U.S. patent application number 10/693688 was filed with the patent office on 2004-12-30 for adenovirus-mediated intratumoral delivery of an angiogenesis antagonist for the treatment of tumors.
Invention is credited to Griscelli, Frank, Legrand, Yves, Li, Hong, Lu, He, Mabilat, Christelle, Opolon, Paule, Perricaudet, Michel, Ragot, Thierry, Soria, Claudine, Soria, Jeannette, Yeh, Patrice.
Application Number | 20040265273 10/693688 |
Document ID | / |
Family ID | 21935358 |
Filed Date | 2004-12-30 |
United States Patent
Application |
20040265273 |
Kind Code |
A1 |
Li, Hong ; et al. |
December 30, 2004 |
Adenovirus-mediated intratumoral delivery of an angiogenesis
antagonist for the treatment of tumors
Abstract
The present invention relates to gene therapy for the treatment
of tumors. The invention more particularly relates to introduction
of a gene encoding an anti-angiogenic factor into cells of a tumor,
for example with a defective adenovirus vector, to inhibit growth
or metastasis, or both, of the tumor. In a specific embodiment,
delivery of a defective adenovirus that expresses the amino
terminal fragment of urokinase (ATF) inhibited growth and
metastasis of tumors. These effects were correlated with a
remarkable inhibition of neovascularization within, and at the
immediate vicinity of, the injection site. Delivery of a defective
adenovirus vector that expresses kringles 1 to 3 of angiostatin
inhibited tumor growth and tumorigenicity, and induced apoptosis of
tumor cells. The invention further provides viral vectors for use
in the methods of the invention.
Inventors: |
Li, Hong; (Epinay Sur Seine,
FR) ; Lu, He; (Epinay Sur Seine, FR) ;
Griscelli, Frank; (Paris, FR) ; Opolon, Paule;
(Paris, FR) ; Soria, Claudine; (Taverny, FR)
; Ragot, Thierry; (Meudon, FR) ; Legrand,
Yves; (Paris, FR) ; Soria, Jeannette;
(Taverny, FR) ; Mabilat, Christelle; (Corbeil
Essonnes, FR) ; Perricaudet, Michel; (Ecrosnes,
FR) ; Yeh, Patrice; (Gif Sur Yvette, FR) |
Correspondence
Address: |
WILEY, REIN & FIELDING, LLP
ATTN: PATENT ADMINISTRATION
1776 K. STREET N.W.
WASHINGTON
DC
20006
US
|
Family ID: |
21935358 |
Appl. No.: |
10/693688 |
Filed: |
October 27, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10693688 |
Oct 27, 2003 |
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09403736 |
Jun 29, 2000 |
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6638502 |
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09403736 |
Jun 29, 2000 |
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PCT/EP98/02491 |
Apr 27, 1998 |
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60044980 |
Apr 28, 1997 |
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Current U.S.
Class: |
424/93.2 ;
435/456 |
Current CPC
Class: |
A61P 35/00 20180101;
A61P 35/04 20180101; C12N 2799/022 20130101; A61K 48/00 20130101;
A61K 38/49 20130101; A61K 38/484 20130101 |
Class at
Publication: |
424/093.2 ;
435/456 |
International
Class: |
A61K 048/00; C12N
015/86 |
Claims
What is claimed is:
1. A method for inhibiting growth of a tumor comprising introducing
into the tumor a defective adenovirus vector comprising a gene
encoding an anti-angiogenic factor operably associated with an
expression control sequence that provides for expression of the
anti-angiogenic factor in a cell of the tumor.
2. The method according to claim 1, wherein the tumor is a lung
carcinoma or a breast carcinoma.
3. The method according to claim 1, wherein the anti-angiogenic
factor comprises a sequence of an amino terminal fragment of
urokinase having an EGF-like domain, with the proviso that the
factor is not urokinase.
4. The method according to claim 3, wherein the anti-angiogenic
factor is an amino terminal fragment of urokinase having an amino
acid sequence of urokinase from about amino acid residue 1 to about
residue 135.
5. The method according to claim 4, wherein the urokinase is murine
urokinase.
6. The method according to claim 4, wherein the urokinase is human
urokinase.
7. The method according to claim 1, wherein the anti-angiogenic
factor is angiostatin.
8. The method according to claim 7, wherein the angiostatin
comprises kringles 1 to 3.
9. The method according to claim 7, wherein the angiostatin is an
amino terminal fragment of plasminogen (Plg) having an amino acid
sequence of plasminogen from about amino acid residue 1 to about
residue 333.
10. The method according to claim 9, wherein the plasminogen is
human plasminogen.
11. A method for inhibiting growth or metastasis, or both, of a
tumor comprising introducing a vector comprising a gene encoding an
amino terminal fragment of urokinase having an EGF-like domain into
the tumor, with the proviso that the gene does not encode
urokinase, wherein the gene is operably associated with an
expression control sequence that provides for expression of the
gene in a cell of the tumor.
12. The method according to claim 11, wherein the amino terminal
fragment of urokinase has an amino acid sequence of urokinase from
about amino acid residue 1 to about residue 135.
13. The method according to claim 12, wherein the urokinase in
murine urokinase.
14. The method according to claim 12, wherein the urokinase in
human urokinase.
15. A defective adenovirus vector comprising a gene encoding an
anti-angiogenic factor operably associated with an expression
control sequence.
16. The virus vector according to claim 15, wherein the
anti-angiogenic factor comprises a nucleic acid sequence of an
amino terminal fragment of urokinase having an EGF-like domain,
with the proviso that the factor is not urokinase.
17. A defective adenovirus vector comprising a gene encoding an
amino terminal fragment of urokinase having an EGF-like domain,
with the proviso that the gene does not encode urokinase.
18. The virus vector according to claim 17, wherein the amino
terminal fragment of urokinase has an amino acid sequence of
urokinase from amino acid residue 1 to about residue 135.
19. The virus vector according to claim 18, wherein the urokinase
is murine urokinase.
20. The virus vector according to claim 18, wherein the urokinase
is human urokinase.
21. The virus vector according to claim 15, wherein the
anti-angiogenic factor is angiostatin.
22. The virus vector according to claim 21, wherein the angiostatin
comprises kringles 1 to 3.
23. The virus vector according to claim 21, wherein the angiostatin
comprises a nucleic acid sequence of an amino terminal fragment of
plasminogen having an amino acid sequence of plasminogen from amino
acid residue 1 to about residue 333.
24. The virus vector according to claim 23, wherein the plasminogen
is human plasminogen.
25. A pharmaceutical composition comprising a virus vector of any
one of claims 15-24 and a pharmaceutically acceptable carrier.
26. Use of the virus vector of any one of claims 15-24 in the
manufacture of a medicament for inhibiting growth of a tumor.
27. Use of the virus vector of any one of claims 16-20 in the
manufacture of a medicament for inhibiting growth, or metastasis,
or both of a tumor.
28. Use of the virus vector of any one of claims 21-24 in the
manufacture of a medicament for inhibiting tumor growth and
inducing apoptosis.
29. Use of a vector comprising a gene encoding an amino-terminal
fragment of urokinase having an EGF-like domain, with the proviso
that the gene does not encode urokinase, operably associated with
an expression control sequence that provides for expression of the
anti-angiogenic factor in the manufacture of a medicament for
inhibiting growth or metastasis, or both, of a tumor.
30. The use according to any of claims 26-29, wherein the tumor is
a lung carcinoma or a breast carcinoma.
33. The method according to claim 31, wherein the anti-angiogenic
factor comprises an amino terminal fragment of urokinase comprising
an EGF-like domain, with the exception that the anti-angiogenic
factor is not urokinase.
34. The method according to claim 33, wherein the anti-angiogenic
factor is an amino terminal fragment of urokinase comprising an
amino acid sequence of urokinase from about amino acid residue 1 to
about residue 135.
35. The method according to claim 34, wherein the urokinase is
murine urokinase.
36. The method according to claim 34, wherein the urokinase is
human urokinase.
37. The method according to claim 31, wherein the anti-angiogenic
factor is angiostatin.
38. The method according to claim 37, wherein the angiostatin
comprises kringles 1 to 3.
39. The method according to claim 37, wherein the angiostatin is an
amino terminal fragment of plasminogen (Plg) comprising an amino
acid sequence of plasminogen from about amino acid residue 1 to
about residue 333.
40. The method according to claim 39, wherein the plasminogen is
human plasminogen.
41. A method for inhibiting growth and/or metastasis of a tumor
comprising introducing a vector comprising a gene encoding an amino
terminal fragment of urokinase comprising an EGF-like domain into
the tumor, with the exception that the gene does not encode
urokinase, wherein the gene is operably associated with an
expression control sequence that provides for expression of the
gene in a cell of the tumor.
42. The method according to claim 41, wherein the amino terminal
fragment of urokinase comprises an amino acid sequence of urokinase
from about amino acid residue 1 to about residue 135.
43. The method according to claim 42, wherein the urokinase is
murine urokinase.
44. The method according to claim 42, wherein the urokinase is
human urokinase.
45. A defective adenovirus vector comprising a gene encoding an
anti-angiogenic factor operably associated with an expression
control sequence.
46. The defective adenovirus vector according to claim 45, wherein
the anti-angiogenic factor comprises an amino terminal fragment of
urokinase comprising an EGF-like domain, with the exception that
the anti-angiogenic factor is not urokinase.
47. A defective adenovirus vector comprising a gene encoding an
amino terminal fragment of urokinase comprising an EGF-like domain,
with the exception that the gene does not encode urokinase.
48. The defective adenovirus vector according to claim 47, wherein
the amino terminal fragment of urokinase comprises an amino acid
sequence of urokinase from about amino acid residue 1 to about
residue 135.
49. The defective adenovirus vector according to claim 48, wherein
the urokinase is murine urokinase.
50. The defective adenovirus vector according to claim 48, wherein
the urokinase is human urokinase.
51. The defective adenovirus vector according to claim 45, wherein
the anti-angiogenic factor is angiostatin.
52. The defective adenovirus vector according to claim 51, wherein
the angiostatin comprises kringles 1 to 3.
53. The defective adenovirus vector according to claim 51, wherein
the angiostatin comprises an amino terminal fragment of plasminogen
comprising an amino acid sequence of plasminogen from about amino
acid residue 1 to about residue 333.
54. The defective adenovirus vector according to claim 53, wherein
the plasminogen is human plasminogen.
55. A pharmaceutical composition comprising the defective
adenovirus vector according to claim 45 and a pharmaceutically
acceptable carrier.
56. A pharmaceutical composition comprising the defective
adenovirus vector according to claim 47 and a pharmaceutically
acceptable carrier.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to gene therapy for the
treatment of tumors. The invention more particularly relates to
introduction of a gene encoding an anti-angiogenic factor into
cells of a tumor, for example with an adenovirus vector, to inhibit
growth or metastasis, or both, of the tumor.
BACKGROUND OF THE INVENTION
[0002] Cell migration is a coordinated process that bridges
cellular activation and adhesion whereas the equilibrium between
pericellular proteolysis and its inhibition (e.g., triggered by
plasminogen activator inhibitors and tissue inhibitors of
metalloproteinases) is disrupted (1-3). Urokinase plasminogen
activator (uPA) is a pivotal player in this process because it
initiates a proteolytic cascade at the surface of migrating cells
by binding to its cell surface receptor (uPAR) (4, 5). Binding of
uPA to its receptor greatly potentiates plasminogen/plasmin
conversion at the cell surface (6). Plasmin is a broadly specific
serine protease which can directly degrade components of the
extracellular matrix such as fibronectin, vitronectin or laminin.
Plasmin also indirectly promotes a localized degradation of the
stroma by converting inactive zymogens into active
metalloproteinases (7). The selective distribution of uPAR at the
leading edge of migrating cells (invadopodes) apparently
concentrates uPA secreted by themselves or by neighboring stroma
cells (8). uPAR is also directly involved in cellular adhesion to
the extracellular matrix as illustrated by its uPA-dependent
binding to vitronectin (9), and because uPAR modulates the binding
properties of several integrin molecules (10). Finally, uPA and
plasmin are somehow involved in cell morphogenesis by activating or
inducing the release of morphogenic factors such as vascular
endothelial growth factor (VEGF), hepatocyte growth factor (HGF),
fibroblast growth factors (FGFs) and transforming growth factor B
(TGF.beta.) (11, 12).
[0003] Taken together, these observations indicate that the
uPA/uPAR system controls cell migration by coordinating cellular
activation, adhesion and motility. This statement is supported by
clinical observations that correlate the presence of enhanced uPA
activity at the invasive edge of the tumors (13, 14). That melanoma
induced by DMBA and croton oil do not progress to a malignant stage
in uPA-deficient mice also support a role of uPA in tumor
establishment and progression (15).
[0004] uPA binds to uPAR by its light chain fragment, also known as
amino-terminal fragment (ATF, amino acid 1-135). This interaction
is species restricted (16) and involves the EGF-like domain of ATF
(residues 146), in which amino acid 19-32, which are not conserved
between mice and human, are critical (17, 18). ATF-mediated
disruption of the uPA/uPAR complex inhibits tumor cell migration
and invasion in vitro (19). Intraperitoneal bolus injection of a
chimeric human ATF-based antagonist has also been used to inhibit
lung metastases of human tumor cells implanted within athymic mice,
without significantly affecting primary tumor growth (20). A
further study reported that intraperitoneal injection of synthetic
peptides derived from murine ATF was effective in inhibiting both
primary tumor growth and lung metastases (21). These results are
consistent with a role of the uPA/uPAR complex in controlling the
motility of both tumor and endothelial cells (22). That a chimeric
murine ATF-based antagonist could inhibit vessel growth in an
artificial bFGF-enriched extracellular matrix (23) further supports
uPA/uPAR involvement in controlling angiogenesis in vivo.
[0005] The formation of blood vessels, or angiogenesis, results
from the capillary growth of pre-existing vessels. Angiogenesis is
essential for a number of physiological processes such as embryonic
development, wound healing and tissue or organ regeneration.
Abnormal growth of new blood vessels occurs in pathological
conditions such as diabetic retinopathy and tumor growth, as well
as tumor dissemination to distant sites [38,24]. Both experimental
and clinical studies have showed that primary tumors as well as
metastasis remain dormant due to a balanced rate of proliferation
and apoptosis unless the angiogenesis process is switched on
[39].
[0006] The growth of endothelial cells is tightly regulated by both
positive and negative factors. The onset of tumor angiogenesis
could be triggered either by an upregulation of tumor-released
angiogenic factors such as vascular endothelial growth factor
(VEGF) and acid or/and basic fibroblast growth factor (bFGFs), or
by a downregulation of angiostatic factors such as thrombospondin
and angiostatin [39]. Both the reconstitution of angiostatic
factors and the removal of angiogenic stimulating factors thus
constitute plausible clinical strategies to suppress tumor
angiogenesis [40, 41]. Angiostatic-based therapies should also
apply to all solid tumors because endothelial cells do not vary
from one tumor type to the other, further emphasizing the clinical
relevance of such an anti-cancer approach. Thus, the therapy
targeting angiogenesis appears to be highly relevant to clinical
practice.
