U.S. patent application number 10/824058 was filed with the patent office on 2004-11-25 for combined tumor suppressor gene therapy and chemotherapy in the treatment of neoplasms.
This patent application is currently assigned to CANJI, INC.. Invention is credited to Demers, G. William, Horowitz, Jo Ann, Maneval, Daniel C., Nielsen, Loretta, Resnick, Gene, Rybak, Mary Ellen.
Application Number | 20040235736 10/824058 |
Document ID | / |
Family ID | 46279448 |
Filed Date | 2004-11-25 |
United States Patent
Application |
20040235736 |
Kind Code |
A1 |
Nielsen, Loretta ; et
al. |
November 25, 2004 |
Combined tumor suppressor gene therapy and chemotherapy in the
treatment of neoplasms
Abstract
In one embodiment, this invention provides methods of treating
mammalian cancer or hyperproliferative cells, said method
comprising contacting said cells with a tumor suppressor protein or
tumor suppressor nucleic acid and also contacting said cell with at
least one adjunctive anti-cancer agent. The invention also provides
for a pharmacological composition comprising a tumor suppressor
protein or a tumor suppressor nucleic acid and at least one
adjunctive anti-cancer agent, and a kit for the treatment of
mammalian cancer or hyperproliferative cells.
Inventors: |
Nielsen, Loretta;
(Millington, NJ) ; Horowitz, Jo Ann; (Kenilworth,
NJ) ; Maneval, Daniel C.; (San Diego, CA) ;
Demers, G. William; (San Diego, CA) ; Rybak, Mary
Ellen; (Warren, NJ) ; Resnick, Gene;
(Scarsdale, NY) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER
EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Assignee: |
CANJI, INC.
SAN DIEGO
CA
|
Family ID: |
46279448 |
Appl. No.: |
10/824058 |
Filed: |
April 13, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10824058 |
Apr 13, 2004 |
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09311772 |
May 13, 1999 |
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09311772 |
May 13, 1999 |
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09024932 |
Feb 17, 1998 |
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60038065 |
Feb 18, 1997 |
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60047834 |
May 28, 1997 |
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Current U.S.
Class: |
514/44R ;
514/19.4; 514/19.5; 514/19.6; 514/44A |
Current CPC
Class: |
A61K 48/0083 20130101;
A61K 48/00 20130101; A61K 33/243 20190101; A61K 9/0019 20130101;
A61K 48/005 20130101; C12N 15/86 20130101; C12N 2799/022 20130101;
A61K 31/337 20130101; A61K 31/555 20130101; C12N 2710/10343
20130101; A61K 38/1709 20130101; C07K 14/4746 20130101; A61K 31/28
20130101; A61K 31/28 20130101; A61K 2300/00 20130101; A61K 31/337
20130101; A61K 2300/00 20130101; A61K 31/555 20130101; A61K 2300/00
20130101; A61K 33/24 20130101; A61K 2300/00 20130101; A61K 38/1709
20130101; A61K 2300/00 20130101 |
Class at
Publication: |
514/012 ;
514/044 |
International
Class: |
A61K 038/17; A61K
048/00 |
Claims
1. A kit for the treatment of mammalian cancer or
hyperproliferative cells, said kit comprising: a first container
comprising a tumor suppressor protein or nucleic acid selected from
the group consisting of wild-type p53 protein or nucleic acid, or a
retinoblastoma (RB) protein or nucleic acid; and a second container
comprising at least one adjunctive anti-cancer agent.
2. The kit of claim 1, wherein said tumor suppressor nucleic acid
encodes a wild-type p53 protein.
3. The kit of claim 1, wherein said adjunctive anti-cancer agent is
paclitaxel or a paclitaxel derivative.
4. The kit of claim 1, further comprising instructions describing
the administration of both said tumor suppressor protein or nucleic
acid and said adjunctive anti-cancer agent to inhibit the growth or
proliferation of said cell.
5. The kit of claim 1, wherein said tumor suppressor protein or
tumor suppressor nucleic acid is selected from the group consisting
of p53, p110.sup.RB, and p56.sup.RB.
6. The kit of claim 1, wherein said first container contains a
nucleic acid that is contained in a recombinant adenoviral
vector.
7. The kit of claim 6, wherein said nucleic acid is contained in a
recombinant adenoviral vector comprising a partial or total
deletion of a protein IX DNA and comprising a nucleic acid encoding
a p53 protein.
8. The kit of claim 7, wherein said deletion of the protein IX gene
sequence extends from about 3500 bp for the 5' viral termini to
about 4000 bp from the 5' viral termini.
9. The kit of claim 8, further comprising a deletion of DNA
sequence designated E1a and E1b.
10. The kit of claim 6, wherein said recombinant adenoviral vector
comprises the adenovirus type 2 major late promoter or the human
CMV promoter, the adenovirus type 2 tripartite leader cDNA and a
human p53 cDNA.
11. The kit of claim 6, wherein said vector is A/C/N/53.
12. A pharmacological composition comprising a tumor suppressor
protein or a tumor suppressor nucleic acid and at least one
adjunctive anti-cancer agent.
13. The composition of claim 12, wherein said adjunctive
anti-cancer agent is paclitaxel or a paclitaxel derivative.
14. The composition of claim 12, wherein said tumor suppressor
protein or tumor suppressor nucleic acid is selected from the group
consisting of a nucleic acid that encodes a wild-type p53 protein,
a nucleic acid that encodes a retinoblastoma (RB) protein, a
wild-type p53 protein, and a retinoblastoma (RB) protein.
15. The composition of claim 12, wherein said nucleic acid encodes
a wild-type p53 protein.
16. The composition of claim 12, wherein said nucleic acid encodes
a said retinoblastoma p110.sup.RB or a p56.sup.RB.
17. The composition of claim 12, wherein said nucleic acid is
contained in recombinant adenoviral vector.
18. The composition of claim 17, wherein said nucleic acid is
contained in a recombinant adenoviral vector comprising a partial
or total deletion of a protein IX DNA and comprising a nucleic acid
encoding a P53 protein.
19. The composition of claim 18, wherein said deletion of the
protein IX gene sequence extends from about 3500 bp for the 5'
viral termini to about 4000 bp from the 5' viral termini.
20. The composition of claim 19, further comprising a deletion of
DNA sequence designated E1a and E1b.
21. The composition of claim 17, wherein said recombinant
adenoviral vector comprises the adenovirus type 2 major late
promoter or the human CMV promoter, the adenovirus type 2
tripartite leader cDNA and a human p53 cDNA.
22. The composition of claim 17, wherein said vector is
A/C/N/53.
23. The composition of claim 13, wherein said paclitaxel or
paclitaxel derivative is paclitaxel.
24. A composition comprising a mammalian cancer or
hyperproliferative cell, wherein said cell contains an exogenous a
tumor suppressor nucleic acid or a tumor suppressor protein and
paclitaxel or a paclitaxel derivative.
25. The composition of claim 24, wherein said tumor suppressor
nucleic acid is a nucleic acid that encodes a tumor suppressor
protein selected from the group consisting of wild-type p53
protein, and a retinoblastoma (RB) protein.
26. The composition of claim 24, wherein said tumor suppressor
nucleic acid encodes a wild-type p53 protein.
27. The composition of claim 25, wherein said retinoblastoma
protein is a p110.sup.RB or a p56.sup.RB.
28. The composition of claim 24, wherein said cells are a present
in a mammal.
29. The composition of claim 24, wherein said cells are neoplastic
cells.
30. The composition of claim 29, wherein said neoplastic cells
comprise a cancer selected from the group consisting of an ovarian
cancer, pancreatic cancer, a non-small cell lung cancer, small cell
lung cancer, hepatocarcinoma, melanoma, retinoblastoma, breast
tumor, colorectal carcinoma, leukemia, lymphoma, brain tumor,
cervical carcinoma, sarcoma, prostate tumor, bladder tumor, tumor
of the reticuloendothelial tissues, Wilm's tumor, astrocytoma,
glioblastoma, neuroblastoma, osteosarcoma, renal cancer, and head
and neck cancer.
Description
[0001] The present application is a Continuation-In-Part
application ("CIP") of U.S. patent application Ser. No. ______,
attorney docket no. 016930-002560US, filed Dec. 22, 1997; U.S. Ser.
No. 08/801,285, filed Feb. 18, 1997; U.S. Ser. No. 08/801,681,
filed Feb. 18, 1997; U.S. Ser. No. 08/801,755, filed Feb. 18, 1997;
U.S. Ser. No. 08/801,765, filed Feb. 18, 1997; U.S. Provisional
Application No. 60/038,065, filed Feb. 18, 1997; and, U.S.
Provisional Application No. 60/047,834, filed May 28, 1997. Each of
the aforementioned applications is explicitly incorporated herein
by reference in their entirety and for all purposes.
FIELD OF THE INVENTION
[0002] This invention describes novel methods of treating subjects
afflicted with hyperproliferative diseases such as tumors or
metastatic disease. In particular, this invention provides methods
of inhibiting the hyperproliferation of cells, more specifically
neoplastic cells, comprising the combined use of a tumor suppressor
gene or gene product and an adjunctive anti-cancer agent.
BACKGROUND OF TH INVENTION
[0003] Chromosome abnormalities are often associated with genetic
disorders, degenerative diseases, and cancer. In particular, the
deletion or multiplication of copies of whole chromosomes or
chromosomal segments, and higher level amplifications of specific
regions of the genome are common occurrences in cancer. See, for
example Smith (1991) Breast Cancer Res. Treat., 18: Suppl. 1: 5-14;
van de Viler (1991) Became. Beefiest. Acta. 1072: 33-50, Sato
(1990) Cancer. Res., 50: 7184-7189. In fact, the amplification of
DNA sequences containing proto-oncogenes and the deletion of DNA
sequences containing tumor-suppressor genes, are each frequently
characteristic of tumorigenesis. Dutrillaux (1990) Cancer Genet.
Cytogenet. 49: 203-217.
[0004] Mutation of the p53 gene is the most common genetic
alteration in human cancers (Bartek (1991) Oncogene 6: 1699-1703,
Hollstein (1991) Science, 253: 49-53). Moreover, introduction of
wild-type p53 in mammalian cancer cells lacking endogenous
wild-type p53 protein suppresses the neoplastic phenotype of those
cells (see, e.g., U.S. Pat. No. 5,532,220).
[0005] Of the many available chemotherapeutic drugs, paclitaxel,
available commercially as Taxol.RTM. (NSC number: 125973) has
generated interest because of its efficacy in clinical trials
against drug-refractory tumors, including ovarian and mammary gland
tumors (Hawkins (1992) Oncology, 6: 17-23, Horwitz (1992) Trends
Phamacol. Sci. 13: 134-146, Rowinsky (1990) J. Natl. Canc. Inst.
82: 1247-1259). Recent studies on the interaction of paclitaxel and
tumor suppressor gene therapy show that reduced levels of tumor
suppressor (i.e., p53) correlated with increased G2/M phase arrest,
micronucleation, and p53 independent paclitaxel-induced apoptosis.
In contrast, surviving cells with intact p53 progressed through
mitosis and transiently accumulated in the subsequent G1 phase,
coincident with increased p53 and p21.sup.cip1,waf1 protein levels
(Wahl (1996) Nature Med. 2:72-79). Similarly, Hawkins (1996) Canc.
Res. 56: 892-898, showed that inactivation of p53 enhanced
sensitivity to certain antimitotic agents including paclitaxel. The
authors suggested that p53 may play a role in DNA repair, thereby
allowing cells to progress more readily through S phase even in the
presence of drugs. These studies thus suggest that tumor suppressor
gene therapy and drug therapy with anti-mitotic agents (especially
paclitaxel therapy) act at cross purposes.
SUMMARY OF THE INVENTION
[0006] This invention provides methods of treating
hyperproliferative mammalian cells. The invention is premised, in
part, on the surprising discovery that adjunctive anti-cancer
agents in combination with tumor suppressor (e.g., p53) gene
therapy provide an enhanced effect in inhibiting proliferation of
neoplastic or other cells having deficient tumor suppressor
activity.
[0007] Thus, in one embodiment, this invention provides methods of
treating cancer or hyperproliferative cells by contacting the cells
with a tumor suppressor protein or tumor suppressor nucleic acid
and with at least one adjunctive anti-cancer agent. In some
embodiments, the methods include co-administration of the tumor
suppressor protein or nucleic acid and the adjunctive anti-cancer
agent with at least one chemotherapeutic agent. For example, a
tumor suppressor nucleic acid (e.g., a nucleic acid encoding p53)
can be administered with an adjunctive anti-cancer agent (e.g.,
paclitaxel) and a DNA damaging agent such as cisplatin,
carboplatin, navelbine (vinorelbine tartate).
[0008] The cancer or hyperproliferative cells are often neoplastic
cells. When the cells are present in a tumor the method inhibits
tumor growth and thereby provides a method of treating a cancer.
Such cancers include, but are not limited to, an ovarian cancer,
pancreatic cancer, a non-small cell lung cancer, small cell lung
cancer, hepatocarcinoma, melanoma, retinoblastoma, breast tumor,
colorectal carcinoma, leukemia, lymphoma, brain tumor, cervical
carcinoma, sarcoma, prostate tumor, bladder tumor, tumor of the
reticuloendothelial tissues, Wilm's tumor, astrocytoma,
glioblastoma, neuroblastoma, ovarian carcinoma, osteosarcoma, renal
cancer, or head and neck cancer.
[0009] A preferred adjunctive anti-cancer agent is paclitaxel or a
paclitaxel derivative while a preferred tumor suppressor nucleic
acid is a nucleic acid that encodes a tumor suppressor protein
selected from the group consisting of p53 protein and its
analogues, and a retinoblastoma (RB) protein. A particularly
preferred tumor suppressor nucleic acid encodes a wild-type p53
protein and a particularly preferred retinoblastoma protein is a
p110.sup.RB or a p56.sup.RB.
[0010] The tumor suppressor nucleic acid is preferably delivered to
the target cell by a vector. Such vectors' viruses have been
modified by recombinant DNA technology to enable the expression of
the tumor suppressor nucleic acid in the target cell. These vectors
may be derived from vectors of non-viral (e.g., plasmids) or viral
(e.g., adenovirus, adenoassociated virus, retrovirus, herpes virus,
vaccinia virus) origin. In the preferred practice of the invention,
the vector is a recombinantly modified adenoviral vector. Non-viral
vectors are preferably complexed with agents to facilitate the
entry of the DNA across the cellular membrane. Examples of such
non-viral vector complexes include the formulation with
polycationic agents which facilitate the condensation of the DNA
and lipid-based delivery systems. An example of a lipid-based
delivery system would include liposome based delivery of nucleic
acids.
[0011] Particularly suitable adenoviral vectors (e.g., for delivery
of a nucleic acid encoding a wild-type p53 protein) comprise a
partial or total deletion of a protein 1.times.DNA. In one
embodiment, the deletion of the protein IX gene sequence extends
from about 3500 bp from the 5' viral termini to about 4000 bp from
the 5' viral termini. The vector may also comprise a deletion of a
non-essential DNA sequence in adenovirus early region 3 and/or in
adenovirus early region 4 and in one embodiment the deletion is the
DNA sequence E1a and/or E1b. A particularly preferred recombinant
adenoviral vector for delivery of a human p53 cDNA comprises the
adenovirus type 2 major late promoter or the human CMV promoter,
and the adenovirus type 2 tripartite leader cDNA. One such
preferred adenoviral vector is ACN53.
[0012] Preferred paclitaxel or paclitaxel derivatives include
paclitaxel and/or Taxotere.RTM. with paclitaxel (Taxol.RTM. being
most preferred. Another preferred adjunctive anti-cancer is
Epothilone. In one particularly preferred embodiment, the tumor
suppressor is A/C/N/53 and the adjunctive anti-cancer agent is
paclitaxel.
[0013] The tumor suppressor protein or tumor suppressor nucleic
acid can be dispersed in a pharmacologically acceptable excipient.
Similarly, the adjunctive anti-cancer (e.g., paclitaxel or
paclitaxel derivative) can be dispersed in a pharmacologically
acceptable excipient. The tumor suppressor protein or tumor
suppressor nucleic acid and said paclitaxel or paclitaxel
derivative can both be dispersed in a single composition
(comprising one or multiple excipient(s)).
[0014] The tumor suppressor (protein or nucleic acid) and/or the
adjunctive anti-cancer can be administered intra-arterially,
intravenously (e.g., injected), intraperitoneally and/or
intratumorally, together or sequentially. Preferred sites of
administration include intra-hepatic-artery, intraperitoneal, or,
where it is desired to treat cells in the head (e.g, neurological
cells), into the carotid system of arteries.
[0015] The tumor suppressor protein or nucleic acid can be
administered in a single dose or a multiplicity of treatments,
e.g., each separated by at least about 6 hours, more preferably in
least three treatments separated by about 24 hours.
[0016] In another preferred embodiment, the tumor suppressor
protein or tumor suppressor nucleic acid is administered (with or
without an adjunctive anti-cancer agent) in a total dose ranging
from about 1.times.10.sup.9 to about 1.times.10.sup.14, or about
1.times.10.sup.9 to about 7.5.times.10.sup.15, preferably about
1.times.10.sup.11 to about 7.5.times.10.sup.13, adenovirus
particles in a treatment regimen selected from the group consisting
of: the total dose in a single dose, the total dose administered
daily over 5 days, the total dose administered daily over 15 days,
and the total dose administered daily over 30 days. The dose can
also be administered continuously for 1 to 30 days. The paclitaxel
or paclitaxel derivative is administered in a total dose ranging
from 75-350 mg/m.sup.2 over 1 hour, 3 hours, 6 hours, or 24 hours
in a treatment regimen selected from the group consisting of
administration in a single dose, in the total dose administered
daily on each of day 1 and day 2, in the total dose administered
daily on each of day 1, day 2, and day 3, on a daily dosage for 15
days, on a daily dosage for 30 days, on daily continuous infusion
for 15 days, on daily continuous infusion for 30 days. A preferred
dose is 100-250 mg/m.sup.2 in 24 hours. Alternatively, the
paclitaxel or derivative can be administered weekly at 60
mg/m.sup.2. This method of administration can be repeated for two
or more cycles (more preferably for three cycles) and the two or
more cycles are can be spaced apart by three or four weeks.
[0017] In some preferred embodiments, a daily dose in the range of
7.5.times.10.sup.9 to about 7.5.times.10.sup.15, preferably about
1.times.10.sup.12 to about 7.5.times.10.sup.13, adenovirus
particles can be administered each day for up to 30 days (e.g., a
regimen of 2 days or 2 to 5 days to 14 days or 30 days with the
same dose being administered each day). The multiple regimen can be
repeated in recurring cycles of 21 to 28 days. Preferred routes of
administration include intra-arterial (e.g., intra-hepatic artery),
intratumorally, and intraperitoneally.
[0018] When the tumor suppressor nucleic acid (e.g., p53) is
administered in an adenoviral vector with an adjunctive anti-cancer
agent (e.g., paclitaxel) and a DNA damaging agent (e.g., cisplatin,
carboplatin, or navelbine), the adenoviral vector is administered
for 5-14 days at about 7.5.times.10.sup.12 to about
7.5.times.10.sup.13 adenoviral particles per day. If the adenoviral
vector and paclitaxel is administered with carboplatin, the dose is
typically 7.5.times.10.sup.13 adenoviral particles per day. For
example, a daily dose of about 7.5.times.10.sup.12 adenoviral
particles can be used for administration to the lung.
[0019] This invention also provides for kits for the treatment of
mammalian cancer or hyperproliferative cells. The kits include a
tumor suppressor protein or nucleic acid described herein (more
preferably a wild-type p53 protein or nucleic acid (e.g., in a
viral or non-viral vector), or a retinoblastoma (RB) protein or
nucleic acid); and an adjunctive anti-cancer agent described herein
(e.g., paclitaxel or a paclitaxel derivative) and/or optionally any
of the other chemotherapeutic agents described herein. The kit can
optionally further include instructions describing the
administration of both the tumor suppressor protein or nucleic acid
and the adjunctive anti-cancer agent (and optionally an other
chemotherapeutic agent) to inhibit the growth or proliferation of
the cancer or hyperproliferative cells. One particularly preferred
kit includes A/C/N/53 and paclitaxel.
[0020] In another embodiment this invention provides
pharmacological compositions comprising a tumor suppressor protein
or a tumor suppressor nucleic acid and an adjunctive anti-cancer
agent. In various embodiments, the pharmacological composition can
optionally include any of the other chemotherapeutic compounds
described herein. One particularly preferred composition includes a
p53 nucleic acid (e.g., A/C/N/53) and paclitaxel. The tumor
suppressor nucleic acid or protein and the chemotherapeutic agent
(e.g., paclitaxel) can be in different excipients or can be
contained in a single excipient as described herein. Where there
are multiple excipients, the excipients can be intermixed or held
separately (e.g., as in microcapsules).
[0021] In still another embodiment, this invention provides a
composition comprising a mammalian cancer or hyperproliferative
cell, wherein said cell contains an exogenous tumor suppressor
nucleic acid or a tumor suppressor protein. The cell may
additionally include an adjunctive anti-cancer agent such as
paclitaxel or a paclitaxel derivative. The exogenous tumor
suppressor nucleic acid or tumor suppressor protein may be any one
or more of the tumor suppressor nucleic acids and/or proteins
described herein. Similarly the cell can be any one or more of the
hyperproliferative and/or cancerous cells described herein.
[0022] In yet another embodiment, this invention provides a method
of treating a metastatic cell. The method involves contacting the
cell with a tumor suppressor nucleic acid or tumor suppressor
polypeptide. Suitable tumor suppressor nucleic acids or
polypeptides include any of the tumor suppressors nucleic acids
and/or polypeptides disclosed herein. The method can additionally
include contacting the cell with any of the the adjunctive
anti-cancer agents disclosed herein. In a particularly preferred
embodiment, the method involves topical administration of the tumor
suppressor nucleic acid and/or polypeptide to a surgical wound.
[0023] In another embodiment, this invention provides particularly
preferred dosage regimen. Thus, in one embodiment, this invention
provides a method of treating mammalian cells, where the method
involves administering to the cells a total dose of a tumor
suppressor protein or tumor suppressor nucleic acid, wherein said
total dose is administered in a multiplicity of administrations of
incremental doses of said tumor suppressor protein or tumor
suppressor nucleic acid. Preferred multiple administrations are
each separated by at least about 6 hours. One preferred
administration is in least three treatments separated by about 24
hours.
[0024] In another embodiment, this invention provides a method of
treating a mammalian cell. The method involves administering to the
cell a total dose of a tumor suppressor protein or tumor suppressor
nucleic acid, wherein the total dose is administered in a
multiplicity of administrations of incremental doses of tumor
suppressor protein or tumor suppressor nucleic acid. The
administrations may be spaced by at least about six hours. The
method can involve at least comprising at least three incremental
doses and the doses can be administered daily. In one embodiment,
the method can comprise at least three treatments separated by
about 24 hours. In another embodiment the method can involve tumor
administering the tumor suppressor nucleic acid is administered in
a total dose ranging from about 1.times.10.sup.9 to about
7.5.times.10.sup.15, or about 1.times.10.sup.11 to about
7.5.times.10.sup.13, adenovirus particles in a treatment regimen
selected from the group consisting of: the total dose in a single
dose, the total dose administered daily over 5 days, the total dose
administered daily over 15 days, and the total dose administered
daily over 30 days. The method may further comprise administering
paclitaxel or a paclitaxel derivative in a total dose ranging from
about 75 mg/m.sup.2 to about 350 mg/m.sup.2 over 24 hours in a
treatment regimen selected from the group consisting of
administration in a single dose, in a dose administered daily on
day 1 and day 2, in a dose administered daily on day 1, day 2, and
day 3, on a daily dosage for 15 days, on a daily dosage for 30
days, on daily continuous infusion for 15 days, on daily continuous
infusion for 30 days. These treatment regimens may be is repeated
for two or more cycles and the two or more cycles can be spaced
apart by three or four weeks. The cells thus treated include
neoplastic cells comprising a cancer selected from the group
consisting of an ovarian cancer, mesothelioma, pancreatic cancer, a
non-small cell lung cancer, small cell lung cancer,
hepatocarcinoma, melanoma, retinoblastoma, breast tumor, colorectal
carcinoma, leukemia, lymphoma, brain tumor, cervical carcinoma,
sarcoma, prostate tumor, bladder tumor, tumor of the
reticuloendothelial tissues, Wilm's tumor, astrocytoma,
glioblastoma, neuroblastoma, osteosarcoma, renal cancer, and head
and neck cancer. The treatment treating preferably results in
inhibition of growth or proliferation of a tumor as assayed by
measurement of the volume of the tumor.
[0025] The invention also provides for a pharmacological
composition comprising a tumor suppressor protein or a tumor
suppressor nucleic acid and at least one adjunctive anti-cancer
agent. The adjunctive anti-cancer agent can be paclitaxel or a
paclitaxel derivative. The tumor suppressor protein or tumor
suppressor nucleic acid can be selected from the group consisting
of a nucleic acid that encodes a wild-type p53 protein, a nucleic
acid that encodes a retinoblastoma (RB) protein, a wild-type p53
protein, and a retinoblastoma (RB) protein.
[0026] The retinoblastoma protein can be p110.sup.RB or a
p56.sup.RB. The nucleic acid can be contained in a recombinant
adenoviral vector. The nucleic acid can be contained in a
recombinant adenoviral vector comprising a partial or total
deletion of a protein IX DNA and comprising a nucleic acid encoding
a P53 protein. In one embodiment, the deletion of the protein IX
gene sequence can extend from about 3500 bp for the 5' viral
termini to about 4000 bp from the 5' viral termini. The deletion of
DNA can include sequence designated E1a and E1b. The recombinant
adenoviral vector can further comprise the adenovirus type 2 major
late promoter or the human CMV promoter, the adenovirus type 2
tripartite leader cDNA and a human p53 cDNA. In a preferred
embodiment, the vector is A/C/N/53. The composition can be
paclitaxel, or a paclitaxel derivative or a paclitaxel
analogue.
[0027] The invention further provides for a composition comprising
a mammalian cancer or hyperproliferative cell, wherein said cell
contains an exogenous a tumor suppressor nucleic acid or a tumor
suppressor protein and an adjunctive anti-cancer agent. The tumor
suppressor nucleic acid can be a nucleic acid that encodes a tumor
suppressor protein selected from the group consisting of wild-type
p53 protein, and a retinoblastoma (RB) protein. The retinoblastoma
protein can be a p110.sup.RB or a p56.sup.RB. The cells can be
present in a mammal. The cells can be neoplastic cells and the
neoplastic cells can comprise a cancer selected from the group
consisting of an ovarian cancer, pancreatic cancer, a non-small
cell lung cancer, small cell lung cancer, hepatocarcinoma,
melanoma, retinoblastoma, breast tumor, colorectal carcinoma,
leukemia, lymphoma, brain tumor, cervical carcinoma, sarcoma,
prostate tumor, bladder tumor, tumor of the reticuloendothelial
tissues, Wilm's tumor, astrocytoma, glioblastoma, neuroblastoma,
osteosarcoma, renal cancer, and head and neck cancer.
