Note: Descriptions are shown in the official language in which they were submitted.
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METHODS OF TREATING NEOPLASIA WITH COMBINATIONS OF TARGET
CELL-SPECIFIC ADENOVIRUS, CHEMOTHERAPY AND RADIATION
TECHI\TICAL FIELD
This invention relates to cell transfection, and in particular methods of
using
adenoviral vectors for the suppression of tumor growth in conjunction with
chemotherapy,
radiation therapy or combinations thereof.
BACKGROUND ART
Neoplasia, also known as cancer, is the second most common cause of death in
the
United States. While the survival rates for individuals with cancer have
increased
considerably in the last few decades, survival of the disease is far from
assured. Cancer is a
catch-all term for over 100 different diseases, each of which are each
fundamentally
characterized by the unchecked proliferation of cells. Individual cancer cells
are also able
to break off from the main tumor, or metastasize, creating additional tumors
in other
regions of the body.
Due to the mortality rate and incidence of neoplasia in the general
population,
research into potential cures has been high on the national agenda for
decades. 'This
research has led to the development a number of treatments, both systemic and
regional
(local). Regional treatments include radiation therapy, some types of
chemotherapy and
surgery. Chemotherapy has most often been used in systemic treatment. Each of
these
treatment regimes has significant disadvantages and limitations. Chemotherapy
and
radiation treatments will be discussed below.
Chemotherapy
Chemotherapy refers to the use of chemical compounds or drugs in the treatment
of
disease, though the term chemotherapy is most often associated with the
treatment of
cancer. Cancer chemotherapeutic agents are also commonly referred to as
antineoplastic
agents. There are a number of classes of chemotherapeutic compounds,
encompassing
nearly 100 individual drugs, as well as numerous drug combination therapies,
methods of
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delivery and schedules of treatment. Each of these chemotherapeutic agents may
be
classified according to several criteria, such as class of compound and
disease state treated.
Certain agents have been developed to take advantage of the rapid division of
cancer cells
and target specific phases in the cell cycle, providing another method of
classification.
Agents can also be grouped according to the type and severity of their side
effects or
method of delivery. However, the most common classification of
chemotherapeutic agents
is by class of compound, which broadly encompasses the mechanism of action of
these
compounds.
Depending on the reference source consulted, there are slight differences in
the
classification of antineoplastics. The classes of compounds are described in
the Physician's
Desk Reference as follows: alkaloids; alkylating agents; anti-tumor
antibiotics;
antimetabolites; hormones and hormone analogs; immunomodulators;
photosensitizing
agents; and miscellaneous other agents. Examples of these antineoplastics are
listed in
Table 1.
The alkaloid class of compounds are also referred to as mitotic inhibitors, as
they
are cell cycle phase specific and serve to inhibit mitosis or inhibit the
enzymes required for
mitosis. They are derived generally from plant alkaloids and other natuxal
products and
work during the M-phase of the cell cycle. This class of compounds is often
used to treat
neoplasias such as acute lymphoblastic leukemia, Hodgkin's and non-Hodgkin's
lymphoma; neuroblastomas and cancers of the lung, breast and testes.
Alkylating agents make up a large class of chemotherapeutic agents, including
of
the following sub-classes, which each represent a number of individual drugs:
alkyl
sulfonates; aziridines; ethylenimines and methylmelamines; nitrogen mustards;
nitrosoureas; and others. Alkylating agents attack neoplastic cells by
directly alkylating the
DNA of cells and therefore causing the DNA to be replication incompetent. This
class of
compounds is commonly used to treat a variety of diseases, including chronic
Ieukemias,
non-Hodgkin's lymphoma, Hodgkin's lymphoma, multiple myeloma and certain lung,
breast and ovarian cancers.
Nitrosoureas axe often categorized as alkylating agents, and have a similar
mechanism of action, but instead of directly alkylating DNA, they inhibit DNA
repair
enzymes causing replication failure. These compounds have the advantage of
being able to
cross the blood-brain barrier and therefore can be used to treat brain tumors.
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Antitumor antibiotics have antimicrobial and cytotoxic activity and also
interfere
with DNA by chemically inhibiting enzymes and mitosis or by altering cell
membranes.
They are not cell cycle phase specific and are widely used to treat a variety
of cancers.
The antimetabolite class of antineoplastics interfere with the growth of DNA
and
RNA and are specific to the S-phase of the cell-cycle. They can be broken down
further by
type of compound, which include folic acid analogs, purine analogs, and
pyrimidine
analogs. They are often employed in the treatment of chronic leukemia, breast,
ovary, and
gastrointestinal tumors.
There are two classes of hormones or hormone analogs used as antineoplastic
agents, the corticosteroid hormones and sex hormones. While some
corticosteroid
hormones can both kill cancer cells and slow the growth of tumors, and are
used in the
treatment of lymphoma, leukemias, etc., sex hormones function primarily to
slow the
growth of breast, prostate and endometrial cancers. There axe numerous
subclasses of
hormones and hormone analogs, including, androgens, antiadrenals,
antiandrogens,
antiestrogens, aromatase inhibitors, estrogens, leutenizing hormone releasing
hormone
(LHRH) analogs and progestins.
An additional smaller class of antineoplastics is classified as immunotherapy.
These are agents which are intended to stimulate the immune system to more
effectively
attack the neoplastic cells. This therapy is often used in combination with
other therapies.
''' There are also a number of compounds, such as campothectins, which are
generally
listed as 'other' antineoplastic agents and can be used to treat a variety of
neoplasias.
While there is a plethora of antineoplastic agents, the efficacy of these
compounds
is often outweighed by the severity of the side effects produced by the agent.
This
comparison is often referred to as the therapeutic index, which describes the
balance
between the required dose to accomplish the destruction of the cancer cells
compared to the
dose at which the substance is unacceptably toxic to the individual. The
drawback to most
antineoplastic agents is the relatively small range of the therapeutic index,
(i.e, the narrow
dosage range in which cancer cells are destroyed without unacceptable toxicity
to the
individual). This characteristic limits the frequency and dosage where an
agent is useful,
and often the side effects become intolerable before the cancer can be fully
eradicated.
The severe side effects experienced with the majority of cancer
chemotherapeutics
are a result of the non-specific nature of these drugs, which do not
distinguish between
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healthy and cancerous cells, and instead destroy both. The cell cycle specific
drugs attempt
to lessen these effects, targeting phases of the cell cycle involved in cell
replication and
division. These drugs do not, however, distinguish between cancerous cells and
healthy
cells which are undergoing normal cell division. The cells most at risk from
these types of
chemotherapy are those which undergo cell division often, including blood
cells, hair
follicle cells, and cells of the reproductive and digestive tracts.
The most common side effects of antineoplastic agents are nausea and
vomiting.. A
large proportion of individuals also suffer from myelosuppression, or
suppression of the
bone marrow, which produces red blood cells, white blood cells and platelets.
These and
other side effects are also exacerbated by the suppression of the immune
system
concomitant with the destruction and lack of production of white blood cells,
and
associated risk of opportunistic infection.
Other side effects common to a wide range of antineoplastic agents include:
hair
loss (alopecia); appetite loss; weight loss; taste changes; stomatitis and
esophagitis
(inflammation and sores); constipation; diarrhea; fatigue; heart damage;
nervous system
changes; lung damage; reproductive tissue damage; liver damage; kidney and
urinary
system damage.
The wide range of the side effects associated with most antineoplastic agents
and
their severity in individuals who are already debilitated with disease and
possibly immune
compromised has led researches to search for mechanisms by which they can
alleviate
some of the side effects while maintaining the efficacy of the treatment.
Several
approaches to this problem have been taken. They include combination
chemotherapy,
where multiple antineoplastics are administered together; adjuvant therapies,
where
additional agents are prescribed along with the antineoplastic agent to fight
the side effects
of the antineoplastic; alternative delivery vehicles for the administration of
chemotherapeutics, such as the encapsulation of antineoplastic agents in
liposomes; and
combined modality treatments, where chemotherapy is combined with radiation
and/or
surgery.
One difficulty with respect to combination chemotherapy is that many
antineoplastic agents have similar side effects, so while their toxicity
profiles are different,
the individual will still suffer greatly and may not be able to finish the
recommended
course of treatment.
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Another aspect of combination chemotherapy is the addition of hormones to the
combination of drugs administered. While the hormone or hormonal analog
treatment is
generally not cytotoxic, hormonal manipulation helps to prevent or slow cell
division and
therefore slows the growth of the tumor. This type of therapy is often used
for hormone
dependent tumors of, for instance, the prostate, breast or ovaries. One well
known example
is the treatment of breast cancer with tamoxifen.