[0007] Many physiological angiostatic factors are derived upon
proteolytic cleavage of circulating proteins. This is the case for
angiostatin [32], endostatin [42], the 16 kDa fragment of prolactin
[43], or platelet factor-4 [44]. Angiostatin was initially isolated
from mice bearing a Lewis lung carcinoma (LLC), and was identified
as a 38 kDa internal fragment of plasminogen (Plg) (aa 98-440) that
encompasses the first four kringles of the molecule [32;
WO95/29242; U.S. Pat. No. 5,639,725]. Angiostatin has been shown to
be generated following hydrolysis of Plg by a metalloelastase from
GM-CSF-stimulated tumor-infiltrating macrophages [45].
Intraperitoneal bolus injections of purified angiostatin in six
different tumor models have proved to be very effective in
suppressing primary tumor growth, with no apparent toxicity [46].
Angiostatin-mediated suppression of tumor angiogenesis apparently
drove the tumor cells into a higher apoptotic rate that
counterbalanced their proliferation rate. In this study, tumor
growth usually resumed following removal of the angiostatin
molecule, emphasizing the importance of achieving long-term
delivery for optimal clinical benefits [46]. In vitro studies with
recombinant proteins indicated that the angiostatic activity of
angiostatin was mostly mediated by kringles 1-3, thus leaving a
minor activity for kringle 4 [47]. As for most angiostatic factors,
little is known about the molecular pathway by which angiostatin
exerts its effect.
[0008] As angiostatic therapy will require a prolonged maintenance
of therapeutic levels in vivo, the continuous delivery of a
recombinant protein will be expensive and cumbersome. Direct in
vivo delivery of the corresponding genes with viral vectors
constitutes an attractive solution to this problem. Because most
cancer gene therapies currently rely on destructive strategies that
target the tumor cells [48], viral-mediated gene delivery of an
angiostatic factor represents a conceptually different, and
possibly synergistic, approach to fight cancer.
[0009] Despite these results, there remains a need to develop
effective treatments for tumors, particularly
chemotherapy-resistant tumors.
[0010] The present invention addresses this need by establishing an
effective mode for treating a tumor.
[0011] Various references are cited in this specification by
number, which are fully set forth after the Examples. None of the
references cited herein should be construed as describing or
suggesting the invention disclosed herein.
SUMMARY OF THE INVENTION
[0012] The present invention advantageously provides a highly
effective gene therapy for tumors. Indeed, in a specific embodiment
of the invention murine urokinase ATF expressed by human tumor
cells in an athymic murine model unexpectedly effectively inhibits
tumorigenicity. In another embodiment, angiostatin expressed in
tumor cells in a murine model inhibited tumor growth and
tumorigenesis, and induced tumor cell apoptosis, in addition to
blocking angiogenesis.
[0013] In a broad aspect, the present invention provides a method
for inhibiting growth or metastasis, or both, of a tumor comprising
introducing a vector comprising a gene encoding an anti-angiogenic
factor operably associated with an expression control sequence that
provides for expression of the anti-angiogenic factor into a cell
or cells of the tumor. Preferably, the vector is a virus vector;
more preferably the virus vector is an adenovirus vector. In a
specific embodiment exemplified infra, the adenovirus vector is a
defective adenovirus vector.
[0014] The methods of the invention are useful in the treatment of
many tumors, as set forth in detail herein. For example, in
specific embodiments, the tumor is a lung carcinoma or a breast
carcinoma.
[0015] In addition, the invention demonstrates for the first time
the advantages of expression of an anti-angiogenic factor by the
transduced tumor cells. Accordingly, a gene encoding any
anti-angiogenic factor, such as a soluble receptor for an
angiogenic protein, or an angiogenesis antagonist, can be delivered
in the practice of the invention. In a specific embodiment, the
anti-angiogenic factor comprises a sequence of an amino terminal
fragment of urokinase having an EGF-like domain, with the proviso
that the factor is not urokinase. For example, the anti-angiogenic
factor may be a chimeric protein comprising ATF-immunoglobulin or
ATF-human serum albumin. In a preferred embodiment, exemplified
infra, the anti-angiogenic factor is an amino terminal fragment of
urokinase having an amino acid sequence of urokinase from about
amino acid residue 1 to about residue 135. In a specific aspect,
the urokinase is murine urokinase. In a more preferred aspect, the
urokinase is human urokinase.
[0016] In an alternative embodiment, the anti-angiogenic factor is
angiostatin, in particular, kringles 1 to 3 of angiostatin. In a
particularly preferred embodiment, the anti-angiogenic factor is
the amino-terminal fragment of plasminogen (Plg) having an amino
acid sequence of plasminogen from about amino acid residue 1 to
about residue 333. In another preferred embodiment, the
anti-angiogenic factor is the amino-terminal fragment (angiostatin)
from human plasminogen.
[0017] In a related embodiment, the invention is directed to use of
a vector comprising a gene encoding an anti-angiogenic factor
operably associated with an expression control sequence that
provides for expression of the anti-angiogenic factor in the
manufacture of a medicament for inhibiting growth or metastasis, or
both, of a tumor. More particularly, the invention provides for use
of a virus vector of the invention, e.g., as set out below, in the
manufacture of a medicament for inhibiting growth or metastasis, or
both, of a tumor.
[0018] Naturally, in addition to the foregoing methods and uses,
the invention provides a novel virus vector comprising a gene
encoding an anti-angiogenic factor operably associated with an
expression control sequence. In a preferred embodiment, the virus
vector is an adenovirus vector. In a more preferred embodiment, the
virus vector is a defective adenovirus vector. The virus vectors of
the invention can provide a gene encoding any anti-angiogenic
factor, as set forth above. For example, the anti-angiogenic factor
may comprise a sequence of an amino terminal fragment of urokinase
having an EGF-like domain, with the proviso that the factor is not
urokinase. In a preferred embodiment, the anti-angiogenic factor is
an amino terminal fragment of urokinase having an amino acid
sequence of urokinase from amino acid residue 1 to about residue
135. In this embodiment, the urokinase may be murine urokinase or,
preferably, human urokinase.
[0019] The invention further provides a pharmaceutical composition
any of the virus vectors of the invention and a pharmaceutically
acceptable carrier.
[0020] Thus, one object of the invention is to provide gene therapy
by delivery of anti-angiogenic factors for treating tumors.
[0021] Another object of the invention is to provide a viral vector
for delivery of an anti-tumorigenic factor.
[0022] Still another object of the invention is to provide an amino
terminal fragment of urokinase (ATF) by gene therapy for treatment
of a tumor.
[0023] Further, another object of the invention is to provide
angiostatin by gene therapy for treatment of a tumor.
[0024] Yet another object of the invention is to provide
angiostatin, particularly kringles 1 to 3 of angiostatin, by gene
therapy for treatment of a tumor.
[0025] These and other objects of the invention are further
elaborated in the following Detailed Description and Examples, and
the accompanying drawings.
DESCRIPTION OF THE DRAWINGS
[0026] FIG. 1. Molecular characterization of virus AdmATF. Panel A:
Structure of AdmATF and AdCO1. The Ad5 chromosome is 36 kb long and
bordered by inverted terminal repeats. Y refers to the
encapsidation signal. Both viruses are defective for growth because
they lack the Ad5 E1 genes. They also carry a 1.9 kb XbaI deletion
within region E3. A schematic representation of the mATF expression
cassette of virus AdmATF is indicated under the Ad5 chromosome (not
drawn to scale). For a review on adenoviral vectors see (38). Panel
B: analysis of mATF expression. MDA-MB-231 cells were infected for
24 hr by AdCO1 (lane 2) or AdmATF (lane 3), or mock-infected (lane
1), and total poly(A+) RNAs were submitted to northern blot
analysis. The ATF-encoding RNA (0.5 kb) is indicated (arrow). A 1.7
kb molecule is also detected (asterisk), a size in agreement with
the utilization of the polyadenylation signal from the adenovirus
pIX gene. Panel C: analysis of ATF secretion by 293-infected cells.
The culture media of mock-infected cells (lane 1), or infected with
AdCO1 (lane 2) or AdmATF (lane 3) were submitted to a western blot
analysis with a polyclonal anti-mouse uPA antibody.
[0027] FIG. 2. Functional characterization of virus AdmATF. Panel
A: The culture medium of AdmATF-infected cells inhibits plasmin
conversion at the surface of LLC cells (see Methods section of the
Example). 293 refers to the supernatant of non infected cells.
Panel B: Infection of LLC cells with AdmATF (right panel)
specifically inhibits cell invasiveness as compared to that of LLC
cells infected with AdCO1 (left panel). The 1.2 mm pores of the
membranes are visible.
[0028] FIG. 3. Intratumoral injection of AdmATF inhibits LLC tumor
growth in syngeneic mice. Tumor cells (2.times.10.sup.6 cells) were
subcutaneously injected into C57BL/6 mice. After 6 days, the
animals received an intratumoral injection of PBS, or 10.sup.9 PFU
of AdCO1 or AdmATF and tumor growth was monitored. The mean values
are represented with their standard variations (n=10). Statistics
were done with the Student test.
[0029] FIG. 4. Intratumoral injection of AdmATF inhibits LLC tumor
vascularization. Panel A: a representative tumor from the AdCO
1-treated (left) and AdmATF-treated groups extracted at day 10 p.i.
is shown. A representative tumor extracted at day 20 p.i. is shown
in panel B (injection with AdCO1) and panel C (injection with
AdmATF). All photographs were taken at the same magnification. Note
that the AdmATF-injected tumors are much smaller that their
AdCO1-injected controls, especially at the latest time p.i.
(compare panels B and C).
[0030] FIG. 5. Intratumoral injection of AdmATF inhibits MDA-MB-231
tumor growth in nude mice. Tumors were implanted by subcutaneous
injection of 3.times.10.sup.6 MDA-MB-231 cells. At day 11 post
implantation, the mice received an intratumoral injection of PBS,
or 10.sup.9 PFU of AdmATF or AdCO1, and the tumor growth was
monitored. The mean values are represented with their standard
variations.
[0031] FIG. 6. Intratumoral injection of AdmATF inhibits
intratumoral and peritumoral angiogenesis. Panels A and B: vWF
immunostaining of tumor sections. Paraffin embedded MDA-MB-231
tumor sections prepared from the AdCO1-- (A) and AdmATF-treated
groups (B) were revealed with a polyclonal anti-vWF serum at day 52
p.i. Panels C and D: Macroscopic evaluation of peritumoral
vascularization within the skin of tumors injected with AdCO1(C) or
AdmATF (D) at day 20 p.i.
[0032] FIG. 7. (A) Recombinant adenoviruses. The Ad5 genome is a 36
kb-long chromosome. Viruses AdK3 and AdCO1 were derived by a lethal
deletion of the E1 genes (nucleotide position 382 to 3446); they
also carry a non-lethal 1.9 kb XbaI deletion within region E3 (for
a review see [37]). The angiostatin expression cassette is shown
under the Ad5 chromosome. The plasminogen secretion signal is
represented by a blackened box; +1 refers to the CMV-driven
transcription start; AATAAA refers to the SV40 late polyadenylation
signal. (B) Analysis of angiostatin secretion from infected-cells.
100 ng of human Plg (lane 1), culture medium from HMEC-1 infected
with AdK3 (lane 2) or AdCO1 (lane 3), C6 infected with AdK3 (lane
4) or AdCO1 (lane 5), and from MDA-MB-231 infected with AdK3 (lane
6) or AdCO1 (lane 7) were submitted to Western blot analysis. (C)
Immuno-detection of angiostatin within C6 tumor extracts; Tumors
were established in nude mice and received 10.sup.9 PFU of AdCO1
(lane 1) or AdK3 (lane 2) and Western blot analysis was performed
10 days p.i. The signal corresponding to angiostatin (36-38 kDa)
and Plg (92 kDa) are indicated (arrow and asterisk
respectively).
[0033] FIG. 8. (A) Inhibition of endothelial cell proliferation. C6
(panel 1), MDA-MB-231 (panel 2) and HMEC-1 (panel 3) were injected
with AdK3 (.diamond-solid.) or Ad-CO1 (D). HMEC-1 cells (panel 4)
cultured with the supernatant from AdK3-(.diamond-solid.) or
AdCO1-infected C6 glioma cells (.quadrature.). (B) Detection of
MPM-2 phosphoepitope in HMEC-1 cells. Mock-infected cells (lane 1),
AdCO1-infected cells (lane 2), and AdK3-infected cells (lane 3).
(C) MPM-2 epitope were detected in HMEC-1 infected with AdCO1
(panel 1) or AdK3 (panel 2) by indirect immunostaining and DNA
content by propidium iodide staining, and quantified by flow
cytometry (see Methods). A Student's t-test was used for
statistical analysis.
[0034] FIG. 9. AdK3 inhibits tumor growth. C6 glioma (panel A) and
MDA-MB-231 carcinoma (panel B) were subcutaneously implanted into
athymic mice (see Methods). When the tumor had reached a volume of
20 mm.sup.3 (day 0), mice received an intratumoral injection of PBS
(.quadrature.), or 10.sup.9 PFU or AdK3 (.cndot.) or AdCO1
(.diamond-solid.). Mean values are represented with their standard
deviations.
[0035] FIG. 10. AdK3 inhibits C6 tumor growth and angiogenesis.
Tumors from AdCO1-treated (panel A) and AdK3-treated groups (panel
B) are shown 10 days p.i. The extent of vascularization at the
periphery of a representative tumor injected with AdCO1 (panel C)
or AdK3 (panel -D) is shown at day 5 p.i. Paraffin-embedded C6
sections from an AdCO1-injected (panel E) or an AdK3-injected tumor
(panel F) were submitted to vWF-immunostaining at day 10 p.i. The
proportion of apoptotic cells was detected by the TUNEL method
within sections from an AdCO1-injected (panel G) or an
AdK3-injected tumor (panel H). The same magnification was used for
AdCO1- and AdK3-injected tumors.
[0036] FIG. 11. Dose dependent effect of AdK3. C6 cells were
infected in vitro, 24 hours with AdCO1 (panel A) or Ad3K (panel B)
and mixed with a ratio of 1 (.quadrature.), 1:2 (.diamond-solid.)
and 1:4 (.cndot.) with non-infected C6 cells, prior to C6 cells
implantation into athymic mice. Tumor volumes were measured during
20 days. Mean values are represented with their standard
deviations.
DETAILED DESCRIPTION OF THE INVENTION
[0037] As disclosed above, the present invention is directed to
methods and vectors for gene therapy of tumors. The methods and
vectors of the invention inhibit tumor growth or tumor metastasis,
or both. These methods and vectors act by inhibiting angiogenesis
of the tumor to an unexpectedly advantageous degree.