[0028] The invention provides for a method of treating a metastatic
cell, said method comprising contacting said cell with a tumor
suppressor nucleic acid or tumor suppressor polypeptide and an
adjunctive anti-cancer agent. The contacting can comprise topical
administration of a tumor suppressor nucleic acid to a surgical
wound. The method can further include co-administration of a
chemotherapeutic agent, and the chemotherapeutic agent can be
cisplatin, carboplatin, or navelbine.
DEFINITIONS
[0029] The term "adjunctive anti-cancer agent" refers to an agent
which has at least one of the following activities: the ability to
modulate of microtubule formation or action, the ability to inhibit
polyprenyl-protein transferase activity, the ability to inhibit
angiogenesis, or the ability to inhibit endocrine activity.
Adjunctive anti-cancer agents useful in the invention are described
in more detail below. As used herein, adjunctive anti-cancer agents
of the invention do not include compounds with DNA damaging
activity.
[0030] "Tumor suppressor genes" are nucleic acids for which
loss-of-function mutations are oncogenic. Thus, the absence,
mutation, or disruption of normal expression of a tumor suppressor
gene in an otherwise healthy cell increases the likelihood of, or
results in, the cell attaining a neoplastic state. Conversely, when
a functional tumor suppressor gene or protein is present in a cell,
its presence suppresses the tumorigenicity, malignancy or
hyperproliferative phenotype of the host cell. Examples of tumor
suppressor nucleic acids within this definition include, but are
not limited to p110.sup.RB, p56.sup.RB, p53, and other tumor
suppressors described herein and in copending application U.S. Ser.
No. 08/328,673 filed on Oct. 25, 1994. Tumor suppressor nucleic
acids include tumor suppressor genes, or nucleic acids derived
therefrom (e.g., cDNAs, cRNAs, mRNAs, and subsequences thereof
encoding active fragments of the respective tumor suppressor
polypeptide), as well as vectors comprising these sequences.
[0031] A "tumor suppressor polypeptide or protein" refers to a
polypeptide that, when present in a cell, reduces the
tumorigenicity, malignancy, or hyperproliferative phenotype of the
cell.
[0032] The term "viral particle" refers to an intact virion. The
concentration of infectious adenovirus viral particles is typically
determined by spectrophotometric detection of. DNA, as described,
for instance, by Huyghe (1995) Human Gene Ther. 6:1403-1416.
[0033] The terms "neoplasia" or "neoplastic" are intended to
describe a cell growing and/or dividing at a rate beyond the normal
limitations of growth for that cell type.
[0034] The term "tumorigenic" or "tumorigenicity" are intended to
mean having the ability to form tumors or capable of causing tumor
formation.
[0035] The phrase "treating a cell" refers to the inhibition or
amelioration of one or more disease characteristics of a diseased
cell. When used in reference to a cancer cell that is neoplastic
(e.g., a mammalian cancer cell lacking an endogenous wild-type
tumor suppressor protein), the phrase "treating a cell" refers to
mitigation or elimination of the neoplastic phenotype. Typically
such treatment results in inhibition (a reduction or cessation of
growth and/or proliferation) of the cell as compared to the same
cell under the same conditions but for the treatment (e.g.,
adjunctive anti-cancer agent and or tumor suppressor nucleic acid
or polypeptide). Such inhibition may include cell death (e.g.,
apoptosis). These terms when used with reference to a tumor refer
to inhibition of growth or proliferation of the tumor mass (e.g.,
as measured volumetrically). Such inhibition may be mediated via
reduction in growth rate and/or proliferation rate and/or death of
cells comprising the tumor mass. The inhibition of growth or
inhibition of proliferation can be accompanied by an alteration in
cellular phenotype (e.g., restoration of morphology characteristic
of healthy cells, restoration of contact inhibition, loss of
invasive phenotype, inhibition of anchorage independent growth,
etc.). For the purposes of this disclosure, a diseased cell will
have one or more pathological traits. These traits in a diseased
cell may include, inter alia, defective expression of one or more
tumor suppressor proteins. Defective expression may be
characterized by complete loss of one or more functional tumor
suppressor proteins or a reduction in the level of expression of
one or more functional tumor suppressor proteins. Such cells are
often neoplastic and/or tumorigenic.
[0036] The term "systemic administration" refers to administration
of a composition or drug, such as the recombinant adenoviral
vectors of the invention or the adjunctive anti-cancer or
chemotherapeutic compounds described herein, in a manner that
results in the introduction of the composition or drug into the
circulatory system. The term "regional administration" refers to
administration of a composition or drug into a specific anatomical
space, such as intraperitoneal, intrathecal, subdural, or to a
specific organ, and the like. For example, regional administration
includes administration of the composition or drug into the hepatic
artery for regional administration to the liver. The term "local
administration" refers to administration of a composition or drug
into a limited, or circumscribed, anatomic space, such as
intratumoral injection into a tumor mass, subcutaneous injections,
intramuscular injections, and the like. Any one of skill in the art
would understand that local administration or regional
administration may also result in entry of the composition or drug
into the circulatory system.
[0037] The term "reduced tumorigenicity" is used herein to refer to
the conversion of hyperproliferative (e.g. neoplastic) cells to a
less proliferative state. In the case of tumor cells, "reduced
tumorigenicity" is intended to mean tumor cells that have become
less tumorigenic or non-tumorigenic or non-tumor cells whose
ability to convert into tumor cells is reduced or eliminated. Cells
with reduced tumorigenicity either form no tumors in vivo or have
an extended lag time of weeks to months before the appearance of in
vivo tumor growth. Cells with reduced tumorigenicity may also
result in slower growing three dimensional tumor mass compared to
the same type of cells having fully inactivated or non-functional
tumor suppressor gene growing in the same physiological milieu
(e.g., tissue, organism age, organism sex, time in menstrual cycle,
etc.).
[0038] As used herein an "active fragment" of a gene or polypeptide
includes smaller portion(s) (subsequences) of the gene or nucleic
acid derived therefrom (e.g., cDNA) that retain the ability to
encode proteins having tumor suppressing activity. Similarly, an
active fragment of a polypeptide refers to a subsequence of a
polypeptide that has tumor suppressing protein. One example of an
active fragment is p56RB as described, e.g., in copending U.S. Ser.
No. 08/328,673 filed on Oct. 25, 1994.
[0039] The term "malignancy" is intended to describe a tumorigenic
cell having the ability to metastasize.
[0040] "Nucleic acids", as used herein, may be DNA or RNA. Nucleic
acids may also include modified nucleotides that permit correct
read through by a polymerase and do not alter expression of a
polypeptide encoded by that nucleic acid.
[0041] The phrase "nucleotide sequence" includes both the sense and
antisense strands as either individual single strands or in the
duplex.
[0042] The phrase "DNA sequence" refers to a single or double
stranded DNA molecule composed of the nucleotide bases, adenosine,
thymidine, cytosine and guanosine.
[0043] The phrase "nucleic acid sequence encoding" refers to a
nucleic acid which directs the expression of a specific protein or
peptide. The nucleic acid sequences include both the DNA strand
sequence that is transcribed into RNA and the RNA sequence that is
translated into protein. The nucleic acid sequences include both
the full length nucleic acid sequences as well as non-full length
sequences derived from the full length sequences. It being further
understood that the sequence includes the degenerate codons of the
native sequence or sequences which may be introduced to provide
codon preference in a specific host cell.
[0044] The phrase "expression cassette", refers to nucleotide
sequences which are capable of affecting expression of a structural
gene in hosts compatible with such sequences. Such cassettes
include at least promoters and optionally, transcription
termination signals. Additional factors necessary or helpful in
effecting expression may also be used as described herein.
[0045] The term "operably linked" as used herein refers to linkage
of a promoter upstream from a DNA sequence such that the promoter
mediates transcription of the DNA sequence.
[0046] "Isolated" or "substantially pure" when referring to nucleic
acid sequences encoding tumor suppressor protein or polypeptide or
fragments thereof refers to isolated nucleic acids which do not
encode proteins or peptides other than the tumor suppressor protein
or polypeptide or fragments thereof.
[0047] The term "recombinant" refers to DNA which has been isolated
from its native or endogenous source and modified either chemically
or enzymatically to delete naturally-occurring flanking nucleotides
or provide flanking nucleotides that do not naturally occur.
Flanking nucleotides are those nucleotides which are either
upstream or downstream from the described sequence or sub-sequence
of nucleotides.
[0048] A "vector" comprises a nucleic acid which can infect,
transfect, transiently or permanently transduce a cell. It will be
recognized that a vector can be a naked nucleic acid, or a nucleic
acid complexed with protein or lipid. The vector optionally
comprises viral or bacterial nucleic acids and/or proteins, and/or
membranes (e.g., a cell membrane, a viral lipid envelope, etc.). It
is recognized that vectors often include an expression cassette
placing the nucleic acid of interest under the control of a
promoter. Vectors include, but are not limited to replicons (e.g.,
plasmids, bacteriophages) to which fragments of DNA may be attached
and become replicated. Vectors thus include, but are not limited to
RNA, autonomous self-replicating circular DNA (plasmids), and
includes both the expression and nonexpression plasmids. Where a
recombinant microorganism or cell culture is described as hosting
an "expression vector" this includes both extrachromosomal circular
DNA and DNA that has been incorporated into the host chromosome(s).
Where a vector is being maintained by a host cell, the vector may
either be stably replicated by the cells during mitosis as an
autonomous structure, or is incorporated within the host's
genome.
[0049] The term effective amount is intended to mean the amount of
vector or drug which achieves a positive outcome on controlling
cell growth and/or proliferation.
[0050] The abbreviation "C.I.U." as used herein, stands for
"cellular infectious units." The C.I.U. is calculated by measuring
viral hexon protein positive cells (e.g., -293 cells) after a 48
hr. infection period (Huyghe (1995) Human Gene Ther. 6:
1403-1416).
[0051] The abbreviation "m.o.i." as used herein refers to
"multiplicity of infection" and is the C.I.U. per cell.
[0052] The term "paclitaxel" as used herein refers to the drug
commercially known as Taxol.RTM.. Taxol.RTM. inhibits eukaryotic
cell replication by enhancing polymerization of tubulin moieties
into stabilized microtubule bundles that are unable to reorganize
into the proper structures for mitosis.
[0053] The term "contacting a cell" when referring to contacting
with a drug and/or nucleic acid is used herein to refer to
contacting in a manner such that the drug and/or nucleic acid is
internalized into the cell. In this context, contacting a cell with
a nucleic is equivalent to transfecting a cell with a nucleic acid.
Where the drug is lipophilic or the nucleic acid is complexed with
a lipid (e.g., a cationic lipid) simple contacting will result in
transport (active, passive and/or diffusive) into the cell.
Alternatively the drug and/or nucleic acid may be itself, or in
combination with a carrier composition be actively transported into
the cell. Thus, for example, where the nucleic acid is present in
an infective vector (e.g., an adenovirus) the vector may mediate
uptake of the nucleic acid into the cell. The nucleic acid may be
complexed to agents which interact specifically with extracellular
receptors to facilitate delivery of the nucleic acid into the cell,
examples include ligand/polycation/DNA complexes as described in
U.S. Pat. Nos. 5,166,320 and 5,635,383. Additionally, viral
delivery may be enhanced by recombinant modification of the knob or
fiber domains of the viral genome to incorporate cell targeting
moieties.
[0054] The constructs designated herein as "A/C/N/53", "A/M/N/53",
p110.sup.RB, p56.sup.RB, refer to the constructs so designated in
copending application U.S. Ser. No. 08/328,673, filed on Oct. 25,
1994, International Application WO 95/11984.
[0055] A "conservative substitution", when describing a protein
refers to a change in the amino acid composition of the protein
that does not substantially alter the protein's activity. Thus,
"conservatively modified variations" of a particular amino acid
sequence refers to amino acid substitutions of those amino acids
that are not critical for protein activity or substitution of amino
acids with other amino acids having similar properties (e.g.,
acidic, basic, positively or negatively charged, polar or
non-polar, etc.) such that the substitutions of even critical amino
acids do not substantially alter activity. Conservative
substitution tables providing functionally similar amino acids are
well known in the art. For example, the following six groups each
contain amino acids that are conservative substitutions for one
another:
[0056] 1) Alanine (A), Serine (S), Threonine (T);
[0057] 2) Aspartic acid (D), Glutamic acid (E);
[0058] 3) Asparagine (N), Glutamine (Q);
[0059] 4) Arginine (R), Lysine (K);
[0060] 5) Isoleucine (1), Leucine (L), Methionine (M), Valine (V);
and
[0061] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
[0062] See also, Creighton (1984) Proteins W.H. Freeman and
Company. In addition, individual substitutions, deletions or
additions which alter, add or delete a single amino acid or a small
percentage of amino acids in an encoded sequence are also
"conservatively modified variations".
BRIEF DESCRIPTION OF THE DRAWINGS
[0063] FIG. 1 illustrates the in vitro inhibition of SK-OV-3
ovarian tumor cells by various concentrations of p53 (A/C/N/53)
and/or Taxol.RTM..
[0064] FIG. 2 provides an isobologram analysis for the experiments
illustrated in FIG. 1. Synergism between Taxol.RTM. and p53
(A/C/N/53) was observed when the cells were pretreated with
Taxol.RTM. 24 hours before p53 treatment.
[0065] FIGS. 3a, 3b, and 3c illustrate the efficacy of p53 Ad
against human breast cancer xenografts in nude mice. Mice were
given a total dose of 2.2.times.10.sup.9 C.I.U. adenovirus
(A/C/N/53 or Ad) per mouse split into 10 injections on days 0-4 and
7-11. Mice were treated with p53 Ad, beta-gal Ad, or vehicle alone.
FIG. 3a illustrates results with MDA-MB-231 tumors. FIG. 3b
illustrates results with MDA-MB468 (468) tumors, and FIG. 3c
illustrates results with MDA-MB435 (435) tumors.
[0066] FIGS. 4a and 4b provide p53 Ad (A/C/N/53) dose response
curves for MDA-MB-231 (-231) tumors (FIG. 4a) and for MDA-MB468
tumors (FIG. 4b). Mice were dosed with
1.times.10.sup.7-1.times.10.sup.9 C.I.U. p53 Ad (A/C/N/53) split
into 10 doses administered peritumorally on days 0-4 and 7-11.
Average percent inhibitions were calculated by comparing the tumor
volumes at each p53 Ad dose with buffer-treated tumors on the days
14/15, 18, 21, 24, 28, 30/32, and 35 (MDA-MB468 tumors only on day
35). The -231 tumors averaged 22.5.+-.2 mm.sup.3 on day 0, while
the -468 tumors averaged 33.1.+-.1.8 mm.sup.3 on day 0.
[0067] FIG. 5 provides a comparison of the efficacy of the
therapeutic agent when administered as a single bolus or as split
doses. Tumors (MDA-MB-231) were dosed with a total of
2.2.times.10.sup.8 C.I.U. p53 Ad per week given during weeks 1 and
3.
[0068] FIG. 6 illustrates the efficacy of multiple cycles of low
dose p53 Ad against large, well-established tumors. A total of
1.32.times.10.sup.9 C.I.U. p53 Ad was given over 6 weeks to
MDA-MB-468 xenografts. (P=plateau in control tumor growth rate;
E=end of dosing.).
[0069] FIGS. 7a, 7b, and 7c illustrate the in vivo inhibition of
MDA-MB-468 tumors in nude mice administered 1.times.10.sup.9 C.I.U.
p53 Ad (A/C/N/53) as a single bolus injection (FIG. 7a) or split
into 3 injections (FIG. 7b) or 5 injections (FIG. 7c).
[0070] FIG. 8 illustrates of the ability of low dose dexamethasone
to suppress the inhibition of tumor growth mediated by NK cells in
scid mice. MDA-MB-231 tumors were dosed with a total of
2.times.10.sup.9 C.I.U. beta-gal Ad (1.1.times.1011 viral
particles) split into 10 injections given on days 14-18 and 21-25.
Subcutaneous dexamethasone (or placebo) pellets released 83.3 .mu.g
steroid per day.
[0071] FIG. 9. Comparison of combination p53 and cisplatin therapy
on normal and tumor cells.
DETAILED DESCRIPTION
[0072] This invention provides new methods of inhibiting the growth
and/or proliferation of cells, more particularly the growth and
proliferation of cancer cells. In one embodiment, the methods
involve contacting the cells with a tumor suppressor nucleic acid,
or tumor suppressor protein and with an adjunctive anti-cancer
agent. Typically, the tumor suppressor protein or nucleic acid used
will be the same species as the tumor suppressor protein that is
lacking. Thus, where the cell lacks endogenous p53 activity, a p53
protein or p53 nucleic acid will be used.
[0073] It was a surprising discovery of this invention that,
contrary to the results described in previous studies (see, e.g.,
Wahl et al. (1996) Nature Med., 2(1): 72-79, and Hawkins et al.
(1996) Canc. Res. 56: 892-898), the treatment of mammalian cells
lacking or deficient in endogenous wild-type tumor suppressor
protein (i.e., many neoplastic cells), with both an adjunctive
anti-cancer agent (e.g., paclitaxel (Taxol.RTM.) and a tumor
suppressor gene or polypeptide (e.g., p53) results in inhibition of
proliferation and/or growth of the cells greater than that observed
with either the chemical treatment or the tumor suppressor
construct alone. Moreover, it was a discovery of this invention
that pretreatment with adjunctive anti-cancer agents dramatically
increases the anti-proliferative effect of a tumor suppressor
nucleic acid. Without being bound by a particular theory, it is
believed that possible means by which an adjunctive anti-cancer
agent may contribute to this enhanced effect is: to increase the
transfection efficiency of various gene therapy vectors (e.g.,
adenovirus vectors); or, to increase expression levels of the tumor
suppressor gene; or, to stabilize microtubules to assist in
intracellular virus transport; or, to provide enhanced effect
through the interaction of various intracellular mechanisms (e.g.,
signaling pathways, apoptotic pathways, cell cycling pathways).
[0074] Thus, in one embodiment, this invention provides methods of
inhibiting diseased mammalian cells lacking, or deficient in, an
endogenous wild-type tumor suppressor protein of cells by
contacting them with an adjunctive anti-cancer agent and with a
tumor suppressor nucleic acid and/or tumor suppressor polypeptide.
When the cells are present in a tumor the method inhibits tumor
growth and thereby provides a method of treating a cancer.
Particularly preferred tumor suppressor nucleic acids or
polypeptides include p53, RB, h-NUC (see, e.g., Chen (1995) Cell
Growth Differ. 6:199-210) or active fragments thereof (e.g.,
p110RB, p56RB), while particularly preferred adjunctive anti-cancer
agents (compounds) include paclitaxel and compounds with
paclitaxel-like activity such as paclitaxel derivatives (e.g.,
analogues).
[0075] It was also a discovery of this invention that contacting of
cells with a tumor suppressor nucleic acid and/or polypeptide can
inhibit metastatic cells. Such inhibition can take the form of
inhibition of the formation, growth, migration, or reproduction of
metastatic cells. In one embodiment, the inhibition can be
characterized by the inhibition (e.g., reduction and/or
elimination) in the appearance of neoplasms remote from the primary
tumor. This invention thus provides methods for treating
(mitigating or eliminating) the progression of metastatic disease.
The methods involve contacting metastatic cells with a tumor
suppressor nucleic acid and/or polypeptide. In a particularly
preferred embodiment, this method may involve contacting the cells
in a surgical wound site (e.g., after removal (debulking) of a
tumor mass) with a tumor suppressor nucleic acid and/or tumor
suppressor polypeptide in combination with adjunctive anti-cancer
agent. The cells can additionally be contacted with an adjunctive
anti-cancer agent as described herein.
[0076] In still another embodiment, this invention provides for
advantageous treatment regimens utilizing tumor suppressor genes
and gene products. In part, these treatment regimens are based on
the surprising discovery that tumor suppressor nucleic acids and/or
polypeptides are more effective in inhibiting cell or tumor growth
when delivered in multiple administrations rather than in a single
bolus.
[0077] The order in which the tumor suppressor and adjunctive
anti-cancer agents are administered is not critical to the
invention. Thus the composition(s) can be administered
simultaneously or sequentially. For instance, in one embodiment,
pretreatment of a cell with at least one adjunctive anti-cancer
agent (alone or in combination with a chemotherapeutic agent)
increases the efficacy of a subsequently administered tumor
suppressor nucleic acid and/or polypeptide. In one embodiment, the
chemotherapeutic agent is administered before the adjunctive
anti-cancer agent and the tumor suppressor nucleic acid and/or
polypeptide. In another embodiment, the adjunctive anti-cancer
agent (alone or in combination with a chemotherapeutic agent) is
administered simultaneously with the tumor suppressor nucleic acid
and/or polypeptide. In a further embodiment, the tumor suppressor
nucleic acid and/or polypeptide is administered after the tumor
suppressor nucleic acid and/or polypeptide.
[0078] The anti-tumor effect of administering the composition and
methods of the invention also includes an anti-tumor, non-specific
effect, the so-called "bystander effect," (see, e.g., Zhang (1996)
Cancer Metastasis Rev. 15:385-401 and Okada (1996) Gene Ther.
3:957-96). Furthermore, the immune system can also be manipulated
to selectively accentuate (or depress) the humoral or the cellular
arm of the immune system, i.e., modulate the B cell and/or T cell
(e.g., a cytotoxic lymphocyte (CTL) or tumor infiltrating
lymphocyte (TIL)) response. For example, an increase in TILs is
observed upon administration of a p53-expressing adenovirus to
humans. Specifically, an increase in TILs (phenotypically T helper
cells, CD3.sup.+ and CD4.sup.+) is observed upon intra-hepatic
arterial administration of a p53-expressing adenovirus for the
treatment of metastatic hepatic carcinoma, as described in detail
below.
[0079] It is recognized that the methods of this invention are not
restricted to the use of a single adjunctive anti-cancer agent or
even the use of a single chemotherapeutic. Thus this invention
provides for methods of inhibiting diseased mammalian cells lacking
an endogenous tumor suppressor protein, or a tumor comprising such
cells, by contacting the cells or tumor with a tumor suppressor
nucleic acid and one or more adjunctive anti-cancer agent as
described herein.
[0080] I. Adjunctive Anti-Cancer Agents
[0081] A) Microtubule Affecting Agents
[0082] As explained above, in one embodiment, this invention
provides methods of inhibiting diseased cells lacking an endogenous
tumor suppressor protein by contacting the cells with a tumor
suppressor protein or tumor suppressor nucleic acid and an
adjunctive anti-cancer agent such as a microtubule affecting agent
(e.g., paclitaxel, a paclitaxel derivative or a paclitaxel-like
compound). As used herein, a microtubule affecting agent is a
compound that interferes with cellular mitosis, i.e., having an
anti-mitotic effect, by affecting microtubule formation and/or
action. Such agents can be, for instance, microtubule stabilizing
agents or agents which disrupt microtubule formation.
[0083] Microtubule affecting agents useful in the invention are
well known to those of skill in the art and include, but are not
limited to allocolchicine (NSC 406042), Halichondrin B (NSC
609395), colchicine (NSC 757), colchicine derivatives (e.g., NSC
33410), dolastatin 10 (NSC 376128), maytansine (NSC 153858),
rhizoxin (NSC 332598), paclitaxel (Taxol.RTM., NSC 125973),
Taxol.RTM. derivatives (e.g., NSC 608832), thiocolchicine (NSC
361792), trityl cysteine (NSC 83265), vinblastine sulfate (NSC
49842), vincristine sulfate (NSC 67574), epothilone A, epothilone,
and discodermolide (see Service, (1996) Science, 274: 2009)
estramustine, nocodazole, MAP4, and the like. Examples of such
agents are also described in the scientific and patent literature,
see, e.g., Bulinski (1997) J. Cell Sci. 110:3055-3064; Panda (1997)
Proc. Natl. Acad. Sci. USA 94:10560-10564; Muhlradt (1997) Cancer
Res. 57:3344-3346; Nicolaou (1997) Nature 387:268-272; Vasquez
(1997) Mol. Biol. Cell. 8:973-985; Panda (1996) J. Biol. Chem.
271:29807-29812.
[0084] Particularly preferred agents are compounds with
paclitaxel-like activity. These include, but are not limited to
paclitaxel and paclitaxel derivatives (paclitaxel-like compounds)
and analogues. Paclitaxel and its derivatives are available
commercially. In addition, methods of making paclitaxel and
paclitaxel derivatives and analogues are well known to those of
skill in the art (see, e.g., U.S. Pat. Nos. 5,569,729; 5,565,478;
5,530,020; 5,527,924; 5,508,447; 5,489,589; 5,488,116; 5,484,809;
5,478,854; 5,478,736; 5,475,120; 5,468,769; 5,461,169; 5,440,057;
5,422,364; 5,411,984; 5,405,972; and 5,296,506).
[0085] Additional microtubule affecting agents can be assessed
using one of many such assays known in the art, e.g., a
semiautomated assay which measures the tubulin-polymerizing
activity of paclitaxel analogs in combination with a cellular assay
to measure the potential of these compounds to block cells in
mitosis (see Lopes (1997) Cancer Chemother. Pharmacol.
41:3747).
[0086] Generally, activity of a test compound is determined by
contacting a cell with that compound and determining whether or not
the cell cycle is disrupted, in particular, through the inhibition
of a mitotic event. Such inhibition may be mediated by disruption
of the mitotic apparatus, e.g., disruption of normal spindle
formation. Cells in which mitosis is interrupted may be
characterized by altered morphology (e.g., microtubule compaction,
increased chromosome number, etc.).
[0087] In a preferred embodiment, compounds with possible tubulin
polymerization activity are screened in vitro. In a preferred
embodiment, the compounds are screened against cultured WR21 cells
(derived from line 69-2 wap-ras mice) for inhibition of
proliferation and/or for altered cellular morphology, in particular
for microtubule compaction. In vivo screening of positive-testing
compounds can then be performed using nude mice bearing the WR21
tumor cells. Detailed protocols for this screening method are
described by Porter (1995) Lab. Anim. Sci., 45(2):145-150.