An additional method of combating the side effects associated with
antineoplastics
and, more importantly, extending the therapeutic dosage of these agents is
adjuvant
therapy, where additional agents are co-administered to the individual in
order to
ameliorate the side effects or toxicity of the antineoplastic agent. Examples
of such
adjuvant therapy includes the administration of chemoprotective agents, such
as the
uroprotective agent mesna, the antimetastatic agent batimastat, the folic acid
replenisher
folinic acid. Additional therapies include the administration of granulocyte
colony
stimulating factors, granulocyte-macrophage colony stimulating factor and even
the
I 5 transplantation of hematopoietic stem cells. These last three therapies
aim to treat lessen
the chance of opportunistic infection due to myelosuppression concomitant with
many
chemotherapy regimens. However, despite the recent advances in antineoplastic
and
adjuvant therapy there are still numerous cancers, for example ovarian cancer,
that are
resistant to current treatments, and leave the individual at risk for
potentially serious
infection.
Radiation Therapy
Along with chemotherapy and surgery, radiation is one of the most commonly
used
treatment modalities, used in approximately 60% of treatment regimens.
Radiation, in any
of several forms, is often used as the primary therapy for basal cell
carcinomas of the skin,
head and neck, prostate cancers, bladder cancers, and others. Often combined
with
chemotherapy and/or surgery, radiation therapy encompasses both local and
total body
administration as well as a number of new advances, including
radioimmunotherapy.
The cytotoxic effect of radiation on neoplastic cells arises from the ability
of
radiation to cause a break in one or both strands of the DNA molecule inside
the cells.
Cells in all phases of the cell cycle are susceptible to this effect. However,
the DNA
damage is more likely to be lethal in cancerous cells because they are less
capable of
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repairing DNA damage. Healthy cells, with functioning cell cycle check
proteins and
repair enzymes, are far more likely to be able to repair the radiation damage
and function
normally after treatment.
Tumors and tissues themselves are also characterized by a range of
susceptibilities
to radiation therapy. Lymphoma and leukemias are very sensitive to radiation
therapy,
while renal cancer and gland tumors are fairly insensitive to radiation. A
tumor that is
considered radiosensitive is one which can be eradicated by a doses) of
radiation that is
also well tolerated by the surrounding tissues. Unsurprisingly, different
tissue types within
- the body tolerate radiation at different doses. Tissues that undergo
frequent cell division
are most effected by treatment, similar to their sensitivity to certain cell
cycle specific
chemotherapy agents.
The radiosensitivity of tumors is also effected by hypoxia, or a Iack of
oxygen in the
interiors of larger tumors. Hypoxic tumors can be 2-3 times less responsive to
radiation
treatment. Certain agents used in conjunction with radiation treatment, such
as some of the
radiosensitizing agents, work by increasing the singlet oxygen species in the
vicinity of the
tumor and therefore increasing its radiosensitivity. Other compounds used in
conjunction
with radiation therapy include radioprotectants which are designed to protect
surrounding
tissue from some of the effects of radiation therapy. Sources of radiation
include:
Americium, chromic phosphate, radioactive, Cobalt, 1311-ethiodized oil, Gold
radioactive, colloidal) iobenguane, Radium, Radon, sodium iodide (radioactive)
, sodium
phosphate (radioactive).
Radiation therapy itself can be classified according to two primary types,
internal
and external radiation thexapy. External therapy involves the administration
of radiation
via a machine capable of producing high-energy external beam radiation. This
therapy can
include either total body irradiation, or can be localized to the region of
the tumor. With
external radiation treatments, the bodily secretions of the individual are not
radioactive
after treatment. The radiation itself can be either electromagnetic (X-ray or
gamma
radiation) or particulate (a or (3 particles). The treatment requirements will
differ
depending upon the characteristics of the tumor. External radiation is often
used pre- or
post-operatively; either to shrink the tumor before surgery, or to mop up
remaining cancer
cells after surgery.
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Internal radiation therapy, also termed brachytherapy, involves implantation
of a
radioactive isotope as the source of the radiation. There a variety of methods
of delivery,
including permanent, temporary, sealed, unsealed, intracavity or interstitial
implants. The
choice of implant is determined by a variety of factors, including the
location and extent of
S the tumor.
A third, but still experimental, type of radiation therapy is often termed
radioimmunotherapy. This involves the attachment of radioisotopes to
monoclonal
antibodies specific for the tumor cells. Upon administration the antibodies
specifically
seek out and destroy the cancer cells.
The side effects of radiation are similar to those of chemotherapy and arise
for the
same reason, the damage of healthy tissue. Radiation is usually more localized
than
chemotherapy, but treatment is still accompanied by damage to previously
healthy tissue.
Many of the side effects are unpleasant, and radiation also shares with
chemotherapy the
disadvantage of being mutagenic, carcinogenic and teratogenic in its own
right. While
1 S normal cells usually begin to recover from treatment within two hours of
treatment,
mutations may be induced in the genes of the healthy cells. These risks are
elevated in
certaiwtissues, such as those in the reproductive system. It has also been
found that people
tolerate radiation differently. Doses that may not lead to new cancers in one
individual
rnay in fact spawn additional cancers in another individual. This could be due
to pre-
existing mutations in cell cycle check proteins or repair enzymes, but current
practice
would not be able to predict at what dose a particular individual is at risk.
Common side
effects of radiation include: bladder irritation; fatigue; diarrhea; low blood
counts; mouth
irritation; taste alteration; loss of appetite; alopecia; skin irritation;
change in pulmonary
function; enteritis; sleep disorders; and others.
2S
Adenovirus Vectors
Until relatively recently, the virtually exclusive focus in development of
adenoviral
vectors for gene therapy has been use of adenovirus merely as a vehicle for
introducing the
gene of interest, not as an effector in itself. Replication of adenovirus had
previously been
viewed as an undesirable result, largely due to the host immune response. More
recently,
however, the use of adenovirus vectors as effectors has been described.
International
Patent Application Nos. PCT/IJS98104084, PCT/IIS98/04080; PCT/LTS98/04133,
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PCTlUS98/04132, PCTlLJS98/16312, PCT/US95/00845, PCT/US96/10838,
PCT/EP98/07380, U.S. Pat. No. 5,998,205 and U.S. Pat. No. 5,698,443. The use
of IRES
in vectors have been described. See, for example, International Patent
Application No.
PCT/LTS98/03699 and International Patent Application No. PCT/EP98107380.
Adenovirus
ElA and E1B genes are disclosed in Rao et al. (1992, Proc. Natl. Acad. Sci.
USA vol. 89:
7742-7746).
Publications describing various aspects of adenovirus biology and/or
techniques
relating to adenovirus include the following. PCT/US95/14461; Graham and Van
de Eb
(1973) Virology 52:456-467; Takiff et al. (1981) Lancet ii:832-834; Berkner
and Sharp
(1983) Nucleic Acid Research 6003-6020; Graham (1984) EMBO J 3:2917-2922; Bett
et
al: (1993) J. Virology 67:591 I-5921; and Bett et al. (1994) Proc. Natl. Acad.
Sci. USA
91:8802-8806 describe adenoviruses that have been genetically modified to
produce
replication-defective gene transfer vehicles. In these vehicles, the early
adenovirus gene
products ElA and E 1 B are deleted and provided in t~aus by the packaging cell
line 293
developed by Frank Graham (Graham et al. (1987) J. Gen. Bi~ol. 36:59-72 and
Graham
(1977) J. Genetic hirology 68:937-940). The gene to be transduced is commonly
inserted
into adenovirus in the deleted ElA and ElB region of the virus genome Bett et
al. (1994),
supra. Adenovirus vectors as vehicles for efficient transduction of genes have
been
described by Stratford-Perricaudet (1990) Human Gene Therapy 1:2-256;
Rosenfeld (1991)
Science 252:431-434; Wang et al. (1991) Adv. Exp. Med. Biol. 309:61-66; Jaffe
et al.
(1992) Nat Gent. 1:372-378; Quantin et al. (1992) Proc Natl. Acad. Sci. USA
89:2581-
2584; Rosenfeld et al. (1992) Cell 68:143-155; Stratford-Perricaudet et al.
(1992) J. Clin.
Invest. 90:626-630; Le Gal La Salle et al. (1993) Science 259:988-990;
Mastrangeli et al.
(1993) J. Clin. Invest. 91:225-234; Ragot et al. (1993) Nature 361:647-650;
Hayaski et al.
(1994) J. Biol. Chem. 269:23872-23875.
There are several other experimental cancer therapies which utilize various
aspects
of adenovirus or adenovirus vectors. See, U.S. Pat. No. 5,776,743; U.S. Pat.