[0038] The invention is based, in part, on experiments involving
gene therapy delivery of the amino terminal fragment of urokinase
(ATF) and angiostatin. ATF is an antagonist of urokinase (uPA)
binding to its cell surface receptor (uPAR), and an inhibitor of
endothelial cell migration. To assess the importance of the
uPA/uPAR interaction for tumor growth and dissemination, a
defective adenovirus expressing murine ATF from the CMV promoter
(AdmATF) was constructed. A single intratumoral injection of AdmATF
inhibited growth of pre-established tumors in two different murine
models, and delayed tumor dissemination. These effects were
correlated with a remarkable inhibition of neovascularization
within, and at the immediate vicinity of, the injection site. The
magnitude of this effect was particularly remarkable in the ability
of murine ATF to inhibit angiogenesis of a human-derived tumor. In
a specific example, a defective adenovirus that expresses the
N-terminal fragment (aa 1-333) from human Plg, including the
pre-activation peptide and kringles 1 to 3 [47] was constructed
(AdK3) and its in vitro and in vivo activity in different murine
tumor models was assessed. The AdK3 vector inhibited tumor growth,
tumor angiogenesis, and tumorigenesis, and induced tumor cell
apoptosis.
[0039] Intratumoral adenovirus-mediated delivery of antagonist
displays potent antitumoral properties by targeting
angiogenesis.
Definitions
[0040] The following defined terms are used throughout the present
specification, and should be helpful in understanding the scope and
practice of the present invention.
[0041] In a specific embodiment, the term "about" or
"approximately" means within 20%, preferably within 10%, and more
preferably within 5% of a given value or range.
[0042] An "anti-angiogenic" factor is a molecule that inhibits
angiogenesis, particularly by blocking endothelial cell migration.
Such factors include fragments of angiogenic proteins that are
inhibitory (such as the ATF of urokinase), angiogenesis inhibitory
factors, such as angiostatin and endostatin; and soluble receptors
of angiogenic factors, such as the urokinase receptor or FGF/VEGF
receptor. The term "angiostatin", which is derived from the
amino-terminal fragment of plasinogen, includes the anti-angiogenic
fragment of angiostatin having kringles 1 to 3. Generally, an
anti-angiogenic factor for use in the invention is a protein or
polypeptide encoded by a gene transfected into tumors using the
vectors of the invention.
[0043] A "variant" of a polypeptide or protein is any analogue,
fragment, derivative, or mutant which is derived from a polypeptide
or protein and which retains at least one biological property of
the polypeptide or protein. Different variants of the polypeptide
or protein may exist in nature. These variants may be allelic
variations characterized by differences in the nucleotide sequences
of the structural gene coding for the protein, or may involve
differential splicing or post-translational modification. The
skilled artisan can produce variants having single or multiple
amino acid substitutions, deletions, additions, or replacements.
These variants may include, inter alia: (a) variants in which one
or more amino acid residues are substituted with conservative or
non-conservative amino acids, (b) variants in which one or more
amino acids are added to the polypeptide or protein, (c) variants
in which one or more of the amino acids includes a substituent
group, and (d) variants in which the polypeptide or protein is
fused with another polypeptide such as serum albumin. The
techniques for obtaining these variants, including genetic
(suppressions, deletions, mutations, etc.), chemical, and enzymatic
techniques, are known to persons having ordinary skill in the
art.
[0044] If such allelic variations, analogues, fragments,
derivatives, mutants, and modifications, including alternative mRNA
splicing forms and alternative post-translational modification
forms result in derivatives of the polypeptide which retain any of
the biological properties of the polypeptide, they are intended to
be included within the scope of this invention.
General Molecular Biology
[0045] In accordance with the present invention there may be
employed conventional molecular biology, microbiology, and
recombinant DNA techniques within the skill of the art. Such
techniques are explained fully in the literature. See, e.g., Sam
brook, Fritsch & Maniatis, Molecular Cloning: A Laboratory
Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y. (herein "Sambrook et al., 1989"); DNA
Cloning: A Practical Approach, Volumes I and II (D. N. Glover ed.
1985); Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic
Acid Hybridization [B. D. Hames & S. J. Higgins eds. (1985)];
Transcription And Translation [B. D. Hames & S. J. Higgins,
eds. (1984)]; Animal Cell Culture [R. I. Freshney, ed. (1986)];
Immobilized Cells And Enzymes [IRL Press, (1986)]; B. Perbal, A
Practical Guide To Molecular Cloning (1984); F. M. Ausubel et al.
(eds.), Current Protocols in Molecular Biology, John Wiley &
Sons, Inc. (1994).
[0046] Therefore, if appearing herein, the following terms shall
have the definitions set out below.
[0047] A "vector" is any means for the transfer of a nucleic acid
according to the invention into a host cell. The term "vector"
includes both viral and nonviral means for introducing the nucleic
acid into a cell in vitro, ex vivo or in vivo. Non-viral vectors
include plasmids, liposomes, electrically charged lipids
(cytofectins), DNA-protein complexes, and biopolymers. Viral
vectors include retrovirus, adeno-associated virus, pox,
baculovirus, vaccinia, herpes simplex, Epstein-Barr and adenovirus
vectors, as set forth in greater detail below. In addition to a
nucleic acid according to the invention, a vector may also contain
one or more regulatory regions, and/or selectable markers useful in
selecting, measuring, and monitoring nucleic acid transfer results
(transfer to which tissues, duration of expression, etc.).
[0048] "Regulatory region" means a nucleic acid sequence which
regulates the expression of a second nucleic acid sequence. A
regulatory region may include sequences which are naturally
responsible for expressing a particular nucleic acid (a homologous
region) or may include sequences of a different origin (responsible
for expressing different proteins or even synthetic proteins). In
particular, the sequences can be sequences of eukaryotic or viral
genes or derived sequences which stimulate or repress transcription
of a gene in a specific or non-specific manner and in an inducible
or non-inducible manner. Regulatory regions include origins of
replication, RNA splice sites, enhancers, transcriptional
termination sequences, signal sequences which direct the
polypeptide into the secretory pathways of the target cell, and
promoters.
[0049] A regulatory region from a "heterologous source" is a
regulatory region which is not naturally associated with the
expressed nucleic acid. Included among the heterologous regulatory
regions are regulatory regions from a different species, regulatory
regions from a different gene, hybrid regulatory sequences, and
regulatory sequences which do not occur in nature, but which are
designed by one having ordinary skill in the art.
[0050] A "cassette" refers to a segment of DNA that can be inserted
into a vector at specific restriction sites. The segment of DNA
encodes a polypeptide of interest, and the cassette and restriction
sites are designed to ensure insertion of the cassette in the
proper reading frame for transcription and translation.
[0051] A cell has been "transfected" by exogenous or heterologous
DNA when such DNA has been introduced inside the cell. A cell has
been "transformed" or "transduced" by exogenous or heterologous DNA
when the transfected DNA effects a phenotypic change.
[0052] "Heterologous" DNA refers to DNA not naturally located in
the cell, or in a chromosomal site of the cell. Preferably, the
heterologous DNA includes a gene foreign to the cell.
[0053] A "nucleic acid" is a polymeric compound comprised of
covalently linked subunits called nucleotides. Nucleic acid
includes polyribonucleic acid (RNA) and polydeoxyribonucleic acid
(DNA), both of which may be single-stranded or double-stranded. DNA
includes cDNA, genomic DNA, synthetic DNA, and semi-synthetic DNA.
The sequence of nucleotides or nucleic acid sequence that encodes a
protein is called the sense sequence. A "recombinant DNA molecule"
is a DNA molecule that has undergone a molecular biological
manipulation.
[0054] A DNA "coding sequence" is a double-stranded DNA sequence
which is transcribed and translated into a polypeptide in a cell in
vitro or in vivo when placed under the control of appropriate
regulatory sequences. The boundaries of the coding sequence are
determined by a start codon at the 5' (amino) terminus and a
translation stop codon at the 3' (carboxyl) terminus. A
polyadenylation signal and transcription termination sequence will
usually be located 3' to the coding sequence.
[0055] Transcriptional and translational control sequences are DNA
regulatory sequences, such as promoters, enhancers, terminators,
and the like, that provide for the expression of a coding sequence
in a host cell. In eukaryotic cells, polyadenylation signals are
control sequences.
[0056] A "promoter sequence" is a DNA regulatory region capable of
binding RNA polymerase in a cell and initiating transcription of a
downstream (3' direction) coding sequence. For purposes of defining
the present invention, the promoter sequence is bounded at its 3'
terminus by the transcription initiation site and extends upstream
(5' direction) to include the minimum number of bases or elements
necessary to initiate transcription at levels detectable above
background. Within the promoter sequence will be found a
transcription initiation site (conveniently defined for example, by
mapping with nuclease S1), as well as protein binding domains
(consensus sequences) responsible for the binding of RNA
polymerase.
[0057] A coding sequence is "under the control" of transcriptional
and translational control sequences in a cell when RNA polymerase
transcribes the coding sequence into mRNA, which is then optionally
trans-RNA spliced and translated into the protein encoded by the
coding sequence.
[0058] A "signal sequence" is included at the beginning of the
coding sequence of a protein to be expressed on the surface of a
cell. This sequence encodes a signal peptide, N-terminal to the
mature polypeptide, that directs the host cell to translocate the
polypeptide. The term "translocation signal sequence" is used
herein to refer to this sort of signal sequence. Translocation
signal sequences can be found associated with a variety of proteins
native to eukaryotes and prokaryotes, and are often functional in
both types of organisms.
[0059] The term "corresponding to" is used herein to refer similar
or homologous sequences, whether the exact position is identical or
different from the molecule to which the similarity or homology is
measured. A nucleic acid or amino acid sequence alignment may
include spaces. Thus, the term "corresponding to" refers to the
sequence similarity, and not the numbering of the amino acid
residues or nucleotide bases.
[0060] The various aspects of the invention will be set forth in
greater detail in the following sections, directed to suitable gene
therapy vectors and promoters, anti-angiogenic proteins, and
therapeutic strategies. This organization into various sections is
intended to facilitate understanding of the invention, and is in no
way intended to be limiting thereof.
Gene Therapy Vectors
[0061] As discussed above, a "vector" is any means for the transfer
of a nucleic acid according to the invention into a host cell.
Preferred vectors are viral vectors, such as retroviruses, herpes
viruses, adenoviruses and adeno-associated viruses. Thus, a gene or
nucleic acid sequence encoding an anti-angiogenic protein or
polypeptide domain fragment thereof is introduced in vivo, ex vivo,
or in vitro using a viral vector or through direct introduction of
DNA. Expression in targeted tissues can be effected by targeting
the transgenic vector to specific cells, such as with a viral
vector or a receptor ligand, or by using a tissue-specific
promoter, or both.
[0062] Viral vectors commonly used for in vivo or ex vivo targeting
and therapy procedures are DNA-based vectors and retroviral
vectors. Methods for constructing and using viral vectors are known
in the art [see, e.g., Miller and Rosman, BioTechniques 7:980-990
(1992)]. Preferably, the viral vectors are replication defective,
that is, they are unable to replicate autonomously in the target
cell. In general, the genome of the replication defective viral
vectors which are used within the scope of the present invention
lack at least one region which is necessary for the replication of
the virus in the infected cell. These regions can either be
eliminated (in whole or in part), be rendered non-functional by any
technique known to a person skilled in the art. These techniques
include the total removal, substitution (by other sequences, in
particular by the inserted nucleic acid), partial deletion or
addition of one or more bases to an essential (for replication)
region. Such techniques may be performed in vitro (on the isolated
DNA) or in situ, using the techniques of genetic manipulation or by
treatment with mutagenic agents. Preferably, the replication
defective virus retains the sequences of its genome which are
necessary for encapsulating the viral particles.
[0063] DNA viral vectors include an attenuated or defective DNA
virus, such as but not limited to herpes simplex virus (HSV),
papillomavirus, Epstein-Barr virus (EBV), adenovirus,
adeno-associated virus (AAV), and the like. Defective viruses,
which entirely or almost entirely lack viral genes, are preferred.
Defective virus is not infective after introduction into a cell.
Use of defective viral vectors allows for administration to cells
in a specific, localized area, without concern that the vector can
infect other cells. Thus, a specific tissue can be specifically
targeted. Examples of particular vectors include, but are not
limited to, a defective herpes virus 1 (HSV1) vector [Kaplitt et
al., Molec. Cell. Neurosci. 2:320-330 (1991)], defective herpes
virus vector lacking a glyco-protein L gene [Patent Publication RD
371005 A], or other defective herpes virus vectors [International
Patent Publication No. WO 94/21807, published Sep. 29, 1994;
International Patent Publication No. WO 92/05263, published Apr. 2,
1994]; an attenuated adenovirus vector, such as the vector
described by Stratford-Perricaudet et al. [J. Clin. Invest.
90:626-630 (1992); see also La Salle et al., Science 259:988-990
(1993)]; and a defective adeno-associated virus vector [Samulski et
al., J. Virol. 61:3096-3101 (1987); Samulski et al., J. Virol.
63:3822-3828 (1989); Lebkowski et al., Mol. Cell. Biol. 8:3988-3996
(1988)].
[0064] Preferably, for in vivo administration, an appropriate
immunosuppressive treatment is employed in conjunction with the
viral vector, e.g., adenovirus vector, to avoid immuno-deactivation
of the viral vector and transfected cells. For example,
immunosuppressive cytokines, such as interleukin-12 (IL-12),
interferon-.gamma. (IFN-.gamma.), or anti-CD4 antibody, can be
administered to block humoral or cellular immune responses to the
viral vectors [see, e.g., Wilson, Nature Medicine (1995)]. In
addition, it is advantageous to employ a viral vector that is
engineered to express a minimal number of antigens.
[0065] Adenovirus vectors. In a preferred embodiment, the vector is
an adenovirus vector. As shown in the Examples, defective
adenovirus vectors have shown themselves to be particularly
effective for delivery of the angiogenesis inhibitors ATF and
angiostatin, as shown by the unexpectedly efficient effects of
inhibiting tumor growth and tumorigenesis. Adenoviruses are
eukaryotic DNA viruses that can be modified to efficiently deliver
a nucleic acid of the invention to a variety of cell types. Various
serotypes of adenovirus exist. Of these serotypes, preference is
given, within the scope of the present invention, to using type 2
or type 5 human adenoviruses (Ad 2 or Ad 5) or adenoviruses of
animal origin (see WO94/26914). Those adenoviruses of animal origin
which can be used within the scope of the present invention include
adenoviruses of canine, bovine, murine (example: Mav1, Beard et
al., Virology 75 (1990) 81), ovine, porcine, avian, and simian
(example: SAV) origin. Preferably, the adenovirus of animal origin
is a canine adenovirus, more preferably a CAV2 adenovirus (e.g.,
Manhattan or A26/61 strain (ATCC VR-800), for example).