[0088] Other methods of screening compounds for desired activity
are well known to those of skill in the art. Typically such assays
involve assays for inhibition of microtubule assembly and/or
disassembly. Assays for microtubule assembly are described, for
example, by Gaskin et al. (1974) J. Molec. Biol., 89: 737-758. U.S.
Pat. No. 5,569,720 also provides in vitro and in vivo assays for
compounds with paclitaxel-like activity.
[0089] B) Polyprenyl-Protein Transferase Inhibitors
[0090] In still another embodiment, this invention provides for the
combined use of tumor suppressor nucleic acids and/or polypeptides
and polyprenyl-protein transferase inhibitors. Particularly
preferred polyprenyl-protein transferase inhibitors include, but
are not limited to farnesyl-protein transferase (FPT) inhibitors,
geranylgeranyl-protein transferase inhibitors, and other
monoterpene protein transferases. Examples of compounds that are
polyprenyl-protein transferase inhibitors are well known in the
scientific and patent literature, see, e.g., Zhang (1997) J. Biol.
Chem. 272:10232-10239; Njoroge (1997) J. Med. Chem. 40:4290-4301;
Mallams (1997) Bioorg. Med. Chem. 5:93-99.
[0091] Exemplary compounds that are farnesyl-protein transferase
inhibitors are given below:
[0092] The FPT inhibitor, designated "FPT39," as described in
International Application WO 97/23478, filed Dec. 19, 1996, where
FPT39 is designated compound "39.0," see pg 95 of WO 97/23478.
1
[0093] As described infra, when FPT39 is used in combination
therapy with a p53 expressing adenovirus of the invention against
prostate tumor cells and mammary tumor cells, the combination was
more effective at killing tumor cells than either agent alone.
[0094] Oligopeptides (mostly tetrapeptides, but also pentapeptides
including the formula Cys-Xaa1-Xaa2-Xaa3: EPA 461,489; EPA 520,823;
EPA 528,486; and WO 95/11917).
[0095] Peptido-mimetic compounds, especially Cys-Xaa-Xaa-Xaa
mimetics: EPA 535,730, EPA 535,731; EPA 618,221; WO 94/09766; WO
94/10138; WO 94/07966; U.S. Pat. No. 5,326,773, U.S. Pat. No.
5,340,828, U.S. Pat. No. 5,420,245; WO 95/20396; U.S. Pat. No.
5,439,918; and WO 95/20396.
[0096] Farnesylated peptide mimetic compounds--specifically
farnesylated Cys-Xaa-Xaa-Xaa mimetic: GB-A2.276,618.
[0097] Other peptido-mimetic compounds: U.S. Pat. No. 5,352,705, WO
94/00419; WO 95100497; WO 95/09000; WO 95/09001; WO 91/12612; WO
95/25086; EPA 675,112, and FR-A 2,718,149.
[0098] Fused-ring tricyclic benzocycloheptapyridines: WO 95/10514;
WO 95/10515; WO 95/10516; WO 96/30363; WO 96/30018; WO 96/30017; WO
96/30362; WO 96/31111; WO 96/31478; WO 96/31477; WO 9631505;
International Patent Application No. PCT/US96/19603, WO 97/23478;
U.S. application Ser. No. 08/728,104, U.S. application Ser. No.
08/712,989, U.S. application Ser. No. 08/713,326, U.S. application
Ser. No. 08/713,908, U.S. application Ser. No. 08/713,705, U.S.
application Ser. No. 08/713,703; U.S. application Ser. No.
08/710,225, U.S. application Ser. No. 08/711,925, U.S. application
Ser. No. 08/712,924; U.S. application Ser. No. 08/713,323; and U.S.
application Ser. No. 08/713,297.
[0099] Farnesyl derivatives: EPA 534,546; WO 94/19357; WO 95/08546,
EPA 537,007; and WO 95/13059.
[0100] Natural products and derivatives: WO 94/18157; U.S. Pat. No.
5,430,055; GB-A 2,261,373, GB-A 2,261,374, GB-A 2,261,375; U.S.
Pat. No. 5,420,334, U.S. Pat. No. 5,436,263.
[0101] Other compounds: WO 94/26723; WO 95/08542; U.S. Pat. No.
5,420,157; WO 95/21815; and WO 96/31501.
[0102] C) Anti-Angiogenic Compounds.
[0103] The tumor suppressor proteins or nucleic acids of this
invention can also be administered in conjunction with
antiangiogenic compounds. Preferred antiangiogenic compositions
inhibit the formation or proliferation of blood vessels, more
preferably the formation and/or proliferation of blood vessels to
tumors.
[0104] Suitable antiangiogenic compositions include, but are not
limited to Galardin (GM6001, Glycomed, Inc., Alameda, Calif.),
endothelial response inhibitors (e.g., agents such as interferon
alpha, TNP470, and vascular endothelial growth factor inhibitors),
agents that prompt the breakdown of the cellular matrix (e.g.,
Vitaxin (human LM-609 antibody, Ixsys Co., San Diego, Calif.;
Metastat, CollaGenex, Newtown, Pa.; and Marimastat BB2516, British
Biotech), and agents that act directly on vessel growth (e.g.,
CM-101, which is derived from exotoxin of Group A Streptococcus
antigen and binds to new blood vessels inducing an intense host
inflammatory response; and Thalidomide).
[0105] Several kinds of steroids have also been noted to exert
antiangiogenic activity. In particular, several reports have
indicated that medroxyprogesterone acetate (MPA), a synthetic
progesterone, potently inhibited neovascularization in the rabbit
corneal assay (Oikawa (1988) Cancer Lett. 43: 85). A pro-drug of
5FU, 5'-deoxy-5-fluorouridine (5'DFUR), might be also characterized
as an antiangiogenic compound, because 5'DFUR is converted to 5-FU
by the thymidine phosphorylase activity of PD-ECGF/TP. 5'DFUR might
be selectively active for PD-ECGF/TP positive tumor cells with high
angiogenesis potential. Recent clinical investigations in showed
that 5'DFUR is likely to be effective for PD-ECGF/TP-positive
tumors. It was showed that a dramatic enhancement of antitumor
effect of 5'DFUR appeared in PD-ECGF/TP transfected cells compared
with untransfected wild-type cells (Haraguchi (1993) Cancer Res.
53: 5680-5682). In addition, combined 5'DFUR+MPA compounds are also
effective antiangiogenics (Yayoi (1994) Int J Oncol. 5: 27-32). The
combination of the 5'DFUR+MPA might be categorized as a combination
of two angiogenesis inhibitors with different spectrums, an
endothelial growth factor inhibitor and a protease inhibitor.
Furthermore, in in-vivo experiments using DMBA-induced rat mammary
carcinomas, 5'DFUR exhibited a combination effect with AGM-1470
(Yamamoto (1995) Oncol Reports 2:793-796).
[0106] Another group of antiangiogenic compounds for use in this
invention include polysaccharides capable of interfering with the
function of heparin-binding growth factors that promote
angiogenesis (e.g., pentosan polysulfate).
[0107] Other modulators of angiogenesis include platelet factor IV,
and AGM 1470. Still others are derived from natural sources
collagenase inhibitor, vitamin D3-analogues, fumigallin, herbimycin
A, and isoflavones.
[0108] D) Endocrine Therapy.
[0109] Endocrine therapy, which is already established and a
representative cytostatic treatment, can lead hormone-dependent
cells to be quiescent and can reduce tumor cell number in-vivo and
can inhibit tumor growth in patients with hormone-dependent tumors.
Such therapies are expected to augment the effect of tumor
suppressors in the treatment of hyperproliferative cells. Thus, in
another embodiment, this invention provides, e.g., for the combined
use of a tumor suppressor nucleic acid and/or polypeptide and an
anti-estrogen, anti-androgen, or anti-progesterone. Endocrine
therapeutics are well known to those of skill in the art are
include, but are not limited to tamoxifen, toremifene (see, e.g.,
U.S. Pat. No. 4,696,949), flutamide, megace, and lupron, see, also,
e.g., WO 91/00732, WO 93/10741, WO 96/26201, and Gauthier et al. J.
Med. Chem. 40: 2117-2122 (1997).
[0110] E) Delivery of Adjunctive Anti-Cancer Agents: Pharmaceutical
Compositions
[0111] Pharmaceutical Compositions
[0112] The adjunctive anti-cancer agents used in the methods of the
invention are typically combined with a pharmaceutically acceptable
carrier (excipient) to form a pharmacological composition. The
pharmaceutical composition of the invention can comprise one or
more adjunctive anti-cancer agents with or without a tumor
suppressor gene or polypeptide, e.g., p53 or RB.
[0113] Pharmaceutically acceptable carriers can contain a
physiologically acceptable compound that acts, e.g., to stabilize
the composition or to increase or decrease the absorption of the
agent and/or pharmaceutical composition. Physiologically acceptable
compounds can include, for example, carbohydrates, such as glucose,
sucrose, or dextrans, antioxidants, such as ascorbic acid or
glutathione, chelating agents, low molecular weight proteins,
compositions that reduce the clearance or hydrolysis of the
adjunctive anti-cancer agents, or excipients or other stabilizers
and/or buffers. Detergents can also used to stabilize the
composition or to increase or decrease the absorption of the
pharmaceutical composition (see infra for exemplary
detergents).
[0114] Other physiologically acceptable compounds include wetting
agents, emulsifying agents, dispersing agents or preservatives
which are particularly useful for preventing the growth or action
of microorganisms. Various preservatives are well known and
include, for example, phenol and ascorbic acid. One skilled in the
art would appreciate that the choice of a pharmaceutically
acceptable carrier, including a physiologically acceptable compound
depends, for example, on the route of administration of the
adjunctive anti-cancer agent and on the particular physio-chemical
characteristics of the adjunctive anti-cancer agent.
[0115] The compositions for administration will commonly comprise a
solution of the adjunctive anti-cancer agent dissolved in a
pharmaceutically acceptable carrier, preferably an aqueous carrier
for water-soluble adjunctive anti-cancer agents. A variety of
carriers can be used, e.g., buffered saline and the like. These
solutions are sterile and generally free of undesirable matter.
These compositions may be sterilized by conventional, well known
sterilization techniques. The compositions may contain
pharmaceutically acceptable auxiliary substances as required to
approximate physiological conditions such as pH adjusting and
buffering agents, toxicity adjusting agents and the like, for
example, sodium acetate, sodium chloride, potassium chloride,
calcium chloride, sodium lactate and the like. The concentration of
adjunctive anti-cancer agent in these formulations can vary widely,
and will be selected primarily based on fluid volumes, viscosities,
body weight and the like in accordance with the particular mode of
administration selected and the patient's needs.
[0116] Routes of Delivery
[0117] The adjunctive anti-cancer agents used in the methods of the
invention are useful for and can be delivered alone or as
pharmaceutical compositions (with or without a tumor suppressor,
e.g., p53) by any means known in the art, e.g., systemically,
regionally, or locally; by intraarterial, intratumoral, intravenous
(IV), parenteral, intra-pleural cavity, topical, oral, or local
administration, as subcutaneous, intra-tracheal (e.g., by aerosol)
or transmucosal (e.g., buccal, bladder, vaginal, uterine, rectal,
nasal mucosa), intra-tumoral (e.g., transdermal application or
local injection). Particularly preferred modes of administration
include intra-arterial injections, especially when it is desired to
have a "regional effect," e.g., to focus on a specific organ (e.g.,
brain, liver, spleen, lungs). For example, intra-hepatic artery
injection is preferred if the anti-tumor regional effect is desired
in the liver; or, intra-carotid artery injection, where it is
desired to deliver a composition to the brain, (e.g., for treatment
of brain tumors) a carotid artery or an artery of the carotid
system of arteries (e.g., occipital artery, auricular artery,
temporal artery, cerebral artery, maxillary artery, etc.).
[0118] Paclitaxel and certain paclitaxel derivatives are only
marginally soluble in aqueous solutions. In a preferred embodiment,
these compositions are either delivered directly to the tumor
locale (e.g. by injection, canalization, or direct application
during a surgical procedure) or they are solubilized in an
acceptable excipient. Methods of administering paclitaxel and its
derivatives are well known to those of skill in the art (see, e.g.,
U.S. Pat. Nos. 5,583,153, 5,565,478, 5,496,804, 45,484,809. Other
paclitaxel derivatives are water soluble analogues and/or prodrugs
(see, U.S. Pat. Nos. 5,411,984 and 5,422,364) and are conveniently
administered by any of a variety of methods as described above.
[0119] The pharmaceutical compositions of this invention are
particularly useful for topical administration e.g., in surgical
wounds to treat incipient tumors, neoplastic and metastatic cells
and their precursors In another embodiment, the compositions are
useful for parenteral administration, such as intravenous
administration or administration into a body cavity or lumen of an
organ.
[0120] Treatment Regimens
[0121] The pharmaceutical compositions can be administered in a
variety of unit dosage forms depending upon the method of
administration. For example, unit dosage forms suitable for oral
administration include powder, tablets, pills, capsules and
lozenges. It is recognized that the adjunctive anti-cancer
compounds (e.g., paclitaxel and related compounds described of,
when administered orally, must be protected from digestion. This is
typically accomplished either by complexing the adjunctive
anti-cancer agent with a composition to render it resistant to
acidic and enzymatic hydrolysis or by packaging the adjunctive
anti-cancer agent in an appropriately resistant carrier such as a
liposome. Means of protecting compounds from digestion are well
known in the art (see, e.g., U.S. Pat. No. 5,391,377 describing
lipid compositions for oral delivery of therapeutic agents).
[0122] Dosages for typical chemotherapeutics are well known to
those of skill in the art. Moreover, such dosages are typically
advisorial in nature and may be adjusted depending on the
particular therapeutic context, patient tolerance, etc. Thus, for
example, a typical pharmaceutical composition (e.g., paclitaxel)
dosage for intravenous (IV) administration would be about 135
mg/m.sup.2 administered over 1-24 hours (typically at 1, 3, or 6
hours, more preferably 3 hours) and more preferably repeated every
three weeks for 3 to 6 cycles. To decrease the frequency and
severity of hypersensitivity reactions, patients may also receive
about 20 mg of dexamethasone (Decadron, and others) orally about 12
hours and 6 hours before, and about 50 mg of diphenhydramine
(Benadryl.RTM., and others) plus about 300 mg of cimetidine
(Tagamet.RTM.) or 50 mg of rantidine (Zantac.RTM.) IV 30 to 60
minutes before treatment with paclitaxel. Considerably higher
dosages (e.g., ranging up to up to about 350 mg/m.sup.2 per day may
be used, particularly when the drug is administered to a secluded
site and not into the blood stream, such as into a body cavity or
into a lumen of an organ. Substantially higher dosages are possible
by any selected route, for example, topical administration. Actual
methods for preparing parenterally administrable compositions will
be known or apparent to those skilled in the art and are described
in more detail in such publications as Remington's Pharmaceutical
Science, 15th ed., Mack Publishing Company, Easton, Pa. (1980) and
U.S. Pat. Nos. 5,583,153, 5,565,478, 5,496,804, and 5,484,809.
Typical doses, e.g., for intraperitoneal administration, will be
20-150 mg/m.sup.2 weekly, or about 250 mg/m.sup.2 every 3
weeks.
[0123] The compositions containing the adjunctive anti-cancer
agents can be administered for therapeutic treatments. In
therapeutic applications, compositions are administered to a
patient suffering from a disease characterized by
hyperproliferation of one or more cell types in an amount
sufficient to cure or at least partially arrest the disease and/or
its complications. An amount adequate to accomplish this is defined
as a "therapeutically effective dose." Amounts effective for this
use will depend upon the severity of the disease and the general
state of the patient's health.
[0124] Single or multiple administrations of the compositions may
be administered depending on the dosage and frequency as required
and tolerated by the patient. In any event, the composition should
provide a sufficient quantity of the adjunctive anti-cancer agents
of this invention to effectively treat the patient.
[0125] II. Tumor Suppressor Genes and Gene Products
[0126] A) Preferred Known Tumor Suppressors.
[0127] As explained above, in one embodiment, this invention
provides methods of inhibiting the growth and/or proliferation of
cells by contacting the cells with a tumor suppressor nucleic acid
and an adjunctive anti-cancer agent (e.g., paclitaxel, a paclitaxel
derivative or a paclitaxel-like compound).
[0128] Tumor suppressor genes are well known to those of skill in
the art and include, but are not limited to RB, p53, APC, FHIT
(see, e.g., Siprashvili (1997) Proc. Natl. Acad. Sci. USA
94:13771-13776), BRACA1 and BRCA2, VHL, WT, DCC, FAP, NF, MEN,
E-cadherin, nm23, MMACI, and PTC. The RB or retinoblastoma gene is
the prototypical tumor suppressor and has been well characterized
(see, e.g., Bookstein (1990) Science 247: 712-715, Benedict (1980)
Cancer Invest., 8: 535-540, Riley (1990) Ann. Rev. Cell Biol.
10-1-29, and Wienberg (1992) Science 254: 1138-1146. Perhaps the
best characterized tumor suppressor is p53 which has been
implicated in many neoblastomas as well as in the genetic
predisposition to the development of diverse tumors in families
with Li-Fraumeni syndrome (see, e.g., Wills (1994) Hum. Gene
Therap. 5:1079-1088, U.S. Pat. No. 5,532,220, WO 95/289048, and
Harris (1996) J. Nat. Canc. Inst. 88(20): 1442) which describe the
cloning expression and use of p53 in gene therapy). Other tumor
suppressors include WT (i.e., WT1 at 11p13) gene characteristic of
Wilms' tumor (see Call et al. (1990) Cell, 60: 60: 509-520, Gessler
(1990) Nature 343: 774-778, and Rose et al. (1990) Cell, 60:
495-508). The tumor suppressor gene called FHIT, for Fragile
Histidine Triad, was found in a region on chromosome 3 (3p14.2,
also reported at 3p21) that is known to be particularly prone to
translocations, breaks, and gaps is believed to lead to esophageal,
stomach and colon cancers (see, e.g., Ohta et al. (1996) Cell, 84:
587-597, GenBank Accession No: U469227). The tumor suppressor genes
DCC (18q21) and FAP are associated with colon carcinoma (see, e.g.,
Hedrick et al. (1994) Genes Dev., 8(10): 1174-1183, GenBank
Accession No: X76132 for DCC, and Wienberg (1992) Science, 254:
1138-1146 for FAP). The NF tumor suppressors (NF1 at 17q11 and NF2
at 22q12) are associated with neurological tumors (e.g.,
neurofribromatosis for NF1 see, e.g., Caivthon et al. (1990) Cell,
62: 193-201, Viskochil et al. (1990) Cell, 62: 187-192, Wallace et
al. (1990) Science, 249: 181-186, and Xug et al. (1990) Cell, 62:
599-608; and Meningioma and schwannoma for NF2). The MEN tumor
suppressor is associated with tumors of the multiple endocrine
neoplasia syndrome (see, e.g., Wienberg Science, 254: 1138-1146,
and Marshall (1991) Cell, 64: 313-326). The VHL tumor suppressor is
associated with von Hippel-Landau disease (Latif (1993) Science
260: 1317-1320, GenBank Accession No: L15409). The widely
publicized BRCA1 and BRCA2 genes are associated with breast cancer
(see, e.g., Skolnick (1994) Science, 266: 66-71, GenBank Accession
No: U14680 for BRCA1, and Teng (1996) Nature Genet. 13:241-244,
GenBank Accession No: U43746)). In addition, the E-cadherin gene is
associated with the invasive phenotype of prostate cancer (Umbas
(1992) Cancer Res. 52: 5104-5109, Bussemakers (1992) Cancer Res.
52: 2916-2999, GenBank Accession No: 272397). The NM23 gene is
associated with tumor metastasis (Dooley (1994) Hum. Genet., 93(1):
63-66, GenBank Accession No: X75598). Other tumor suppressors
include DPC4 (identified at 18q21) associated with pancreatic
cancer, hMLH1 (3p) and hMSH2 (2p) associated with colon cancers,
and CDKN2 (p16) and (9p) associated with melanoma, pancreatic and
esophageal cancers. Finally, the human PTC gene (a homologue of the
drosophila patched (ptc) gene) is associated with nevoid basal cell
carcinoma syndrome (NBCCS) and with somatic basal cell carcinomas
(see, e.g., see Hahn et al. (1996) Cell, 85: 841-851). This list of
tumor suppressor genes is neither exhaustive nor intended to be
limiting and is meant simply to illustrate the wide variety of
known tumor suppressors.
[0129] B) Identification and Screening of Previously Unknown Tumor
Suppressors.
[0130] Methods of identifying or assaying for tumor suppressor
genes are well known to those of skill in the art. Typically
hyperproliferative cells are screened for gene loss of which, or
mutation of which, is associated (correlated) with the
hyperproliferative state. The most stringent test for a gene to
qualify as a tumor suppressor gene (TSG) is its ability to suppress
the tumorigenic phenotype of a tumor or of cells derived from a
tumor. The tumor suppressor nucleic acid is preferably introduced
into tumor cells as a cloned cDNA in an appropriate expression
vector, or an individual chromosome harboring a candidate tumor
suppressor gene is introduced into tumor cells by microcell
transfer technique. Alternatively, the tumor suppressor gene
product (e.g., a tumor suppressor polypeptide) is introduced into
the cell(s) and the proliferation rate of the cells is measured
(e.g., by counting cells or measuring tumor volume, etc.). Complete
or partial inhibition of proliferation (e.g., decrease of
proliferation rate), contact inhibition, loss of invasive
phenotype, cell differentiation, and apoptosis, are all indicators
of suppression of the tumorigenic phenotype (reduced susceptibility
to the neoplastic state).
[0131] Methods of screening tumors to identify altered or
underexpressed nucleic acids are well known to those of skill in
the art. Such methods include, but are not limited to subtractive
hybridization (see, e.g., Hampson (1992) Nucleic Acids Res.
20:2899), comparative genomic hybridization ((CGH), see, e.g., WO
93/18186, Kallioniemi (1992) Science, 258: 818), and expression
monitoring using high density arrays of nucleic acid probes (see,
e.g., Lockhart (1996) Nature Biotechnology, 14(13): 1675-1680).
[0132] C) Preparation of p53 and Other Tumor Suppressors.
[0133] As indicated above, this invention involves contacting a
cell, e.g., in vitro, in physiological solution (e.g., blood), in a
tissue organ, or organism with a tumor suppressor nucleic acid or a
tumor suppressor gene product such as a polypeptide. The tumor
suppressor nucleic acid or polypeptide can be a nucleic acid or
polypeptide of any known tumor suppressor including, but not
limited to RB, p53, h-NUC (Chen (1995) supra), APC, FHIT, BRACA1,
BRCA2, VHL, WT, DCC, FAP, NF, MEN, E-cadherin, nm23, MMACI, and PTC
as described above. In a preferred embodiment, the tumor suppressor
is an RB nucleic acid or polypeptide or a p53 nucleic acid or
polypeptide or active fragment(s) thereof.
[0134] In a most preferred embodiment, the p53 or RB tumor
suppressor nucleic acid is present in an expression cassette under
control of a promoter that expresses the tumor suppressor gene or
cDNA when it is located in the target (e.g., tumor) cell. Methods
of constructing expression cassettes and/or vectors encoding tumor
suppressor genes are well known to those of skill in the art as
described below.
[0135] 1. Preparation of Tumor Suppressor Nucleic Acids.
[0136] DNA encoding the tumor suppressor proteins or protein
subsequences of this invention may be prepared by any suitable
method including, for example, cloning and restriction of
appropriate sequences or direct chemical synthesis (e.g., using
existing sequence information as indicated above) by methods such
as the phosphotriester method of Narang (1979) Meth. Enymol. 68:
90-99; the phosphodiester method of Brown et al., Meth. Enzymol.
68: 109-151 (1979); the diethylphosphoramidite method of Beaucage
et al., Tetra. Lett., 22: 1859-1862 (1981); and the solid support
method of U.S. Pat. No. 4,458,066.
[0137] Chemical synthesis produces a single stranded
oligonucleotide. This may be converted into double stranded DNA by
hybridization with a complementary sequence, or by polymerization
with a DNA polymerase using the single strand as a template. One of
skill would recognize that while chemical synthesis of DNA is
limited to sequences of about 100 bases, longer sequences may be
obtained by the ligation of shorter sequences.
[0138] Alternatively, subsequences may be cloned and the
appropriate subsequences cleaved using appropriate restriction
enzymes. The fragments may then be ligated to produce the desired
DNA sequence.
[0139] In one embodiment, tumor suppressor nucleic acids of this
invention may be cloned using DNA amplification methods such as
polymerase chain reaction (PCR). Thus, for example, the nucleic
acid sequence or subsequence is PCR amplified, using a sense primer
containing one restriction site (e.g., NdeI) and an antisense
primer containing another restriction site (e.g., HindIII). This
will produce a nucleic acid encoding the desired tumor suppressor
sequence or subsequence and having terminal restriction sites. This
nucleic acid can then be easily ligated into a vector containing a
nucleic acid encoding the second molecule and having the
appropriate corresponding restriction sites. Suitable PCR primers
can be determined by one of skill in the art using the published
sequence information for any particular known tumor suppressor
gene, cDNA, or protein. Appropriate restriction sites can also be
added to the nucleic acid encoding the tumor suppressor protein or
protein subsequence by site-directed mutagenesis. The plasmid
containing the tumor suppressor sequence or subsequence is cleaved
with the appropriate restriction endonuclease and then ligated into
the vector encoding the second molecule according to standard
methods.
[0140] As indicated above, the nucleic acid sequences of many tumor
suppressor genes are known. Thus, for example, the nucleic acid
sequence of p53 is found in Lamb et al. (1986) Mol. Cell Biol. 6:
1379-1385, GenBank Accession No: M13111). Similarly, the nucleic
acid sequence of RB is described by Lee et al. (1987) Nature, 329:
642-645 (GenBank Accession No: M28419). The nucleic acid sequences
of other tumor suppressors are available as indicated above in
Section II(a). Using the available sequence information one of
ordinary skill in the art can clone the tumor suppressor genes into
vectors suitable for practice in this invention.
[0141] The p53 and RB tumor suppressors are particularly preferred
for use in the methods of this invention. Methods of cloning p53
and RB into vectors suitable for expression of the respective tumor
suppressor proteins or for gene therapy applications are well known
to those of skill in the art. Thus, for example, the cloning and
use of p53 is described in detail by Wills (1994) supra; in U.S.
Pat. No. 5,532,220, in copending U.S. Ser. No. 08/328,673 filed on
Oct. 25, 1994, and in WO 95/11984. Typically the expression
cassette is constructed with the tumor suppressor cDNA operably
linked to a promoter, more preferably to a strong promoter (e.g.,
the Ad2 major late promoter (Ad2 MLP), or the human cytomegalovirus
immediate early gene promoter (CMV)). In a particularly preferred
embodiment, the promoter is followed by the tripartite leader cDNA
and the tumor suppressor cDNA is followed by a polyadenylation site
(e.g., the E1b polyadenylation site) (see, e.g., copending U.S.