No: 5,846,945; U.S. Pat. No. 5,801,029; PCT/LTS99108592; U.S. Pat. No.
5,747,469;
PCT/US98/03514; and PCT/US97/22036.
Of particular interest is the development of more specific, targeted forms of
cancer
therapy, especially in cancers that are difficult to treat successfully, such
as prostate,
bladder or ovarian cancer. In contrast to conventional cancer therapies, which
result in
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relatively non-specific and often serious toxicity, more specific treatment
modalities
attempt to inhibit or kill malignant cells selectively while leaving healthy
cells intact.
There is, therefore a serious need for developing specific, less toxic cancer
therapies.
All references cited herein are hereby incorporated by reference in their
entirety.
SUMMARY OF THE INVENTION
The invention provides methods for the administration of combinations of a
target
cell-specific adenoviral vector and at least one antineoplastic agents) and/or
radiation to an
individual in need thereof, such as, an individual with neoplasia.
Accordingly, in one aspect, the invention provides methods. of suppressing
tumor
growth in an individual comprising the steps of a) administering to the
individual a
' composition comprising a replication-competent target cell-specific
adenoviral vector
wherein said vector comprises an adenovirus gene essential for replication
(preferably an
early gene) under transcriptional control of a target cell specific
transcriptional regulatory
element (TRE); and b) administering an antineoplastic agent to the individual,
wherein the
adenoviral vector and antineoplastic agent are administered in amounts
sufficient to
suppress tumor growth. In some embodiments, the amount of adenovirus vector
and/or
anitneoplastic agent administered is less than that known in the art to be
effective for
suppressing tumor growth when either is administered alone. In one embodiment,
the
antineoplastic agent includes alkaloids, alkylating agents, antibiotics,
antimetabolites,
immunomodulators, nitrosoureas, hormone antagonists/agonists and analogs, or
photosensitizing agents.
In another aspect, the invention provides methods of suppressing tumor growth
in
an individual comprising the following steps: a) administering to the
individual a
composition comprising a replication-competent target cell-specific adenoviral
vector
wherein said vector comprises an adenovirus gene essential for replication
(preferably an
early gene) under transcriptional control of a target cell specific
transcriptional regulatory
element (TRE); and b) administering an effective amount of radiation. In some
embodiments, the amount of adenovirus vector and/or radiation administered is
less than
that known in the art to be effective for suppressing tumor growth when
administered
alone. In one embodiment, the radiation includes X-rays, gamma rays, alpha
particles, beta
particles, electrons, photons, neutrons, other ionizing radiation or
radioactive isotopes.
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In yet another aspect, the present invention provides methods for suppressing
tumor
growth in an individual comprising the following steps, in any order: a)
administering to
the individual an effective amount of a replication-competent target cell-
specific adenoviral
vector and an effective amount of at least one antineoplastic agent; and b)
administering an
effective amount of an appropriate course of radiation therapy to the
individual. In one
embodiment, the method may fiu-ther comprise, c) administering to the
individual an
additional dose of the adenoviral/chemotherapeutic solution or radiation as
necessary to
treat the individual's neoplasia. In another embodiment, the method may
further comprise
a delay between any of steps a), b) and c). In some embodiments, the amount of
adenovirus vector and/or anitneoplastic agent and/or radiation administered
will be less
than that known in the art to be effective for suppressing tumor growth when
either is
administered alone.
Any THE which directs cell-specific expression can be used in the disclosed
adenovirus vectors. In one embodiment, TREs include, for example, TREs
specific for
prostate cancer cells, breast cancer cells, hepatoma cells, melanoma cells,
bladder cells or
colorectal cancer cells. In another embodiment, the TREs include, probasin
(PB) TRE;
prostate-specific antigen (PSA) TRE; mucin (MUCI ) TRE; oc-fetoprotein (AFP)
TRE;
hKLK2 TRE; tyrosinase TRE; human uroplakin II THE (hUPII) or carcinoembryonic
antigen (CEA) TRE. In other embodiments, the target cell-specific THE is a
cell status-
specific TRE. In yet other embodiments, the target cell-specific THE is a
tissue specific
TRE.
In one aspect, the adenovirus vectors comprise adenovirus genes essential for
viral
replication. An essential gene can be an early viral gene, including for
example, EIA;
E1B; E2; and/or E4, or a late viral gene. In another aspect, the adenovirus
vector
comprises E3.
In some embodiments, the adenovirus vectors comprise an adenovirus gene having
an inactivation of its endogenous promoter. In one embodiment, the adenovirus
gene is
essential for viral replication under control of a target cell-specific TRE.
In another
embodiment, the adenovirus gene is EIA wherein the EIA promoter is inactivated
and
wherein the ElA gene is under transcriptional control of a heterologous cell-
specific TRE.
In another embodiment, the adenovirus gene is E 1 B wherein the E 1 B promoter
is
inactivated and wherein the E1B gene is under transcriptional control of a
heterologous
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cell-specific TRE. In other embodiments, the adenovirus vectors comprise the
adenovirus
gene, ElB, having a deletion of the 19-kDa region.
In other embodiments, an enhancer element for the adenovirus genes is
inactivated,
such as an inactivation of ElA enhancer. In yet other embodiments, the ElA
promoter is
inactivated and the ElA enhancer I is inactivated. In further embodiments, the
THE has its
endogenous silencer element inactivated.
In another embodiment, the replication competent adenovirus vectors comprise
co-
transcribed first and second genes under transcriptional control of a
heterologous, target
cell-specific transcriptional regulatory element (TRE), wherein the second
gene is under
translational control of an internal ribosome entry site (IRES). In one
aspect, the first
andlor second genes are adenovirus genes and in another aspect, the first
and/or second
adenovirus genes axe essential for viral replication. An essential gene can be
an early viral
gene, including for example, ElA; E1B; E2; and/or E4, or a late viral gene. In
another
aspect an early gene is E3.
In one embodiment, the first gene is an adenovirus gene and the second gene is
a
therapeutic gene. In another embodiment, both genes are adenovirus genes. In
an
additional embodiment, the first adenovirus gene is EIA, and the second
adenovirus gene is
E1B. Optionally, the endogenous promoter for one of the co-transcribed
adenovirus gene
essential for viral replication, such as for example, EIA, is inactivated,
placing the gene
under sole transcriptional control of a target cell-specific TRE.
In additional embodiments, the adenovirus vector comprises at least one
additional
co-transcribed gene under the control of the cell-specific TRE. In another
embodiment, an
additional co-transcribed gene is under the translational control of an IRES.
In another aspect of the present invention, adenovirus vectors further
comprise a
transgene such as, for example, a cytotoxic gene. In one embodiment, the
transgene is
under the transcriptional control of the same THE as the first gene and second
genes and
optionally under the translational control of an internal ribosome entry site.
In another
embodiment, the transgene is under the transcriptional control of a different
THE that is
functional in the same cell as the THE regulating transcription of the first
and second genes
and optionally under the translational control of an IRES.
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BRIEF DESCRIPTION OF THE DRAWINGS
Figures lA-1B is a schematic depicting target cell-specific adenovirus vectors
described in the Examples.
Figure 2 is a graph depicting percent viable LNCaP prostate tumor cells
treated with
CV787 adenovirus vector (solid circles; MOI 0.01); CV787 and TAXOLTM
(paclitaxel;
solid squares); TAXOLTM alone (solid triangles; 6.25 nM) and mock infected
control
(diamonds). For the combined administration of CV787 and TAXOLTM , TAXOLTM was
administered first, 24 hrs prior to CV787.
Figure 3 is a graph depicting percent viable LNCaP prostate tumor cells
treated with
CV787 adenovirus vector (solid circles; MOI 0.01); CV787 and TAXOTERETM
(docetaxel; solid squares;); TAXOTERETM alone (triangles; 3.12 nM); and mock
infected
control (diamonds) In the combination administration, TAXOTERETM was
administered
f rst.
Figure 4 is a graph depicting percent viable LNCaP prostate tumor cells
treated with
IS CV787 adenovirus vector (solid circles; MOI 0.01); CV787 and TAXOTERE~
(docetaxel; solid squares); TAXOTERETM alone (triangles; 3.12 nM); and mock
infected
control '(diamonds) For the combination administration, CV787 was added first.
Figure 5 is a graph depicting percent viable LNCaP prostate tumor cells
treated with
CV787 adenovirus vector (solid circles; MOI 0.1); CV787 and mitoxantrone (MTX;
solid
squares;); MTX alone (solid circles with "X"; 100 nM); and mock infected
control
(diamonds). For the combination administration , Mitoxantrone was administered
first, 24
hrs prior to CV787.