[0066] Preferably, the replication defective adenoviral vectors of
the invention comprise the ITRs, an encapsidation sequence and the
nucleic acid of interest. Still more preferably, at least the E1
region of the adenoviral vector is non-functional. The deletion in
the E1 region preferably extends from nucleotides 455 to 3329 in
the sequence of the AdS adenovirus (PvuII-BglII fragment) or 382 to
3446 (HinfII-Sau3A fragment). Other regions may also be modified,
in particular the E3 region (WO95/02697), the E2 region
(WO94/28938), the E4 region (WO94/28152, WO94/12649 and
WO95/02697), or in any of the late genes L1-L5.
[0067] In a preferred embodiment, the adenoviral vector has a
deletion in the E1 region (Ad 1.0). Examples of E1-deleted
adenoviruses are disclosed in EP 185,573, the contents of which are
incorporated herein by reference. In another preferred embodiment,
the adenoviral vector has a deletion in the E1 and E4 regions (Ad
3.0). Examples of E1/E4-deleted adenoviruses are disclosed in
WO95/02697 and WO96/22378, the contents of which are incorporated
herein by reference. In still another preferred embodiment, the
adenoviral vector has a deletion in the E1 region into which the E4
region and the nucleic acid sequence are inserted (see FR94 13355,
the contents of which are incorporated herein by reference).
[0068] The replication defective recombinant adenoviruses according
to the invention can be prepared by any technique known to the
person skilled in the art (Levrero et al., Gene 101 (1991) 195, EP
185 573; Graham, EMBO J. 3 (1984) 2917). In particular, they can be
prepared by homologous recombination between an adenovirus and a
plasmid which carries, inter alia, the DNA sequence of interest.
The homologous recombination is effected following cotransfection
of the said adenovirus and plasmid into an appropriate cell line.
The cell line which is employed should preferably (i) be
transformable by the said elements, and (ii) contain the sequences
which are able to complement the part of the genome of the
replication defective adenovirus, preferably in integrated form in
order to avoid the risks of recombination. Examples of cell lines
which may be used are the human embryonic kidney cell line 293
(Graham et al., J. Gen. Virol. 36 (1977) 59) which contains the
left-hand portion of the genome of an AdS adenovirus (12%)
integrated into its genome, and cell lines which are able to
complement the E1 and E4 functions, as described in applications
WO94/26914 and WO95/02697. Recombinant adenoviruses are recovered
and purified using standard molecular biological techniques, which
are well known to one of ordinary skill in the art.
[0069] Adeno-associated viruses. The adeno-associated viruses (AAV)
are DNA viruses of relatively small size which can integrate, in a
stable and site-specific manner, into the genome of the cells which
they infect. They are able to infect a wide spectrum of cells
without inducing any effects on cellular growth, morphology or
differentiation, and they do not appear to be involved in human
pathologies. The AAV genome has been cloned, sequenced and
characterized. It encompasses approximately 4700 bases and contains
an inverted terminal repeat (ITR) region of approximately 145 bases
at each end, which serves as an origin of replication for the
virus. The remainder of the genome is divided into two essential
regions which carry the encapsidation functions: the left-hand part
of the genome, which contains the rep gene involved in viral
replication and expression of the viral genes; and the right-hand
part of the genome, which contains the cap gene encoding the capsid
proteins of the virus.
[0070] The use of vectors derived from the AAVs for transferring
genes in vitro and in vivo has been described (see WO 91/18088; WO
93/09239; U.S. Pat. No. 4,797,368, U.S. Pat. No. 5,139,941, EP 488
528). These publications describe various AAV-derived constructs in
which the rep and/or cap genes are deleted and replaced by a gene
of interest, and the use of these constructs for transferring the
said gene of interest in vitro (into cultured cells) or in vivo,
(directly into an organism). The replication defective recombinant
AAVs according to the invention can be prepared by cotransfecting a
plasmid containing the nucleic acid sequence of interest flanked by
two AAV inverted terminal repeat (ITR) regions, and a plasmid
carrying the AAV encapsidation genes (rep and cap genes), into a
cell line which is infected with a human helper virus (for example
an adenovirus). The AAV recombinants which are produced are then
purified by standard techniques.
[0071] The invention also relates, therefore, to an AAV-derived
recombinant virus whose genome encompasses a sequence encoding a
nucleic acid encoding an anti-angiogenic factor flanked by the AAV
ITRs. The invention also relates to a plasmid encompassing a
sequence encoding a nucleic acid encoding an anti-angiogenic factor
flanked by two ITRs from an AAV. Such a plasmid can be used as it
is for transferring the nucleic acid sequence, with the plasmid,
where appropriate, being incorporated into a liposomal vector
(pseudo-virus).
[0072] Retrovirus vectors. In another embodiment the gene can be
introduced in a retroviral vector, e.g., as described in Anderson
et al., U.S. Pat. No. 5,399,346; Mann et al., 1983, Cell 33:153;
Temin et al., U.S. Pat. No. 4,650,764; Temin et al., U.S. Pat. No.
4,980,289; Markowitz et al., 1988, J. Virol. 62:1120; Temin et al.,
U.S. Pat. No. 5,124,263; EP453242, EP178220; Bernstein et al.
Genet. Eng. 7 (1985) 235; McCormick, BioTechnology 3 (1985) 689;
International Patent Publication No. WO 95/07358, published Mar.
16, 1995, by Dougherty et al.; and Kuo et al., 1993, Blood 82:845.
The retroviruses are integrating viruses which infect dividing
cells. The retrovirus genome includes two LTRs, an encapsidation
sequence and three coding regions (gag, pol and env). In
recombinant retroviral vectors, the gag, pol and env genes are
generally deleted, in whole or in part, and replaced with a
heterologous nucleic acid sequence of interest. These vectors can
be constructed from different types of retrovirus, such as, HIV,
MoMuLV ("murine Moloney leukaemia virus" MSV ("murine Moloney
sarcoma virus"), HaSV ("Harvey sarcoma virus"); SNV ("spleen
necrosis virus"); RSV ("Rous sarcoma virus") and Friend virus.
Defective retroviral vectors are disclosed in WO95/02697.
[0073] In general, in order to construct recombinant retroviruses
containing a nucleic acid sequence, a plasmid is constructed which
contains the LTRs, the encapsidation sequence and the coding
sequence. This construct is used to transfect a packaging cell
line, which cell line is able to supply in trans the retroviral
functions which are deficient in the plasmid. In general, the
packaging cell lines are thus able to express the gag, pol and env
genes. Such packaging cell lines have been described in the prior
art, in particular the cell line PA317 (U.S. Pat. No. 4,861,719);
the PsiCRIP cell line (WO90/02806) and the GP+envAm-12 cell line
(WO89/07150). In addition, the recombinant retroviral vectors can
contain modifications within the LTRs for suppressing
transcriptional activity as well as extensive encapsidation
sequences which may include a part of the gag gene (Bender et al.,
J. Virol. 61 (1987) 1639). Recombinant retroviral vectors are
purified by standard techniques known to those having ordinary
skill in the art.
[0074] Retroviral vectors can be constructed to function as
infectious particles or to undergo a single round of transfection.
In the former case, the virus is modified to retain all of its
genes except for those responsible for oncogenic transformation
properties, and to express the heterologous gene. Non-infectious
viral vectors are prepared to destroy the viral packaging signal,
but retain the structural genes required to package the
co-introduced virus engineered to contain the heterologous gene and
the packaging signals. Thus, the viral particles that are produced
are not capable of producing additional virus.
[0075] Targeted gene delivery is described in International Patent
Publication WO 95/28494, published October 1995.
[0076] Non-viral Vectors. Alternatively, the vector can be
introduced in vivo as nucleic acid free of transfecting excipients,
or with transfection facilitating agents, e.g., lipofection. For
the past decade, there has been increasing use of liposomes for
encapsulation and transfection of nucleic acids in vitro. Synthetic
cationic lipids designed to limit the difficulties and dangers
encountered with liposome mediated transfection can be used to
prepare liposomes for in vivo transfection of a gene encoding a
marker [Felgner, et. al., Proc. Natl. Acad. Sci. U.S.A.
84:7413-7417 (1987); see Mackey, et al., Proc. Natl. Acad. Sci.
U.S.A. 85:8027-8031 (1988); Ulmer et al., Science 259:1745-1748
(1993)]. The use of cationic lipids may promote encapsulation of
negatively charged nucleic acids, and also promote fusion with
negatively charged cell membranes [Felgner and Ringold, Science
337:387-388 (1989)]. Particularly useful lipid compounds and
compositions for transfer of nucleic acids are described in
International Patent Publications WO95/18863 and WO96/17823, and in
U.S. Pat. No. 5,459,127. The use of lipofection to introduce
exogenous genes into the specific organs in vivo has certain
practical advantages. Molecular targeting of liposomes to specific
cells represents one area of benefit. It is clear that directing
transfection to particular cell types would be particularly
advantageous in a tissue with cellular heterogeneity, such as
pancreas, liver, kidney, and the brain. Lipids may be chemically
coupled to other molecules for the purpose of targeting [see
Mackey, et. al., supra]. Targeted peptides, e.g., hormones or
neurotransmitters, and proteins such as antibodies, or non-peptide
molecules could be coupled to liposomes chemically.
[0077] Other molecules are also useful for facilitating
transfection of a nucleic acid in vivo, such as a cationic
oligopeptide (e.g., International Patent Publication WO95/21931),
peptides derived from DNA binding proteins (e.g., International
Patent Publication WO96/25508), or a cationic polymer (e.g.,
International Patent Publication WO95/21931).
[0078] It is also possible to introduce the vector in vivo as a
naked DNA plasmid. Naked DNA vectors for gene therapy can be
introduced into the desired host cells by methods known in the art,
e.g., transfection, electroporation, microinjection, transduction,
cell fusion, DEAE dextran, calcium phosphate precipitation, use of
a gene gun, or use of a DNA vector transporter [see, e.g., Wu et
al., J. Biol. Chem. 267:963-967 (1992); Wu and Wu, J. Biol. Chem.
263:14621-14624 (1988); Hartmut et al., Canadian Patent Application
No. 2,012,311, filed Mar. 15, 1990; Williams et al., Proc. Natl.
Acad. Sci. USA 88:2726-2730 (1991)]. Receptor-mediated DNA delivery
approaches can also be sued [Curiel et al., Hum. Gene Ther.
3:147-154 (1992); Wu and Wu, J. Biol. Chem. 262:4429-4432
(1987)].
[0079] The nucleic acid can also be administered as a naked DNA.
Methods for formulating and administering naked DNA to mammalian
muscle tissue are disclosed in U.S. Pat. Nos. 5,580,859 and
5,589,466, the contents of which are incorporated herein by
reference.
[0080] Regulatory Regions. Expression of an anti-angiogenic factor
from a vector of the invention may be controlled by any regulatory
region, i.e., promoter/enhancer element known in the art, but these
regulatory elements must be functional in the host target tumor
selected for expression.
[0081] The regulatory regions may comprise a promoter region for
functional transcription in the tumor, as well as a region situated
in 3' of the gene of interest, and which specifies a signal for
termination of transcription and a polyadenylation site. All these
elements constitute an expression cassette.
[0082] Promoters that may be used in the present invention include
both constitutive promoters and regulated (inducible) promoters.
The promoter may be naturally responsible for the expression of the
nucleic acid. It may also be from a heterologous source. In
particular, it may be promoter sequences of eukaryotic or viral
genes. For example, it may be promoter sequences derived from the
genome of the cell which it is desired to infect. Likewise, it may
be promoter sequences derived from the genome of a virus, including
the adenovirus used. In this regard, there may be mentioned, for
example, the promoters of the EIA, MLP, CMV and RSV genes and the
like.
[0083] In addition, the promoter may be modified by addition of
activating or regulatory sequences or sequences allowing a
tissue-specific or predominant expression (enolase and GFAP
promoters and the like). Moreover, when the nucleic acid does not
contain promoter sequences, it may be inserted, such as into the
virus genome downstream of such a sequence.
[0084] Some promoters useful for practice of this invention are
ubiquitous promoters (e.g., HPRT, vimentin, actin, tubulin),
intermediate filament promoters (e.g., desmin, neurofilaments,
keratin, GFAP), therapeutic gene promoters (e.g., MDR type, CFTR,
factor VIII), tissue-specific promoters (e.g., actin promoter in
smooth muscle cells), promoters which are preferentially activated
in dividing cells, promoters which respond to a stimulus (e.g.,
steroid hormone receptor, retinoic acid receptor),
tetracycline-regulated transcriptional modulators, cytomegalovirus
immediate-early, retroviral LTR, metallothionein, SV-40, E1a, and
MLP promoters. Tetracycline-regulated transcriptional modulators
and CMV promoters are described in WO 96/01313, U.S. Pat. Nos.
5,168,062 and 5,385,839, the contents of which are incorporated
herein by reference.
[0085] Thus, the promoters which may be used to control gene
expression include, but are not limited to, the cytomegalovirus
(CMV) promoter, the SV40 early promoter region (Benoist and
Chambon, 1981, Nature 290:304-310), the promoter contained in the
3' long terminal repeat of Rous sarcoma virus (Yamamoto, et al.,
1980, Cell 22:787-797), the herpes thymidine kinase promoter
(Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445),
the regulatory sequences of the metallothionein gene (Brinster et
al., 1982, Nature 296:39-42); prokaryotic expression vectors such
as the b-lactamase promoter (Villa-Kamaroff, et al., 1978, Proc.
Natl. Acad. Sci. U.S.A. 75:3727-3731), or the tac promoter (DeBoer,
et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:21-25); see also
"Useful proteins from recombinant bacteria" in Scientific American,
1980, 242:74-94; promoter elements from yeast or other fungi such
as the Gal 4 promoter, the ADC (alcohol dehydrogenase) promoter,
PGK (phosphoglycerol kinase) promoter, alkaline phosphatase
promoter; and the animal transcriptional control regions, which
exhibit tissue specificity and have been utilized in transgenic
animals: elastase I gene control region which is active in
pancreatic acinar cells (Swift et al., 1984, Cell 38:639-646;
Ornitz et al., 1986, Cold Spring Harbor Symp. Quant. Biol.
50:399-409; MacDonald, 1987, Hepatology 7:425-515); insulin gene
control region which is active in pancreatic beta cells (Hanahan,
1985, Nature 315:115-122), immunoglobulin gene control region which
is active in lymphoid cells (Grosschedl et al., 1984, Cell
38:647-658; Adames et al., 1985, Nature 318:533-538; Alexander et
al., 1987, Mol. Cell. Biol. 7:1436-1444), mouse mammary tumor virus
control region which is active in testicular, breast, lymphoid and
mast cells (Leder et al., 1986, Cell 45:485-495), albumin gene
control region which is active in liver (Pinkert et al., 1987,
Genes and Devel. 1:268-276), alpha-fetoprotein gene control region
which is active in liver (Krumlauf et al., 1985, Mol. Cell. Biol.