Ser. No. 08/328,673, WO 95/11984 and Wills (1994) supra). It will
be appreciated that various tissue-specific promoters are also
suitable. Thus, for example, a tyrosinase promoter can be used to
target expression to melanomas (see, e.g., Siders (1996) Cancer
Res. 56:5638-5646). In a particularly preferred embodiment, the
tumor suppressor cDNA is expressed in a vector suitable for gene
therapy as described below.
[0142] 2. Preparation of Tumor Suppressor Protein.
[0143] a) De Novo Chemical Synthesis.
[0144] Using known sequences of tumor suppressor polypeptides, the
tumor suppressor proteins or subsequences thereof may be
synthesized using standard chemical peptide synthesis techniques.
Where the desired subsequences are relatively short (e.g., when a
particular antigenic determinant is desired) the molecule may be
synthesized as a single contiguous polypeptide. Where larger
molecules are desired, subsequences can be synthesized separately
(in one or more units) and then fused by condensation of the amino
terminus of one molecule with the carboxyl terminus of the other
molecule thereby forming a peptide bond.
[0145] Solid phase synthesis in which the C-terminal amino acid of
the sequence is attached to an insoluble support followed by
sequential addition of the remaining amino acids in the sequence is
the preferred method for the chemical synthesis of the polypeptides
of this invention. Techniques for solid phase synthesis are
described by Barany and Merrifield, Solid-Phase Peptide Synthesis;
pp. 3-284 in The Peptides: Analysis, Synthesis, Biology. Vol. 2:
Special Methods in Peptide Synthesis, Part a., Merrifield, et al.
J. Am. Chem. Soc., 85: 2149-2156 (1963), and Stewart et al., Solid
Phase Peptide Synthesis, 2nd ed. Pierce Chem. Co., Rockford, Ill.
(1984).
[0146] b) Recombinant Expression.
[0147] In a preferred embodiment, the tumor suppressor proteins or
subsequences thereof, are synthesized using recombinant DNA
methodology. Generally this involves creating a DNA sequence that
encodes the fusion protein, placing the DNA in an expression
cassette under the control of a particular promoter, expressing the
protein in a host, isolating the expressed protein and, if
required, renaturing the protein.
[0148] Methods of cloning the tumor suppressor nucleic acids into a
particular vector are described above. The nucleic acid sequences
encoding tumor suppressor proteins or protein subsequences may then
be expressed in a variety of host cells, including E. coli, other
bacterial hosts, yeast, and various higher eukaryotic cells such as
the COS, CHO and HeLa cells lines and myeloma cell lines. As the
tumor suppressor proteins are typically found in eukaryotes, a
eukaryote host is preferred. The recombinant protein gene will be
operably linked to appropriate expression control sequences for
each host. For E. coli this includes a promoter such as the T7,
trp, or lambda promoters, a ribosome binding site and preferably a
transcription termination signal. For eukaryotic cells, the control
sequences will include a promoter and preferably an enhancer
derived from immunoglobulin genes, SV40, cytomegalovirus, etc., and
a polyadenylation sequence, and may include splice donor and
acceptor sequences.
[0149] The plasmids of the invention can be transferred into the
chosen host cell by well-known methods such as calcium chloride
transformation for E. coli and calcium phosphate treatment or
electroporation for mammalian cells. Cells transformed by the
plasmids can be selected by resistance to antibiotics conferred by
genes contained on the plasmids, such as the amp, gpt, neo and hyg
genes.
[0150] Once expressed, the recombinant tumor suppressor proteins
can be purified according to standard procedures of the art,
including ammonium sulfate precipitation, affinity columns, column
chromatography, gel electrophoresis and the like (see, generally,
R. Scopes, Protein Purification, Springer-Verlag, N.Y. (1982),
Deutscher, Methods in Enzymology Vol. 182: Guide to Protein
Purification., Academic Press, Inc. N.Y. (1990)). Substantially
pure compositions of at least about 90 to 95% homogeneity are
preferred, and 98 to 99% or more homogeneity are most preferred.
Once purified, partially or to homogeneity as desired, the
polypeptides may then be used (e.g., as immunogens for antibody
production).
[0151] One of skill in the art would recognize that after chemical
synthesis, biological expression, or purification, the tumor
suppressor protein(s) may possess a conformation substantially
different than the native conformations of the constituent
polypeptides. In this case, it may be necessary to denature and
reduce the polypeptide and then to cause the polypeptide to re-fold
into the preferred conformation. Methods of reducing and denaturing
proteins and inducing re-folding are well known to those of skill
in the art (See, Debinski (1993) J. Biol. Chem. 268: 14065-14070;
Kreitman (1993) Bioconjug. Chem. 4: 581-585; and Buchner (1992)
Anal. Biochem. 205: 263-270). Debinski (1993) supra, for example,
describes the denaturation and reduction of inclusion body proteins
in guanidine-DTE. The protein is then refolded in a redox buffer
containing oxidized glutathione and L-arginine.
[0152] One of skill will appreciate that many conservative
variations of the nucleic acid and polypeptide sequences described
herein yield functionally identical products. For example, due to
the degeneracy of the genetic code, "silent substitutions" (i.e.,
substitutions of a nucleic acid sequence which do not result in an
alteration in an encoded polypeptide) are an implied feature of
every nucleic acid sequence which encodes an amino acid. Similarly,
"conservative amino acid substitutions," in one or a few amino
acids in an amino acid sequence are substituted with different
amino acids with highly similar properties (see, the definitions
section, supra), are also readily identified as being highly
similar to a disclosed amino acid sequence, or to a disclosed
nucleic acid sequence which encodes an amino acid. Such
conservatively substituted variations of each explicitly described
sequence are a feature of the present invention.
[0153] One of skill would recognize that modifications can be made
to the tumor suppressor proteins without diminishing their
biological activity. Some modifications may be made to facilitate
the cloning, expression, or incorporation of the targeting molecule
into a fusion protein. Such modifications are well known to those
of skill in the art and include, for example, a methionine added at
the amino terminus to provide an initiation site, or additional
amino acids (e.g., poly His) placed on either terminus to create
conveniently located restriction sites or termination codons or
purification sequences.
[0154] Modifications to nucleic acids and polypeptides may be
evaluated by routine screening techniques in suitable assays for
the desired characteristic. For instance, changes in the
immunological character of a polypeptide can be detected by an
appropriate immunological assay. Modifications of other properties
such as nucleic acid hybridization to a target nucleic acid, redox
or thermal stability of a protein, hydrophobicity, susceptibility
to proteolysis, or the tendency to aggregate are all assayed
according to standard techniques.
[0155] D) Delivery of Tumor Suppressors to Target Cells.
[0156] The tumor suppressors used in the methods of this invention
can be introduced to the cells either as a protein or as a nucleic
acid. Where the tumor suppressor is provided as a protein, a tumor
suppressor gene expression product (e.g., a p53 or an RB
polypeptide or fragment thereof possessing tumor suppressor
activity) is delivered to the target cell using standard methods
for protein delivery (see discussion, below). Alternatively, where
the tumor suppressor is a tumor suppressor nucleic acid (e.g., a
gene, a cDNA, an mRNA, etc.) the nucleic acid is introduced into
the cell using conventional methods of delivering nucleic acids to
cells. These methods typically involve delivery methods of in vivo
or ex vivo gene therapy as described below. Particularly preferred
methods of delivering p53 or RB include lipid or liposome delivery
and/or the use of retroviral or adenoviral vectors.
[0157] 1. In Vivo Gene Therapy.
[0158] In a more preferred embodiment, the tumor suppressor nucleic
acids (e.g., cDNA(s) encoding the tumor suppressor protein) are
cloned into gene therapy vectors that are competent to transfect
cells (such as human or other mammalian cells) in vitro and/or in
vivo.
[0159] Several approaches for introducing nucleic acids into cells
in vivo, ex vivo and in vitro have been used. These include lipid
or liposome based gene delivery (WO 96/18372; WO 93/24640; Mannino
(1988) BioTechniques 6(7): 682-691; Rose, U.S. Pat. No. 5,279,833;
WO 91/06309; and Felgner (1987) Proc. Natl. Acad. Sci. USA 84:
7413-7414) and replication-defective retroviral vectors harboring a
therapeutic polynucleotide sequence as part of the retroviral
genome (see, e.g., Miller (1990) Mol. Cell. Biol. 10:4239 (1990);
Kolberg (1992) J. NIH Res. 4: 43, and Cornetta (1991) Hum. Gene
Ther. 2: 215).
[0160] For a review of gene therapy procedures, see, e.g., Zhang
(1996) Cancer Metastasis Rev. 15:385-401; Anderson, Science (1992)
256: 808-813; Nabel (1993) TIBTECH 11: 211-217; Mitani (1993)
TIBTECH 11: 162-166; Mulligan (1993) Science, 926-932; Dillon
(1993) TIBTECH 11: 167-175; Miller (1992) Nature 357: 455-460; Van
Brunt (1988) Biotechnology 6(10): 0.1149-1154; Vigne (1995)
Restorative Neurology and Neuroscience 8: 35-36; Kremer (1995)
British Medical Bulletin 51(1) 31-44; Haddada (1995) in Current
Topics in Microbiology and Immunology, Doerfler and Bohm (eds)
Springer-Verlag, Heidelberg Germany; and Yu (1994) Gene Therapy,
1:13-26.
[0161] The vectors useful in the practice of the present invention
are typically derived from viral genomes. Vectors which may be
employed include recombinantly modified enveloped or non-enveloped
DNA and RNA viruses, preferably selected from baculoviridiae,
parvoviridiae, picornoviridiae, herpesveridiae, poxyiridae,
adenoviridiae, or picornnaviridiae. Chimeric vectors may also be
employed which exploit advantageous ments of each of the parent
vector properties (See e.g., Feng (1997) Nature Biotechnology
15:866-870. Such viral genomes may be modified by recombinant DNA
techniques to include the tumor suppressor gene and may be
engineered to be replication deficient, conditionally replicating
or replication competent. In the preferred practice of the
invention, the vectors are replication deficient or conditionally
replicating. Preferred vectors are derived from the adenoviral,
adeno-associated viral and retroviral genomes. In the most
preferred practice of the invention, the vectors are replication
incompetent vectors derived from the human adenovirus genome.
[0162] Conditionally replicating viral vectors are used to achieve
selective expression in particular cell types while avoiding
untoward broad spectrum infection. Examples of conditionally
replicating vectors are described in Bischoff, et al. (1996)
Science 274:373-376; Pennisi, E. (1996) Science 274:342-343;
Russell, S. J. (1994) Eur. J. of Cancer 30A(8):1165-1171.
Additionally, the viral genome may be modified to include inducible
promoters which achieve replication or expression of the transgene
only under certain conditions. Examples of inducible promoters are
known in the scientific literature (See, e.g. Yoshida and Hamada
(1997) Biochem. Biophys. Res. Comm. 230:426-430; lida, et al.
(1996) J. Virol. 70(9):6054-6059; Hwang, et al. (1997) J. Virol
71(9):7128-7131; Lee, et al. (1997) Mol. Cell. Biol.
17(9):5097-5105; and Dreher, et al. (1997) J. Biol. Chem 272(46);
29364-29371. The transgene may also be under control of a tissue
specific promoter region allowing expression of the transgene only
in particular cell types.
[0163] Widely used retroviral vectors include those based upon
murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV),
Simian Immuno deficiency virus (SIV), human immuno deficiency virus
(HIV), and combinations thereof. See, e.g., Buchscher (1992) J.
Virol. 66(5) 2731-2739; Johann (1992) J. Virol. 66 (5):1635-1640
(1992); Sommerfelt (1990) Virol. 176:58-59; Wilson (1989) J. Virol.
63:2374-2378; Miller (1991) J. Virol. 65:2220-2224; Wong-Staal et
al., PCT/US94/05700, and Rosenburg and Fauci (1993) in Fundamental
Immunology, Third Edition Paul (ed) Raven Press, Ltd., New York and
the references therein, and Yu (1994) supra). The vectors are
optionally pseudotyped to extend the host range of the vector to
cells which are not infected by the retrovirus corresponding to the
vector. The vesicular stomatitis virus envelope glycoprotein
(VSV-G) has been used to construct VSV-G-pseudotyped HIV vectors
which can infect hematopoietic stem cells (Naldini et al. (1996)
Science 272:263, and Akkina (1996) J Virol 70:2581).
[0164] Adeno-associated virus (AAV)-based vectors are also used to
transduce cells with target nucleic acids, e.g., in the in vitro
production of nucleic acids and peptides, and in in vivo and ex
vivo gene therapy procedures. See, Okada (1996) Gene Ther.
3:957-964; West (1987) Virology 160:38-47; Carter (1989) U.S. Pat.
No. 4,797,368; Carter et al. WO 93/24641 (1993); Kotin (1994) Human
Gene Therapy 5:793-801; Muzyczka (1994) J. Clin. Invst. 94:1351,
for an overview of AAV vectors. Construction of recombinant AAV
vectors are described in a number of publications, including
Lebkowski, U.S. Pat. No. 5,173,414; Tratschin (1985) Mol. Cell.
Biol. 5(11):3251-3260; Tratschin (1984) Mol. Cell. Biol. 4:
2072-2081; Hermonat (1984) Proc. Natl. Acad. Sci. USA 81:
6466-6470; McLaughlin (1988) and Samulski (1989) J. Virol.,
63:03822-3828. Cell lines that can be transformed by rAAV include
those described in Lebkowski (1988) Mol. Cell. Biol., 8:3988-3996.
Other suitable viral vectors include herpes virus and vaccinia
virus.
[0165] In a particularly preferred embodiment, the tumor suppressor
gene is expressed in an adenoviral vector suitable for gene
therapy. The use of adenoviral vectors in vivo, and for gene
therapy, is well described in the patent and scientific literature,
e.g., see, Hermens (1997) J. Neurosci. Methods., Jan., 71(1):
85-98; Zeiger (1996) Surgery 120:921-925; Channon (1996) Cardiovasc
Res. 32:962-972; Huang (1996) Gene Ther. 3:980-987; Zepeda (1996)
Gene Ther. 3:973-979; Yang (1996) Hum. Mol. Genet. 5:1703-1712;
Caruso (1996) Proc. Natl. Acad. Sci. USA 93:11302-11306; Rothmann
(1996) Gene Ther. 3:919-926; Haecker (1996) Hum. Gene Ther.
7:1907-1914. The use of adenoviral vectors is described in detail
in WO 96/25507. Particularly preferred adenoviral vectors are
described by Wills (1994) supra; in copending U.S. Ser. No.
08/328,673, and WO 95/11984.
[0166] Particularly preferred adenoviral vectors include a deletion
of some or all of the protein IX gene. In one embodiment, the
adenoviral vectors include deletions of the E1a and/or E1b
sequences. In a most preferred embodiment, the adenoviral construct
is a p53 encoding construct such as A/C/N/53 or A/M/N/53 (see,
e.g., U.S. Ser. No. 08/328,673, and WO 95/11984).
[0167] Also preferred are vectors derived from the human adenovirus
type 2 or type 5. Such vectors are preferably are replication
deficient by modifications or deletions in the E1a and/or E1b
coding regions. Other modifications to the viral genome to achieve
particular expression characteristics or permit repeat
administration or lower immune response are preferred. More
preferred are recombinant adenoviral vectors having complete or
partial deletions of the E4 coding region, optionally retaining E4
ORF6 and ORF 6/7. The E3 coding sequence may be deleted but is
preferably retained. In particular, it is preferred that the
promoter operator region of E3 be modified to increase expression
of E3 to achieve a more favorable immunological profile for the
therapeutic vectors. Most preferred are human adenoviral type 5
vectors containing a DNA sequence encoding p53 under control of the
cytomegalovirus promoter region and the tripartite leader sequence
having E3 under control of the CMV promoter and deletion of E4
coding regions while retaining E4 ORF6 and ORF 6/7. In the most
preferred practice of the invention as exemplified herein, the
vector is ACN53.
[0168] In a particularly preferred embodiment, the tumor suppressor
gene is p53 or RB. As explained above. the cloning and use of p53
is described in detail by Wills (1994) supra; in copending U.S.
Ser. No. 08/328,673 filed on Oct. 25, 1994, and in WO 95/11984.
[0169] 2. Ex Vivo Gene Therapy.
[0170] In one embodiment, the methods of this invention are used to
inhibit hyperproliferative (e.g., neoplastic) cells in a subject
(e.g., a mammal including but not limited to rat, murine, bovine,
porcine, equine, canine, feline, largomorph, or human). Pathologic
hyperproliferative cells are characteristic off disease states
including, but not limited to Grave's disease, psoriasis, benign
prostatic hypertrophy, Li-Fraumeni syndrome, breast cancer,
sarcomas, bladder cancer, colon cancer, lung cancer various
leukemia and lymphomas and other neoplasms.
[0171] Ex vivo application of the methods of this invention, in
particular, provide means for depleting a suitable sample of
pathologic hyperproliferative cells. Thus, for example
hyperproliferative cells contaminating hematopoietic precursors
during bone marrow reconstitution can be eliminated by the ex vivo
application of the methods of this invention. Typically such
methods involve obtaining a sample from the subject organism. The
sample is typically a heterogenous cell preparation containing both
phenotypically normal and pathogenic (hyperproliferative) cells.
The sample is contacted with the tumor suppressor nucleic acids or
proteins and the adjunctive anti-cancer agent according to the
methods of this invention. The tumor suppressor gene can be
delivered, e.g., in a viral vector, such as a retroviral vector or
an adenoviral vector. The treatment reduces the proliferation of
the pathogenic cells thereby providing a sample containing a higher
ratio of normal to pathogenic cells which can be reintroduced into
the subject organism.
[0172] Ex vivo cell transformation for diagnostics, research, or
for gene therapy (e.g., via re-infusion of the transformed cells
into the host organism) is well known to those of skill in the art.
In a preferred embodiment, cells are isolated from the subject
organism, transfected with the tumor suppressor gene or cDNA of
this invention, and re-infused back into the subject organism
(e.g., patient). Various cell types suitable for ex vivo
transformation are well known to those of skill in the art.
Particular preferred cells are progenitor or stem cells (see, e.g.,
Freshney (1994) Culture of Animal Cells, a Manual of Basic
Technique, third edition Wiley-Liss, New York, and the references
cited therein for a discussion of how to isolate and culture cells
from patients). Transformed cells are cultured by means well known
in the art. See, also Kuchler (1977) Biochemical Methods in Cell
Culture and Virology, Kuchler, R. J., Dowden, Hutchinson and Ross,
Inc., and Atlas (1993) CRC Handbook of Microbiological Media (Parks
ed) CRC press, Boca Raton, Fla. Mammalian cell systems often will
be in the form of monolayers of cells, although mammalian cell
suspensions are also used. Alternatively, cells can be derived from
those stored in a cell bank (e.g., a blood bank). Illustrative
examples of mammalian cell lines include the HEC-1-B cell line,
VERO and Hela cells, Chinese hamster ovary (CHO) cell lines, W138,
BHK, Cos-7 or MDCK cell lines (see, e.g., Freshney, supra).
[0173] In one particularly preferred embodiment, stem cells are
used in ex-vivo procedures for cell transformation and gene
therapy. The advantage to using stem cells is that they can be
differentiated into other cell types in vitro, or can be introduced
into a mammal (such as the donor of the cells) where they will
engraft in the bone marrow. Methods for differentiating stem cells
(e.g., CD34.sup.+) stem cells in vitro into clinically important
immune cell types using cytokines such a GM-CSF, IFN-gamma and
TNF-alpha are known (see, e.g., Inaba (1992) J. Exp. Med.
176:1693-1702; Szabolcs (1995) 154:5851-5861).
[0174] Rather than using stem cells, T cells or B cells are also
used in some embodiments in ex vivo procedures. Several techniques
are known for isolating T and B cells. The expression of surface
markers facilitates identification and purification of such cells.
Methods of identification and isolation of cells include FACS,
incubation in flasks with fixed antibodies which bind the
particular cell type and panning with magnetic beads.
[0175] Stem cells are isolated for transduction and differentiation
using known methods. For example, in mice, bone marrow cells are
isolated by sacrificing the mouse and cutting the leg bones with a
pair of scissors. Stem cells are isolated from bone marrow cells by
panning the bone marrow cells with antibodies which bind unwanted
cells, such as CD4.sup.+ and CD8+(T cells), CD45+(panB cells), GR-1
(granulocytes), and Ia.sup.d (differentiated antigen presenting
cells). For an example of this protocol see, e.g., Inaba (1992)
supra.
[0176] In humans, bone marrow aspirations from iliac crests are
performed e.g., under general anesthesia in the operating room. The
bone marrow aspirations is approximately 1,000 ml in quantity and
is collected from the posterior iliac bones and crests. If the
total number of cells collected is less than about
2.times.10.sup.8/kg, a second aspiration using the sternum and
anterior iliac crests in addition to posterior crests is performed.
During the operation, irradiated packed red cells are administered
to replace the volume of marrow taken by the aspiration. Human
hematopoietic progenitor and stem cells are characterized by the
presence of a CD34 surface membrane antigen. This antigen is used
for purification, e.g., on affinity columns which bind CD34. After
the bone marrow is harvested, the mononuclear cells are separated
from the other components by means of ficol gradient
centrifugation. This can be performed by a semi-automated method
using a cell separator (e.g., a Baxter Fenwal CS3000+ or Terumo
machine). The light density cells, composed mostly of mononuclear
cells are collected and the cells are incubated in plastic flasks
at about 37.degree. C. for about 1.5 hours. The adherent cells
(monocytes, macrophages and B-Cells) are discarded. The
non-adherent cells are then collected and incubated with a
monoclonal anti-CD34 antibody (e.g., the murine antibody 9C5) at
4.degree. C. for 30 minutes with gentle rotation. The final
concentration for the anti-CD34 antibody is preferably about 10
.mu.g/ml. After two washes, paramagnetic microspheres (e.g., Dyna
Beads, supplied by Baxter Immunotherapy Group, Santa Ana, Calif.)
coated with sheep antimouse IgG (Fc) antibody are added to the cell
suspension at a ratio of about 2 cells/bead. After a further
incubation period of about 30 minutes at about 4.degree. C., the
rosetted cells with magnetic beads are collected with a magnet.
Chymopapain (Baxter Immunotherapy Group, Santa Ana, Calif.) at a
final concentration of 200 U/ml can be added to release the beads
from the CD34+ cells.
[0177] Alternatively, and preferably, an affinity column isolation
procedure can be used which binds to CD34, or to antibodies bound
to CD34 (see, e.g., Ho (1995) Stem Cells 13 (suppl. 3): 100-105 and
Brenner (1993) Journal of Hematotherapy 2: 7-17).
[0178] In another embodiment, hematopoietic stem cells can be
isolated from fetal cord blood. Yu (1995) Proc. Natl. Acad. Sci.
USA, 92: 699-703 describe a preferred method of transducing
CD34.sup.+ cells from human fetal cord blood using retroviral
vectors.
[0179] 3. Administration of Tumor Suppressor-Expressing Nucleic
Acid: Vectors and Expression Cassettes
[0180] Routes of Administration
[0181] Expression cassettes and vectors (e.g., retroviruses,
adenoviruses, liposomes, etc.) containing the therapeutic, tumor
suppressor-expressing nucleic acids of the invention, can be
administered directly to the organism for transduction of cells in
vivo. Administration is by any of the routes normally used for
introducing a molecule into ultimate contact with blood or tissue
cells, e.g., systemically, regionally, or locally, as discussed in
detail, sup ra, for the administration of adjunctive anti-cancer
agents. The "packaged" nucleic acids (at a minimum, a tumor
suppressor coding sequence with a promoter) are administered in any
suitable manner, preferably with pharmaceutically acceptable
carriers, also discussed supra. Suitable methods of administering
such packaged nucleic acids are available and well known to those
of skill in the art, and, although more than one route can be used
to administer a particular composition, a particular route can
often provide a more immediate and more effective reaction than
another route.
[0182] For example, administration of a recombinant adenovirus
vector engineered to express a tumor suppressor gene can elicit an
immune response, specifically, an antibody response, against the
adenoviral vector. Some patients may have pre-existing
anti-adenoviral reacting antibodies. Thus, in some circumstances,
regional or local, rather than systemic, administration, of the
tumor-suppressor expressing adenoviral vector is optimal and most
effective. For example, as discussed below, ovarian cancer limited
to the abdominal cavity is one clinical scenario in which regional
p53 gene therapy, i.e., intraperitoneal (IP) administration, should
be considered as a preferred treatment plan. Administration of
recombinant adenoviruses IP also results in infection of the
peritoneal lining and absorption of the adenoviral vector into the
systemic circulation (other means of regional administration can
also result in introduction of the adenoviral vector into the
systemic circulation). The extent of this effect may depend on the
concentration and/or total amount of viral particles administered
IP. If the systemic effect is desired, a higher concentration over
several consecutive days may be preferred.
[0183] Local administration of the tumor suppressor-expressing
adenoviral vector of the invention is also preferred in some
circumstances, e.g., when the patient has pre-existing
anti-adenoviral reactive antibodies. Such "local administration"
can be, e.g., by intra-tumoral injection, if internal, or mucosal
application, if external. Alternatively, a "local administration"
effect can be effected by targeting the adenoviral vector to the
tumor using, e.g., tumor specific antigen-recognizing reagents (as
antibodies) on liposomes or on the adenovirus itself.
[0184] Formulations
[0185] Pharmaceutically acceptable carriers are determined in part
by the particular composition being administered, as well as by the
particular method used to administer the composition. Accordingly,
there is a wide variety of suitable formulations of pharmaceutical
compositions of the present invention.
[0186] Formulations suitable for oral administration of
pharmaceutical compositions comprising the tumor
suppressor-expressing nucleic acids can consist of (a) liquid
solutions, such as an effective amount of the packaged nucleic acid
suspended in diluents, such as water, saline or PEG 400; (b)
capsules, sachets or tablets, each containing a predetermined
amount of the active ingredient, as liquids, solids, granules or
gelatin; (c) suspensions in an appropriate liquid; and (d) suitable
emulsions. Tablet forms can include one or more of lactose,
sucrose, mannitol, sorbitol, calcium phosphates, corn starch,
potato starch, tragacanth, microcrystalline cellulose, acacia,
gelatin, colloidal silicon dioxide, croscarmellose sodium, talc,
magnesium stearate, stearic acid, and other excipients, colorants,
fillers, binders, diluents, buffering agents, moistening agents,
preservatives, flavoring agents, dyes, disintegrating agents, and
pharmaceutically compatible carriers. Lozenge forms can comprise
the active ingredient in a flavor, usually sucrose and acacia or
tragacanth, as well as pastilles comprising the active ingredient
in an inert base, such as gelatin and glycerin or sucrose and
acacia emulsions, gels, and the like containing, in addition to the
active ingredient, carriers known in the art.
