used in our experiments. Diarrhea, a
common sign of vinblastine toxicity when doses of 10 mg/m.sup.2 are
used, was generally not observed, except for the above mentioned
episode.
[0068] Histopathologic analysis: To further elucidate the
mechanisms involved in the tumor regression following treatment
with vinblastine, DC101, or the combined therapy, tissue
histopathology assessment was undertaken. Cancer cells with high
nuclear to cytoplasmic ratio form cuffs around central vessels, and
apoptotic cells characterized by pyknotic nuclei and cytoplasmic
blebbing, are only evident as a thin rim at the periphery of the
cuffs. The nuclei of these cells stain strongly for terminal
deoxynucleotidyl transferase (TUNEL) reactivity, as expected for
cells undergoing apoptosis. Vinblastine alone or DC101 treatment
alone both show an increase in the width of the apoptotic rims,
suggesting the cells most distal to the tumor vasculature are
primarily affected, but a large percentage of viable tumor cells
still survive in the center of the cuff. In contrast, histology of
the combined therapy group, as would be predicted by the regression
in tumor size in this treatment group at the time of analysis,
shows overwhelming loss of both cell viability and pre-existing
tumor architecture. There is a close similarity of the appearance
of H/E and TUNEL stain. Interestingly, we observed signs of
endothelial cell toxicity in all of the treatment groups. Rather
than a typical single layer of flattened endothelial cells
surrounding the vascular lumen in untreated group, we observed
edema, and detachment from surrounding basement membrane and
leading to complete vascular wall disintegration and tumor cell
death.
[0069] Tumor perfusion by assessment of intravascular fluorescence:
To further explore the possibility that tumor regression induced
with treatment using DC101 and vinblastine was indeed due to the
vascular injury, rather than a direct anti-tumor cell effect, we
assessed tumor perfusion directly by using a FITC-Dextran
fluorescence method. Mice carrying established subcutaneous SK-
N-AS human neuroblastoma xenografts (.about.0.75 cm.sup.3) were
randomized into four groups and treated systemically with either
saline control, DC101, vinblastine or combination therapy for 10
days. FITC-Dextran was injected into the lateral tail vein and
equilibrated throughout the vascular compartment. The majority of
the blood-borne dextran, because of its 150 kDa size, remains
intravascular, and despite some perivascular losses due to changes
in vascular permeability and the possibility of interstitial
hemorrhages, the fluorescence is reflective of the overall volume
of blood passing through the tumor vasculature. Since our therapy
is chronic in its nature, changes in intra-tumoral vascular/blood
volume are likely to represent structural changes rather than
transient fluxes in vascular permeability. By these criteria DC101
alone caused a 47 % decrease in tumor perfusion, whereas
vinblastine alone resulted in a 41% decrease, and the combination
of the two drugs resulted in 65% perfusion inhibition. Of interest
is the appreciable difference in gross vascularity in the
corresponding tumor specimens.
[0070] Effects of chemotherapy treatments on in vivo angiogenesis:
The direct assessment of tumor vasculature does not provide any
clues as to whether the apparent vascular inhibition within the
tumor is a primary cause or a secondary consequence of the tumor
regression. Evidence for the former would provide support for the
hypothesis that low-dose vinblastine treatment alone is potentially
anti-angiogenic, and the extent of this anti-angiogenic effect may
be further enhanced by concurrent treatment with DC101. Again, the
ratio between intra- and extra-vascular volume within the tumor
could be also somewhat affected by transient changes in vascular
permeability (27). To address these questions, we repeated the same
fluorescence measurement using an in vivo Matrigel plug
angiogenesis assay. Four treatment groups were treated with an
identical therapeutic regimen as in the tumor perfusion experiment
of example 11. The regression of vascularity in subcutaneously
implanted Matrigel pellets was quantitatively assessed by measuring
the fluorescence of circulating FITC-labeled dextran. DC101
administration inhibited bFGF induced vascularization to 50% of the
positive control group, and vinblastine administration inhibited
vascularization to 62.5% of the positive control group. There was
again an enhanced effect with combination therapy, which reduced
the Matrigel pellet fluorescence, and by inference vascularization
to 29.2% of control, a level only marginally different to the
negative control (Matrigel not supplemented with growth
factors).
[0071] Thus, large (0.75 cm.sup.3) established human neuroblastoma
xenografts could be induced to completely regress with this
combination strategy, whereas either agent alone caused only
partial and temporary regressions with relapses observed in all
animals treated at between 30-50 days after initiation of the
individual therapy treatments. In striking contrast, a fully
regressed state could be induced and maintained for as long as the
combination therapy was maintained, which in our case was 200 days,
in the absence of any significant toxicity, as assessed by lack of
weight loss. No myelosuppression has been observed.