5:1639-1648; Hammer et al., 1987, Science 235:53-58), alpha
1-antitrypsin gene control region which is active in the liver
(Kelsey et al., 1987, Genes and Devel. 1:161-171), beta-globin gene
control region which is active in myeloid cells (Mogram et al.,
1985, Nature 315:338-340; Kollias et al., 1986, Cell 46:89-94),
myelin basic protein gene control region which is active in
oligodendrocyte cells in the brain (Readhead et al., 1987, Cell
48:703-712), myosin light chain-2 gene control region which is
active in skeletal muscle (Sani, 1985, Nature 314:283-286), and
gonadotropic releasing hormone gene control region which is active
in the hypothalamus (Mason et al., 1986, Science
234:1372-1378).
Genes Encoding Anti-Angiogenic Proteins
[0086] The vectors of the invention can be used to deliver a gene
encoding an anti-angiogenic protein into a tumor in accordance with
the invention. In a preferred embodiment, the anti-angiogenic
factor is the amino terminal fragment (ATF) of urokinase,
containing the EGF-like domain. Such fragment corresponds to amino
acid residues about 1 to about 135 of ATF.
[0087] In another embodiment, ATF may be provided as a fusion
protein, e.g., with immunoglobulin or human serum albumin
[WO93/15199], which is specifically incorporated herein by
reference in its entirety.
[0088] An effective ATF for use in the invention can be derived
from any urokinase, such as murine urokinase, although human
urokinase ATF is preferred. In addition, the invention contemplates
administration of a non-human urokinase ATF modified by
substitution of specific amino acid residues with the corresponding
residues from human ATF. For example, murine ATF can be modified at
one or more, and preferably all, amino acid residues as follows:
tyrosine-23 to asparagine; arginine-28 to asparagine; arginine-30
to histidine; and arginine-31 to tryptophan. Thus, urokinase ATF
from any source can be humanized. This is easily accomplished by
modifying the coding sequence using routine molecular biological
techniques.
[0089] Genes encoding other anti-angiogenesis protein can also be
used according to the invention. Such genes include, but are not
limited to, genes encoding angiostatin [O'Reilly et al., Cell
79:315-328 (1994); WO95/29242; U.S. Pat. No. 5,639,725], including
angiostatin comprising kringles 1 to 3; tissue inhibition of
metalloproteinase [Johnson et al., J. Cell. Physiol. 160:194-202
(1994)]; inhibitors of FGF or VEGF; and endostatin [WO97/15666]. In
a preferred embodiment, the anti-angiogenic factor is angiostatin,
particularly kringles 1 to 3 of angiostatin. In a particularly
preferred embodiment, the anti-angiogenic factor is the
amino-terminal fragment of plasminogen (Plg) having an amino acid
sequence of plasminogen from about amino acid residue I to about
residue 333. In another preferred embodiment, the amino terminal
fragment of plasminogen/angiostatin is human plasminogen
(angiostatin).
[0090] In another embodiment, the invention provides for
administration of genes encoding soluble forms of receptors for
angiogenic factors, including but not limited to soluble VGF/VEGF
receptor, and soluble urokinase receptor [Wilhem et al., FEBS
Letters 337:131-134 (1994)].
[0091] In general, any gene encoding a protein or soluble receptor
that antagonizes endothelial cell activation and migration, which
is involved in angiogenesis, can be employed in the gene therapy
vectors and methods of the invention. Notwithstanding, it is
particularly noteworthy that gene therapy delivery of ATF or
angiostatin is especially effective in this regard, for reasons
pointed out above and exemplified below.
[0092] A gene encoding an anti-angiogenic factor, whether genomic
DNA or cDNA, can be isolated from any source, particularly from a
human cDNA or genomic library. Methods for obtaining such genes are
well known in the art, as described above [see, e.g., Sambrook et
al., 1989, supra].
[0093] Due to the degeneracy of nucleotide coding sequences, other
nucleic acid sequences which encode substantially the same amino
acid sequence as an anti-angiogenic factor gene may be used in the
practice of the present invention and these are contemplated as
falling within the scope of the claimed invention. These include
but are not limited to allelic genes, homologous genes from other
species, and nucleotide sequences comprising all or portions of
anti-angiogenic factor genes which are altered by the substitution
of different codons that encode the same amino acid residue within
the sequence, thus producing a silent change. Likewise, the
anti-angiogenic factor derivatives of the invention include, but
are not limited to, those containing, as a primary amino acid
sequence, all or part of the amino acid sequence of an
anti-angiogenic factor protein including altered sequences in which
functionally equivalent amino acid residues are substituted for
residues within the sequence resulting in a conservative amino acid
substitution. For example, one or more amino acid residues within
the sequence can be substituted by another amino acid of a similar
polarity, which acts as a functional equivalent, resulting in a
silent alteration. Substitutes for an amino acid within the
sequence may be selected from other members of the class to which
the amino acid belongs. For example, the nonpolar (hydrophobic)
amino acids include alanine, leucine, isoleucine, valine, proline,
phenylalanine, tryptophan and methionine. Amino acids containing
aromatic ring structures are phenylalanine, tryptophan, and
tyrosine. The polar neutral amino acids include glycine, serine,
threonine, cysteine, tyrosine, asparagine, and glutamine. The
positively charged (basic) amino acids include arginine, lysine and
histidine. The negatively charged (acidic) amino acids include
aspartic acid and glutamic acid. Such alterations will not be
expected to affect apparent molecular weight as determined by
polyacrylamide gel electrophoresis, or isoelectric point.
[0094] Particularly preferred substitutions are:
[0095] Lys for Arg and vice versa such that a positive charge may
be maintained;
[0096] Glu for Asp and vice versa such that a negative charge may
be maintained;
[0097] Ser for Thr such that a free --OH can be maintained; and
[0098] Gln for Asn such that a free CONH.sub.2 can be
maintained.
[0099] The genes encoding anti-angiogenic factor derivatives and
analogs of the invention can be produced by various methods known
in the art. The manipulations which result in their production can
occur at the gene or protein level. For example, the cloned
anti-angiogenic factor gene sequence can be modified by any of
numerous strategies known in the art (Sambrook et al., 1989,
supra). The sequence can be cleaved at appropriate sites with
restriction endonuclease(s), followed by further enzymatic
modification if desired, isolated, and ligated in vitro. In the
production of the gene encoding a derivative or analog of
anti-angiogenic factor, care should be taken to ensure that the
modified gene remains within the same translational reading frame
as the anti-angiogenic factor gene, uninterrupted by translational
stop signals, in the gene region where the desired activity is
encoded.
[0100] Additionally, the anti-angiogenic factor-encoding nucleic
acid sequence can be mutated in vitro or in vivo, to create and/or
destroy translation, initiation, and/or termination sequences, or
to create variations in coding regions and/or form new restriction
endonuclease sites or destroy preexisting ones, to facilitate
further in vitro modification, such as to form a chimeric gene.
Preferably, such utations enhance the functional activity of the
mutated anti-angiogenic factor gene product. Any technique for
mutagenesis known in the art can be used, including but not limited
to, in vitro site-directed mutagenesis (Hutchinson, C., et al.,
1978, J. Biol. Chem. 253:6551; Zoller and Smith, 1984, DNA
3:479-488; Oliphant et al., 1986, Gene 44:177; Hutchinson et al.,
1986, Proc. Natl. Acad. Sci. U.S.A. 83:710), use of TAB.RTM.
linkers (Pharmacia), etc. PCR techniques are preferred for site
directed mutagenesis (see Higuchi, 1989, "Using PCR to Engineer
DNA", in PCR Technology: Principles and Applications for DNA
Amplification, H. Erlich, ed., Stockton Press, Chapter 6, pp.
61-70).
Therapeutic Targets and Strategies
[0101] The process according to the present invention enables one
to treat tumors. According to the present invention, it is now
possible, by a judicious choice of various injections, infusions,
direct application, etc., to infect specifically and unilaterally a
large number of tumor cells.
[0102] Pharmaceutical Compositions. For their use according to the
present invention, the vectors, either in the form of a virus
vector, nucleic acid-lipid composition, or naked DNA, are
preferably combined with one or more pharmaceutically acceptable
carriers for an injectable formulation. The phrase
"pharmaceutically acceptable" refers to molecular entities and
compositions that are physiologically tolerable and do not
typically produce an allergic or similar untoward reaction, such as
gastric upset, dizziness and the like, when administered to a
human. Preferably, as used herein, the term "pharmaceutically
acceptable" means approved by a regulatory agency of the Federal or
a state government or listed in the U.S. Pharmacopoeia or other
generally recognized pharmacopoeia for use in animals, and more
particularly in humans. The term "carrier" refers to a diluent,
adjuvant, excipient, or vehicle with which the compound is
administered. Such pharmaceutical carriers can be sterile liquids,
such as water and oils, including those of petroleum, animal,
vegetable or synthetic origin, such as peanut oil, soybean oil,
mineral oil, sesame oil and the like. Water or aqueous solution
saline solutions and aqueous dextrose and glycerol solutions are
preferably employed as carriers, particularly for injectable
solutions. Suitable pharmaceutical carriers are described in
"Remington's Pharmaceutical Sciences" by E. W. Martin. These may be
in particular isotonic, sterile, saline solutions (monosodium or
disodium phosphate, sodium, potassium, calcium or magnesium
chloride and the like or mixtures of such salts), or dry,
especially freeze-dried compositions which upon addition, depending
on the case, of sterilized water or physiological saline, allow the
constitution of injectable solutions.
[0103] The preferred sterile injectable preparations can be a
solution or suspension in a nontoxic parenterally acceptable
solvent or diluent. Examples of pharmaceutically acceptable
carriers are saline, buffered saline, isotonic saline (e.g.,
monosodium or disodium phosphate, sodium, potassium, calcium or
magnesium chloride, or mixtures of such salts), Ringer's solution,
dextrose, water, sterile water, glycerol, ethanol, and combinations
thereof. 1,3-butanediol and sterile fixed oils are conveniently
employed as solvents or suspending media. Any bland fixed oil can
be employed including synthetic mono- or di-glycerides. Fatty acids
such as oleic acid also find use in the preparation of
injectables.
[0104] The phrase "therapeutically effective amount" is used herein
to mean an amount sufficient to reduce by at least about 15
percent, preferably by at least 50 percent, more preferably by at
least 90 percent, and most preferably prevent, a clinically
significant deficit in the activity, function and response of the
host. Alternatively, a therapeutically effective amount is
sufficient to cause an improvement in a clinically significant
condition in the host.
[0105] The virus doses used for the administration may be adapted
as a function of various parameters, and in particular as a
function of the site (tumor) of administration considered, the
number of injections, the gene to be expressed or alternatively the
desired duration of treatment. In general, the recombinant
adenoviruses according to the invention are formulated and
administered in the form of doses of between 10.sup.4 and 10.sup.14
pfu, and preferably 10.sup.6 to 10.sup.11 pfu. The term pfu (plaque
forming unit) corresponds to the infectivity of a virus solution,
and is determined by infecting an appropriate cell culture and
measuring, generally after 15 days, the number of plaques of
infected cells. The technique for determining the pfu titre of a
viral solution are well documented in the literature.
[0106] In a preferred embodiment, the composition comprises an
adenovirus comprising the anti-angiogenic factor gene, e.g., ATF
gene (AdATF) or angiostatin (AdK3), in a concentration of about
1.times.10.sup.9 pfu/100 .mu.l.
[0107] The compositions according to the invention are particularly
useful for administration to tumors.
[0108] Tumors. The present invention is directed the treatment of
tumors, particularly solid tumors. Examples of solid tumors that
can be treated according to the invention include sarcomas and
carcinomas such as, but not limited to: fibrosarcoma, myxosarcoma,
liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma,
angiosarcoma, endotheliosarcoma, lymphangiosarcoma,
lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's
tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,
pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,
squamous cell carcinoma, basal cell carcinoma, adenocarcinoma,
sweat gland carcinoma, sebaceous gland carcinoma, papillary
carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary
carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma,
bile duct carcinoma, choriocarcinoma, seminoma, embryonal
carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung
carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial
carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma,
ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,
oligodendroglioma, meningioma, melanoma, neuroblastoma, and
retinoblastoma.
[0109] In another embodiment, dysproliferative changes (such as
metaplasias and dysplasias) are treated or prevented in epithelial
tissues such as those in the cervix, esophagus, and lung. Thus, the
present invention provides for treatment of conditions known or
suspected of preceding progression to neoplasia or cancer, in
particular, where non-neoplastic cell growth consisting of
hyperplasia, metaplasia, or most particularly, dysplasia has
occurred (for review of such abnormal growth conditions, see
Robbins and Angell, 1976, Basic Pathology, 2d Ed., W.B. Saunders
Co., Philadelphia, pp. 68-79). Hyperplasia is a form of controlled
cell proliferation involving an increase in cell number in a tissue
or organ, without significant alteration in structure or function.
As but one example, endometrial hyperplasia often precedes
endometrial cancer. Metaplasia is a form of controlled cell growth
in which one type of adult or fully differentiated cell substitutes
for another type of adult cell. Metaplasia can occur in epithelial
or connective tissue cells. Atypical metaplasia involves a somewhat
disorderly metaplastic epithelium. Dysplasia is frequently a
forerunner of cancer, and is found mainly in the epithelia; it is
the most disorderly form of non-neoplastic cell growth, involving a
loss in individual cell uniformity and in the architectural
orientation of cells. Dysplastic cells often have abnormally large,
deeply stained nuclei, and exhibit pleomorphism. Dysplasia
characteristically occurs where there exists chronic irritation or
inflammation, and is often found in the cervix, respiratory
passages, oral cavity, and gall bladder. For a review of such
disorders, see Fishman et al., 1985, Medicine, 2d Ed., J. B.
Lippincott Co., Philadelphia.
[0110] The present invention is also directed to treatment of
non-malignant tumors and other disorders involving inappropriate
cell or tissue growth augmented by angiogenesis by administering a
therapeutically effective amount of a vector of the invention to
the tissue undergoing inappropriate growth. For example, it is
contemplated that the invention is useful for the treatment of
arteriovenous (AV) malformations, particularly in intracranial
sites. The invention may also be used to treat psoriasis, a
dermatologic condition that is characterized by inflammation and
vascular proliferation; and benign prostatic hypertrophy, a
condition associated with inflammation and possibly vascular
proliferation. Treatment of other hyperproliferative disorders is
also contemplated.