[0187] The packaged nucleic acids, alone or in combination with
other suitable components, can be made into aerosol formulations
(i.e., they can be "nebulized") to be administered via inhalation.
Aerosol formulations can be placed into pressurized acceptable
propellants, such as dichlorodifluoromethane, propane, nitrogen,
and the like.
[0188] Suitable formulations for rectal administration include, for
example, suppositories, which consist of the packaged nucleic acid
with a suppository base. Suitable suppository bases include natural
or synthetic triglycerides or paraffin hydrocarbons. In addition,
it is also possible to use gelatin rectal capsules which consist of
a combination of the packaged nucleic acid with a base, including,
for example, liquid triglycerides, polyethylene glycols, and
paraffin hydrocarbons.
[0189] Formulations suitable for parenteral administration, such
as, for example, by intraarticular (in the joints), intravenous,
intramuscular, intradermal, intraperitoneal, and subcutaneous
routes, include aqueous and non-aqueous, isotonic sterile injection
solutions, which can contain antioxidants, buffers, bacteriostats,
and solutes that render the formulation isotonic with the blood of
the intended recipient, and aqueous and non-aqueous sterile
suspensions that can include suspending agents, solubilizers,
thickening agents, stabilizers, and preservatives. In the practice
of this invention, compositions can be administered, for example,
by intravenous infusion, orally, topically, intraperitoneally,
intravesically or intrathecally. Parenteral administration and
intravenous administration are the preferred methods of
administration. The formulations of packaged nucleic acid can be
presented in unit-dose or multi-dose sealed containers, such as
ampules and vials.
[0190] Formulations of the invention as injection solutions and
suspensions can be prepared from sterile powders, granules, and
tablets of the kind previously described. The exact composition of
the formulation, the concentration of the reagents and nucleic acid
in the formulation, its pH, buffers, and other parameters will vary
depending on the mode and site of administration (e.g., whether
systemic, regional or local administration) and needs related to
storage, handling, shipping, and shelf life of the particular
pharmaceutical composition. Optimization of these parameters
depending on the particular need of the formulation can be done by
routine methods; and any of ingredients and parameters for known
injectable formulations can be used. One example of a suitable
formulation is, e.g., a recombinant wild type p53-expressing
adenovirus vector of the invention (rAd5/p53) at a concentration of
about 7.5.times.10.sup.11 to 7.5.times.10.sup.10 particles per ml,
sodium phosphate monohydrate at 0.42 mg/ml, sodium phosphate
dibasic anhydride at 2.48 mg/ml, sodium chloride at sodium
phosphate monohydrate at 5.8 mg/ml, sucrose at 20.0 mg/ml,
magnesium chloride hexahydrate at 0.40 mg/ml, typically stored in
1.0 ml dosages. An exemplary formulation for enhanced stability
during storage and distribution, especially at refrigeration
temperatures, uses rAd5/p53 (at also about 7.5.times.10.sup.11 to
7.5.times.10.sup.10 particles per ml), sodium phosphate monobasic
dihydrate at 1.7 mg/ml, tromethamine (Trizma, or, Tris base, Sigma
Chemical Co., St. Louis, Mo.) at 1.7 mg/ml, magnesium chloride
hexahydrate at 0.4 mg/ml, sucrose at 20 mg/ml, polysorbate 80 at
0.15 mg/ml, glycerol at 100 mg/ml, typically stored in 1.0 ml
dosages.
[0191] Cells transduced by the packaged nucleic acid as described
above in the context of ex vivo therapy can also be administered
intravenously or parenterally as described above.
[0192] The dose administered to a patient, in the context of the
present invention, should be sufficient to effect a beneficial
therapeutic response in the patient over time. The dose will be
determined by the efficacy of the particular vector employed and
the condition of the patient, as well as the body weight or surface
area of the patient to be treated. The size of the dose also will
be determined by the existence, nature, and extent of any adverse
side-effects that accompany the administration of a particular
vector, or transduced cell type in a particular patient.
[0193] In determining the effective amount of the vector to be
administered in the treatment, the physician evaluates circulating
plasma levels of the vector, vector toxicities, progression of the
disease, and the production of anti-vector antibodies. The typical
dose for a nucleic acid is highly dependent on route of
administration and gene delivery system. Depending on delivery
method the dosage can easily range from about 1 .mu.g to 100 mg or
more. In general, the dose equivalent of a naked nucleic acid from
a vector is from about 1 .mu.g to 100 .mu.g for a typical 70
kilogram patient, and doses of vectors which include a viral
particle are calculated to yield an equivalent amount of
therapeutic nucleic acid.
[0194] For administration, transduced cells of the present
invention can be administered at a rate determined by the LD.sub.50
of the vector, or transduced cell type, and the side-effects of the
vector or cell type at various concentrations, as applied to the
mass and overall health of the patient. Administration can be
accomplished via single or divided doses as described below.
[0195] In a preferred embodiment, prior to infusion, blood samples
are obtained and saved for analysis. Vital signs and oxygen
saturation by pulse oximetry are closely monitored. Blood samples
are preferably obtained 5 minutes and 1 hour following infusion and
saved for subsequent analysis. In ex vivo therapy, leukopheresis,
transduction and reinfusion can be repeated are repeated every 2 to
3 months. After the first treatment, infusions can be performed on
a outpatient basis at the discretion of the clinician. If the
reinfusion is given as an outpatient, the participant is monitored
for at least 4, and preferably 8 hours following the therapy.
[0196] As described above, the adenoviral constructs can be
administered systemically (e.g., intravenously), regionally (e.g.,
intraperitoneally) or locally (e.g., intra- or peri-tumoral or
intracystic injection, e.g., to treat bladder cancer). Particularly
preferred modes of administration include intra-arterial injection,
more preferably intra-hepatic artery injection (e.g., for treatment
of liver tumors), or, where it is desired to deliver a composition
to a brain tumor, a carotid artery or an artery of the carotid
system of arteries (e.g., occipital artery, auricular artery,
temporal artery, cerebral artery, maxillary artery, etc.). Delivery
for treatment of lung cancer can be accomplished, e.g., by use of a
bronchoscope. Typically such administration is in an aqueous
pharmacologically acceptable buffer as described above. However, on
one particularly preferred embodiment, the adenoviral constructs or
the tumor suppressor expression cassettes are administered in a
lipid formulation, more particularly either complexed with
liposomes to for lipid/nucleic acid complexes (e.g., as described
by Debs and Zhu (1993) WO 93/24640; Mannino (1988) supra; Rose,
U.S. Pat. No. 5,279,833; Brigham (1991) WO 91/06309; and Felgner
(1987) supra) or encapsulated in liposomes, more preferably in
immunoliposomes directed to specific tumor markers. It will be
appreciated that such lipid formulations can also be administered
topically, systemically, or delivered via aerosol.
[0197] 4. Enhancing Tumor Suppressor Delivery.
[0198] Tumor suppressor delivery can be enhanced by the use of one
or more "delivery-enhancing agents". A "delivery-enhancing agent"
refers to any agent which enhances delivery of a therapeutic gene,
such as a tumor suppressor gene to a cancerous tissue or organ.
Such enhanced delivery may be achieved by various mechanisms. One
such mechanism may involve the disruption of the protective
glycosaminoglycan layer on the epithelial surface of an organ or
tissue (e.g., the bladder). Examples of such delivery-enhancing
agents are detergents, alcohols, glycols, surfactants, bile salts,
heparin antagonists, cyclooxygenase inhibitors, hypertonic salt
solutions, and acetates. Alcohols include for example the aliphatic
alcohols such as ethanol, N-propanol, isopropanol, butyl alcohol,
acetyl alcohol. Glycols include glycerine, propyleneglycol,
polyethyleneglycol and other low molecular weight glycols such as
glycerol and thioglycerol. Acetates such as acetic acid, gluconol
acetate, and sodium acetate are further examples of
delivery-enhancing agents. Hypertonic salt solutions like 1M NaCl
are also examples of delivery-enhancing agents. Examples of
surfactants are sodium dodecyl sulfate (SDS) and lysolecithin,
polysorbate 80, nonylphenoxypolyoxyethylene,
lysophosphatidylcholine, polyethylenglycol 400, polysorbate 80,
polyoxyethylene ethers, polyglycol ether surfactants and DMSO. Bile
salts such as taurocholate, sodium tauro-deoxycholate,
deoxycholate, chenodesoxycholate, glycocholic acid,
glycochenodeoxycholic acid and other astringents like silver
nitrate may be used. Heparin-antagonists like quaternary amines
such as prolamine sulfate may also be used. Cyclooxygenase
inhibitors such as sodium salicylate, salicylic acid, and
non-steroidal anti-inflammatory drug (NSAIDS) like indomethacin,
naproxen, diclofenac may be used.
[0199] Detergents include anionic, cationic, zwitterionic, and
nonionic detergents. Exemplary detergents include but are not
limited to taurocholate, deoxycholate, taurodeoxycholate,
cetylpyridium, benalkonium chloride, ZWITTERGENT.RTM.3-14
detergent, CHAPS (3-[(3-Cholamidopropyl)di-
methylammoniol]-1-propanesulfonate hydrate, Aldrich), Big CHAP (as
described in U.S. Ser. No. 08/889,355, filed Jul. 8, 1997; and,
International Application WO 97/25072, Jul. 17, 1997), Deoxy Big
CHAP (ibid), TRITON.RTM.-X-100 detergent, C12E8,
Octyl-B-D-Glucopyranoside, PLURONIC.RTM.-F68 detergent, TWEEN.RTM.
20 detergent, and TWEEN.RTM. 80 detergent (CALBIOCHEM.RTM.
Biochemicals).
[0200] In an embodiment, the delivery-enhancing agent is included
in the buffer in which the recombinant adenoviral vector delivery
system is formulated. The delivery-enhancing agent may be
administered prior to the recombinant virus or concomitant with the
virus. In some embodiments, the delivery-enhancing agent is
provided with the virus by mixing a virus preparation with a
delivery-enhancing agent formulation just prior to administration
to the patient. In other embodiments, the delivery-enhancing agent
and virus are provided in a single vial to the care giver for
administration.
[0201] In the case of a pharmaceutical composition comprising a
tumor suppressor gene contained in a recombinant adenoviral vector
delivery system formulated in a buffer which further comprises a
delivery-enhancing agent, the pharmaceutical composition is
preferably be administered over time in the range of about 5
minutes to 3 hours, preferably about 10 minutes to 120 minutes, and
most preferably about 15 minutes to 90 minutes. In another
embodiment the delivery-enhancing agent may be administered prior
to administration of the recombinant adenoviral vector delivery
system containing the tumor suppressor gene. The prior
administration of the delivery-enhancing agent may be in the range
of about 30 seconds to 1 hour, preferably about 1 minute to 10
minutes, and most preferably about 1 minute to 5 minutes prior to
administration of the adenoviral vector delivery system containing
the tumor suppressor gene.
[0202] The concentration of the delivery-enhancing agent will
depend on a number of factors known to one of ordinary skill in the
art such as the particular delivery-enhancing agent being used, the
buffer, pH, target tissue or organ and mode of administration. The
concentration of the delivery-enhancing agent will be in the range
of 1% to 50% (v/v), preferably 10% to 40% (v/v) and most preferably
15% to 30% (v/v). Preferably, the detergent concentration in the
final formulation administered to the patient is about 0.5-2.times.
the critical micellization concentration (CMC). A preferred
concentration of Big CHAP is about 2-20 mM, more preferable about
3.5-7 mM.
[0203] The buffer containing the delivery-enhancing agent may be
any pharmaceutical buffer such as phosphate buffered saline or
sodium phosphate/sodium sulfate, Tris buffer, glycine buffer,
sterile water and other buffers known to the ordinarily skilled
artisan such as those described by Good et al. (1966) Biochemistry
5:467. The pH of the buffer in the pharmaceutical composition
comprising the tumor suppressor gene contained in the adenoviral
vector delivery system, may be in the range of 6.4 to 8.4,
preferably 7 to 7.5, and most preferably 7.2 to 7.4.
[0204] A preferred formulation for administration of a recombinant
adenovirus is about 10.sup.9-10.sup.11 PN/ml virus, about 2-10 mM
Big CHAP or about 0.1-1.0 mM TRITON.RTM.-X-100 detergent, in
phosphate buffered saline (PBS), plus about 2-3% sucrose (w/v) and
about 1-3 mM MgCl.sub.2, at about pH 6.4-8.4. The use of
delivery-enhancing agents is described in detail in copending in
copending application U.S. Ser. No. 08/779,627 filed on Jan. 7,
1997.
[0205] In order to facilitate the improved gene transfer for
nucleic acid formulations comprising commercial Big-CHAP
preparations, the concentration of Big CHAP will vary based on its
commercial source. When the Big CHAP is sourced from CALBIOCHEM, it
is preferred that the concentration be in a range of 2 to 10
millimolar. More preferred is 4 to 8 millimolar. Most preferred is
approximately 7 millimolar.
[0206] When the Big CHAP is sourced from Sigma, it is preferred
that the concentration of Big CHAP be in a range of 15 to 35
millimolar. More preferred is 20 to 30 millimolar. Most preferred
is approximately 25 millimolar.
[0207] In a further embodiment of the invention, delivery-enhancing
agents having Formula I are provided: 2
[0208] wherein n is an integer from 2-8, X.sub.1 is a cholic acid
group or deoxycholic acid group, and X.sub.2 and X.sub.3 are each
independently selected from the group consisting of a cholic acid
group, a deoxycholic acid group, and a saccharide group. At least
one of X.sub.2 and X.sub.3 is a saccharide group. The saccharide
group may be selected from the group consisting of pentose
monosaccharide groups, hexose monosaccharide groups,
pentose-pentose disaccharide groups, hexose-hexose disaccharide
groups, pentose-hexose disaccharide groups, and hexose-pentose
disaccharide groups. In one preferred embodiment, the compounds of
the present invention have the Formula II: 3
[0209] wherein X.sub.1 and X.sub.2 are selected from the group
consisting of a cholic acid group and a deoxycholic acid group and
X.sub.3 is a saccharide group.
[0210] These compounds are preferably used in the range of about
0.002 to 2 mg/ml, more preferably about 0.02 to 2 mg/ml, most
preferably about 0.2 to 2 mg/ml in the formulations of the
invention. Most preferred is approximately 2 mg/ml.
[0211] Phosphate buffered saline (PBS) is the preferred
solubilizing agent for these compounds. However, one of ordinary
skill in the art will recognize that certain additional excipients
and additives may be desirable to achieve solubility
characteristics of these agents for various pharmaceutical
formulations. For examples, the addition of well known solubilizing
agents such as detergents, fatty acid esters, surfactants may be
added in appropriate concentrations so as to facilitate the
solubilization of the compounds in the various solvents to be
employed. When the solvent is PBS, a preferred solubilizing agent
is Tween 80 at a concentration of approximately 0.15%.
[0212] 5. Administration of Tumor Suppressor Proteins.
[0213] Tumor suppressor proteins (polypeptides) can be delivered
directly to the tumor site by injection or administered
systemically as described above. In a preferred embodiment, the
tumor suppressor proteins are combined with a pharmaceutically
acceptable carrier (excipient) to form a pharmacological
composition as described above. The tumor suppressor polypeptide
will be administered in a therapeutically effective dose. Thus the
compositions will be administered in an amount sufficient to cure
or at least partially arrest the disease and/or its complications.
Amounts effective for this use will depend upon the severity of the
disease and the general state of the patient's health.
[0214] It will be recognized that tumor suppressor polypeptides,
when administered orally, must be protected from digestion. This is
typically accomplished either by complexing the polypeptide with a
composition to render it resistant to acidic and enzymatic
hydrolysis or by packaging the polypeptide in an appropriately
resistant carrier such as a liposome as described above. Means of
protecting polypeptides for oral delivery are well known in the art
(see, e.g., U.S. Pat. No. 5,391,377 describing lipid compositions
for oral delivery of therapeutic agents).
[0215] III. Combination Pharmaceuticals
[0216] The tumor suppressor and the adjunctive anti-cancer agent
can be administered individually with either the tumor suppressor
nucleic acid or polypeptide being administered before the
adjunctive anti-cancer (tumor suppressor pretreatment) or the
adjunctive anti-cancer being administered before the tumor
suppressor nucleic acid and/or polypeptide (cancer drug
pretreatment). Of course the tumor suppressor nucleic acid and/or
polypeptide and the adjunctive anti-cancer agent can be
administered simultaneously.
[0217] In one embodiment, the tumor suppressor nucleic acid and/or
polypeptide and the adjunctive anti-cancer agent are administered
as a single pharmacological composition. In this embodiment, the
tumor suppressor nucleic acid and/or polypeptide and the adjunctive
anti-cancer agent can be suspended or solubilized in a single
homogeneous delivery vehicle. Alternatively the tumor suppressor
nucleic acid and/or polypeptide and the adjunctive anti-cancer
agent can each be suspended or solubilized in different delivery
vehicles which in turn are suspended (disbursed) in single
excipient either at the time of administration or continuously.
Thus, for example, an adjunctive anti-cancer agent may be
solubilized in a polar solvent (e.g., paclitaxel in ethanol) and
the tumor suppressor nucleic acid may be complexed with a lipid
which are then either stored together in a suspension or,
alternatively are combined at the time of administration. Various
suitable delivery vehicles, excipients, etc., are described
above.
[0218] IV. Treatment Regimen: Combined and Individual Therapy
[0219] A) Tumor Suppressor Treatment Regimen.
[0220] It was a discovery of this invention that tumor suppressor
nucleic acids or polypeptides, more particularly tumor suppressor
nucleic acids show greater efficacy in inhibiting tumor growth when
administered in multiple doses rather than in a single dose. Thus
this invention provides a treatment regimen for a tumor suppressor
gene or polypeptide that comprises multiple administrations of the
tumor suppressor nucleic acid or polypeptide.
[0221] The tumor suppressor protein or tumor suppressor nucleic
acid may be administered (with or without an adjunctive anti-cancer
agent) in a total dose ranging from about 1.times.10.sup.9 to about
1.times.10.sup.14, about 1.times.10.sup.9 to about
7.5.times.10.sup.15, preferably about 1.times.10.sup.11 to about
7.5.times.10.sup.13, adenovirus particles in a treatment regimen
selected from the group consisting of: the total dose in a single
dose, the total dose divided over 5 days or administered daily for
5 days, the total dose divided over 15 days or administered daily
for 15 days, and the total dose divided over 30 days or
administered daily for 30 days. This method of administration can
be repeated for two or more cycles (more preferably for three
cycles) and the two or more cycles are can be spaced apart by three
or four weeks. The treatment may consist of a single dosage cycle
or dosage cycles may range from about 2 to about 12, more
preferably from about 2 to about 6 cycles.
[0222] Particularly preferred treatment regimen include the total
dose divided over 5 days and administered daily, the total dose
divided over 15 days and administered daily, and the total dose
divided over 30 days and administered daily.
[0223] In some preferred embodiments, a daily dose in the range of
7.5.times.10.sup.9 to about 7.5.times.10.sup.15, preferably about
1.times.10.sup.12 to about 7.5.times.10.sup.13, adenovirus
particles can be administered each day for up to 30 days (e.g., a
regimen of 2 days, 2 to 5 days, 7 days, 14 days, or 30 days with
the same dose being administered each day). The multiple regimen
can be repeated in recurring cycles of 21 to 28 days.
[0224] In some embodiments, different routes of administration will
result in use of different preferred dosage ranges. For instance,
for intra-hepatic arterial delivery, a preferred range will
typically be between 7.5.times.10.sup.9 and about
1.times.10.sup.15, more preferably about 1.times.10.sup.11 to about
7.5.times.10.sup.13, adenovirus particles per day for 5 to 14 days.
These regimens can further include administration of adjunctive
anti-cancer agents, FUDR or 5'-deoxy-5-fluorouridine (5'-DFUR), or
irinotecan hydrochloride (CPT-11;
7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin).
For intratumoral delivery, a preferred range will typically be
between 7.5.times.10.sup.9 and about 1.times.10.sup.13, more
preferably about 1.times.10.sup.11 to about 7.5.times.10.sup.12,
adenovirus particles per day. For intraperitoneal delivery, a
preferred range will typically be between 7.5.times.10.sup.9 and
about 1.times.10.sup.15, more preferably about 1.times.10.sup.11 to
about 7.5.times.10.sup.13, adenovirus particles per day for 5-10
days.
[0225] B) Combination Therapy Treatment Regimen
[0226] Where the tumor suppressor is used in combination with an
adjunctive anti-cancer agent the tumor suppressor nucleic acid is
administered in total dose as described above. In combination, the
adjunctive anti-cancer agent is administered in a total dose
dependent upon the agent used. For instance, paclitaxel or a
paclitaxel derivative is administered in a total dose ranging from
75-350 mg/m.sup.2 over 1 hour, 3 hours, 6 hours, or 24 hours in a
treatment regimen selected from the group consisting of
administration in a single dose, in a dose administered daily on
day 1 and day 2, in a dose administered daily on day 1, day 2, and
day 3, on a daily dosage for 15 days, on a daily dosage for 30
days, on daily continuous infusion for 15 days, on daily continuous
infusion for 30 days. A preferred dose is 100-250 mg/m.sup.2 in 24
hours.
[0227] Pretreatment with an adjunctive anti-cancer agent (e.g.,
paclitaxel) prior to treatment with a tumor suppressor nucleic acid
enhances the efficacy of the tumor suppressor. Thus, in one
particularly preferred embodiment the cell, tissue, or organism is
treated with the adjunctive anti-cancer agent prior to the tumor
suppressor nucleic acid. The adjunctive anti-cancer agent treatment
preferably precedes the tumor suppressor nucleic acid treatment by
about twenty four hours although longer or shorter periods are
acceptable.
[0228] The pretreatment is particularly efficacious when the
adjunctive anti-cancer agent is a paclitaxel-like compound, more
preferably paclitaxel or a paclitaxel derivative (e.g., Taxol.RTM.
or Taxotere.RTM.). Particularly preferred tumor suppressors are RB
and p53 with p53 being most preferred, in particular p53 in an
adenoviral vector (e.g., A/C/N/53).
[0229] V. Treatment of and Prophylaxis for Metastases
[0230] As illustrated in Examples 2 and 3, tumor suppressor (e.g.,
p53) gene replacement therapy has been demonstrated to have
efficacy against human tumor cells in vitro, human tumor xenografts
in immunocompromised hosts, and human lung tumors (in vivo).
Surgical debulking of primary tumors in patients often results in
tumor regrowth at the primary site and tumor metastasis from that
site due to microscopic "nests" of tumor cells which are missed by
the surgeon. Alternatively, in order to make sure that all the
tumor is removed from a primary site, the patient may be subjected
to disfiguring surgery which removes a large amount of normal
tissue surrounding the primary tumor site.
[0231] In another embodiment, this invention provides methods of
inhibiting the growth and/or proliferation of metastases
(metastatic cells). The method generally involves either systemic
or topical administration of a tumor suppressor, more preferably
topical administration of p53 or RB.
[0232] A) Systemic Treatment.
[0233] As explained in Examples 2 and 3, systemic treatment (e.g.,
intravenous injection) of tumor suppressor vectors (e.g., A/C/N/53)
inhibited the progression of metastases in vivo. Thus, in one
embodiment, this invention provides methods for inhibiting the
progression of metastatic disease by administering to an organism a
tumor suppressor nucleic acid and/or a tumor suppressor polypeptide
as described above. The tumor suppressor is preferably a tumor
suppressor nucleic acid, more preferably a p53 tumor suppressor
nucleic acid and most preferably a p53 nucleic acid in an
adenoviral vector (e.g. A/C/N/53). In another preferred embodiment,
the tumor suppressor nucleic acid is provided encapsulated in a
liposome or complexed to a lipid (see, e.g., Debs and Zhu (1993) WO
93/24640; Mannino and Gould-Fogerite (1988) BioTechniques 6(7):
682-691; Rose U.S. Pat. No. 5,279,833; Brigham (1991) WO 91/06309;
and Felgner et al. (1987) Proc. Natl. Acad. Sci. USA 84:
7413-7414).
[0234] B) Topical Treatment
[0235] In another embodiment, topical application of the tumor
suppressor protein or tumor suppressor nucleic acid is preferred in
conjunction with surgery. In this embodiment, the tumor suppressor,
preferably in the form of an infectious vector, is applied along
the surface of the wound cavity after tumor removal. The infectious
particles will carry p53 into any residual tumor cells at the wound
site, inducing their apoptosis (programmed cell death). This
treatment will impact long-term patient survival and/or reduce the
amount of normal tissue surrounding the tumor site which needs to
be removed during surgery.
[0236] The tumor suppressor is preferably compounded in one of the
many formulations known by those of skill in the art to be suitable
for topical application. Thus, for example, an infections
preparation of the human p53 tumor suppressor gene (e.g., A/C/N/53)
is suspended in a suitable vehicle (e.g., petroleum jelly or other
cream or ointment) which is suitable for spreading along the
surface of the wound cavity. Alternatively, the tumor suppressor
can be prepared in an aerosol vehicle for application as a spray
inside the wound cavity. In other embodiments, the tumor suppressor
can be prepared in degradable (resorbable) materials, e.g,
resorbable sponges, that can be packed into the wound cavity and
which release the tumor suppressor protein or vector in a
time-dependent manner.
[0237] Preferred embodiments for application of recombinant
adenoviral vectors to certain defined topical areas, e.g., cornea,
gastro-intestinal tract, tumoral resection sites use solid carriers
to support a longer incubation time and facilitate viral infection.
Carriers can be gauze or ointments soaked with the recombinant
adenovirus solution. The virus can be applied via the gauze support
to the cornea to achieve improved transgene effects. The drained
gauze can also be prophylactically applied to resected tumor areas
in order to avoid recurrence. Ointments can be applied topically to
areas of the gastrointestinal tract, or topically to areas of the
pancreas for tumor suppressor gene therapy.
[0238] Exemplary ointment carriers include petroleum based
Puralube.RTM. or water soluble KJ-Jelly.RTM.. In an exemplary
method, sterile gauze pads (5.times.5 cm) or tear flow test strips
can be soaked in an adenoviral vector solution (e.g.,
1.times.10.sup.9 PN/ml) until totally wet. The pads or strips are
layered on top of the target tissue and incubated at 37 degrees C.
for 30 minutes. One of skill will recognize that other fabrics,
gelatins, or ointments can be included that can take up or be
mixable with water. In addition, other excipients may be added that
can enhance gene transfer as described above.