[0072] The dose of vinblastine used in our experiments was in the
range of 1.5 mg/m.sup.2, every 3 days, which is approximately 3
times the MTD of this drug in humans, and {fraction (1/16)}-
{fraction (1/20)} of the MTD in mice, given the fact that the MTD
of vinblastine in mice is 4-5 times higher than in humans (33,34).
Using the Matrigel plug assay, we demonstrated that continuous low
dose vinblastine administration can cause a direct anti-angiogenic
effect in vivo. The combined effect with DC101 was significant.
Sequence CWU 1
1
16 1 10 PRT Mouse 1 Gly Phe Asn Ile Lys Asp Phe Tyr Met His 1 5 10
2 17 PRT Mouse 2 Trp Ile Asp Pro Glu Asn Gly Asp Ser Asp Tyr Ala
Pro Lys Phe Gln Gly 1 5 10 15 3 8 PRT Mouse 3 Tyr Tyr Gly Asp Tyr
Glu Gly Tyr 1 5 4 10 PRT Mouse 4 Ser Ala Ser Ser Ser Val Ser Tyr
Met His 1 5 10 5 7 PRT Mouse 5 Ser Thr Ser Asn Leu Ala Ser 1 5 6 9
PRT Mouse 6 Gln Gln Arg Ser Ser Tyr Pro Phe Thr 1 5 7 117 PRT Mouse
7 Gln Val Lys Leu Gln Gln Ser Gly Ala Glu Leu Val Gly Ser Gly Ala 1
5 10 15 Ser Val Lys Leu Ser Cys Thr Thr Ser Gly Phe Asn Ile Lys Asp
Phe 20 25 30 Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu
Glu Trp Ile 35 40 45 Gly Trp Ile Asp Pro Glu Asn Gly Asp Ser Asp
Tyr Ala Pro Lys Phe 50 55 60 Gln Gly Lys Ala Thr Met Thr Ala Asp
Ser Ser Ser Asn Thr Ala Tyr 65 70 75 80 Leu Gln Leu Ser Ser Leu Thr
Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Asn Ala Tyr Tyr Gly
Asp Tyr Glu Gly Tyr Trp Gly Gln Gly Thr Thr 100 105 110 Val Thr Val
Ser Ser 115 8 108 PRT Mouse 8 Asp Ile Glu Leu Thr Gln Ser Pro Ala
Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Ile Thr Cys
Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 His Trp Phe Gln Gln
Lys Pro Gly Thr Ser Pro Lys Leu Trp Ile Tyr 35 40 45 Ser Thr Ser
Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly
Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Met Glu Ala Glu 65 70
75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Phe
Thr 85 90 95 Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg Ala 100
105 9 30 DNA Mouse 9 ggcttcaaca ttaaagactt ctatatgcac 30 10 51 DNA
Mouse 10 tggattgatc ctgagaatgg tgattctgat tatgccccga agttccaggg c
51 11 24 DNA Mouse 11 tactatggtg actacgaagg ctac 24 12 30 DNA Mouse
12 agtgccagct caagtgtaag ttacatgcac 30 13 21 DNA Mouse 13
agcacatcca acctggcttc t 21 14 27 DNA Mouse 14 cagcaaagga gtagttaccc
attcacg 27 15 351 DNA Mouse 15 caggtcaagc tgcagcagtc tggggcagag
cttgtggggt caggggcctc agtcaaattg 60 tcctgcacaa cttctggctt
caacattaaa gacttctata tgcactgggt gaagcagagg 120 cctgaacagg
gcctggagtg gattggatgg attgatcctg agaatggtga ttctgattat 180
gccccgaagt tccagggcaa ggccaccatg actgcagact catcctccaa cacagcctac
240 ctgcagctca gcagcctgac atctgaggac actgccgtct attactgtaa
tgcatactat 300 ggtgactacg aaggctactg gggccaaggg accacggtca
ccgtctcctc a 351 16 324 DNA Mouse 16 gacatcgagc tcactcagtc
tccagcaatc atgtctgcat ctccagggga gaaggtcacc 60 ataacctgca
gtgccagctc aagtgtaagt tacatgcact ggttccagca gaagccaggc 120
acttctccca aactctggat ttatagcaca tccaacctgg cttctggagt ccctgctcgc
180 ttcagtggca gtggatctgg gacctcttac tctctcacaa tcagccgaat
ggaggctgaa 240 gatgctgcca cttattactg ccagcaaagg agtagttacc
cattcacgtt cggctcgggg 300 accaagctgg aaataaaacg ggcg 324
* * * * *