Figure 6 is a bar graph depicting percent viable LNCaP prostate tumor cells
with no
treatment (mock); CV787 treatment (MOI 0.01); etoposide~treatment (S00 ng/ml);
and
CV787 plus etoposide (Eto) treatment on day 8 (Etoposide was administered
first).
Figure 7 is a bar graph depicting percent viable LNCaP prostate tumor cells
with no
treatment; CV787 treatment (MOI 0.01); doxorubicin treatment (50 nglml); and
CV787
plus doxorubicin (Doxo) treatment on day 8 (CV787 was administered first).
Figure 8 is a bar graph depicting percent viable LNCaP prostate tumor cells
with no
treatment; CV787 treatment (MOI 0.1); cisplatin treatment (8.25 ~,M); and
CV787 plus
cisplatin (Cis) treatment on day 5 (Cisplatin was administered first).
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Figure 9 is a bar graph depicting percent viable LNCaP prostate tumor cells
with no
treatment; CV787 treatment (MOI 0.01); S-fluorouracil (5-FLT; 35 ~M)
treatment; and
CV787 plus 5-fluorouracil treatment on day 8 (5-fluorouracil was administered
first).
Figure 10 is a graph depicting percent viable LNCaP prostate tumor cells
treated
with CV787 adenovirus vector (solid circles; MOI 0.01); CV787 and radiation
(solid
squares); radiation alone (solid triangles; l3~Cs; 2 Gy); and mock infected
control
(diamonds). For combination administration, CV787 was administered first, 24
hours prior
to radiation.
Figure 11 is a graph depicting CV787 adenovirus yield in LNCaP prostate tumor
cells treated with CV787 (MOI 0.1) and mock infected control; CV787 and
TAXOLTM
(6.25 nM); CV787 and mitoxantrone (Mito; 100 nM); CV787 and doxorubicin (Dox;
50
ng/ml); and CV787 and etoposide (500 ng/mI), on day 6 of treatment. For all
combination
administration, CV787 was administered first.
Figure 12 is a bar graph depicting CV787 adenovirus yield in LNCaP prostate
1 S tumor cells (dashed shading); HBL-100 breast epithelial cells (horizontal
shading); and PA-
1 ovary cells (solid shading) when treated with CV787 adenovirus vector (MOT
0. I );
CV787 and TAXOLTM (6.25 nM); CV787 and mitoxantrone (MTX; 100 nM);and CV787
and doxorubicin (Doxo; 50 ng/ml). For combination administration, CV787 was
administered first with virus yield measured at 72 hours after infection.
Figure 13 is a graph depicting relative percent viable cells for combination
treatment compared to chemotherapeutic agent alone over time when treated with
CV787
adenovirus vector (MOI 0.01 ) and TAXOLTM (6.25 nM). LNCaP, prostate tumor
cells
(solid circles); HBL-100, breast epithelial tissue cells (solid triangles);
OVCAR-3, ovarian
cancer cells (solid diamonds); and 293, human embryonic kidney cells (solid
squares), ElA
and E1B permissible. For combination administration, CV787 was administered
first.
Figure 14 is a bar graph depicting percent viable cells when treated with
CV787
adenovirus vector (dark shading; MOI 0.1); CV787 and mitoxantrone (MTX;
outlined; 100
nM) and mitoxantrone alone (horizontal shading) on day 7 of treatment. LNCaP,
prostate
tumor cells; HBL-100, breast epithelial tissue cells; OVCAR-3, ovarian cancer
cells; and
293, human embryonic kidney cells, EIA and E1B permissible. For combination
administration, CV787 was administered first.
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Figure 15 is a graph depicting percent viable Hep3B (3B) and HepG2 (G2)
hepatoma cells treated with CV790 adenovirus vector (diamonds; MOI 0.01);
CV790 and
doxorubicin (triangles); and doxorubicin alone (squares; 10 ng/ml). For
combination
administration, CV790 was administered first.
Figure 16 is a graph depicting percent viable HepG2 (G2) hepatoma cells
treated
with CV790 adenovirus vector (solid diamonds; MOI 0.01); CV790 and doxorubicin
(solid
triangles); and doxorubicin alone (solid squares; 10 ng/ml). For combination
administration, Doxorubicin was administered first.
Figure 17 is a graph depicting percent viable HepG2 (G2) hepatoma cells
treated
with CV790 adenovirus vector (solid diamonds; MOI 0.01); CV790 and doxorubicin
(solid
triangles); and doxorubicin alone (solid squares; 10 ng/ml). For combination
administration, CV790 and doxorubicin were administered together.
Figure 18 is a graph depicting percent viable HepG2 (G2) and Hep3B (3B)
hepatoma cells treated with CV790 adenovirus vector (diamonds; MOI 0.1); CV790
and
cisplatin (triangles); and cisplatin alone (squares; 1 pglml). For combination
administration, CV790 was administered first.
Figure 19 is a graph depicting percent viable HepG2 (G2) and Hep3B (3B)
hepatoma cells treated with CV790 adenovirus vector (diamonds; MOI 0.1); CV790
and
TAXOLTM (paclitaxel; triangles); and TAXOLTM alone (squares; 0.5 ng/ml). For
combination administration, CV790 was administered first.
Figure 20 is a graph depicting percent viable HepG2 (G2) and Hep3B (3B)
hepatoma cells treated with CV790 adenovirus vector (diamonds; MOI 0.1); CV790
and 5-.
fluorouracil (triangles); and S-fluorouracil alone (squares; 10 ng/ml). For
combination
administration, CV790 was administered first.
Figure 21 is a graph depicting percent viable HepG2 (G2) and Hep3B (3B)
hepatoma cells treated with CV790 adenovirus vector (diamonds; MOI 0.1); CV790
and
mitoxantrone (triangles); and mitoxantrone alone (squares; 4 ng/ml). For
combination
administration, CV790 was administered first.
Figure 22 is a graph depicting percent viable HepG2 (G2) and Hep3B (3B)
hepatoma cells treated with CV790 adenovirus vector (diamonds; MOI 0.1); CV790
and
mitomycin C (triangles); and mitomycin C alone (squares; 10 ng/ml). For
combination
administration, CV790 was administered first.
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Figure 23 is a graph depicting the tumor volume of LNCaP prostate tumor
xenografts treated with CV787 adenovirus vector (triangles; 1 x 10'
particles/mm3); CV787
and TAXOL~ (solid squares); TAXOL~ alone (paclitaxel; solid circles; 15
mg/kg); and
mock infected control (solid diamonds). For combination administration, CV787
was
administered first on day 0 via intra-tumor injection. T.AXOLTM was
administered on day
1 , 2, 3, and 4.
Figure 24 is a graph depicting the relative percent of tumor volume of LNCaP
prostate tumor xenografts treated with TAXOL~ and CV787 adenovirus vector
(triangles;
TAXOLTM 2 mglkg; 1 x I01° particles); CV787 and TAXOL~ (solid squares;
TAXOL~
10 mg/kg); TAXOL~ alone (solid circles; 10 mg/kg); and TAXOLTT'1 alone (solid
diamonds; 2 mg/leg). For combination administration, TAXOLTM was administered
first
via intravenous administration.
Figure 25 is a graph depicting the tumor volume of LNCaP prostate tumor
xenografts treated with CV787 adenovirus vector (triangles; 1 x 101°
particles); CV787 and
TAXOL~1~'1 (solid squares); TAXOL~1~1 alone (solid circles; 20 mg/kg); mock
infected
control (vehicle; solid diamonds). For combination administration, CV787 was
administered first via intravenous delivery.
Figure 26 is a graph depicting the relative percent tumor volume of LNCaP
prostate
tumor xenografts treated with CV787 adenovirus vector (shaded squares; 1 x
1011
particles); CV787 (solid circles; 1 x I01° particles); CV787 (1 x
101° particles) and
mitoxantrone (Mito; "X"; 3 mg/lcg); CV787 (1 x 1011 particles) and
mitoxantrone (solid
diamonds; 3 mg/kg); mitoxantrone alone (solid triangles; 3 mg/kg) and mock
infected
control (vehicle; ""). For combination administration, CV787 was administered
first.
Figure 27 is a graph depicting the relative percent tumor volume of LNCaP
prostate
tumor xenografts treated with CV787 adenovirus vector (solid circles; 1 x
101° particles);
CV787 and estramustine (solid squares); estramustine alone (triangles); and
mock infected
control (solid diamonds). For combination administration, CV787 was
administered first.
Estramustine was administered at 14 mg/kg on days 2-5, 7-11, 13-17 and 20-24.