[0239] VI. Combination Treatments with Other Chemotherapeutics
[0240] A) Tumor Suppressors Administered in Combination with
Multiple Chemotherapeutic Combinations
[0241] It will be appreciated that the methods of this invention
are not limited to combination of a tumor suppressor with a single
adjunctive anti-cancer agent. While methods typically involve
contacting a cell with a tumor suppressor (e.g., p53) and an
adjunctive anti-cancer agent such as paclitaxel, the methods of the
invention also entail contacting the cell with a combination of a
tumor suppressor gene or polypeptide and two, three or a
multiplicity of adjunctive anti-cancer agents and optionally other
chemotherapeutic drugs. In addition, one of skill will recognize
that a chemotherapeutic agent(s) can also be used with tumor
suppressor proteins or genes, in the absence of an adjunctive
anti-cancer agent(s).
[0242] Many chemotherapeutic drugs are well known in the scientific
and patent literature; exemplary drugs that can be used in the
methods of the invention include but are not limited to: DNA
damaging agents (including DNA alkylating agents) e.g., cisplatin,
carboplatin (see, e.g., Duffull (1997) Clin Pharmacokinet.
33:161-183); Droz (1996) Ann Oncol. 7:997-1003), navelbine
(vinorelbine), Asaley, AZQ, BCNU, Busulfan,
carboxyphthalatoplatinum, CBDCA, CCNU, CHIP, chlorambucil,
chlorozotocin, cis-platinum, clomesone,
cyanomorpholino-doxorubicin, cyclodisone, cytoxan,
dianhydrogalactitol, fluorodopan, hepsulfam, hycanthone, melphalan,
methyl CCNU, mitomycin C, mitozolamide, nitrogen mustard, PCNU,
piperazine alkylator, piperazinedione, pipobroman, porfiromycin,
spirohydantoin mustard, teroxirone, tetraplatin, thio-tepa,
triethylenemelamine, uracil nitrogen mustard, Yoshi-864);
topoisomerase I inhibitors (e.g., topotecan hydrochloride,
irinotecan hydrochloride (CPT 11), camptothecin, camptothecin Na
salt, aminocamptothecin, CPT-11 and other camptothecin
derivatives); topoisomerase II inhibitors (doxorubicin, including
doxorubicin encapsulated in liposomes (see, U.S. Pat. Nos.
5,013,556 and 5,213,804) amonafide, m-AMSA, anthrapyrazole
derivatives, pyrazoloacridine, bisantrene HCL, daunorubicin,
deoxydoxorubicin, mitoxantrone, menogaril, N,N-dibenzyl daunomycin,
oxanthrazole, rubidazone, VM-26, and VP-16); RNA/DNA
antimetabolites (e.g., L-alanosine, 5-azacytidine, 5-fluorouracil,
acivicin, aminopterin, aminopterin derivatives, an antifol, Baker's
soluble antifol, dichlorallyl lawsone, brequinar, ftorafur
(pro-drug), 5,6-dihydro-5-azacytidine, methotrexate, methotrexate
derivatives, N-(phosphonoacetyl)-L-aspartate (PALA), pyrazofurin,
and trimetrexate); and, DNA antimetabolites (e.g., 3-HP,
2'-deoxy-5-fluorouridine, 5-HP, alpha-TGDR, aphidicolin glycinate,
ara-C, 5-aza-2'-deoxycytidine, beta-TGDR, cyclocytidine, guanazole,
hydroxyurea, inosine, glycodialdehyde, macbecin II,
pyrazoloimidazole, thioguanine, and thiopurine). The tumor
suppressor nucleic acid and/or polypeptide can also be administered
in combination with chemotherapeutic agents such as vincristine,
temozolomide (see, e.g., U.S. Pat. No. 5,260,291), and toremifene
(see, e.g., U.S. Pat. No. 4,696,949 for information on
toremifene).
[0243] Preclinical studies in relevant animal models have shown
that p53 adenovirus combined with cisplatin, carboplatin,
navelbine, doxorubicin, 5-fluorouracil, methotrexate, or etoposide,
inhibited cell proliferation more effectively than chemotherapy
alone treating the tumors: SSC-9 head and neck, SSC-15 head and
neck, SSC-25 head and neck, SK-OV-3 ovarian, DU-145 prostate,
MDA-MB-468 breast and MDA-MB-231 breast tumor cells. In another
embodiment, an enhanced anti-tumor efficacy is seen using a three
drug combination of p53 gene (expressed, e.g., in an adenovirus
vector), an adjuctive anti-cancer agent (e.g., paclitaxel) and a
DNA damaging agent (e.g., cisplatin). The combination of p53,
paclitaxel and cisplatin has been shown to be effective in an
ovarian tumor model. These data support the combination of p53 gene
therapy with chemotherapy in clinical trials.
[0244] These other chemotherapeutic drugs can be used in
combination with the tumor suppressor nucleic acid and/or
polypeptide with or without the presence of an adjunctive
anti-cancer agent. This invention also contemplates the use of
radiation therapy in combination with any of the tumor suppressors
described above or in conjunction with the tumor suppressors
described above combined with an adjunctive anti-cancer agent.
[0245] It will also be appreciated that any of these
chemotherapeutics can be used individually in combination with a
tumor suppressor nucleic acid or polypeptide according to the
methods of this invention.
[0246] When the tumor suppressor nucleic acid (e.g., p53) is
administered in an adenoviral vector with an adjunctive anti-cancer
agent (e.g., paclitaxel) and a DNA damaging agent (e.g., cisplatin,
carboplatin, or navelbine), the adenoviral vector is typically
administered for 5 to 14 days at about 7.5.times.10.sup.12 to about
7.5.times.10.sup.13 adenoviral particles per day. For example, a
daily dose of about 7.5.times.10.sup.13 adenoviral particles in
combination with carboplatin can be used. In one embodiment, a
daily dose of about 7.5.times.10.sup.12 adenoviral particles can be
used for administration to the lung. In another embodiment, p53 is
administered with topotecan.
[0247] Typically, the DNA damaging agent will be administered at
the recommended dose, see, e.g., Physician's Desk Reference, 51st
ed. (Medical Economics, Montvale, N.J. 1997). For instance,
carboplatin is administered to achieve an AUC (an area under the
curve) of about 6-7.5 mg/ml/min.
[0248] Protease Inhibitors
[0249] In still another embodiment, this invention provides for the
combined use of tumor suppressor nucleic acids and/or polypeptides
and protease inhibitors. Particularly preferred protease inhibitors
include, but are not limited to collagenase inhibitors, matrix
metalloproteinase (MMP) inhibitors (see, e.g., Chambers (1997) J.
Natl. Cancer Inst. 89:1260-1270). In a preferred embodiment, the
methods comprise administering concurrently or sequentially, an
effective amount of a protease inhibitor and an effective amount of
a tumor suppressor polypeptide and/or nucleic acid. Examples of
compounds that are protease inhibitors are well known in the
scientific and patent literature.
[0250] Immunomodulators
[0251] The tumor suppressor proteins and nucleic acids of this
invention can be used in conjunction with immunomodulators where
the immunomodulators either upregulate an immune response directed
against the hyperproliferative or cancer cell (e.g., an immune
response directed against a tumor specific antigen) or downregulate
an immune response directed against the tumor suppressor protein,
tumor suppressor nucleic acid, tumor suppressor vector (e.g.,
anti-adenoviral reaction), and/or combined chemotherapeutic.
[0252] Thus, for example, this invention provides for the combined
sequential or concurrent administration of an effective amount of a
tumor suppressor nucleic acid and/or tumor suppressor polypeptide
with an effective amount of an immunomodulator. Immunomodulators
include, but are not limited to cytokines such as IL-2, IL-4, IL-10
(U.S. Pat. No. 5,231,012; Lalani (1997) Ann. Allergy Asthma
Immunol. 79:469-483; Geissler (1996) Curr. Opin. Hematol.
3:203-208), IL-12 (see, e.g., Branson (1996) Human Gene Ther. 1:
1995-2002), and gamma-interferon.
[0253] Immunomodulators that function as immunosuppressants can be
utilized to mitigate an immune response targeted against the
therapeutic (e.g., tumor suppressor protein or nucleic acid or
adjunctive anticancer agent, etc.). Immunosuppressants are well
known to those of skill in the art. Suitable immunosuppressants
include, but are not limited to cyclo-phosphamide, dexamethasone,
cyclosporin, FK506 (tacrolimus) (Lochmuller (1996) Gene Therapy
3:706-716) IL-10, and the like. Antibodies against cell surface
receptors which modulate the immune response can also be used. For
instance, antibodies that block ligand binding to cellular
receptors on B cells, T cells, NK cells, macrophages, and tumor
cells can be used for this purpose. For examples of this strategy
see, e.g., Yang (1996) Gene Therapy 3:412-420; Lei (1996) Human
Gene Therapy 7:2273-2279; Yang (1996) Science 275:1862-1867.
[0254] VII. Therapeutic Kits.
[0255] In another embodiment, this invention provides for
therapeutic kits. The kits include, but are not limited to a tumor
suppressor nucleic acid or polypeptide or a pharmaceutical
composition thereof. The kits may also include an adjunctive
anti-cancer agent or a pharmaceutical composition thereof or
pharmaceutical composition thereof. The various compositions may be
provided in separate containers for individual administration or
for combination before administration. Alternatively the various
compositions may be provided in a single container. The kits may
also include various devices, buffers, assay reagents and the like
for practice of the methods of this invention. In addition, the
kits may contain instructional materials teaching the use of the
kit in the various methods of this invention (e.g., in the
treatment of tumors, in the prophylaxis and/or treatment of
metastases, and the like).
[0256] The kit can optionally include one or more immunomodulators
(e.g., immunosuppressants). Particularly preferred immunomodulators
include any of the immunomodulators described herein.
[0257] VIII. Cells Containing Heterologous Tumor Suppressor Nucleic
Acids or Polypeptides, and Other Agents
[0258] Further provided by this invention is a transfected or
otherwise treated prokaryotic or eukaryotic host cell, for example
an animal cell (e.g., a mammalian cell) containing a heterologous
tumor suppressor nucleic acid and/or tumor suppressor polypeptide.
The cell may optionally additionally contain an adjunctive
anti-cancer agent, e.g. paclitaxel or other microtubule affecting
agent.
[0259] Suitable prokaryotic cells include, but are not limited to
bacterial cells such as E. coli cells. Suitable animal cells
preferably include mammalian, more preferably human cells. Host
cells include, but are not limited to any mammalian cell, more
preferably any neoplastic or tumor cell such as any of the cells
described herein.
[0260] The transfected host cells described herein are useful as
compositions for diagnosis or therapy. When used pharmaceutically,
they can be combined with various pharmaceutically acceptable
carriers as described above for ex vivo gene therapy. The cells can
be administered therapeutically or prophylactically in effective
amounts described in detail above. In a diagnostic context, the
cells may be used for teaching or other reference purposes and
provide suitable models for identification of cells thus
transfected and/or treated.
[0261] IX. Preclinical and Clinical Efficacy of p.sup.53 Adenovirus
Gene Therapy
[0262] Adenovirus-mediated p53 gene therapy is currently undergoing
phase I/II clinical trials in several countries. The pharmaceutical
composition used in these clinical trials included an exemplary
wild type p53-expressing adenovirus of the invention (rAd/p53)
consisting of a replication-deficient, type 5 adenovirus vector
expressing the human tumor suppressor gene under the control of the
cytomegalovirus promoter ("rAd5/p53"), as described herein (see
Wills (1994) supra).
[0263] Regional Administration
[0264] Ovarian cancer limited to the abdominal cavity is one
clinical scenario in which regional p53 gene therapy, i.e.,
intraperitoneal administration, should be considered as a preferred
treatment plan.
EXAMPLES
[0265] The following examples are offered to illustrate, but not to
limit the claimed invention.
Example 1
Combination Therapy with p53 and Taxol.RTM.
[0266] The invention provides for the combined administration of
nucleic acid expressing a tumor suppressor polypeptide and
paclitaxel in the treatment of neoplasms. The following example
details the ability of a p53 expressing adenovirus of the invention
in combination with Taxol.RTM. to treat neoplasms, and that the
combination therapy was more effective at killing tumor cells than
either agent alone.
[0267] Combination Therapy In Vitro.
[0268] The cells were subjected to one of three treatment regimes:
In treatment 1, the cells were pretreated with Taxol.RTM.
twenty-four hours before exposure to the p53 adenovirus construct
A/C/N/53. In treatment two, the cells were pretreated with the p53
adenovirus construct and then later contacted with Taxol.RTM.. In
treatment three, the cells were contacted simultaneously with both
the Taxol.RTM. and the p53 adenovirus. Thus, the p53 Ad and
Taxol.RTM. can be administered within the same twenty four (24)
hour period or concurrently.
[0269] Approximately 1.5.times.10.sup.4 cells in culture medium
(head and neck cell lines SCC-9, SCC-15, and SCC-25 in 1:1 mix of
DMEM+Ham's F12 media with 0;4 .mu.g/ml cortisol and 10% FBS and 1%
non-essential amino acids, prostate DU-145 and Ovarian SK-OV-3 in
Eagles essential medium plus 10% FBS) were added to each well on a
96 well microtitre plate and cultured for about 4 hours at
37.degree. C. and 5% CO.sub.2. The drug (Taxol.RTM.), the p53
adenovirus, or the appropriate vehicle/buffer was added to each
well. As paclitaxel is not water soluble, the drug was dissolved in
ethyl alcohol prior to administration. Cells were then cultured
overnight at 37.degree. C. and 5% CO.sub.2. p53 adenoviruses were
administered in phosphate buffer (20 mM NaH.sub.2PO.sub.4, pH 8.0,
130 mM NaCl, 2 mM MgCl.sub.2, 2% sucrose).
[0270] Cell death was then quantitated according to the method of
Mosmann (1983) J. Immunol. Meth., 65: 55-63. Briefly, approximately
25 .mu.l of 5 mg/ml MTT vital dye [3-(4,5
dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide] was added to
each well and allowed to incubate for 3-4 hrs. at 37.degree. C. and
5% CO.sub.2. Then 100 .mu.l of 10% SDS detergent was added to each
well and allowed to incubate overnight at 37.degree. C. and 5%
CO.sub.2. Signal in each well was then quantitated using a
Molecular devices microtiter plate reader (Thermo-Max). The
particular cell lines used and the results obtained therefrom are
listed in Table 1.
1TABLE 1 In vitro evaluation of the adjunctive anti-cancer agent
Taxol .RTM. combined with tumor suppressor nucleic acid. Taxol
A/C/N/ Cell line dose 53 dose Treatment Cancer (.mu.g/ml) (m.o.i.)
Taxol .RTM. pretreatment p53 pretreatment simultaneous SK-OV-3 0.37
40 additive effect no effect additive effect Ovarian p .ltoreq.
0.0001 p > 0.2000 p .ltoreq. 0.0001 cancer SCC-25 0.10 or 2.5 or
additive effect very small effect additive effect Head and 0.01 5.0
p .ltoreq. 0.0001 p = 0.0606 p .ltoreq. 0.0001 neck cancer SCC-15
0.10 or 2.5 or additive effect additive effect additive effect Head
and 0.01 5.0 p .ltoreq. 0.002 p .ltoreq. 0.0001 p .ltoreq. 0.0001
neck cancer DU-145 0.36 or 2.5 or additive effect additive effect
additive effect prostate 0.036 or 5.0 p .ltoreq. 0.03 p .ltoreq.
0.0001 p .ltoreq. 0.0001 cancer 0.0036 SCC-9 0.12 or 2.5 or
additive effect additive effect additive effect Head and 0.012 or
5.0 p .ltoreq. 0.01 p .ltoreq. 0.0001 p .ltoreq. 0.0001 neck 0.0012
cancer
[0271] In general, the p53 adenovirus was more effective when added
after or concurrently with Taxol.RTM. than when it was added first.
These results suggest a synergistic interaction between A/C/N/53
and Taxol.RTM..
[0272] Isobologram Analysis Establishes Synergistic Effect.
[0273] SK-OV-3 (p53 null) ovarian tumor cells were treated with
combinations of Taxol.RTM. and p53/adenovirus (A/CIN/53) as
illustrated in Table 2. Dosing was performed as described above.
Cell death was quantitated on day 3 using the MTT assay as
described above. In addition, a dose response curve for p53 Ad
alone (using the doses listed in Table 2) was generated (after 2
day cell exposure to the drug) and a dose response curve for
Taxol.RTM. alone was performed using the doses listed above (3 day
cell exposure to the drug).
2TABLE 2 Treatment groups for combined Taxol .RTM. and p53 Ad
(A/C/N/53) treatments. Taxol .RTM. p53 AD Group (.mu.g/ml) (m.o.i.)
1 0.001 0.5 2 0.01 0.5 3 0.1 0.5 4 0.5 0.5 5 1 0.5 6 5 0.5 7 10 0.5
8 20 0.5 9 0.001 1 10 0.01 1 11 0.1 1 12 0.5 1 13 1 1 14 5 1 15 10
1 16 20 1 17 0.001 5 18 0.01 5 19 0.1 5 20 0.5 5 21 1 5 22 5 5 23
10 5 24 20 5 25 0.001 10 26 0.01 10 27 0.1 10 28 0.5 10 29 1 10 30
5 10 31 10 10 32 20 10 33 0.001 25 34 0.01 25 35 0.1 25 36 0.5 25
37 1 25 38 5 25 39 10 25 40 20 25 41 0.001 50 42 0.01 50 43 0.1 50
44 0.5 50 45 1 50 46 5 50 47 10 50 48 20 50
[0274] FIG. 1 illustrates the inhibition of cell proliferation (as
compared to the buffer control) as a function of treatment. In
general increasing doses of either Taxol.RTM. or p53 decreased the
rate of cell proliferation with the combination of p53 and
Taxol.RTM. having a greater effect than either drug alone.
[0275] FIG. 2 illustrates an isobologram analysis of these data
using the Isobole method as reviewed by Berenbaum (1989) Pharmacol.
Rev. 93-141. Synergism between Taxol.RTM. and p53 (A/C/N/53) was
observed when cells were pretreated with Taxol.RTM. 24 hours before
p53 (A/C/N/53) treatment. In FIG. 2, the straight line (isobole for
ED.sub.30) represents the effects on cell proliferation which would
be expected if treatment with the two drugs were merely additive.
In fact, the observed effects fall to the lower left of the isobole
line indicating that lower than predicted concentrations of each
drug were needed and a synergistic interaction between the two
drugs has occurred.
Example 2
p53 Adenovirus-Mediated Gene Therapy Against Metastases
[0276] The invention provides for the administration of nucleic
acid expressing a tumor suppressor polypeptide in the treatment of
metastases. The following example details the ability of a p53
expressing adenovirus of the invention to infect various tissues in
the body and to treat metastases.
[0277] Female scid mice (mice homozygous for the SCID mutation lack
both T and B cells due to a defect in V(D)J recombination) were
injected with 5.times.10.sup.6 MDA-MB-231 mammary carcinoma cells
into their mammary fat pads. After the primary tumors were well
established and had time to metastasize to the lungs, the primary
tumors were surgically removed (on day 11). Mice were treated with
intravenous A/C/N/53 or with control buffer on days 23, 30, 37, 44
(1qW) with control buffer or with A/C/N/53 (a p53 in an adenovirus)
at 4.times.10.sup.8 C.I.U./injection. On day 49, the lungs were
harvested, fixed, stained and examined microscopically.
[0278] The results are illustrated below in Table 3.
3TABLE 3 Inhibition of MDA-MB-231 lung metastases using A/C/N/53
Treatment No Metastases .ltoreq.6 Metastases .gtoreq.84 Metastases
Buffer 11 (65%) 1 (6%) 5 (29%) n = 17 A/C/N/53 5 (50%) 4 (40%) 1
(10%) n = 10
[0279] The A/C/N/53 treatment decreased the number of metastases in
the mice that had them.
[0280] In a second experiment, 231 tumors in the mammary fat pads
of scid or scid-beige mice were given peritumoral injections with
A/C/N/53. A total dose of 2-4.times.10.sup.9 C.I.U. given in 10
injections decreased the number of mice with lung metastases by 80%
in scid mice and 60% in scid-beige mice. Also the number of
metastases per mouse was dramatically reduced in mice with any lung
tumors at all. As indicated above, intravenous dosing with A/C/N/53
also demonstrated efficacy against lung metastases in scid mice.
These data indicate that cancer gene therapy with A/C/N/53 may
impact the severity of metastatic disease in addition to decreasing
primary tumor burden.
[0281] In another experiment, female scid mice were injected with
5.times.10.sup.6 MDA-MB-231 mammary tumor cells/mouse into the
mammary fat pad on day 0. The primary (mammary) tumors were
surgically removed on day 18. The mice were treated with
intravenous injections of buffer, beta-gal AD, or p53 Ad (A/C/N/53)
on days 21, 24, 32, 39, and 36. The virus dose per injection was
4.times.10.sup.8 C.I.U. (A/C/N/53) (PN/C.I.U. =23.3) and
9.3.times.10.sup.9 particles beta-gal Ad (PN/C.I.U. =55.6;
1.7.times.10.sup.8 C.I.U.).
[0282] Lungs and livers were harvested on day 51 and fixed in
formalin. Tissue sections were evaluated for lung tumors and for
liver damage. Major organs from 2 buffer and 2 beta-gal Ad mice
were flash-frozen for cryosectioning and analysis of
B-galactosidase enzyme activity.
4TABLE 4 Inhibition of MDA-MB-231 lung metastases using A/C/N/53
Lung Metastases per Beta-gal Ad p53 Ad Mouse Buffer Gp.* Gp.* Grp.
.ltoreq.20 11% (1) 8% (1) 21% (3) >20 and .ltoreq.100 11% (1)
33% (4) 79% (11) >100 and .ltoreq.200 33% (3) 33% (4) 0% (0)
>200 and .ltoreq.300 33% (3) 17% (2) 0% (0) >300 11% (1) 8%
(1) 0% (0) Total number 9 12 14 evaluated Regrowth of primary 82%
(9/11) 88% (14/16) 100% (14/14) tumor *Number of metastases is
under-estimated. Multiple tumors had grown together in these
lungs.
[0283] The number of metastases per lung in the buffer and beta-gal
Ad groups was not significantly different (p=0.268, see Table
4).
[0284] p53 Ad treatment significantly reduced the number of
metastases per lung when compared to either the buffer of beta-gal
Ad groups (p<0.001 and p<0.002, respectively). In addition to
the reduction in the number of metastases, there was also a
dramatic reduction in the size of lung metastases in the p53 Ad
group. In the control groups, tissue sections from most lungs were
>50% occupied by neoplastic tissue and individual tumors were no
longer recognizable over large areas of the lungs. In contrast,
lung metastases in most of the p53 Ad group were small and easily
distinguishable as individual tumors.
[0285] Adenovirus Tissue Distribution
[0286] Liver tissues had the highest number of infected cells
(about 50%) and beta-galactosidase activity was intense. Lung had
scattered patches of infected cells evenly distributed throughout
the tissue. Intestines and stomach had periodic infection of cells
in the outer smooth muscle wall surrounding the organs. There was
also beta-galactosidase activity in scattered microvilli along the
lumen. The smooth outer muscle wall surrounding the uterus had
periodic infection of cells similar to that seen in the intestines.
Most stromal cells in the ovary were infected. The spleen had
scattered beta-galactosidase activity in the smooth muscle
components of the organ. There were very few infected cells
(<1%) inside the main bulk of striated heart muscle. There were
almost no infected cells in primary tumors in the mammary fat pad,
nor in the underlying striated muscle. There were no infected cells
in the kidney.
[0287] Liver Pathology.
[0288] All livers were grossly normal when necropsied. There was no
overt necrosis in any liver. However, mice treated with adenovirus
did have hepatocellular abnormalities (not present in the buffer
group) which included elevated numbers of cells in mitosis,
cellular inclusions, and changes in hepatocyte size and shape.
Example 3
p53 Adenovirus-Mediated Gene Therapy Against Human Breast Cancer
Xenografts
[0289] The invention provides for the treatment of various cancers
by the administration of nucleic acid expressing a tumor suppressor
polypeptide. The following example details the ability of a p53
expressing adenovirus of the invention to treat human breast
cancer.
[0290] Introduction of wild-type p53 into tumors with null or
mutant p53 offers a novel strategy for controlling tumor growth.
Casey (1991) Oncogene 6: 1791-1797, introduced wild-type p53 into
breast cancer cells in vitro via a plasmid DNA vector. The number
of MDA-MB-468 (p53.sup.mut) and T47D (p53.sup.mut) colonies arising
after plasmid transfection was reduced 50% by wild-type p53. Also,
none of the resultant colonies expressed the wild-type p53
transfectant. By contrast, the number of MCF-7 (pS3 wt) colonies
was not affected. Negrini (1994) Cancer Res. 54: 1818-1824,
conducted a similar study using MDA-MB-231 cells. Colony formation
was reduced 50% by transfection with a plasmid containing wild-type
p53 and none of resultant colonies expressed wild-type p53.
Paradoxically, in this study similar results were observed with
MCF-7 cells.
[0291] In the study described in this example the efficacy of a
replication-deficient, recombinant, E1 region-deleted, p53
adenovirus (p53 Ad; (A/C/N/53) Wills (1994) supra) was tested
against three human breast cancer cell lines expressing mutant p53,
MDA-MB-231, MDA-MB-468, and MDA-MB-435. The MDA-MB-231 cells carry
an Arg-to-Lys mutation in codon 280 of the p53 gene (Bartek (1990)
Oncogene 5: 893-899). The MDA-MB-468 cells carry an Arg-to-His
mutation in codon 273 (Id.). The MDA-MB-435 cells carry a
Gly-to-Glu mutation in codon 266 of the p53 gene (Lesoon-Wood
(1995) Hum. Gene Ther. 6:395-405).
[0292] Previous studies have shown high levels of wild-type p53
expression in tumor cells from human breast, ovary, lung,
colorectum, liver, brain, and bladder after infection with p53 Ad
in vitro (Wills (1994) supra., Harris et al. (1996) Cancer Gene
Therapy 3: 121-130). Adenovirus-mediated p53 expression ultimately
resulted in changes in cell morphology and the induction of
apoptosis in p53 null or mutant p53 tumor cells. Infection of 468
breast cancer cells by p53 Ad at 10 m.o.i. (multiplicity of
infection) caused almost 100% inhibition of DNA synthesis by 72
hrs. after infection. In addition, infection with p53 Ad in vitro
inhibited proliferation of MDA-MB-468 and MDA-MB-231 cells with
ED.sub.50 values of 3.+-.2 and 12.+-.10 m.o.i., respectively.