[0111] Methods of administration. According to the invention, the
preferred route of administration to a tumor is by direct injection
into the tumor. The tumor can be imaged using any of the techniques
available in the art, such as magnetic resonance imaging or
computer-assisted tomography, and the therapeutic composition
administered by stereotactic injection, for example.
[0112] Alternatively, if a tumor target is characterized by a
particular antigen, a vector of the invention can be targeted to
the antigen as described above, and administered systemically or
subsystemically, as appropriate, e.g., intravenously,
intraarterioally, intraperitoneally, intraventricularly, etc.
[0113] Combination Therapies. Although the methods of the invention
are effective in inhibiting tumor growth and metastasis, the
vectors and methods of the present invention are advantageously
used with other treatment modalities, including without limitation
surgery, radiation, chemotherapy, and other gene therapies.
[0114] For example, the vectors of the invention can be
administered in combination with nitric oxide inhibitors, which
have vasoconstrictive activity and reduce blood flow to the
tumor.
[0115] In another embodiment, a vector of the invention can be
administered with a chemotherapeutic such as, though not limited
to, taxol, taxotere and other taxoids [e.g., as disclosed in U.S.
Pat. Nos. 4,857,653; 4,814,470; 4,924,011, 5,290,957; 5,292,921;
5,438,072; 5,587,493; European Patent No. 0 253 738; and
International Patent Publication Nos. WO91/17976, WO93/00928,
WO93/00929, and WO9601815], or other chemotherapeutics, such as
cis-platin (and other platin intercalating compounds), etoposide
and etoposide phosphate, bleomycin, mitomycin C, CCNU, doxorubicin,
daunorubicin, idarubicin, ifosfamide, and the like.
[0116] In still another embodiment, a vector of the invention can
be administered in conjunction with another gene therapy for
tumors, such as but by no means limited to p53 or analogues thereof
such as CTS-1 [WO97/04092], thymidine kinase (TK), anti-RAS single
chain antibodies, interferon-.alpha. or interferon-.gamma., etc.,
as described above. Any vector for gene therapy can be used in
conjunction with the present invention, such as a viral vector or
naked DNA. In a preferred embodiment, a single vector (virus or
DNA) is used to deliver genes coding for both an anti-angiogenesis
factor and another tumor therapy gene.
[0117] In another aspect, the present invention provides for
regulated expression of the anti-angiogenic factor gene in concert
with expression of proteins useful in the context of treatment for
proliferative disorders, such as tumors and cancers, when the
heterologous gene encodes a targeting marker or immunomodulatory
cytokine that enhances targeting of the tumor cell by host immune
system mechanisms. Examples of such heterologous genes for
immunomodulatory (or immuno-effector) molecules include, but are
not limited to, interferon-.alpha., interferon-.gamma.,
interferon-.beta., interferon-.omega., interferon-1, tumor necrosis
factor-.alpha., tumor necrosis factor-, interleukin-2,
interleukin-7, interleukin-12, interleukin-15, B7-1 T cell
costimulatory molecule, B7-2 T cell costimulatory molecule, immune
cell adhesion molecule (ICAM)1 T cell costimulatory molecule,
granulocyte colony stimulatory factor, granulocyte-macrophage
colony stimulatory factor, and combinations thereof.
[0118] The present invention will be better understood be reference
to the following Examples, which are provided by way of
exemplification and not by way of limitation.
EXAMPLE 1
Gene Therapy With ATF Inhibits Tumor Growth and Metastasis
[0119] Example 1 demonstrates that expression of the uPA/uPAR
antagonist ATF (amino terminal fragment of urokinase) inhibits
tumor growth and metastasis. A defective adenovirus expressing
murine ATF from the CMV promoter (AdmATF) was constructed. A single
intratumoral injection of AdmATF inhibited growth of
pre-established tumors in two different murine models, and delayed
tumor dissemination. These effects were correlated with a
remarkable inhibition of neovascularization within, and at the
immediate vicinity of, the injection site. The magnitude of this
effect was particularly remarkable in the ability of murine ATF to
inhibit angiogenesis of a human-derived tumor.
Methods
[0120] Recombinant adenoviruses. AdmATF is an E1-defective
recombinant adenovirus that expresses the murine ATF gene from the
CMV promoter. Plasmid pDB 1519 16 was used as starting material to
introduce a stop codon after residue 135 of mature uPA. Briefly,
the uPA-encoding sequences (including its signal peptide) were
isolated, restricted by NsiI, and residues 128 to 135 followed by a
stop codon were reintroduced as a synthetic fragment. The ATF open
reading frame was then inserted between the CMV promoter and the
SV40 late polyadenylation signal sequence, generating plasmid
pEM8-mATF. This plasmid also carries the first 6.3 kb of the AdS
genome except that the ATF expression cassette has been inserted
between position 382 and 3446, in place of the El genes (FIG. 1A).
AdmATF was constructed in 293 cells by homologous recombination
between pEM8-mATF and ClaI-restricted AdRSVbGal DNA 25. Individual
viral plaques were isolated onto 293-derived cell monolayers grown
in soft agar, amplified onto fresh 293 cells and viral DNA was
extracted 26. EcORI, EcORV and AvrII+NdeI restriction analyses
confirmed the identity and clonality of the recombinant adenovirus.
AdCO1 is a defective control adenovirus that is identical to AdmATF
except that it does not carry any transgene expression cassette in
place of E1a. Both viruses were propagated in 293, a human
embryonic kidney cell line that constitutively expresses the E1
genes of AdS 27. Viral stocks were prepared and titrated as
described 25. Unless otherwise stated, MDA-MB-231 cells and Lewis
lung carcinoma (LLC) cells were infected at a multiplicity of
infection (MOI) of 300 PFU/cell. These infection conditions were
previously shown to translate respectively into 80 and 65% of
b-galactosidase-expressing cells when virus AdRSVbGal 25 was
used.
[0121] Northern blot analysis. Subconfluent MDA-MB-231 cell
cultures were infected with AdmATF or AdCO1, and total RNA was
extracted 24 hr post-infection (p.i.) by the RNAZOL procedure
(Biogentex, Inc), and polyadenylated RNAs were purified. The
samples were run in a 1% form aldehyde agarose gel, and transferred
onto Hybond N membranes (Amersham). The membranes were
prehybridized with denatured sonicated salmon sperm DNA (100
.mu.g/ml) for 1 hr at 42.degree. C. in 10 ml of 50% deionized
formamide, 0.2% SDS, 5.times. Denhardt's solution, and incubated
overnight with a random-primed (.sup.32P)-labeled 1.2 kb
XbaI-HindIII fragment from murine uPA cDNA (16). The membranes were
washed twice in 2.times.SSC/0.1% SDS for 1 hr at 50.degree. C.,
once in 0.1.times.SSC for 30 min, and exposed to Kodak XAR-5 films
for 1 hr at room temperature.
[0122] Western blot analysis. Supernatants from virally-infected
cells were collected 24 hr p.i., run in a 12.5% SDS-polyacrylamide
gel (400 .mu.g of protein per lane), prior to transfer onto a
nitrocellulose membrane (Schleicher & Schuell). After
incubation for 1 hr in blocking buffer, the membranes were
incubated for 1 hr with a polyclonal serum raised against murine
uPA (Pr. R Lijnen, Leuven, Belgium), then for an additional hour
with a horse-radish peroxidase-conjugated goat anti-rabbit serum
(Dako). The membranes were washed three times in PBS-Tween buffer,
and incubated with 3-Amino-9-ethyl-Carbazole (AEC) for 5 min.
[0123] Inhibition of cell-associated proteolysis. Native uPA was
first dissociated from its cell surface receptor by submitting LLC
cell monolayers to a 3 min acidification in glycine-HCl (pH 3),
followed by incubation in 0.5 M HEPES buffer. The cells were then
incubated for 20 min at 37.degree. C. with the supernatant of
AdCO1- and AdmATF-infected 293 cells. After 3 washes in PBS/0.1%
BSA, the cells were incubated at 37.degree. C. for 20 min with 1 nM
of murine uPA (Pr. R. Lijnen, Leuven, Belgium). Unbound uPA was
then removed by washing in PBS, and cell-associated uPA was
quantified by adding 0.4 .mu.M of human plasminogen and plasmin
substrate S-2251 (Kabi Vitrum, Sweden).
[0124] In vitro invasion assay. Twenty four hr p.i., LLC cells were
detached with 1 mM EDTA, washed in PBS, and resuspended in FCS-free
MDEM medium supplemented with 0.1% BSA. Invasion assays were
carried out in a transwell unit as described (19). Briefly,
polycarbonate filters of 1.2 .mu.m pore size (Transwell, Costar)
were coated with 160 .mu.g Matrigel (Becton Dickinson) and dried.
The lower chambers of the Transwell units were filled with human
fibroblast-conditioned medium containing 10 ng/ml EGF, and the
upper chambers were seeded with 3.times.10.sup.5 infected cells.
After 24 hr incubation at 37.degree. C., the number of cells that
had reached the lower chamber was determined under a light
microscope following staining with Giesma.
[0125] Syngeneic tumor model. Lewis lung carcinomas were serially
passaged onto C57BL/6 syngeneic mice. Briefly, C57BL/6 implanted
subcutaneously with a LLC tumor were sacrificed when the tumor had
reached a volume of 600-1200 mm.sup.3. Tumor cells were resuspended
in a 0.9% saline solution following filtration through a cotton
sieve, and 2.times.10.sup.6 cells (0.5 ml) were subcutaneously
implanted to the dorsa of 6-7 weeks-old C57BL/6 female mice. After
5 days, the tumors had reached a size of approximately 20 mm.sup.3,
and they were injected with 0.2 ml of PBS (n=8), or 109 PFU (0.2
ml) of AdCO1 (n=10) or AdmATF (n=10). The size of the primary tumor
was measured at day 5, 10 and 15 p.i. At day 16 p.i., the number of
lung metastases was assessed 3 hr after an intraperitoneal
injection of 65 mg BrdU. Lung tissues were removed, fixed overnight
in acetic formaldehyde acid (AFA), and paraffin sections were
incubated 15 min in 4N HCl, neutralized and saturated by washing
twice for 15 min in PBS/0.5% BSA/0.1% Tween 20 prior to incubation
with peroxidase-labeled mouse anti-BrdU monoclonal antibody
(Boehringer) for 45 min at 37.degree. C., and AEC. BrdU-positive
foci were quantified under a light microscope at a magnification of
25.
[0126] Athymic murine model. Cultured MDA-MB-231 cells (ATCC HTB
26) were harvested, washed, resuspended in PBS at
1.5.times.10.sup.7 cells/ml, and 3.times.10.sup.6 cells were
subcutaneously injected in the dorsa of 6-7 weeks old nude mice.
When the tumors had reached a volume of 15-20 mm.sup.3 (i.e., after
11 days), the animals received an intratumoral injection of 109 PFU
of AdmATF (n=5) or AdCO1 (n=5), or PBS (n=5), and the size of the
tumors was monitored until day 52 p.i., after which the animals
were sacrificed and the extent of intratumoral vascularization was
assessed as described (28). Briefly, tumor tissues were fixed
overnight in AFA, transferred to 100% ethanol, embedded in paraffin
and 5 .mu.m thick sections were prepared. After toluene treatment
and rehydration, the sections were permeabilized with 2 .mu.g/ml
proteinase K at 37.degree. C. for 15 min. Endogenous peroxidase
activity was quenched by 0.3% H.sub.2O.sub.2 for 15 min. The
sections were washed with PBS, incubated 15 min in 7.5% BSA, and
incubated 30 min with a rabbit polyclonal serum raised against
human vWF (Dako). After two washes in PBS, the sections were
incubated with biotinylated goat anti-rabbit IgG antibodies for 30
min, washed, and incubated with streptavidin-peroxidase for 15 min
prior to addition of AEC. Neovascular hotspots were first
identified at low magnification and vWF-positive microvessels were
quantified. Meyer's hematoxylin was used for counterstaining as
described (28).
[0127] To evaluate AdmATF infection on tumor establishment,
confluent MDA-MB-231 cells were first infected with AdmATF or AdCO1
at an MOI of 50 PFU/cell. The cells were washed 24 hr p.i.,
resuspended in 120 .mu.l PBS, mixed with 80 .mu.l ice-cold
Matrigel, and 1.3.times.10.sup.6 cells were subcutaneously
implanted into the dorsa of nude mice. Tumor establishment and
growth were followed until day 51 after implantation.
Results
[0128] Molecular and functional characterization of AdmATF. AdmATF
is a defective recombinant adenovirus that expresses murine ATF
from the CMV promoter whereas AdCO1 is an "empty" control
adenovirus (FIG. 1A). In vitro studies were first carried out to
characterize AdmATF with regards to its ability to express a
functional uPA antagonist following infection. ATF gene expression
was demonstrated by northern analysis of poly(A+) RNAs extracted
from MDA-MB-231 cells infected for 24 hr with AdmATF, but not AdCO1
(FIG. 1B). Secretion of ATF by AdmATF-infected cells was
demonstrated for 293, LLC and MDA-MB-231 cells by Western blot
analysis. For example, an ATF-specific polypeptide with a molecular
weight corresponding to that of the mature peptide (15.3 kDa) is
uniquely detected in the medium from 293 cells infected for 24 hr
p.i. with AdmATF (FIG. 1C).
[0129] ATF is a potent antagonist of uPA binding to its cell
surface receptor (uPAR), and disruption of this complex is known to
greatly inhibit the conversion of inactive plasminogen into
plasmin. LLC cell-associated plasmin conversion was thus measured
to assess the functionality of ATF secreted by AdmATF-infected
cells. As a prerequisite, we checked that LLC cells displayed
significant levels of cell-associated uPA activity (data not
shown), implying that they secrete uPA and express uPAR. Plasmin
conversion/activity was significantly reduced when endogenous uPA
had been previously removed from the cell surface by a mild acid
treatment prior to incubation with the supernatant of
AdmATF-infected 293 cells and addition of 1 nM murine uPA (FIG.
2A).
[0130] The uPA/uPAR complex is also crucial to cell motility. An in
vitro cell invasion assay was used to confirm the functionality of
AdmATF. LLCs cells were infected with AdmATF or AdCO1, and the
number of cells that had migrated through a matrix-coated membrane
was determined after 24 hr (FIG. 2B). Quantification of the data
demonstrated that AdmATF infection inhibited LLC invasiveness by
65% (n=5) as compared to AdCO1 control infections.
[0131] Intratumoral injection of AdmATF inhibits tumor growth and
dissemination. We first used the Lewis lung carcinoma-C57BV6
syngeneic model to evaluate the antitumoral effects associated with
a single intratumoral administration of AdmATF. Five days after
subcutaneous implantation, the tumors were injected with 10.sup.9
PFU of AdmATF, 10.sup.9 PFU of AdCO1, or PBS, and tumor growth was
monitored until day 15 p.i. As shown in FIG. 3, an overall
inhibition was specifically observed in the AdmATF-treated group.