Figure 28 is a graph depicting the tumor volume of LNCaP prostate tumor
xenografts treated with CV787 adenovirus vector (solid circles; I x
I01° particles), CV787
and docetaxel (solid squares; 1 x 101° particles, 10 mg/kg); docetaxel
alone (solid triangles;
CA 02404060 2002-09-23
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I O mg/kg); and mock infected control (shaded diamonds). For combination
administration,
CV787 was administered first.
Figure 29 is a graph depicting the tumor volume of LNCaP prostate tumor
xenografts treated with CV787 adenovirus vector (shaded triangles; 1 x
101° particles),
CV787 (unfilled triangles; 1 x 1011 particles); CV787 and docetaxel (solid
squares; 1 x l Olo
particles, 5 mg/kg); docetaxel alone (solid circles; 5 mg/kg); and mock
infected control
(solid diamonds). For combination administration, CV787 was administered
first.
Figure 30 is a graph depicting the relative percent tumor volume of Hep3B
hepatoma xenografts treated with CV790 adenovirus vector (solid circles; 1 x
1011
particles); CV790 and doxorubicin (Doxo; solid squares); doxorubicin alone
(triangles; 10
mg/kg); and mock infected control (solid diamonds). For combination
administration,
CV790 was administered first.
Figure 31 is a graph depicting the relative percent tumor volume of Hep3B
hepatoma xenografts treated with CV890 adenovirus vector (solid circles; 1 x
1011
particles); CV890 and doxorubicin (solid squares); doxorubicin alone
(triangles; IO mg/kg);
and mock infected control (solid diamonds). For combination administration,
CV890 was
administered first.
Figure 32 is a graph depicting percent viable LNCaP prostate tumor cells
treated
with CV787 adenovirus vector (solid circles; MOI 0.1); CV787 and radiation
(solid
squares); radiation alone (solid triangles; 6 Gy); and no treatment
(diamonds). In
combination administration , radiation was administered first.
Figure 33 is a graph depicting percent viable LNCaP prostate tumor cells
treated
with CV787 adenovirus vector (solid circles; MOI 0.1); CV787 and radiation
(solid
squares); radiation alone (solid triangles; 6 Gy); and no treatment
(diamonds). In
combination administration, CV787 was administered first.
Figure 34 is a graph depicting the virus yield of CV787 adenovirus vector over
time
for CV787 administered with radiation first (solid squares; MOI 0.1; 6 Gy) and
CV787
administered without radiation (solid circles).
Figure 35 is a graph depicting the virus yield of CV787 adenovirus vector over
time
for CV787 administered before radiation (solid squares; MOI 0.1; 6 Gy) and
CV787
administered without radiation (solid circles).
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Figure 36 is a graph depicting percent of cell death of LNCaP prostate tumor
cells
treated with CV787 adenovirus vector (MOI 0.01) and increasing doses of
radiation, on day
6 of treatment. CV787 was administered first.
Figure 37 depicts a nucleotide and amino acid sequence for ADP.
Figure 38 depicts an ICSO isobologram of doxorubicin and CV 890 on Hep3B cells
at day 5.
Figure 39 depicts in vivo efficacy of CV890 with doxorubicin. Hep3B nude mouse
xenografts were grouped (n=6) and treated with CV890 alone (1x1011
particles/dose, iv),
doxorubicin alone (lOmg/kg, ip), CV890 and doxorubicin combination (1x1011
particles of
CV890 through tail vein and lOmg/lcg doxorubicin ip), or vehicle control.
Tumor size was
measured weekly and the tumor volume were normalized as 100% at the day of
treatment.
Error bars represent the standard error of the mean.
Figure 40 shows the virus yield of CV802, CV882 and CV884 in cell lines.
Figure 41 are schematic depictions of various adenovirus constructs described
herein.
MODES FOR CARRYING OUT THE INVENTION
We have discovered methods of using replication-competent, target cell-
specific
adenovirus vectors in combination with single chemotherapeutic agents,
combinations of
chemotherapeutic agents, radiation therapy treatment and the combination of
radiation
therapy and chemotherapeutic agents. The target cell-specific replication-
competent
adenovirus vectors comprise an adenovirus gene essential for replication,
preferably an
early gene, under the transcriptional control of a cell type-specific
transcriptional regulatory
element (TRE). By providing for cell type-specific transcription through the
use of one or
more cell type-specific TREs, the adenovirus vectors effect cell-specific
cytotoxicity due to
selective replication. We have observed synergy with respect to these
adenoviral vectors
and various chemotherapeutic agents as well as radiation compared to results
using
adenovirus or chemotherapy or radiation alone.
Although chemotherapeutic agents are used to treat a wide variety of cancers,
the
success rate is highly variable and the chemotherapeutic agents themselves are
highly toxic,
causing highly undesirable side effects and possibly contributing additional
mutagenic or
carcinogenic results in an already immune-compromised individual. Because the
combination of adenoviral vectors and chemotherapeutics can synergistically
enhance the
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efficacy of treatment, this in turn permits a lower effective dose of virus
andlor
chemotherapeutic agent, reducing the toxicity of the treatment and the
suffering of the
individual. An additional potential benefit is reduced length of treatment, as
we have
observed that tumors respond to the combined viral therapy more quickly than
to
chemotherapy or viral therapy alone.
We have also discovered that, in spite of their potential to damage viral DNA
and
thus compromise adenoviral vector function, viral replication is not
appreciably changed in
the presence of chemotherapeutic agents) and/or radiation, and that
simultaneous
administration of target-cell specific adenovirus and chemotherapeutic agents)
is effective
for killing tumor cells.
In some embodiments, the methods are for suppressing tumor growth. In other
embodiments, the methods are for reducing size andlor extent of a tumor. In
other
embodiments, the methods are for delaying development of a tumor. In other
embodiments, the methods are for treating a neoplasia. In still other
embodiments, the
methods are for killing tumor cells.
With respect to all methods described herein, target cells (i.e., neoplastic,
proliferative cells) are contacted with an appropriate adenovirus vector
described herein
(preferably in the form of an adenovirus particle) such that the vector enters
the cell and
viral replication initiates. Target cells) are also contacted with another
agent which kills
tumor cells, such as a chemotherapeutic agents) and/or radiation.
Individuals suitable for treatment by these methods include individuals who
have or
are suspected of having neoplasia, including individuals in the early or late
stages of the
disease, as well as individuals who have previously been treated (e.g., are in
the adjuvant
setting). Other individuals suitable for the methods described herein are
those who are
considered high risk for developing a tumor, such as those who have a genetic
predisposition to development of a neoplasia and/or who have been exposed to
an agents)
which is correlated with development of a neoplasia. Treatment regimes include
both the
eradication of tumors or other forms of the disease as well as palliation of
the disease.
These methods of treatment are suitable for numerous forms of neoplasia,
including, but
not limited to bladder cancer, prostate cancer, liver cancer, breast cancer,
colon cancer,
melanoma, ovarian pancreatic, lung, and brain cancer.
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The presence of neoplasia and the suitability of the individual for receiving
the
methods described herein rnay be determined by any of the techniques known in
the art,
including diagnostic methods such as imaging techniques, analysis of serum
tumor markers
and biopsy.
The various methods of the invention will be described below. Certain
embodiments of the methods use replication-competent target cell-specific
adenoviral
vectors such as CV706 (prostate specific); CV787(prostate specific);
CV790(liver
specific); CV829(bladder specific); CV884 (bladder specific); CV859(melanoma
specific);
CV873(colon/breast specific); CV890 (liver specific); CV874(bladder specific);
CV875(bladder specific); CV876(bladder specific); CV877(bladder specific) and
CV855(melanoma specific) as described herein. A summary of the components of
these
vectors is included in the Examples section as Table 4. Although methods of
tumor
suppression are exemplified in the discussion below, it is understood that the
alternative
methods described above axe equally applicable and suitable for these methods,
and that the
endpoints of these methods are measured using methods standard in the art,
including the
diagnostic and assessment methods described above.
General Techniques
The practice of the present invention will employ, unless otherwise indicated,
conventional techniques of molecular biology (including recombinant
techniques),
microbiology, cell biology, biochemistry and immunology, which are within the
scope of
those of skill in the art. Such techniques are explained fully in the
literature, such as,
"Molecular Cloning: A Laboratory Manual", second edition (Sambrook et al.,
1989);
"Oligonucleotide Synthesis" (M.J. Gait, ed., 1984); "Animal Cell Culture"
(R.I. Freshney,
ed., 1987); "Methods in Enzymology" (Academic Press, Inc.); "Handbook of
Experimental
Immunology" (D.M. Weir & C.C. Blackwell, eds.); "Gene Transfer Vectors for
Mammalian Cells" (J.M. Miller & M.P. Calos, eds., 1987); "Current Protocols in
Molecular Biology" (F.M. Ausubel et al., eds., 1987); "PCR: The Polymerise
Chain
Reaction", (Mullis et al., eds., 1994); and "Current Protocols in Immunology"
(J.E. Coligan
et al., eds., 1991).