Proliferation of three other p53-mutant breast carcinoma lines was
also inhibited at low concentrations of p53 Ad. The ED.sub.50
values were 16.+-.4 m.o.i. for SK-BR-3 cells, 3.+-.3 m.o.i. for
T-47D cells, and 2.+-.2 m.o.i. for BT-549 cells. Infection of
MDA-MB-468 and MDA-MB-231 cells with 30 m.o.i. of an equivalent,
recombinant adenovirus expressing E. coli beta-galactosidase
(beta-gal), instead of p53, resulted in >67% beta-gal positive
MDA-MB-468 cells and 34-66% beta-gal positive MDA-MB-231 cells. By
correlating the percentage of beta-gal positive cells with the p53
anti-proliferative effects in a large panel of tumor cells with
altered p53, Harris et al. (supra.) showed a strong positive
correlation between the degree of p53-induced inhibition and the
degree of adenovirus transduction. In contrast, cell lines
expressing normal levels of wild-type p53 were minimally affected
by p53 transduction, independent of the adenovirus transduction
rate.
[0293] Proliferation of MCF7 and HBL-100 cells, two human mammary
cell lines containing wild-type p53, was relatively unaffected by
p53 Ad concentrations greater than or equal to 99 m.o.i. in vitro.
In other words, growth inhibition of MCF-7 and HBL-100 cells
required p53 Ad concentrations at least 8- and 33-fold higher than
the ED.sub.50 values for -231 and -468 cells, respectively. Using a
similar recombinant p53 Ad, Katayose (1995) Clin. Cancer Res.
1:889-897, demonstrated increased p53 protein expression, decreased
cell proliferation, and increased apoptotic cell death in -231
cells transduced in vitro. This study extends these in vitro
results with -468 and -231 cells to breast cancer xenografts in
vivo. The efficacy of adenovirus-mediated p53 gene therapy is
evaluated in another breast cancer cell line (MDA-MB-435) which is
resistant to adenovirus transduction in vitro.
[0294] Materials and Methods
[0295] Cell Lines and Adenovirus Infections In Vitro
[0296] The human breast cancer cell lines MDA-MB-231, -468, and
-435 were obtained from ATCC (Rockville, Md., USA). The -231 cells
were cultured in DMEM (Life Technologies, Grand Island, N.Y.) with
10% fetal calf serum (FCS; Hyclone, Logan, Utah) at 37.degree. C.
and 5% CO.sub.2. The -468 cells were cultured in Leibovitz's L-15
medium (Life Technologies) containing 10% FCS at 37.degree. C. The
-435 cells were cultured in Leibovitz's L-15 medium with 15% FCS
and 10 .mu.g/ml bovine insulin (Sigma Chem. Co., St. Louis, Mo.) at
37.degree. C.
[0297] Construction and propagation of the human wild-type p53
expressing and E. coli beta-galactosidase (beta-gal) expressing
recombinant adenoviruses (rAd), where transgene expression is
directed by the human cytomegalovirus promoter, have been described
previously (Wills (1994) supra). Adenoviruses were administered in
phosphate buffer (20 mM NaH.sub.2PO.sub.4, pH 8.0, 130 mM NaCl, 2
mM MgCl.sub.2, 2% sucrose). C.I.U. is defined as cellular
infectious units. The concentration of infectious viral particles
was determined by measuring viral hexon protein positive 293 cells
after a 48 hr. infection period (Huyghe (1995) supra).
[0298] For in vitro infection studies with p53 Ad, cells were
plated at a density of 1-5.times.10.sup.4 cells/well in 12-well
tissue culture dishes (Becton Dickinson, Lincoln Park, N.J., USA).
Cells were transduced with 0, 10, or 50 m.o.i. (multiplicity of
infection-C.I.U./cell) p53 Ad and cultured for 72 hrs. as
previously described (Wills (1994) supra.). For in vitro infection
studies with beta-gal Ad, cells were plated at a density of
1.times.10.sup.5 cells/well. The cells were transduced with 0, 10,
50, or 100 m.o.i. beta-gal Ad. After 48 hours, the cells were fixed
with 0.2% glutaraldehyde (Sigma Chemical Co.) then washed 3 times
with PBS (Life Technologies). The cells were then assayed in 1 ml
of X-Gal solution [1.3 mM MgCl.sub.2, 15 mM NaCl, 44 mM Hepes
buffer, pH 7.4, 3 mM potassium ferricyanide, and 1 mg/ml X-Gal in
N,N-dimethylformamide (10% final conc.)]. X-Gal was purchased from
Boehringer Mannheim Corp., Indianapolis, Ind. All other chemicals
were purchased from Sigma.
[0299] To determine the percentage of transduced cells, 5
microscope fields were counted from each culture well and the
average percent expressing beta-galactosidase was calculated for 3
wells at each m.o.i.
[0300] Adenovirus Treatment In Vivo
[0301] Athymic female nude mice were purchased from Charles River
Laboratories (Wilmington, Mass., USA). All mice were maintained in
a VAF-barrier facility and all animal procedures were performed in
accordance with the rules set forth in the N.I.H. Guide for the
Care and Use of Laboratory Animals. Tumor cells were injected
subcutaneously or into the mammary fat pad.
[0302] Cell inoculations were: 5.times.10.sup.6-231 cells/mouse,
1.times.10.sup.7 MDA-MB-468 cells/mouse, or 1.times.10.sup.7
MDA-MB-435 cells/mouse. Tumors were allowed to grow in vivo for
10-11 days before the start of dosing, except for one -468
experiment where the tumors grew for 33 days before treatment
started. Tumor volume was calculated as the product of measurements
in three dimensions. Tumor volumes for different treatment groups
on each day were compared by Student's t test using Statview II
software (Abacus Concepts, Berkeley, Calif.). Average percent
inhibitions for groups dosed on days 0-4 and 7-11 were calculated
using significant values (p<0.05) from day 14 to the end of the
study.
[0303] The specific effects of p53 were distinguished from
adenovirus vector effects by subtracting the average tumor growth
inhibition caused by beta-gal Ad from growth inhibition caused by
p53 Ad. All virus injections were peri/intra-tumoral. In general,
two 5-day courses of tumor therapy (i.e., 5 injections) were given
to each mouse, separated by a 2 day "resting period". In some
cases, this dosing regime was extended for more than 2 weeks and/or
buffer vehicle was substituted for virus for some injections. Tumor
growth curves show mean tumor volume.+-.s.e.m.
[0304] Histology and Apoptag Immunohistochemistry
[0305] Tissue samples were fixed in 10% buffered formalin and
processed overnight in a Miles VIP Tissue Processor, then imbedded
in paraffin. Five micron tissue sections were cut with a Leitz
microtome. The slides were stained with a routine Harris
hematoxylin and eosin stain (Luna et al. (1968), Manual of
Histologic Staining Methods of the Armed Forces Institute of
Pathology. New York: McGraw Hill Book Co.).
[0306] Apoptag in situ apoptosis detection kits were purchased from
Oncor (Gaithersburg, Md., USA). Samples were assayed as per kit
directions. Briefly, deparaffinized, rehydrated tissue sections
were treated with Oncor Protein Digesting Enzyme, incubated with
TdT, and developed using an avidin-peroxidase kit (rabbit IgG-Sigma
Chem. Co. .sup.#EXTRA-3) and DAB (Vector Lab. .sup.#SK4100). Slides
were counterstained with methyl green.
[0307] Beta-Galactosidase Assay
[0308] Tumors were embedded in TBS (Triangle Biomedical Sciences,
Durham, N.C., USA) and flash frozen in a 2-methylbutane/dry ice
bath. Frozen tissue sections (8 .mu.m thick) were fixed in 0.5%
glutaraldehyde at 4.degree. C. for 5 min. and then assayed for
beta-gal expression as described above.
[0309] Integrin FACS Analysis
[0310] Cells were suspended by treatment with 0.02% EDTA, pelleted,
and then washed 2.times. with PBS. Cells were then resuspended at a
concentration of 1.times.10.sup.6 cells/ml and incubated with
primary antibodies (final conc. 1:250/ml) at 4.degree. C. for 1 hr.
Cell suspensions were washed 2.times. with PBS to remove excess
primary antibody. Cells were then incubated with FITC-conjugated
rabbit antimouse adjunctive antibody (final conc. 1:250/ml, Zymed)
at 4.degree. C. for 1 h. Cells were washed as before with PBS and
immediately analyzed. Fluorescence was measured with a FACS Vantage
flow cytometer (Becton Dickinson, Mountain View, Calif., USA). Side
scatter and forward scatter were determined simultaneously, and all
data were collected with a Hewlett Packard computer equipped with
FACS research software (Becton Dickenson). Primary antibodies used
to detect integrin receptors were obtained from the following
suppliers: anti-alpha, (12084-018, Gibco BRL); anti-beta.sub.3
(550036, Becton Dickenson); anti-alpha.sub.vbeta.su- b.3 (MAP1976,
Chemicon); anti-beta.sub.1, (550034, Becton Dickenson); and
anti-alpha.sub.vbeta.sub.5 (MAB 1961, Chemicon).
[0311] Results
[0312] Adenovirus Transduction Efficiency and p53 Growth Inhibition
In Vitro
[0313] The -231 and -468 cells were both highly transduced in vitro
at an m.o.i. of 10. By contrast, -435 cells were rarely transduced,
even at 100 m.o.i. For -231 cells, 8% (10 m.o.i.), 46% (50 m.o.i.),
and 62% (100 m.o.i.) of the cells were transduced by beta-gal Ad.
For 468 cells, 78% (10 m.o.i.), 84% (50 m.o.i.), and 97% (100
m.o.i.) of the cells were transduced by beta-gal Ad. For 435 cells,
0.5% (10 m.o.i.), 1% (50 m.o.i.), and 1.3% (100 m.o.i.) of the
cells were transduced by beta-gal Ad.
[0314] Infection with 50 m.o.i. p53 Ad resulted in nearly complete
cell death in the 231 and 468 cell cultures. By contrast, p53 Ad
had no detectable effect on the growth of 435 cells.
[0315] p53 Ad Efficacy Against Human Breast Cancer Xenografts
[0316] Adenovirus-mediated p53 gene therapy was highly effective
against -231 and -468 xenografts (FIGS. 3a & 3b). In the 231
experiment, 1 mouse in the beta-gal Ad group and 3 mice in the p53
Ad group were tumor-free at the end of the study, and all tumors
regressed during p53 Ad treatment. Inhibition of -231 tumor growth
averaged 86% (p.ltoreq.0.01). The component of growth inhibition
due to p53 averaged 37%, while adenovirus-specific inhibition
averaged 49% (p<0.01). Inhibition of -468 tumor growth averaged
74% (p.ltoreq.0.001). One mouse in the p53 Ad group was tumor-free
at the end of the study and all tumors regressed during p53 Ad
treatment. The component of growth inhibition due to p53 averaged
45% (p<0.001), while adenovirus-specific inhibition averaged 28%
(p<0.05). No side-effects were observed in either experiment.
The ED.sub.50 values for -231 and -468 tumor growth inhibition were
3.times.10.sup.8 C.I.U. (cell infectious units) and
2.times.10.sup.8 C.I.U., respectively (FIG. 4). The -435 tumors
were almost completely resistant to p53 Ad treatment (FIG. 3c).
Growth inhibition in the 435 tumor groups treated with adenovirus
was not significant,
[0317] FIG. 5 shows a comparison of the efficacy of two p53 Ad
dosing regimes against -231 tumors. All mice were given 5
peritumoral injections per week. All mice treated with the
therapeutic agent (p53 Ad) received a total of 2.2.times.10.sup.9
C.I.U./mouse per week. One group received a single bolus injection
containing the entire week's dose of adenovirus. The other 4
injections for the week consisted of buffer vehicle (1.times.
group). The other treated group received the same Ad dose split
into five injections per week (5.times. group). This dosing regime
was given during weeks 1 and 3 (days 0-4, 14-18). Growth inhibition
averaged 73% for the 5.times. group (p-<0.01), but only 44% for
the 1.times. group (p<0.05 for the first three weeks of the
study, not significant after day 21). The first cycle of p53 gene
therapy was more effective than the second cycle. After the first
therapy cycle, 4 mice in the 1.times. group, 5 mice in the 5.times.
group, and 1 mouse in the vehicle control group were tumor-free.
One mouse in the 5.times. group relapsed with a very small tumor by
day 21. No further "cures" were observed after the second cycle of
therapy.
[0318] FIG. 6 shows an experiment using 468 tumors that were
initially 4-fold larger than the 468 tumors shown in FIG. 3b,
treated with a 10-fold lower dose of adenovirus. A total dose of
2.2.times.10.sup.8 C.I.U. p53 Ad/mouse per week was administered.
One group received a single bolus injection of virus, followed by 4
injections of buffer per week (1.times. group). The other treated
group received the same viral dose split into five injections per
week (5.times. group). These dosing schedules were given for 6
weeks. The total viral dose administered over 6 weeks was
approximately half the dose used in FIG. 3. This dosing regime
resulted in a cytostatic effect on tumor volume in mice treated
with p53 Ad (p.ltoreq.0.05). Treatments given early in the study
appeared to be more effective than those given during later weeks.
One mouse in the 5.times. group was tumor-free by day 21. However
when tumor growth inhibition in all mice was compared, the IX
dosing regime (60%) was slightly, but not significantly, more
effective than the 5.times. regime (55%). By 1 week after the end
of dosing, the tumor growth rate in the 5.times. group started to
increase. One month after the start of the study, the vehicle
control tumors started to necrose and growth plateaued.
[0319] In Vivo Infectivity After Repeated Adenovirus Exposure
[0320] At the end of the studies shown in FIGS. 5 and 6, some
tumors were injected with beta-gal Ad. These tumors were harvested
24 hrs. later and frozen tissue sections were assayed for
beta-galactosidase expression. Tumors treated with p53 Ad for 2 or
6 weeks were still transduced by beta-gal Ad, although transduction
was lowest in the -468 tumors treated for 6 weeks with p53 Ad
5.times. per week. Sections from only 1 of the 3-468 tumors
injected in the 5.times. group had cells expressing
beta-galactosidase.
[0321] Induction of Apoptosis In Vivo by p53 Ad
[0322] The MDA-MB-231 and MDA-MB-468 breast cancer xenografts in
nude mice were injected with 1-5.times.10.sup.8 C.I.U. p53Ad or
buffer 48 to 72 hrs. before harvest. The induction of apoptosis by
p53 Ad was assayed using Apoptag immunohistochemistry on tissue
sections. Tumors injected with p53 Ad had areas of extensive
apoptosis along the needle track(s) of tumors injected
intra-tumorally and around the outside border of tumors injected
peritumorally. By contrast, tumors injected with buffer had only a
few scattered apoptotic cells, as expected.
[0323] Comparison of Integrin Expression in MDA-MB-231 and
MDA-MB-435 Cells
[0324] FACS analysis of integrin expression was performed on
MDA-MB-231 and MDA-MB-435 cells to determine whether the low Ad
transduction of MDA-MB-435 cells was due to a deficiency in the
alpha.sub.v integrins needed for internalization of Ad types 2, 3,
and 4 (Wickham et al. (1993) Cell, 73: 309-319; Wickham et al.
(1994) J. Cell Biol., 127: 257-264; and Mathias et al. (1994) J.
Virol. 68: 6811-6814). Both cell types expressed alpha.sub.v,
alpha.sub.vbeta.sub.3, alpha.sub.vbeta.sub.5, and beta.sub.1
integrin moieties at approximately the same levels. Integrin
alpha.sub.vbeta.sub.3 and beta.sub.3 expression were higher on
MDA-MB-435 cells than on MDA-MB-231 cells.
[0325] Discussion: When a total dose of 2.2.times.10.sup.9 C.I.U.
p53 Ad was administered in 10 injections, tumor growth inhibition
was 74% for MDA-MB-468 tumors and 86% for MDA-MB-231 tumors, but
was not significant for MDA-MB-435 tumors. In MDA-MB-468 tumors,
61% of the total response was p53-specific, while in MDA-MB-231
tumors, 43% of the total response was p53-specific. The ability of
beta-gal Ad to transduce MDA-MB-231, MDA-MB-468, and MDA-MB-435
cells in vitro was generally predictive of the in vivo results. At
the same virus concentrations, -468 cells had a slightly higher
transduction rate than MDA-MB-231 cells, while MDA-MB-435 cells
were resistant to adenovirus transduction. The MDA-MB-435 results
in vitro correlated with the very poor response in vivo.
[0326] Systemic treatment of nude mice bearing MDA-MB-435 tumors,
with a p53-liposome vector, has been shown to cause tumor growth
inhibition, and in some cases, recession (Lesoon-Wood et al. (1995)
Hum. Gene Ther., 6: 395-405). P53-liposome treatment also reduced
the number of lung metastases. These results demonstrate that the
lack of MDA-MB-435 tumor response to p53 Ad treatment in this study
was not due to an inability of p53 to inhibit growth and metastasis
of MDA-MB-435 tumors. Rather, these results suggest it was the low
adenovirus transduction efficiency of MDA-MB-435 cells that caused
their nonresponsiveness to p53 Ad treatment.
[0327] The alpha.sub.v integrins have been implicated as cellular
elements required for efficient internalization of type 2, 3, and 4
adenoviruses (Wickham (1993) supra.; Wickham (1994) supra.; and
Mathias (1994) supra.). It is likely that alpha.sub.v integrins
perform the same role for type 5 Ad. Wickham et al. (1994) supra.,
observed 5-10-fold higher internalization of a recombinant type 5
adenovirus in cells transfected with alpha.sub.vbeta.sub.5 as
compared to cells lacking alpha.sub.v expression or transfected
with alpha.sub.vbeta.sub.3. The human embryonic kidney -293 cells
used for production of the p53 Ad used herein express
alpha.sub.vbeta.sub.1, but not alpha.sub.vbeta.sub.3 integrins
(Bodary (1990) J. Biol. Chem. 265: 5938-5941). Therefore, it seemed
prudent to measure -435 cell expression of the alpha.sub.v,
beta.sub.1, beta.sub.3, and alpha.sub.5 integrin subunits. Both
MDA-MB-231 and MDA-MB-435 cells expressed roughly equivalent levels
of the integrin family molecules. Therefore, the lack of Ad
transduction of MDA-MB-435 cells is not due to a deficiency in
alpha integrin expression. Currently, no literature exists on the
identity of the cellular receptor required for Ad binding to target
cells. It is possible that the MDA-MB-435 cells are deficient in
this receptor or that some other component required for viral
binding, internalization, or gene expression is defective.
[0328] The continued efficacy of p53 Ad over multiple cycles of
therapy was examined in the MDA-MB-231 and MDA-MB-468 tumor models.
It appears that efficacy decreased with continued dosing, however
this effect needs to be examined in more detail. Prevailing theory
holds that adenovirus infection generates a rapid inflammatory and
cytolytic response mediated by cytotoxic T cells in hosts with
fully functional immune systems (reviewed by Wilson (1995) Nature
Med. 4: 887-889). This T cell response is stimulated by adenovirus
antigens produced in host cells and presented in conjunction with
MHC moieties on the cell surface. Neutralizing antibodies specific
for cells transduced by adenovirus are produced later in the immune
response and are believed responsible for the reduced ability to
reinfect host cells with adenovirus after the initial inoculations.
The athymic nude mice used in these studies have a defective T cell
immune response to foreign antigens, but are able to generate a B
cell-mediated antibody response (Boven (1991) The Nude Mouse in
Oncology Research. Boston: CRC Press). The production of
neutralizing anti-adenovirus antibodies could explain the reduced
efficacy of p53 adenovirus (p53 Ad) therapy over time in the
present studies. The impaired immune function in nude mice and the
poor blood supply to the interiors of the tumor xenografts could
explain the partial effectiveness of the p53 Ad even after 6 weeks
of dosing and the ability to infect a few tumor cells with beta-gal
Ad even after repeated p53 Ad injections.
[0329] In addition to breast cancer, a number of other cancers have
been treated with recombinant adenoviruses expressing wild-type
p53. These reports include models of cervical cancer (Hamada (1996)
Cancer Res. 56: 3047-3054), prostate cancer (Eastham (1995) Cancer
Res. 55: 5151-5155), head and neck cancer (Clayman (1995) Cancer
Res. 55: 1-6), lung cancer (Wills (1994) supra.), ovarian cancer
(13), glioblastoma (27, 28), and colorectal cancer (13, 29).
Collectively, these data support ongoing clinical investigations
evaluating the effects of adenovirus-mediated p53 gene therapy. The
present results demonstrate the ability of wild-type p53 to curtail
cancerous cell growth in vivo in breast cancer xenografts
expressing mutant p53. The present studies also confirm that
adenovirus appears to be an efficient delivery vehicle for the 53
when target cells express the appropriate viral "receptor(s)".
Example 4
Further Investigations of Treatment Regimen on Tumor Inhibition
[0330] The invention provides for the treatment of various cancers
by the administration of nucleic acid expressing a tumor suppressor
polypeptide using various dosage regimens. The following example
details the increased efficacy of split dosing the administration
of p53 expressing adenovirus of the invention.
[0331] In order to investigate the effect of a single dosage
regimen as compared to split doses administered over a period of
time, scid mice injected with MDA-MB-468 and MDA-MB-231 tumors were
treated with a total dose per mouse of 1.times.10.sup.9 I.U. p53 Ad
(A/C/N/53) administered as a single bolus injection or split into 3
or 5 injections administered once a day over the course of a week
(indicated by arrows in FIG. 7).
[0332] The results obtained with MDA-MB-468 tumors were similar to
those obtained with MDA-MB-23 tumors and are illustrated in FIGS.
7a, 7b, and 7c. In general, split dosing inhibited tumor growth
better than single bolus injections with the 5 injection dosage
regimen having significant improvement over the 3 injection dosage
regimen.
Example 5
Dexamethasone Mutes the Inhibition of Tumor Growth Associated with
NK Cell-Mediated Anti-Adenovirus Immune Response
[0333] It has been demonstrated that repeated administration of
adenovirus vectors can induce an anti-adenovirus immune response.
To investigate whether the immunosuppressant properties of low dose
Dexamethasone (Dex) can inhibit the anti-adenovirus immune response
(e.g., NK cell response) MDA-MB-231 tumors in scid mice were
treated with recombinant virus of the invention in the absence and
in the presence of dexamethasone.
[0334] Approximately 5.times.10.sup.6 MDA-MB-231 cells/mouse were
injected into the mammary fat pads of female scid mice on day 0. On
day 11, dexamethasone or placebo pellets were implanted
subcutaneously. The 5 mg pellets were designed to release 83.3
.mu.g dexamethasone/day continuously for 60 days (Innovative
Research of America, Sarasota, Fla.). All mice received a total of
10 peritumoral injections given once a day on days 14-18 and 21-25
(0.1 ml per injection). The total virus dose was 2.times.10.sup.9
C.I.U./mouse (p53 AD (A/C/N/53 or beta-galactosidase Ad).
Treatments were as listed in Table 5.
5TABLE 5 Treatment of MDA-MB-231 tumors in scid mice. Group Hormone
Gene Therapy 1 placebo buffer 2 placebo beta-gal Ad 3 placebo p53
Ad 4 dexamethasone buffer 5 dexamethasone beta-gal Ad 6
dexamethasone p53 Ad
[0335] Low dosage dexamethasone treatment had no significant effect
on the growth rate of MDA-MB-231 tumors in scid mice (p>0.05).
No adverse side effects of dexamethasone were observed. Treatment
of tumors with beta-galactosidase adenovirus caused significant
inhibition of tumor growth in placebo control tumors (p<0.001,
days 21-30), but not in dexamethasone treated tumors (P>0.05,
see FIG. 8). Tumors treated with placebo and beta-gal Ad grew
slower than tumors treated with placebo and dexamethasone
(p<0.01, days 23-30).
[0336] There was no significant p53-specific inhibition of tumor
growth in placebo control tumors (p>0.05). By contrast, tumors
treated with dexamethasone and p53 Ad did grow significantly slower
than tumors treated with dexamethasone and beta-gal Ad (P<0.02,
days 21-30) or placebo and p53 Ad (p<0.04, days 21-30).
[0337] Low dose dexamethasone treatment thus muted the inhibition
of tumor growth associated with the anti-adenovirus immune response
(e.g., NK cell response) in scid mice without adverse side-effects.
The data also suggest that low dose dexamethasone treatment may
stimulate transgene (e.g., p53) expression driven by the CMV
promoter in recombinant adenoviruses. Conversely, dexamethasone may
increase adenovirus transduction efficiency and thereby increase
tumor cell death.
[0338] The MDA-MB-231 breast cancer model was next used to evaluate
the anti-tumor efficacy of Ad with and without p53 in mice with
differing abilities to mount an immune response to foreign
antigens. Nude mice with non-functional T cells, scid mice with
nonfunctional T and B cells but elevated NK cells, and scid-beige
mice with nonfunctional T, B, and NK cells were studied.
[0339] To study the efficacy of rAd5/p53 (described supra) against
MDA-MD-231 xenografts: nude mice were given a total dose of
2.2.times.10.sup.9 C.I.U. Ad per mouse split into 10 injections on
days 0 to 4 and 7 to 11. SCID mice were given a total virus
dose=4.times.10.sup.9 C.I.U. split into 10 doses given on days 0-4
and 7-11. SCID-Beige mice were given a total virus
dose=1.6.times.10.sup.9 C.I.U. split into 10 doses given on days
0-4 and 7-11. All mice were treated with p53 Ad, beta-gal Ad, or
vehicle alone.
[0340] In nude mice (non-functional T cells) or scid mice
(nonfunctional T and B cells; elevated NK cells), intratumoral
dosing with control Ad vector (no p53 insert) caused some
inhibition of tumor growth. Ad expressing p53 (rAd5/p53) had
greatly enhanced anti-tumor efficacy compared to control Ad. By
contrast, in scid-beige mice (nonfunctional T, B, and NK cells),
anti-tumor efficacy was all due to p53 expression with no Ad vector
component to the tumor growth inhibition. These data demonstrate a
previously unrecognized role for NK cells in Ad-mediated tumor
growth inhibition. The data also suggest that suppression of the
immune system might abrogate some vector-specific, NK cell
mediated, side effects.
Example 6
Combination p53 Adenovirus and Chemotherapy Treatment
[0341] The invention provides for the combined administration of
nucleic acid expressing a tumor suppressor polypeptide and
chemotherapeutic agents in the treatment of neoplasms. The
following example details the ability of a p53 expressing
adenovirus of the invention in combination with various anti-cancer
drugs, cisplatin, doxorubicin, 5-fluorouracil (5-FU), methotrexate,
and etoposide, to treat neoplasms, and that the combination therapy
was more effective, i.e., was synergistic, at killing tumor cells
than either agent alone.