The animals were then sacrificed at day 16 p.i., and lung
metastases were numbered by counting the number of BrdU-positive
foci. Whereas metastases were apparent in all animals injected with
PBS (n=8), 7 out of 9, and only 3 out of 9 scored positive within
the AdCO1- and AdmATF-treated groups, respectively. The average
number of BrdU-positive foci per lung sections was also reduced in
the AdmATF-treated group (2.7) as compared to that in the
AdCO1-treated (6.3) and PBS-treated (6.6) groups. A single
intratumoral administration of AdmATF therefore significantly
inhibited tumor growth and lung dissemination in this highly
aggressive model. In a separate experiment, tumor-bearing animals
were infected with AdCO1 or AdmATF, and the tumors extracted at day
10 and 20 p.i. for macroscopic inspection. While AdCO1-injected LLC
tumors displayed an intense vascularization at both time points,
tumors from the AdmATF-treated group displayed only marginal
vascularization (FIG. 4).
[0132] The antitumoral effects of AdmATF are exerted at the level
of angiogenesis. To specifically evaluate the sole inhibition of
angiogenesis for tumor growth, we studied adenovirus-mediated
delivery of the murine uPA/uPAR antagonist in the human-derived
MDA-MB-231 breast carcinoma model implanted into athymic mice. A
direct action of murine ATF on the tumor cells should be minimal in
this model as murine uPA binds human uPAR 200-fold less efficiently
than murine uPAR. Eleven days after subcutaneous tumor cell
inoculation, the animals received a single intratumoral injection
of 10.sup.9 PFU of AdmATF, 10.sup.9 PFU of AdCO1, or PBS, and tumor
growth was monitored until day 52 p.i. While no significant effect
were apparent until day 32 p.i., an arrest of tumor growth then
became evident in the AdmATF-infected, but not the AdCO1-infected
group (FIG. 5). Mice were sacrificed at day 52 p.i., and
intratumoral angiogenesis was assessed by visualization of von
Willebrand Factor (vWF)-immunoreactive vessels (FIG. 6A). An
average of 4 to 6 vessels were detected within the sections from
the AdmATF-treated tumors as compared to 18 to 20 in the sections
from the AdCO1-injected tumors. Tumors injected with AdmATF also
displayed little peripheral neovascularization as compared to their
AdCO1-treated counterparts (FIG. 6B). When MDA-MB-231 cells were
first infected in vitro before subcutaneous inoculation in the
presence of Matrigel, tumors became apparent in the AdCO1-treated
group as early as 7 days post-implantation. A tumor of limited size
was apparent in only one animal from the AdmATF-treated group
(n=5), in sharp contrast to the larger tumors present in 4 out of 5
animals from the AdCO1-infected group. Again, the tumor that had
developed following inoculation of AdmATF-infected tumor cells was
less vascularized than those that developed following inoculation
of AdCO1-infected cells (data not shown).
Discussion
[0133] We have studied the antitumoral effects associated with the
local delivery of the amino-terminal, non-catalytic, fragment of
urokinase (ATF), a potent antagonist of urokinase binding to its
receptor (uPAR) at the surface of both tumor (19, 20) and
endothelial cells (22, 23). In vivo delivery of ATF was achieved by
intratumoral administration of a defective adenovirus that
expresses a secretable ATF molecule of murine origin from the CMV
promoter (AdmATF). To exclude non-specific cytotoxic effects
consecutive to virus infection (29), an "empty" otherwise isogenic
adenovirus (AdCO1) was used as a control virus throughout the
study. This is an important control also because recombinant
adenoviruses can use the aVb3 integrin for infection (30), a cell
surface receptor somehow involved in tumor growth and angiogenesis
(31).
[0134] A single intratumoral injection of AdmATF is efficient in
reducing tumor growth (FIG. 3) and delaying dissemination to the
lungs in the aggressive LLC-C57BL/6 syngeneic murine model. Murine
ATF apparently partly exerted these effects by inhibiting the
invasiveness of the tumor cell themselves (FIG. 2B), a result
consistent with the inhibition of cell-associated proteolysis
following AdmATF infection (FIG. 2A). ATF-based antagonists are
also potent inhibitors of endothelial cells motility (22, 23),
suggesting that inhibition of tumor angiogenesis may have also
contributed to the effects observed in this model. Indeed, LLC
tumors injected with AdmATF displayed very little vascularization
as compared to AdCO1-infected control tumors (FIG. 4). That
specific AdmATF-mediated tumor growth inhibition became evident at
late time p.i. but not so much at early time likely results from
lesser requirements of smaller tumors (typically below 300 mm3, see
FIG. 3 and FIG. 5) for neovascularization to provide the growth
nutrients (for a review see 24).
[0135] Inhibition of LLC cells dissemination to the lungs was only
transient as the survival rate from the AdmATF-treated group was
only slightly extended (less than 30 days after tumor implantation)
as compared to that from the AdCO1-treated group (less than 25
days). The effects of AdmATF injection on tumor cells dissemination
may be explained either because the tumor cells were frozen
following AdmATF infection, and/or because few vessels were
available for entry into the vasculature. That dissemination did
eventually occur suggests that some tumor cells may have had
already reached the vasculature at the time of AdmATF injection.
Alternatively, infection with E1-deleted adenoviruses is also
typically associated with a rapid clearance of the infected cells
in C57BL/6 mice immunotolerant for the transgene product (29), and
ATF is a small molecule that exhibits a very short half-life in
vivo .
[0136] Preclinical data indicate that the uPA/uPAR complex is
critically involved in controlling cell migration, including that
of endothelial cells. For example, an ATF-IgG fusion protein with
an extended in vivo half-life has been shown to inhibit
angiogenesis and tumor growth in a bFGF-enriched Matrigel murine
model (23). The present study provides evidence that the
antitumoral effects of uPA/uPAR antagonists are essentially exerted
by controlling intratumoral and peripheral angiogenesis: whereas
the antitumoral effects of AdmATF-mediated gene delivery may have
been multifactorial as both tumor and endothelial cells are
potential targets in the syngeneic tumor model, this is not the
case in the MDA-MB-231/athymic murine model because mATF is a poor
antagonist of uPA/uPAR complex formation at the surface of human
cells, including MDA-MB-231 (32). A remarkable feature that emerged
in the MDA-MB-231 model was the efficacy of AdmATF in preventing
tumor growth (FIG. 5) and neovascularization within and at the
vicinity of the tumor (FIG. 6). In contrast, tumors infected with
the control adenovirus were still capable of "attracting" adjacent
vessels. The antitumoral properties of AdmATF are further
illustrated in this model by the reduced efficacy of tumor
establishment following infection.
[0137] Malignant tumors are life-threatening because they invade
and abrogate the function of vital organs at distant sites,
emphasizing the importance of targeting angiogenesis to fight
cancer (33; see also 34). First, growth of primary tumors relies on
neovascularization to provide the required nutrients. Second,
metastases have also been reported to undergo apoptosis in the
absence of neovascularization (35). Furthermore, growing
capillaries within the tumor are "leaky": they exhibit a fragmented
basal membrane (36), a prerequisite for efficient penetration of
the tumor cells into the vasculature (37). The overall results of
this study demonstrate that significant antitumoral effects can be
achieved following a single intratumoral administration of a
recombinant adenovirus expressing a potent antagonist of uPA/uPAR
function at the cell surface, and that these effects mostly result
from an inhibition of angiogenesis. Applying this approach to
invasive solid tumors is certainly attractive for cancer gene
therapy because of the pleiotropic clinical effects expected
following inhibition of tumor angiogenesis.
EXAMPLE 2
Gene Therapy With Angiostatin Inhibits Tumors In Vivo
[0138] Example 2 demonstrates that expression of the amino terminal
fragment of human plasminogen (angiostatin K3) inhibits tumor
growth in vivo by blocking endothelial cell proliferation
associated with a mitosis arrest. The antitumoral effects that
follow the local delivery of the N-terminal fragment of human
plasminogen (angiostatin K3) have been studied in two xenograft
murine models. Angiostatin delivery was achieved by a defective
adenovirus expressing a secretable angiostatin K3 molecule from the
CMV promoter (AdK3). In in vitro studies, AdK3 selectively
inhibited endothelial cell proliferation, and disrupted the G2/M
transition induced by M-phase-promoting factors. AdK3-infected
endothelial cells showed a marked mitosis arrest that correlated
with the downregulation of the M-phase phosphoproteins. A single
intratumoral injection of AdK3 into pre-established rat C6 glioma
or human MDA-MB-231 breast carcinoma grown in athymic mice was
followed by a significant arrest of tumor growth, that was
associated with a suppression of neovascularization within and at
vicinity of the tumors. AdK3 therapy also induced a 10-fold
increase in apoptotic tumor cells as compared to control
adenovirus. The data support the concept that targeted
anti-angiogenesis, using adenovirus-mediated gene transfer,
represents a promising strategy for delivering anti-angiogeneic
factors as bolus injections of anti-angiogenic proteins still
present unsolved pharmacological problems.
Methods
[0139] Construction of AdK3. AdK3 is an E1-defective recombinant
adenovirus that expresses the N-terminal fragment of human
plasminogen (up to residue 333) from the CMV promoter. Human Plg
cDNA was obtained from plasmid PG5NM119. A fragment encoding the 18
aa signal peptide of Plg, followed by the first 326 residues of
mature Plg was first subcloned between the BamHI and ScaI sites of
plasmid pXL2675. A synthetic oligodeoxynucleotide encoding residues
327 to 333 followed by a stop codon was then added, prior to
inclusion between the CMV promoter and the SV40 late
polyadenylation signal. This expression cassette was then inserted
between the EcORV and BamHI sites of plasmid pCO5 to generate
plasmid pCO5-K3. AdK3 was constructed in 293 cells by homologous
recombination between pCO5-K3 and ClaI-restricted AdRSV.beta.gal
DNA [25]. Individual plaques were isolated onto 293-derived cell
monolayers, amplified onto fresh 293 cells and viral stocks were
prepared as described [25]. AdCO1 is a control virus that is
identical to AdK3 except that it does not carry any expression
cassette.
[0140] Cell lines maintenance and infection. C6 glioma cells (ATCC
CCL-107) and MDA-MB 231 cells (ATCC HTB 26) were cultured in DMEM
with 10% of fetal calf serum (FCS). Viral infection was performed
with 5% FCS. Human Microcapillary Endothelial Cells (HMEC-1) [49]
were cultured in MCDB 131 supplemented with 20% of FCS, 1 mM
L-glutamine, 1 .mu.g/ml of hydrocortisone, 10 ng/ml of epithelium
growth factor and infection was performed in the same medium but
with 10% of FCS and 3 ng/ml of recombinant human b-FGF (R&D
system). The multiplicity of infection (MOI) was chosen as to
obtain between 80% to 100% infected cells as judged by X-GAL
staining following infection with virus AdRSV.beta.Gal.
[0141] Western blot analysis. Subconfluent cells were infected with
AdK3 or AdCO1 at an MOI of 300 plaque-forming units (PFU)/cell.
Cell culture supernatants were collected 48 to 96 hr post-infection
(p.i.). For in vivo immunological analysis of the K3 angiostatin
molecule, the tumors were collected at day 10 p.i., frozen in
liquid nitrogen, powdered, extracted with lysis buffer (10 mM NEM,
1% triton X100, 1 mM PMSF, 0.1M NH OH) and centrifuged at 12000 rpm
at 4.degree. C. The samples with 300 .mu.g of protein were run in a
10% SDS-polyacrylamide gel, prior to being transferred onto a
nitrocellulose membrane (Schleicher & Schuell). 100 ng human
Plg (Stago) was run as a control. After 2 hr incubation in blocking
buffer (TBS-5% milk-0.05% Tween 20), the membranes were incubated
for 1 hr with anti-human Plg MAb A1D12 [50], 1 hr with a
horseradish peroxidase-conjugated goat anti-mouse serum (Biosys).
After washing, the membranes were detected with ECL bioluminescence
kit (Amersham, UK). To detect the MPM-2 phosphoepitope, the
extracts were prepared from the HMEC-1 cell 96 hr p.i. and probed
with the specific mitotic MPM-2 MAb (DAKO).
[0142] Proliferation assay. Tumor or HMEC-1 cells were infected
with AdK3 or AdCO1 at the indicated MOI for 12 hr. The cells were
collected with 1 mM EDTA, washed twice with PBS and resuspended.
They were seeded into 96-well culture plates (5000 cells/well) and
cultured for 72 hr. In addition, HMEC-1 cells were cultured in
MCDB131 medium containing 40, 20 or 10% supernatant of AdK3 or
AdCO1-transduced C6 glioma cells. Supernatants from
virally-infected C6 cells were collected 96 hr p.i., heated 30 min
at 56.degree. C. in order to inactivate the virus, concentrated 10
times and dialyzed against PBS. Cells were quantified with a cell
proliferation assay kit using a MTS tetrazolium compound
(Promega).
[0143] Formation of capillary tube in a fibrin matrix model. This
model was devised according to the method of Pepper et al [51]
using Calf Pulmonary Artery Endothelial cells (CPAE) (ATCC CCL 209)
infected for 12 hr with AdK3 or AdCO1 at an MOI of 600.
[0144] Whole blood lysis assay. Whole blood clot lysis was
performed by mixing 80 U/ml of tissue-plasminogen activator, 250
.mu.l of culture supernatant obtained 4 days p.i. with AdK3 or
AdCO1, and 500 .mu.l of citrate-anti-coagulated whole blood
collected from healthy donors. Coagulation was triggered by adding
1 U/ml of thrombin and of 12 mM Ca++. The extent of clot lysis was
determined by lysis time and by following the kinetics of soluble
D-Dimers as described [52].
[0145] Immunoflow cytometry. HMEC-1 were infected for 96 hr with
AdK3 or AdCO1 at an MOI of 300 PFU/cell. The cells were collected,
permeabilized with triton X100, incubated with iodide propidium (20
.mu.g/ml) and ribonuclease A (100 .mu.g/ml) for 30 min at room
temperature to label DNA, prior to incubation with mitotic MPM-2
antibody as described [53]. FITC-conjugated anti-mouse IgG
antibodies were used to detect MPM-2 phosphoepitope. The experiment
was performed in a Coulter EPICS Profile II flow cytometer and the
data were analysed by Multicycle software (Phoenix Flow Systems,
San Diego, Calif.).
[0146] Athymic murine models. Cultured C6 glioma cells and
MDA-MB-231 cells were harvested, washed, resuspended in PBS at
1.5.times.1 07 and 0.25.times.10.sup.7 cells/ml respectively and a
volume of 200 .mu.l was subcutaneously injected into the dorsa of
6-7 weeks old nude mice. When the tumors had reached a volume of 20
mm.sup.3, the animals received an intratumoral injection of
10.sup.9 PFU of either AdK3 (n=6), or AdCOI (n=6), or PBS (n=6).