For techniques related to adenovirus, see, inter alia, Felgner and Ringold
(1989)
Nature 337:387-388; Berkner and Sharp (1983) Nucl. Acids Res. 11:6003-6020;
Graham
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(1984) EMBO J. 3:2917-2922; Bett et al. (1993) J. hirology 67:5911-5921; Bett
et al.
(1994) Proc. Natl. Acad. Sci. USA 91:8802-8806.
Definitions
As used herein, the terms "neoplastic cells", "neoplasia", "tumor", "tumor
cells",
"cancer" and "cancer cells", (used interchangeably) refer to cells which
exhibit relatively
autonomous growth, so that they exhibit an aberrant growth phenotype
characterized by a
significant loss of control of cell proliferation. Neoplastic cells can be
malignant or benign.
The terms "antineoplastic agent", "antineoplastic chemotherapeutic agent",
"chemotherapeutic agent", "antineoplastic" and "chemotherapeutic" are used
interchangeably herein and refer to chemical compounds or drugs which are used
in the
treatment of cancer e.g., to kill cancer cells and/or lessen the spread of the
disease.
"Radiation therapy" is a term commonly used in the art to refer to multiple
types of
radiation therapy including internal and external radiation therapy,
radioimmunotherapy,
and the use of various types of radiation including X-rays, gamma rays, alpha
particles,
beta particles, photons, electrons, neutrons, radioisotopes, and other forms
of ionizing
radiation. As used herein, the term "radiation therapy" is inclusive of all of
these types of
radiation therapy; unless otherwise specified.
As used herein, "suppressing tumor growth" refers to reducing the rate of
growth of
a tumor, halting tumor growth completely, causing a regression in the size of
an existing
tumor, eradicating an existing tumor and/or preventing the occurrence of
additional tumors
upon treatment with the compositions, kits or methods of the present
invention.
"Suppressing" tumor growth indicates a growth state that is curtailed when
compared to
growth without contact with, i.e., transfection by, an adenoviral vector
combined with
administration of chemotherapeutic agents and radiation as described herein.
Tumor cell
growth can be assessed by any means known in the art, including, but not
limited to,
measuring tumor size, determining whether tumor cells are proliferating using
a 3H-
thymidine incorporation assay, or counting tumor cells. "Suppressing" tumor
cell growth
means any or all of the following states: slowing, delaying, and stopping
tumor growth, as
well as tumor shrinkage.
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"Delaying development" of a tumor means to defer, hinder, slow, retard,
stabilize,
andlor postpone development of the disease. This delay can be of varying
lengths of time,
depending on the history of the disease and/or individual being treated.
Aswsed herein, "synergy" or "synergistic effect" when referring to combination
administration of adenovirus vector and antineoplastic agent and/or radiation
means that
the effect of the combination is more than additive when compared to
administration of
adenovirus vector, antineoplastic agent or radiation alone.
An "adenovirus vector" or "adenoviral vector" (used interchangeably) comprises
a
polynucleotide construct of the invention. A polynucleotide construct of this
invention
may be in any of several forms, including, but not limited to, DNA, DNA
encapsulated in
an adenovirus coat, DNA packaged in another viral or viral-like form (such as
herpes
simplex, and AAV), DNA encapsulated in liposomes, DNA complexed with
polylysine,
complexed with synthetic polycationic molecules, conjugated with transferrin,
and
complexed with compounds such as PEG to immunologically "mask" the molecule
and/or
increase half life, and conjugated to a nonviral protein. Preferably, the
polynucleotide is
DNA. As used herein, "DNA" includes not only bases A, T, C, and G, but also
includes
any of their analogs or modified forms of these bases, such as methylated
nucleotides,
internucleotide modifications such as uncharged linkages and thioates, use of
sugar
analogs, and modified andlor alternative backbone structures, such as
polyamides. For
purposes of this invention, adenovirus vectors are replication-competent in a
target cell.
As used herein, a "transcription response element" or "transcriptional
regulatory
element", or "TRE" is a polynucleotide sequence, preferably a DNA sequence,
which
increases transcription of an operably linked polynucleotide sequence in a
host cell that
allows that THE to function. A THE can comprise an enhancer and/or a promoter.
A
"transcriptional regulatory sequence" is a TRE. A "target cell-specific
transcriptional
response element" or "target cell-specific TRE" is a polynucleotide sequence,
preferably a
DNA sequence, which is preferentially functional in a specific type of cell,
that is, a target
cell. Accordingly, a target cell-specific THE transcribes an operably linked
polynucleotide
sequence in a target cell that allows the target cell-specific THE to
function. The term
"target cell-specific", as used herein, is intended to include cell type
specificity, tissue
specificity, developmental stage specificity, and tumor specificity, as well
as specificity for
a cancerous state of a given target cell. "Target cell-specific TRE" includes
cell type-
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specific and cell status-specific TRE, as well as "composite" TREs. The term
"composite
TRE" includes a THE which comprises both a cell type-specific and a cell
status-specific
TRE. A target cell-specific THE can also include a heterologous component,
including, for
example, an SV40 or a cytomegalovirus (CMV) promoter(s). An example of a
target cell
~ specific THE which is tissue specific is a CMV THE which contains both
promoters) and
enhancer(s).
As described in more detail herein, a target cell-specific THE can comprise
any
number of configurations, including, but not limited to, a target cell-
specific promoter; and
target cell-specific enhancer; a heterologous promoter and a target cell-
specific enhancer; a
target cell-specific promoter and a heterologous enhancer; a heterologous
promoter and a
heterologous enhancer; and multimers of the foregoing. The promoter and
enhancer
components of a target cell-specific THE may be in any orientation and/or
distance from
the coding sequence of interest, as long as the desired target cell-specific
transcriptional
activity is obtained. Transcriptional activation can be measured in a number
of ways
known in the art (and described in more detail below), but is generally
measured by
detection and/or quantitation of mRNA or the protein product of the coding
sequence under
control of (i.e., operably linked to) the target cell-specific TRE. As
discussed herein, a
target cell-specific THE can be of varying lengths, and of varying sequence
composition.
As used herein, the term "cell status-specific TRE" is preferentially
functional, i.e., confers
~ transcriptional activation on an operably linked polynucleotide in a cell
which allows a cell
status-specific THE to function, i.e., a cell which exhibits a particular
physiological
condition, including, but not limited to, an aberrant physiological state.
"Cell status" thus
refers to a given, or particular, physiological state (or condition) of a
cell, which is
reversible and/or progressive. The physiological state may be generated
internally or
externally; for example, it may be a metabolic state (such as in response to
conditions of
low oxygen), or it may be generated due to heat or ionizing radiation. "Cell
status" is
distinct from a "cell type", which relates to a differentiation state of a
cell, which under
normal conditions is irreversible. Generally (but not necessarily), as
discussed herein, a
cell status is embodied in an aberrant physiological state, examples of which
are given
below.
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A "functional portion" of a target cell-specific THE is one which confers
target cell-
specific transcription on an operably linked gene or coding region, such that
the operably
linked gene or coding region is preferentially expressed in the target cells.
By "transcriptional activation" or an "increase in transcription," it is
intended that
transcription is increased above basal levels in the target cell (i.e., target
cell) by at least
about 2 fold, preferably at least about 5 fold, preferably at least about 10
fold, more
preferably at least about 20 fold, more preferably at least about 50 fold,
more preferably at
least about 100 fold, more preferably at least about 200 fold, even more
preferably at least
about 400 fold to about 500 fold, even more preferably at least about 1000
fold. Basal
levels are generally the level of activity (if any) in a non-target cell
(i.e., a different cell
type), or the level of activity (if any) of a reporter construct lacking a
target cell-specific
THE as tested in a target cell line.
A "functionally-preserved variant" of a target cell-specific THE is a target
cell-
specific THE which differs from another target cell-specific TRE, but still
retains target
cell-specific transcription activity, although the degree of activation may be
altered (as
discussed below). The difference in a target cell-specific THE can be due to
differences in
linear sequence, arising from, for example, single base mutation(s),
addition(s), deletion(s),
and/or modifications) of the bases. The difference can also arise from changes
in the
sugar(s), and/or linkages) between the bases of a target cell-specific TRE.