[0342] p53 Administered with Chemotherapeutic Drugs In Vitro
[0343] Cisplatin, Doxorubicin, 5-Fluorouracil (5-FU), Methotrexate,
and Etoposide, in Combination with p53
[0344] The effect of the clinically-relevant anti-cancer drugs
cisplatin, doxorubicin, 5-fluorouracil (5-FU), methotrexate, and
etoposide, in combination with a tumor suppressor vector of the
invention (A/C/N/53), was investigated in vitro. SCC-9 head and
neck squamous cell carcinoma, SCC-15 head and neck squamous cell
carcinoma, SCC-25 head and neck squamous cell carcinoma, and DU-145
prostate carcinoma cells were subjected to one of three treatment
regimes: In treatment 1, the cells were pretreated with the
anti-cancer chemotherapeutic twenty-four hours before exposure to a
p53 adenovirus construct A/C/N/53. In treatment 2, the cells were
pretreated with the p53 adenovirus construct and then later
contacted with the anti-cancer chemotherapeutic. In treatment 3,
the cells were contacted simultaneously with both the anti-cancer
chemotherapeutic and the p53 adenovirus.
[0345] All cell lines were obtained from ATCC (Rockville, Md.).
SCC-9, SCC-15, and SCC-25 head and neck tumor cells (p53.sup.null)
were cultured in a 1:1 mixture of DMEM and Ham's F-12 (GIBCO/Life
Technologies, Grand Island, N.Y.) with 10% fetal calf serum (FCS;
Hyclone, Logan, Utah), 0.4 .mu.g/ml hydrocortisone (Sigma Chem.
Co., St. Louis, Mo.), and 1% nonessential amino acids (GIBCO) at
37.degree. C. and 5% CO.sub.2. SK-OV-3 human ovarian tumor cells
(p53.sup.null) and DU-145 human prostate tumor cells (p53.sup.mut)
were cultured in Eagle's MEM plus 10% FCS at 37.degree. C. and 5%
CO.sub.2.MDA-MB-231 human mammary tumor cells (p53.sup.mut) were
cultured in DMEM (GE3CO) with 10% fetal calf serum (Hyclone) at
37.degree. C. and 5% CO.sub.2.MDA-MB-468 human mammary tumor cells
(p53.sup.mut) were cultured in Leibovitz's L-15 medium (GEBCO)
containing 10% FCS at 37.degree. C., no CO.sub.2.
[0346] MDA-MB-231 mammary tumor cells carry an Arg-to-Lys mutation
in codon 280 of the p53 gene and express mutant p53 (Bartek (1990)
supra). DU-145 prostate tumor cells carry two p53 mutations on
different chromosomes, a Pro-to-Leu mutation in codon 223 and a
Val-to-Phe mutation in codon 274 (Isaacs (1991) Cancer Res.
51:4716-4720), and express mutant p53. SK-OV-3 ovarian tumor cells
are p53-null (Yaginuma (1992) Cancer Res. 52:4196-4199). SCC-9
cells have a deletion between codons 274 and 285 resulting in a
frame shift mutation; no immunoreactive p53 protein is detectable
in SCC-9 nuclei (Jung (1992) Cancer Res. 52:6390-6393; Caamano
(1993) Am J. Pathol. 142:1131-1139; Min (1994) Eur. J. Cancer
30B:338-345). SCC-15 cells have an insertion of 5 base pairs
between codons 224 and 225; they produce low levels of p53 mRNA,
but no detectable p53 protein (Min (1994) supra). SCC-25 cells have
loss of heterozygosity (LOH) at chromosome 17 and a 2 base pair
deletion in codon 209 on the remaining allele; p53 mRNA is not
detectable in SCC-25 cells and no immunoreactive p53 protein is
observed in their nuclei (Caamano (1993) supra). Approximately
1.5.times.10.sup.4 cells in culture medium (as described in example
1) were added to each well on a 96 well microtitre plate and
cultured for about 4 hours at 37.degree. C. and 5% CO.sub.2.
[0347] Construction and propagation of the human wild-type p53 and
E. coli galactosidase (beta-gal) adenoviruses (Ad) have been
described previously (Wills (1994) supra). The concentration of
infectious viral particles was determined by measuring the
concentration of viral hexon protein positive 293 cells after a 48
hr. infection period (Huyghe (1995) supra). C.I.U. is defined as
cellular infectious units. p53-expressing adenoviruses were
administered in phosphate buffer (20 mM NaH.sub.2PO.sub.4, pH 8.0,
130 mM NaCl, 2 mM MgCl.sub.2, 2% sucrose). The drug, the p53
adenovirus, or the appropriate vehicle/buffer was added to each
well. For in vitro studies with p53 Ad, cells were plated at a
density of 1.5.times.10.sup.4 cells/well on a 96-well plate and
cultured for 4 hrs. at 37.degree. C. and 5% CO.sub.2. Drug, p53 Ad,
or the appropriate vehicle was added to each well and cell culture
was continued overnight. Then drug, p53 Ad, or the appropriate
vehicle was added to each well. Cell culture was continued for an
additional 2 days.
[0348] Cell death was then quantitated according to the MTT assay
as described by Mosmann (1983) J. Immunol. Meth., 65: 55-63.
Briefly, approximately 25 .mu.l of 5 mg/ml MTT vital dye [3-(4,5
dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide] was added to
each well and allowed to incubate for 3-4 hrs. at 37.degree. C. and
5% CO.sub.2. Then 100 .mu.l of 10% SDS detergent was added to each
well and allowed to incubate overnight at 37.degree. C. and 5%
CO.sub.2. Signal in each well was then quantitated using a
Molecular devices microtiter plate reader.
[0349] In all cases, using cisplatin (see Table 6 for summary
results), doxorubicin (see Table 7 for summary results), 5-FU,
methotrexate, and etoposide, the combination therapy was more
effective at killing tumor cells than either agent alone. The
combination of methotrexate and p53 Ad was tested in one cell line.
When SCC-15 cells were treated with 0.7 .mu.M methotrexate 24 hours
before 5 m.o.i. p53 Ad, the combined antiproliferative effect of
the two drugs was only 5% more than with p53 Ad alone, although
this difference was statistically significant (p.ltoreq.0.003).
Pretreatment of DU-145 cells with 2.6 .mu.M etoposide 24 hours
before 5 or 10 m.o.i. p53 Ad resulted in greater combined efficacy
over either drug alone (p.ltoreq.0.0001). When SCC-15 cells were
treated with 0.3 .mu.M etoposide 24 hours before 5 m.o.i. p53 Ad,
the combined antiproliferative effect of the two drugs was only 5%
more than with p53 Ad alone, although this difference was also
statistically significant (p.ltoreq.0.003). The combination of
tumor suppressor gene therapy and anti-neoplastic agents did not
demonstrate antagonistic effects.
[0350] In a second experiment, the efficacy of treatment of normal
cells (MRC-9 cells) was compared with tumor cells (FIG. 9). In this
experiment, growth was assayed as .sup.3H-thymidine incorporation
rather than the MTT assay. The normal cells (diploid fibroblast
MRC-9 cells) did not show more pronounced effects with combination
treatment. As would be expected, the effect of tumor suppressor
alone was negligible in normal cells and highly significant in
tumor cells. In contrast, the anti-cancer chemotherapeutic alone
(e.g., cisplatin, doxorubicin, 5-FU, methotrexate, and etoposide)
was more effective in normal cells than cancer cells (see FIG.
9).
6TABLE 6 Anti-proliferative effects of p53 Ad in combination with
cisplatin. Greater Combined Efficacy? Cell Line p53 Protein Tissue
Type Cisplatin First p53 Ad First Simultaneous SK-OV-3 Null Ovarian
Yes (p .ltoreq. 0.0001) Yes (p .ltoreq. 0.0001) Yes (p .ltoreq.
0.0001) SCC-9 Null Head & Neck Yes (p .ltoreq. 0.0001) Yes (p
.ltoreq. 0.0001) Yes (p .ltoreq. 0.0001) SCC-15 Null Head &
Neck Yes (p .ltoreq. 0.0001) ND ND SCC-25 Null Head & Neck Yes
(p .ltoreq. 0.0001) Yes (p .ltoreq. 0.0001) Yes (p .ltoreq. 0.0001)
MDA-MB-468 Mutant Breast Yes (p .ltoreq. 0.0001) ND Yes (p .ltoreq.
0.0001) MDA-MB-231 Mutant Breast Yes (p .ltoreq. 0.0002) Yes (p
.ltoreq. 0.0001) Yes (p .ltoreq. 0.0001) ND = not done
[0351]
7TABLE 7 Anti-proliferative effects of p53 Ad in combination with
doxorubicin. Greater Combined Efficacy? Cell Line p53 Protein
Tissue Type Dox First p53 Ad First Simultaneous SK-OV-3 Null
Ovarian Yes (p .ltoreq. 0.0001) Yes (p .ltoreq. 0.0001) Yes (p
.ltoreq. 0.0001) SCC-9 Null Head & Neck Yes (p .ltoreq. 0.0001)
Yes (p .ltoreq. 0.0001) Yes (p .ltoreq. 0.0001) SCC-15 Null Head
& Neck Yes (p .ltoreq. 0.0001) Yes (p .ltoreq. 0.0001) Yes (p
.ltoreq. 0.0001) SCC-25 Null Head & Neck Yes (p .ltoreq.
0.0001) Yes (p .ltoreq. 0.0001) Yes (p .ltoreq. 0.0001) DU-145
Mutant Prostate Yes (p .ltoreq. 0.0001) Yes (p .ltoreq. 0.0001) Yes
(p .ltoreq. 0.0001) MB-231 Mutant Breast Yes (p .ltoreq. 0.0001)
Yes (p .ltoreq. 0.0001) Yes (p .ltoreq. 0.0001)
[0352] Doxorubicin and p53 Effects on Human Hepatocellular
Carcinoma
[0353] The following example details the ability of a p53
expressing adenovirus of the invention in combination with
doxorubicin to treat neoplasms, and that the combination therapy
was more effective, i.e., was synergistic, at killing tumor cells
than either agent alone. The results demonstrate a synergistic
interaction between the p53 expressing vector of the invention
(ACN53) and doxorubicin.
[0354] Doxorubicin (Adriamycin) and p53 (ACN53, the recombinant
adenovirus construct expressing the human wild-type p53 transgene)
were administered to the following cell lines: HLE, a human
hepatocellular carcinoma cell line (Hsu (1993) Carcinogenesis
14:987-992; Farshid (1992) J. Med. Virol. 38:235-239; Dor (1975)
Gann. 66:385-392), with a mutated p53; HLF, a human hepatocellular
carcinoma cell line with a mutated p53 (ibid); Hep 3B, a human
hepatocellular carcinoma with a null p53 (Hasegawa (1995) In Vitro
Cell Dev Biol Anim. 31:55-61); Hep G2, a human hepatocellular
carcinoma with p53 wild-type (ibid); and, SK-HEP-1, a human liver
adenocarcinoma with p53 wild-type (Lee (1995) FEBS Lett.
368:348-352). Cell viability measured with the live cell probe
calcien AM (Molecular Probes) (see, e.g., Poole (1993) J. Cell Sci.
106:685-691). The substrate calcien AM is cleaved by cellular
esterases to generate a fluorescent product.
[0355] Cells were plated in 96 well plates (5.times.10.sup.3
cells/well), allowed to adhere overnight, treated in triplicate
with dilutions of ACN53 and dilutions of doxorubicin on day 0, such
that a dose response curve for doxorubicin treatment was generated
with each dose of ACN53. On day 3 media was aspirated and calcien
AM in PBS was added to the cells. Fluorescent intensity of each
well was determined using fluorescent plate reader. Fluorescent
value from wells with no cells were subtracted and data was
expressed as the percent viability (fluorescent intensity) compared
to untreated control wells. ED.sub.50 values were used to generate
isobologram plots to assess the interaction between ACN53 and
doxorubicin.
[0356] Isobologram analysis for each cell line showed a synergistic
interaction between the p53 expressing vector of the invention
(ACN53) and doxorubicin; this synergy was independent of the p53
status of the cell line. Note, however, the ED.sub.50 for ACN53 in
the absence of doxorubicin is higher in the p53 wild-type cell
lines than in the p53 altered lines.
[0357] In another similar experiment, HLE cells were plated in 96
well plates (5.times.10.sup.3 cells/well); allowed to adhere
overnight; and, treated in triplicate with dilutions of ACN53 and
dilutions of doxorubicin such that a dose response curve for
doxorubicin treatment was generated with each dose of ACN53. Three
groups were used to test the effects on dosing order on the
interaction between ACN53 and doxorubicin.
8 group day 0 day 1 day 2 day 3 simultaneous ACN53, harvest
doxorubicin ACN53 first ACN53 doxorubicin harvest doxorubicin first
doxorubicin ACN 53 harvest
[0358] Cells were incubated for 3 days after first treatment. Media
was aspirated and calcien AM in PBS was added to the cells.
Fluorescent intensity of each well was determined using fluorescent
plate reader. Fluorescent value from wells with no cells were
subtracted and data was expressed as the percent viability
(fluorescent intensity) compared to untreated control wells.
ED.sub.50 values were used to generate isobologram plots to assess
the interaction between ACN53 and doxorubicin. Isobologram analysis
for each dosing regimen showed similar interaction, consistent with
synergy, between ACN53 and doxorubicin in HLE cells that was
independent of the dosing order of the treatment.
[0359] p53 with Chemotherapeutic Drugs In Vivo
[0360] The effect of the clinically-relevant anti-cancer drugs
cisplatin, doxorubicin, and 5-fluorouracil (5-FU), in combination
with a tumor suppressor vector of the invention (A/C/N/53), was
further investigated in vivo.
[0361] C.B. 17/ICR-scid mice were purchased from Taconic Farms
(Germantown, N.Y.) or Charles River Laboratories (Wilmington,
Mass.). Athymic nu/nu mice were purchased from Charles River
Laboratories. All mice were maintained in a VAF-barrier facility
and all animal procedures were performed in accordance with the
rules set forth in the N.I.H. Guide for the Care and Use of
Laboratory Animals. Tumor volumes for different treatment groups on
each day were compared by Student's t test using Statview II
software (Abacus Concepts, Berkeley, Calif.). Tumor growth curves
were constructed to show mean tumor volume.+-.s.e.m. There were
normally ten mice per group.
[0362] SK-OV-3 Ovarian Tumor Model:
[0363] Established intraperitoneal SK-OV-3 tumors were treated with
intraperitoneal doses of vehicles, p53 Ad, cisplatin, or both
drugs. Mice were given six injections of p53 Ad over a period of
two weeks. The total virus dose was 1.5.times.10.sup.9 C.I.U.
(3.1.times.10.sup.10 viral particles).
[0364] Cisplatin efficacy: Female scid mice were injected with
5.times.10.sup.6 SK-OV-3 ovarian tumor cells, I.P., on day 0. Mice
were dosed I.P. on days 6, 8, 10, 13, 15, and 17 (p53 Ad only on
D17). Mice received 0.2 ml total volume (0.1 ml cisplatin vehicle
or cisplatin plus 0.1 ml Ad buffer or p53 Ad). The p53 Ad dose was
2.5.times.10.sup.8 C.I.U./mouse/day (5.2.times.10.sup.9 viral
particles). The cisplatin dose was 2 mg/kg/day. Tumors were
harvested and weighed on day 20.
[0365] Mice in one treatment group received five doses of cisplatin
simultaneously with the first five p53 Ad doses. This dose of
intraperitoneal p53 Ad reduced mouse tumor burden only 17% by day
20 (p<0.01). However, when combined with cisplatin, p53 Ad
caused a 38% decrease in tumor burden as compared to cisplatin
alone (p.ltoreq.0.0008). Mice treated with drug vehicles or with
p53 Ad alone had bloody ascites and invasive tumor nodules in the
diaphragm muscle. These symptoms were absent in the mice treated
with cisplatin alone or cisplatin with p53 Ad.
[0366] Cisplatin/Paclitaxel efficacy: Female scid mice were
injected with 2.5.times.10.sup.6 SK-OV-3 ovarian tumor cells, I.P.,
on day 0. Mice were dosed I.P. on days 7, 9, 11, 16, and 18. Mice
received 0.3 ml total volume (0.1 ml cisplatin vehicle or cisplatin
plus 0.1 ml paclitaxel vehicle or paclitaxel plus 0.1 ml Ad buffer
or p53 Ad). The p53 Ad dose was 2.5.times.10.sup.8 C.I.U./mouse/day
(5.2.times.10.sup.9 viral particles). The cisplatin dose was 0.5
mg/kg/day. The paclitaxel dose was 1 mg/kg/day. Tumors were
harvested and weighed on day 30. n=7=10 mice per group.
[0367] In this second study, SK-OV-3 ovarian tumors were treated
with intraperitoneal doses of vehicles, p53 Ad, cisplatin plus
paclitaxel, or all three drugs simultaneously. The combination of
all three drugs reduced tumor burden 34% more than the combination
of cisplatin plus paclitaxel, demonstrating the enhanced efficacy
of the three drug combination (p.ltoreq.0.0006).
[0368] DU-145 Prostate Tumor Model:
[0369] Cisplatin Efficacy:
[0370] Intraperitoneal DU-145 tumors were treated with
intraperitoneal doses of vehicles, p53 Ad, cisplatin, or both
drugs. Male scid mice were injected with 2.5.times.10.sup.6 DU-145
cells, I.P., on day 0. Mice were dosed I.P. on days 7, 9, 11, 14,
and 16. Mice received 0.2 ml total volume (0.1 ml cisplatin vehicle
or cisplatin plus 0.1 ml Ad buffer or p53 Ad). The p53 Ad dose was
8.3.times.10.sup.8 C.I.U./mouse/day (2.9.times.10.sup.10 PN). The
cisplatin dose was 1 mg/kg/day. Tumors were harvested and weighed
on day 22. The combination of p53 Ad and cisplatin had greatly
enhanced anti-tumor efficacy compared to either drug alone
(p<0.0004).
[0371] MDA-MB-468 Mammary Tumor Model:
[0372] Cisplatin Efficacy:
[0373] Established MDA-MB-468 tumors were treated with vehicles,
p53 Ad, cisplatin, or both drugs. Female scid mice were injected
with 1.times.10.sup.7 MDA-MB-468 cells into the mammary fat pad, 11
days before the start of dosing on day 0. The intraperitoneal
cisplatin dose was 1 mg/kg/day. The intratumoral p53 Ad dose was
8.3.times.10.sup.8 CIU/mouse/day (2.9.times.10.sup.10 viral
particles) given simultaneously on days 0-4. p53 Ad had enhanced
efficacy when combined with cisplatin (days 8-31,
p.ltoreq.0.0004).
[0374] Doxorubicin Efficacy:
[0375] In a second experiment, MDA-MB-468 tumors were treated with
vehicles, p53 Ad, doxorubicin, or both drugs. Female nude mice were
injected with 1.times.10.sup.7 MDA-MB-468 cells subcutaneously 12
days prior to the start of dosing on day 0. The intraperitoneal
doxorubicin dose was 4 mg/kg/day on days 0, 2, 7, and 9. The
intratumoral p53 Ad dose was 5.times.10.sup.8 CIU/mouse/day
(1.03.times.10.sup.10 viral particles) on days 0-4 and 7-11.
[0376] p53 Ad had greater efficacy when administered in combination
with doxorubicin (days 14-24, p.ltoreq.0.05).
[0377] SCC-15 Head and Neck Tumor Model:
[0378] 5-Fluorouracil Efficacy:
[0379] Subcutaneous SCC-15 tumors were treated with vehicles, p53
Ad, 5-fluorouracil (5-FU), or both drugs. Scid mice were injected
with 5.times.10.sup.6 SCC-15 cells subcutaneously 7 days prior to
the start of dosing on day 0. The intraperitoneal 5-fluorouracil
dose was 50 mg/kg/day in 40% hydroxypropyl-beta-cyclodextran
(Cerestar Inc., Hammond, Ind.) given I.P. on days 0, 7, and 14
(once a week for 3 weeks). The p53 Ad dose was 2.times.10.sup.8
CIU/mouse/day (4.times.10.sup.9 viral particles), on days 0, 1, 7,
8, 14, and 15 (6 intratumoral injections over a period of 3 weeks).
The 5-FU dose of 50 mg/kg was given. The combination of p53 Ad and
5-FU resulted in greater antitumor activity than when either drug
was used alone (p<0.04).
[0380] p53 With FPT Inhibitor
[0381] The effect of a farnesyl protein transferase inhibitor in
combination with a tumor suppressor vector designated A/C/N/53 was
investigated in vitro. The following examples detail the ability of
a p53 expressing adenovirus of the invention in combination with
the FPT inhibitor designated "FPT39," as described in International
Application WO 97/23478, filed Dec. 19, 1996, where FPT39 is
designated compound "39.0," see pg 95 of WO 97/23478) to treat
neoplasms, and that for combination therapy against prostate tumor
cells and mammary tumor cells was more effective at killing tumor
cells than either agent alone.
[0382] Anti-Proliferative Efficacy of rAd5/p53 and FPT39 Against
SK-OV-3 Ovarian Tumor
[0383] Methods: SK-OV-3 human ovarian tumor cells (p53.sup.null)
were aliquoted into 96-well plates at a density of 250 cells per
well in Eagle's MEM plus 10% fetal bovine serum. Cells were then
incubated at 37.degree. C. and 5% CO.sub.2 for 4 hours. FPT39 or
drug vehicle was added to each well and cell culture was continued
for 3 days. After 3 days, untreated cells in some wells were
counted in order to calculate the amount of rAd5/p53 to add. Then
rAd5/p53 or drug vehicle was added to each well and cell culture
was continued for another 3 days. Cell proliferation was measured
using the MTT assay. Briefly, 25 ul of 5 mg/ml MTT vital dye
[3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] was
added to each well and allowed to incubate for 3-4 hrs. at
37.degree. C. and 5% CO.sub.2. Then, 100 ul of 10% SDS detergent
was added to each well and the incubation was continued overnight.
Fluorescence in each well was quantitated using a Molecular Devices
microtiter plate reader. Cell proliferation data was analyzed using
the Thin Plate Spline statistical methodology of O'Connell and
Wolfinger (1997) J. Comp. Graph. Stat. 6: 224-241
[0384] Results: rAd5/p53 and FPT39 had additive efficacy in
inhibiting cell growth. Neither synergism (p>0.05) nor
antagonism (p>0.05) were demonstrated in this experiment.
[0385] Anti-Proliferative and Synergistic Efficacy of rAd5/p53 and
FPT39 (FPT Inhibitor) Against DU-145 Prostate Tumor Cells
[0386] Methods: DU-145 human prostate tumor cells (p53.sup.mut)
were treated with FPT39 or drug vehicle, and rAd5/p53, and the cell
cultures were subsequently analyzed, as described above for the
SK-OV-3 human ovarian tumor cells. The experiment was repeated
twice.
[0387] Results: Experiment 1: rAd5/p53 and FPT39 had additive
efficacy in inhibiting cell growth. Neither synergism (p>0.05)
nor antagonism (p>0.05) were demonstrated in this
experiment.
[0388] Experiment 2: rAd5/p53 and FPT39 had synergistic efficacy
(p=0.0192). These results demonstrate that rAd5/p53 and FPT39 can
interact and have synergistic efficacy against prostate tumor cell
proliferation.
[0389] Anti-Proliferative and Synergistic Efficacy of rAd5/p53 and
FPT39 (FPT Inhibitor) Against MDA-MB-231 Mammary Tumor Cells
[0390] Methods: MDA-MB-231 human breast cancer cells (p53.sup.mut)
were treated with FPT39 or drug vehicle, and rAd5/p53, and the cell
cultures were subsequently analyzed, as described above for the
SK-OV-3 human ovarian tumor cells. The experiment was repeated
twice.
[0391] Results: Experiment 1: rAd5/p53 and FPT39 had additive
efficacy. Neither synergism (p>0.05) were demonstrated in this
experiment.
[0392] Experiment 2: rAd5/p53 and FPT39 had additive efficacy over
most of the response surface. However, synergism was evident at
isoboles greater that or equal to 70 (i.e., less than 30% of cells
killed, p=0.0001). These results demonstrate that rAd5/p53 and
FPT39 can interact and have synergistic efficacy against human
breast cancer cell proliferation.
Example 7
Immune Response Profile in Patients with Metastatic Hepatic
Carcinomas Treated with Adenovirus Vector Carrying p53
[0393] The invention provides for the combined in vivo
administration of nucleic acid expressing p53 and other
chemotherapeutic agents in the treatment of neoplasms. The
following example details the ability of the p53 expressing
adenovirus of the invention to increases the levels of
tumor-killing lymphocytes found within a human liver carcinoma.
[0394] The aim of this study was to characterize the genotypes and
phenotypes of the tumor infiltrating lymphocytes (TILs) in
metastatic liver carcinomas from the colon harboring p53 mutations
(for discussion TILs, see, e.g., Wang (1997) Mol. Med. 3:716-731;
Marrogi (1997) Int. J. Cancer 74:492-501). A total of 16 patients
were treated in a dose escalating manner, 10.sup.9-10.sup.11
particles, through hepatic artery canalization with an adenovirus
vector carrying wild type p53 gene. A total of four biopsies from
each patient was obtained 3 to 7 days after administering the
adenoviral vector. Immunohistochemical analysis were performed on
the frozen tissues obtained from normal liver and tumor-host tissue
interface sites. Computer assisted image analysis was performed to
quantitate immunoreactivity to the following monoclonal antibodies:
CD3, CD4, CD8, CD25, CD28, CD56, HLA-DR, IFN-gamma, TNF-alpha and
IL-2. An increase in TILs (CD3.sup.+ and CD4.sup.+) population was
observed with the maximum at 7.5.times.10.sup.10 particles. At
higher doses, a decrease in CD3.sup.+ and CD4+population was
observed. An inverse correlation was observed for CD8+cells. At the
highest dose (2.5.times.10.sup.11) an increase in the CD3.sup.+,
CD4.sup.+ and CD8.sup.+ population was observed in the tumor as
compared to the normal. These results demonstrate that delivery of
high doses of adenovirus particles results in increased TILs
composed of CD4.sup.+ and CD8.sup.+ population.
[0395] It is understood that the examples and embodiments described
herein are for illustrative purposes only and that various
modifications or changes in light thereof will be suggested to
persons skilled in the art and are to be included within the spirit
and purview of this application and scope of the appended claims.
All publications, patents, and patent applications cited herein are
hereby incorporated by reference for all purposes.
* * * * *