Tumor size was monitored until day 10 p.i. for the C6 glioma model,
and day 42 p.i. for the MDA-MB-231 model.
[0147] To assess the effect of AdK3 infection on tumor
establishment and progression, MDA-MB-231 and C6 cells were
infected for 24 hr at an MOI of 50 and 100 PFU/cell, respectively,
prior to subcutaneous inoculation into the dorsa of nude mice
(n=6). Infected MDA-MB-231 cells are less tumorigenic than infected
C6 cells so 80 .mu.l ice-cold Matrigel (Becton Dickinson) had to be
added to 120 .mu.l of PBS prior to subcutaneous implantation
(10.sup.6 MDA-MB-231 or 0.25.times.10.sup.6 C6 cells). Tumor
establishment and growth were followed until day 25 (MDA-MB-213) or
day 22 (C6) p.i. A Student's t-test was used for statistical
analysis.
[0148] Immunohistochemistry. Tumor tissues were fixed in alcohol
formalin acetic acid, embedded in paraffin and 5 pm sections were
prepared. After toluene treatment and rehydration, the sections
were pretreated three times for 5 min in a microwave oven in 10 mM
citrate buffer (pH 6.0), quenched by 3% H.sub.2O.sub.2 for 5 min to
remove endogenous peroxidase activity, washed in PBS, then
incubated with a rabbit polyclonal serum raised against human von
Willebrand factor (vWF; Dako, dilution 1:200) for 60 min. After 3
washes, the sections were incubated with biotinylated goat
anti-rabbit IgG antibodies for 30 min., washed, and incubated with
streptavidin-peroxidase for 30 min. prior to addition of
3-Amino-9-ethyl-carbazole. Meyer's hematoxylin was used for
counterstaining. Apoptotic cells within the section were detected
by a kit using a terminal deoxynucleotidyl transferase-mediated
dUTP-biotin nick end labeling method (TUNEL) (Boehringer Mannheim).
For proliferating cell nuclear antigen (PCNA) staining procedure
included a biotinylated mouse anti-PCNA antibody (Pharmingen,
dilution 1:100) followed by streptavidin peroxidase and substrate
revelation.
Results
[0149] Molecular characterization of AdK3. Recombinant AdK3 carries
a CMV-driven N-terminal fragment of human Pig that includes the
first three kringle domains of the angiostatin molecule [47],
whereas AdCO1 is an "isogenic" control adenovirus that does not
encode any expression cassette (FIG. 7A). Secretion of the K3
molecule in the culture media 2-3 days after infection with AdK3
was demonstrated for HMEC-1, C6 and MDA-MB-231 cells by Mab A1D12
immunoblotting, whereas no signal was detected following infection
with AdCO1 (FIG. 7B). The secreted immunoreactive peptide appeared
as a doublet with a molecular weight of 36 and 38 kDa, most likely
reflecting a different extent of N-glycosylation at Asn.sup.289 as
described for Plg [54, 55].
[0150] Functional characterization of AdK3. Transduction of HMEC-1
by AdK3 resulted in an inhibition of bFGF-stimulated proliferation
in a dose-dependent manner at day 3 p.i.: 30% at an MOI of 50, 74%
at an MOI of 150, and 97% at an MOI of 300, in sharp contrast to
the cells infected with AdCO1 (P<0.005). AdK3 did not affect
MDA-MB-231 and C6 cell proliferation (FIG. 8A). To assess the
paracrine potential of the K3 molecule to exert these effects,
virus-free culture media from virally-infected C6 glioma cells were
added to HMEC-1 cells. As illustrated in FIG. 8A, we did observe a
dose-dependent inhibition of HMEC-1 cell proliferation by C6
cell-secreted angiostatin (p<0.001). The addition of AdK3 also
significantly inhibited the capillary formation of CPAE cells in
fibrin gel with a 55% mean reduction (not shown). Moreover, whole
blood clot lysis induced by tPA was not inhibited by the addition
of cell culture supernatants from AdK3-infected C6 cells, and the
generation of D-Dimers was basically unchanged during the first
three hours (1200 ng/ml versus 1150 ng/ml).
[0151] AdK3 inhibits mitosis of endothelial cells. To determine if
angiostatin is able to block the mitosis of HMEC-1, a flow
immunocytometry analysis was performed with the cells labeled with
MAb MPM-2 that binds to the phosphorylated proteins specifically
present during the M-phase, together with concurrent DNA staining.
The results showed that mitosis of AdK3-infected HMEC-1 cells was
decreased by 82% relative to AdCO 1 infection: only 5% of HMEC-1
cells within the G2/M pic scored positive for MPM-2 following
infection with AdK3 as compared to 27% following AdCO1 control
infection (FIG. 8C). Western blot analysis was performed from
HMEC-1 extracts in order to detect MPM-2 positive proteins as at
least 16 mitotic phosphoproteins were usually revealed by MPM-2
with an apparent molecular weight ranging from 40 to more than 200
kDa. As compared to control extracts from non-infected or
AdCO1-infected HMEC-1 cells, extracts from AdK3-infected cells
exhibited a markedly reduced level of MPM2-reactive phosphoproteins
(FIG. 8B).
[0152] AdK3 inhibits tumor growth. To induce local secretion of
angiostatin, a single dose of 109 PFU of AdK3 was injected into 20
mm.sup.3 pre-established human MDA-MB-231 breast carcinoma and rat
C6 glioma tumors grown in athymic mice, and tumor growth was
monitored. As shown in FIG. 9A, C6 tumors from the AdK3-injected
group were significantly smaller than those from the AdCO1 or the
PBS control groups: at day 10 p.i., AdK3-injected tumors had
reached a mean volume of 278.+-.14 mm.sup.3 versus 1403.+-.142
mm.sup.3 or 1583.+-.259 mm.sup.3 for AdCO1- and PBS-injected
tumors, respectively (p<0.05). This 80% inhibition correlated
with the detection of angiostatin-immunoreactive material (FIG.
7C). As shown in FIG. 9B, tumor growth was similarly inhibited
(85%) in the MDA-MB-231 tumor model at day 42 p.i.: 80.+-.4
mm.sup.3 for AdK3-treated tumors versus 563.+-.137 mm.sup.3 for
AdCO1- and 530.+-.69 mm.sup.3 for PBS-injected tumors respectively
(p<0.05).
[0153] AdK3 inhibits angiogenesis and induces tumor cell apoptosis
in vivo. C6 tumors infected with AdCO1 appeared much more
vascularized than their AdK3-infected counterparts (FIG. 10, panels
A-B). Intratumoral angiogenesis was thus assessed by
vWF-immunostaining of tumor sections as described [28].
vWF-positive hotspots were first localized at low magnification,
and vWF-positive vessels were then counted at 200.times.
magnification (FIG. 10, panels E-F). The results indicated a marked
reduction of intratumoral vascularization within AdK3-injected
tumors (5.+-.2 vWF-positive vessels per field) as compared to the
AdCO1-injected control (14.+-.4; n=5, p<0.005). Tumors in the
PBS-injected group exhibited an identical number of vessels
(14.+-.3) indicating that the infection conditions used did not
interfere with tumor angiogenesis. At the macroscopic level, C6
tumors injected with AdK3 displayed little peripheral
neovascularization as compared to their AdCO1-treated counterparts
(FIG. 10, panels C-D). Similar results were obtained within
MDA-MB-231 tumor sections (4.8.+-.1.2 vWF-immunoreactive
vessels/field for AdK3 versus 15.6.+-.3 for AdCO1, p=0.02).
[0154] Tumor cell apoptosis was then quantified in situ with the C6
tumor samples by the TUNEL method (see Methods). The results
indicated a marked increase of apoptotic cells in the AdK3-injected
C6 tumors 10 days p.i. (20.+-.9 versus 1-2 apoptotic cells per
field for control tumors, p<0.001) (FIG. 10, panels G-H). In
contrast, the tumor cell proliferation rate was not different among
the three animal groups as assessed by PCNA immunostaining (not
shown). Ad-angiostatin therapy induced a 10 fold increase in
apoptotic tumor cells without affecting the proliferation of these
cells, similar to the reported results obtained by daily injections
of purified angiostatin.
[0155] AdK3 inhibits tumorigenesis. To determine whether inhibition
of tumor angiogenesis attenuated tumorigenesis, MDA-MB-231 and C6
cells were first infected for 24 hr prior to injection into the
dorsa of nude mice. After 5 days, all the mice from the
AdCO1-infected group developed hypervascularized C6 tumors with an
average size of 27.4 +3.41 mm.sup.3, whereas 20% of animals from
the AdK3-infected group remained tumor free after 12 days (FIG.
11). The remaining animals exhibited very small tumors (average
size of 0.42.+-.0.05 mm.sup.3) that were hardly vascularized. After
22 days, the tumors that were observed within the AdK3 group were
at least 5-fold smaller than those from the AdCO1 group (n=5,
p<0.005; FIG. 11). Similar observations were made with the
MDA-MB-231 tumor model (not shown).
Discussion
[0156] Angiostatin has been shown to be a physiopathological
inhibitor of angiogenesis secreted by primary tumors, driving the
metastasis into a dormant state. It was therefore tempting to
assess the therapeutic potential of angiostatin on primary tumors.
However, systemic and intraperitoneal bolus injections of human
angiostatin have underlined difficult pharmacological problems
because angiostatin is rapidly cleared from the circulation [46]. A
prolonged exposure of purified angiostatin at high doses was indeed
required to maintain cytostatic intratumoral concentrations of
angiostatin [46]. It was not clear that direct transduction of the
tumor and the surrounding tissue with a recombinant virus encoding
an angiostatin cDNA would represent a more efficient method of
achieving constant intratumoral concentrations of angiostatin.
Adenoviruses are appropriate vectors in such a strategy as they can
efficiently express their transgene at therapeutic levels in both
proliferating and non-proliferating cells (for a review see [37]),
allowing to target a wide area for angiostatin production. Thus, a
defective adenovirus that expresses the N-terminal fragment (aa
1-333) from human Plg, including its pre-activation peptide and
kringles 1 to 3 (AdK3) was constructed.
[0157] The use of Mab A1D12, which is specific to human Plg [50]
first demonstrated an efficient secretion of angiostatin in the
culture media of cells infected with AdK3. The inclusion of the
N-terminal pre-activation peptide in the angiostatin molecule did
not affect its anti-angiogenic activity since AdK3- but not
AdCO1-infected endothelial cells showed a marked, dose-dependent,
arrest in proliferation in vitro (FIG. 8A). Furthermore, the
proliferation of MDA-MB-231 or C6 tumor cells was not affected by
AdK3-infection demonstrating the restricted action of angiostatin
for endothelial cells. Virus-free supernatants from AdK3-infected
tumor cell culture also inhibited endothelial cell proliferation,
illustrating the paracrine effect of angiostatin secreted by
transduced-tumor cells.
[0158] Because the kringle domains are important for Plg binding to
fibrin and fibrin degradation, it was essential to analyze the
effect of this therapy in thrombolysis, a physiological protection
against thrombosis in vivo. The angiostatin secreted in the culture
medium failed to inhibit tPA-induced whole blood clot lysis in
vitro. Although this experiment has not excluded the deleterious
competition between angiostatin and Plg to bind to fibrin during
thrombolysis in vivo, it indicates that an angiostatic effect could
be achieved at a concentration far below that required for
abrogating plasminogen-dependent thrombolysis in vivo. This may
also suggest that endothelial cells exhibit a receptor that
recognizes angiostatin and not intact Pig.
[0159] Flow cytometry analysis of endothelial cells infected with
AdK3 demonstrated a complete disappearance of the mitotic
population positive for MPM-2 MAb [56]. Immunoblot analysis
revealed that M-phase phosphoproteins reactive to MPM-2 MAb were
indeed downregulated in arigiostatin-treated endothelial cells, in
sharp contrast with control endothelial cells. This observation
should be helpful to define the mechanism by which angiostatin
abrogates the proliferation of endothelial cells. We also showed
that angiostatin disrupted the G2/M transition induced by
M-phase-promoting factor (MPF), composed of cdc2 and its associated
regulatory subunit, cyclin B [57]. MPF phosphorylated proteins,
reactive with MPM-2 MAb, are involved in major alterations of
cellular structures and activities for an efficient progression to
mitosis. The reason why active MPF was lacking in AdK3-transduced
endothelial cells must be further investigated.
[0160] A single intratumoral injection of AdK3, but not of AdCO1
was shown to dramatically inhibit primary tumor growth in two
pre-established xenograft murine models. This inhibitory effect on
tumor growth was tightly correlated with a markedly decreased
vascularization within, and at the vicinity of the tumors (FIG.
10), together with the detection of angiostatin-immunoreactive
material in the tumor extracts (FIG. 7C). C6 glioma is a highly
vascularized tumor due to its VEGF overexpression [58].
Interestingly, the AdK3-transduced C6 glioma apparently failed to
establish a vascular network within the tumor mass to support rapid
and extensive growth (FIG. 10), and this failure translated to more
than 80% inhibition of tumor growth. vWF immunostaining of tumor
sections also revealed a significant reduction of neoangiogenesis
in the AdK3-treated tumors: well formed vessels with a mature lumen
were frequently observed in control C6 tumors, but not in
AdK3-treated C6 glioma (FIG. 10). This decrease in vessel density
was associated with a 10-fold increase in tumor cells apoptosis and
no apparent modification of the tumor cell proliferation index,
probably because (i) of the lack of endothelial-derived paracrine
factors, (ii) a reduction in nutrient support, and (iii) hypoxia
triggered p53-dependent apoptosis of the tumor cells [59, 60]. In
the MDA-MB-231 breast carcinoma model, a single intratumoral
injection of AdK3 similarly induced a remarkable inhibition of
tumor angiogenesis and growth.
[0161] In the course of this study, AdK3-transduced C6 and
MDA-MB-231 cells exhibited a lower tumorigenic potential as
reflected by a prolonged delay for AdK3-infected cells to develop
into visible tumors following implantation.
[0162] Angiostatic therapy using recombinant adenoviruses has been
shown to be experimentally plausible and efficient. The possibility
of delivering more than one angiostatic factor could also synergize
to arrest tumor growth. It is also envisioned that its association
with cytotoxic approaches may be particularly potent to improve the
clinical outcome of malignant diseases.
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[0224] The present invention is not to be limited in scope by the
specific embodiments described herein. Indeed, various
modifications of the invention in addition to those described
herein will become apparent to those skilled in the art from the
foregoing description and the accompanying figures. Such
modifications are intended to fall within the scope of the appended
claims.
[0225] It is further to be understood that all base sizes or amino
acid sizes, and all molecular weight or molecular mass values,
given for nucleic acids or polypeptides are approximate, and are
provided for description.
[0226] Various publications are cited herein, the disclosures of
which are incorporated by reference in their entireties.
* * * * *