For example,
certain point mutations within sequences of TREs have been shown to decrease
transcription factor binding and stimulation of transcription. See Blackwood,
et al. (1998)
Science 281:60-63 and Smith et al. (1997) J. Biol. Chem. 272:27493-27496. One
of skill in
the art would recognize that some alterations of bases in and around
transcription factor
binding sites are more likely to negatively affect stimulation of
transcription and cell-
specificity, while alterations in bases which are not involved in
transcription factor binding
are not as likely to have such effects. Certain mutations are also capable of
increasing THE
activity. Testing of the effects of altering bases may be performed i~ vitro
or in vivo by any
method known in the art, such as mobility shift assays, or transfecting
vectors containing
these alterations in THE functional and THE non-functional cells.
Additionally, one of
skill in the art would recognize that point mutations and deletions can be
made to a THE
sequence without altering the ability of the sequence to regulate
transcription.
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As used herein, a THE derived from a specific gene is referred to by the gene
from
which it was derived and is a polynucleotide sequence which regulates
transcription of an
operably linked polynucleotide sequence in a host cell that expresses said
gene. Fox
example, as used herein, a "human glandular kallikrein transcriptional
regulatory element",
or "hKLK2-TRE" is a polynucleotide sequence, preferably a DNA sequence, which
increases transcription of an operably linked polynucleotide sequence in a
host cell that
allows an hKLK2-THE to function, such as a cell (preferably a mammalian cell,
even more
preferably a human cell) that expresses androgen receptor, such as a prostate
cell. An
hKLK2-THE is thus responsive to the binding of androgen receptor and comprises
at least a
portion of an hKLK2 promoter and/or an hKLK2 enhancer (i.e., the ARE or
androgen
receptor binding site).
As used herein, a "probasin (PB) transcriptional regulatory element", or "PB-
TRE"
is a polynucleotide sequence, preferably a DNA sequence, which selectively
increases
transcription of an operably-linked polynucleotide sequence in a host cell
that allows a PB-
THE to function, such as a cell (preferably a mammalian cell, more preferably
a human
cell, even more preferably a prostate cell) that expresses androgen receptor.
A PB-THE is
thus responsive to the binding of androgen receptor and comprises at least a
portion of a PB
promoter and/or a PB enhancer (i.e., the ARE or androgen receptor binding
site).
As used herein, a "prostate-specific antigen (PSA) transcriptional regulatory
element", or "PSA-THE", or "PSE TRE" is a polynucleotide sequence, preferably
a DNA
sequence, which selectively increases transcription of an operably linked
polynucleotide
sequence in a host cell that allows a PSA-THE to function, such as a cell
(preferably a
mammalian cell, more preferably a human cell, even more preferably a prostate
cell) that
expresses androgen receptor. A PSA-THE is thus responsive to the binding of
androgen
receptor and comprises at least a portion of a PSA promoter and/or a PSA
enhancer (i.e., the .
ARE or androgen receptor binding site).
As used herein, a "carcinoembryonic antigen (CEA) transcriptional regulatory
element", or "CEA-TRE" is a polynucleotide sequence, preferably a DNA
sequence, which
selectively increases transcription of an operably linked polynucleotide
sequence in a host
cell that allows a CEA-THE to function, such as a cell (preferably a mammalian
cell, even
more preferably a human cell) that expresses CEA. The CEA-THE is responsive to
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CA 02404060 2002-09-23
WO 01/72341 PCT/USO1/09042
transcription factors and/or co-factor(s) associated with CEA-producing cells
and
comprises at least a portion of the CEA promoter and/or enhancer.
As used herein, an "oc-fetoprotein (AFP) transcriptional regulatory element",
or
"AFP-TRE" is a polynucleotide sequence, preferably a DNA sequence, which
selectively
increases transcription (of an operably linked polynucleotide sequence) in a
host cell that
allows an AFP-THE to function, such as a cell (preferably a mammalian cell,
even more
preferably a human cell) that expresses AFP. The AFP-THE is responsive to
transcription
factors and/or co-factor(s) associated with AFP-producing cells and comprises
at least a
portion of the AFP promoter and/or enhancer.
As used herein, an "a mucin gene (lllUC) transcriptional regulatory element",
or
"MUCl -TRE" is a polynucleotide sequence, preferably a DNA sequence, which
selectively
increases transcription (of an operably-linked polynucleotide sequence) in a
host cell that
allows a MUCI -THE to function, such as a cell (preferably a mammalian cell,
even more
preferably a human cell) that expresses MUC 1. The MUCI -THE is responsive to
transcription factors and/or co-factor(s) associated with MUC1-producing cells
and
comprises at least a portion of the MUCl promoter and/or enhancer.
As used herein, a "urothelial cell-specific transcriptional response element",
or
"urothelial cell-specific TRE" is polynucleotide sequence, preferably a DNA
sequence,
which increases transcription of an operably linked polynucleotide sequence in
a host cell
that allows a urothelial-specific THE to function, i.e., a target cell. A
variety of urothelial
cell-specific TREs are known, are responsive to cellular proteins
(transcription factors
and/or co-faetor(s)) associated with urothelial cells, and comprise at least a
portion of a
urothelial-specific promoter and/or a urothelial-specific enhancer. Methods
are described
herein for measuring the activity of a urothelial cell-specific THE and thus
for determining
whether a given cell allows a urothelial cell-specific THE to function.
As used herein, a "rnelanocyte cell-specific transcriptional response
element", or
"melanocyte cell-specific TRE" is polynucleotide sequence, preferably a DNA
sequence,
which increases transcription of an operably linked polynucleotide sequence in
a host cell
that allows a melanocyte-specific THE to function, i.e., a target cell. A
variety of
melanocyte cell-specific TREs are known, are responsive to cellular proteins
(transcription
factors and/or co-factor(s)) associated with melanocyte cells, and comprise at
least a
portion of a melanocyte-specific promoter and/or a melanocyte-specific
enhancer.
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Methods are described herein for measuring the activity of a melanocyte cell-
specific THE
and thus for determining whether a given cell allows a melanocyte cell-
specific THE to
function.
As used herein, a target cell-specific THE can comprise any number of
configurations, including, but not limited to, a target cell-specific
promoter; a target cell-
specific enhancer; a target cell-specific promoter and a target cell-specific
enhancer; a
target cell-specific promoter and a heterologous enhancer; a heterologous
promoter and a
target cell-specific enhancer; and multimers of the foregoing. The promoter
and enhancer
components of a target cell-specific THE may be in any orientation and/or
distance from
the coding sequence of interest, as long as the desired target cell-specific
transcriptional
activity is obtained. Transcriptional activation can be measured in a number
of ways
known in the art (and described in more detail below), but is generally
measured by
detection and/or quantitation of mRNA or the protein product of the coding
sequence under
control of (i.e., operably linked to) the target cell-specific TRE.
As used herein, an "internal ribosome entry site" or "IRES" refers to an
element
that promotes direct internal ribosome entry to the initiation codon, such as
ATG, of a
cistron (a protein encoding region), thereby leading to the cap-independent
translation of
the gene. Jackson RJ, Howell MT, Kaminski A (1990) Trends Bioehem Sci
15(12):477-83)
and Jackson RJ and Kaminski, A. (1995) RNA 1(10):985-1000). The present
invention
encompasses the use of any IRES element which is able to promote direct
internal
ribosome entry to the initiation codon of a cistron. "Under translational
control of an
IRES" as used herein means that translation is associated with the IRES and
proceeds in a
cap-independent manner. Examples of "IRES" known in the art include, but are
not limited
to IRES obtainable from picornavirus (Jackson et al., 1990, Tends Biochem Sci
15(12):477-483); and IRES obtainable from viral or cellular mRNA sources, such
as for
example, immunogloublin heavy-chain binding protein (BiP), the vascular
endothelial
growth factor (VEGF) (Huez et al. (1998) Mol. Cell. Biol. 18(11):6178-6190),
the
fibroblast growth factor 2, and insulin-like growth factor, the translational
initiation factor
eIF4G, yeast transcription factors TFIID and HAP4. IRES have also been
reported in
different viruses such as cardiovirus, rhinovirus, aphthovirus, HCV, Friend
marine
leukemia virus (FrMLV) and Moloney marine leukemia virus (MoMLV). As used
herein,
"IRES" encompasses functional variations of IRES sequences as long as the
variation is
26
CA 02404060 2002-09-23
WO 01/72341 PCT/USO1/09042
able to promote direct internal ribosome entry to the initiation codon of a
cistron. In
preferred embodiments, the IRES is mammalian. In other embodiments, the IRES
is viral
or protozoan. In one illustrative embodiment disclosed herein, the IRES is
obtainable from
encephelomycarditis virus (ECMV) (commercially available from Novogen, Duke et
al.
(1992) J. Viol 66(3):1602-1609). In another illustrative embodiment disclosed
herein, the
IRES is from VEGF. Table I and Table II disclose a variety of IRES sequences
useful in
the present invention. In some embodiments, an adenovirus vector comprising an
IRES
exhibits greater specificity for the target cell than an adenovirus vector
comprising a target
cell-specific THE operably linked to a gene and lacking an IRES. In some
embodiments,
specificity is conferred by preferential transcription and/or translation of
the first and
second genes due to the presence of a target cell specific TRE. In other
embodiments,
specificity is conferred by preferential replication of the adenovirus vectors
in target cells
due to the target cell-specific THE driving transcription of a gene essential
for replication.
A "multicistronic transcript" refers to an mRNA molecule which contains more
than one protein coding region, or cistron. A mRNA comprising two coding
regions is
denoted a "bicistronic transcript." The "5'-proximal" coding region or cistron
is the coding
region whose translation initiation codon (usually AUG) is closest to the 5'-
end of a
multicistronic mRNA molecule. A "5'-distal" coding region or cistron is one
whose
translation initiation codon (usually AUG) is not the closest initiation codon
to the 5' end
of the mRNA. The terms "5'-distal" and "downstream" are used synonymously to
refer to
coding regions that are not adjacent to the 5' end of a mRNA molecule.
As used herein, "co-transcribed" means that two (or more) coding regions of
polynucleotides are under transcriptional control of single transcriptional
control element.
A "gene" refers to a coding region of a polynucleotide. A "gene" may or may
not
include non-coding sequences and/or regulatory elements.
"Replicating preferentially", as used herein, means that the adenovirus
replicates
more in a target cell than a non-target cell. Preferably, the adenovirus
replicates at a
significantly higher rate in target cells than non target cells; preferably,
at least about 2-fold
higher, preferably, at least about 5-fold higher, more preferably, at least
about 10-fold
higher, still more preferably at least about 50-fold higher, even more
preferably at least
about 100-fold higher, still more preferably at least about 400- to 500-fold
higher, still
more preferably at least about 1000-fold higher, most preferably at least
about 1 x 106
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WO 01/72341 PCT/USO1/09042
higher. Most preferably, the adenovirus replicates solely in the target cells
(that is, does not
replicate or replicates at a very low levels in non-target cells).
As used herein, the term "vector" refers to a polynucleotide construct
designed for
transduction/transfection of one or more cell types. Vectors may be, for
example, "cloning
vectors" which are designed for isolation, propagation and replication of
inserted
nucleotides, "expression vectors" which are designed for expression of a
nucleotide
sequence in a host cell, or a "viral vector" which is designed to result in
the production of a
recombinant virus or virus-like particle, or "shuttle vectors", which comprise
the attributes
of more than one type of vector.
The terms "polynucleotide" and "nucleic acid", used interchangeably herein,
refer
to a polymeric form of nucleotides of any length, either ribonucleotides or
deoxyribonucleotides. These terms include a single-, double- or triple-
stranded DNA,
genomic DNA, cDNA, RNA, DNA-RNA hybrid, or a polymer comprising purine and
pyrimidine bases, or other natural, chemically, biochemically modified, non-
natural or
derivatized nucleotide bases. The backbone of the polynucleotide can comprise
sugars and
phosphate groups (as may typically be found in RNA or DNA), or modified or
substituted
sugar or phosphate groups. Alternatively, the backbone of the polynucleotide
can comprise
a polymer of synthetic subunits such as phosphoramidates and thus can be a
oligodeoxynucleoside phosphoramidate (P-NH2) or a mixed phosphoramidate-
phosphodiester oligomer. Peyrottes et al. (1996) Nucleic Acids Res: 24: 1841-
8; .
Chaturvedi et al. (1996) Nucleic Acids Res. 24: 2318-23; Schultz et al. (1996)
Nucleic
Acids Res. 24: 2966-73. A phosphorothioate linkage can be used in place of a
phosphodiester linkage. Braun et al. (1988) J. Immunol. 141: 2084-9; Latimer
et al. (1995)
Molec. Immuhol. 32: 1057-1064. In addition, a double-stranded polynucleotide
can be
obtained from the single stranded polynucleotide product of chemical synthesis
either by
synthesizing the complementary strand and annealing the strands under
appropriate
conditions, or by synthesizing the complementary strand de novo using a DNA
polymerase
with an appropriate primer. Reference to a polynucleotide sequence (such as
referring to a
SEQ ID NO) also includes the complement sequence.
The following are non-limiting examples of polynucleotides: a gene or gene
fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant
polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of
any
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CA 02404060 2002-09-23
WO 01/72341 PCT/USO1/09042
sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A
polynucleotide may comprise modified nucleotides, such as methylated
nucleotides and
nucleotide analogs, uracyl, other sugars and linking groups such as
fluororibose and
thioate, and nucleotide branches. The sequence of nucleotides may be
interrupted by non-
nucleotide components. A polynucleotide may be further modified after
polymerization,
such as by conjugation with a labeling component. Other types of modifications
included
in this definition are caps, substitution of one or more of the naturally
occurring nucleotides
with an analog, and introduction of means for attaching the polynucleotide to
proteins,
metal ions, labeling components, other polynucleotides, or a solid support.
Preferably, the
polynucleotide is DNA. As used herein, "DNA" includes not only bases A, T, C,
and G,
but also includes any of their analogs or modified forms of these bases, such
as methylated
nucleotides, internucleotide modifications such as uncharged linkages and
thioates, use of
sugar analogs, and modified and/or alternative backbone structures, such as
polyamides.
A polynucleotide or polynucleotide region has a certain percentage (for
example,
80%, 85%, 90%, or 95%) of "sequence identity" to another sequence means that,
when
aligned, that percentage of bases are the same in comparing the two sequences.
This
alignment and the percent homology or sequence identity can be determined
using software
programs known in the art, for example those described in Current Protocols i~
Molecular
Biology (F.M. Ausubel et al., eds., 1987) Supplement 30, section 7.7.18. A
preferred
alignment program is ALIGN Plus (Scientific and Educational Software,
Pennsylvania),
preferably using default parameters, which are as follows: mismatch = 2; open
gap = 0;
extend gap = 2.
"Under transcriptional control" is a term well understood in the art and
indicates
that transcription of a polynucleotide sequence, usually a DNA sequence,
depends on its
being operably (operatively) linked to an element which contributes to the
initiation of, or
promotes, transcription. "Operably linked" refers to a juxtaposition wherein
the elements
are in an arrangement allowing them to function.
An "E3 region" (used interchangeably with "E3") is a term well understood in
the
art and means the region of the adenoviral genome that encodes the E3 products
(discussed
herein). Generally, the E3 region is located between about 28583 and 30470 of
the
adenoviral genome. The E3 region has been described in various publications,
including,
for example, Wold et al. (1995) Curr. Topics Microbiol. Immunol. 199:237-274.
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A "portion" of the E3 region means less than the entire E3 region, and as such
includes polynucleotide deletions as well as polynucleotides encoding one or
more
polypeptide products of the E3 region. As used herein, "cytotoxicity" is a
term well
understood in the art and refers to a state in which a cell's usual
biochemical or biological
activities are compromised (i.e., inhibited). These activities include, but
are not limited to,
metabolism; cellular replication; DNA replication; transcription; translation;
uptake of
molecules. "Cytotoxicity" includes cell death and/or cytolysis. Assays are
known in the
art which indicate cytotoxicity, such as dye exclusion, 3H-thymidine uptake,
and plaque
assays.
An "E 1 B I 9-kDa region" (used interchangeably with "E 1 B 19-kDa genomic
region") refers to the genomic region of the adenovirus E1B gene encoding the
E1B 19-
kDa product. According to wild-type Ad5 , the EIB 19-kDa region is a 261bp
region
located between nucleotide 1714 and nucleotide 2244. The E1B 19-kDa region has
been
described in, for example, Rao et al., Proc. Natl. Acad. Sci. USA, 89: 7742-
7746. The
present invention encompasses deletion of part or all of the E1B 19-kDa region
as well as
embodiments wherein the E1B 19-kDa region is mutated, as long as the deletion
or
mutation lessens or eliminates the inhibition of apoptosis associated with E1B-
l9kDa.
The term "selective cytotoxicity", as used herein, refers to the cytotoxicity
conferred by an adenovirus vector of the present invention on a cell which
allows or
induces a target cell-specific THE to function (a target cell) when compared
to the
cytotoxicity conferred by an adenoviral vector of the present invention on a
cell which does
not allow a target cell-specific THE to function (a non-target cell). Such
cytotoxicity may
be measured, for example, by plaque assays, by reduction or stabilization in
size of a tumor
comprising target cells, or the reduction or stabilization of serum levels of
a marker
characteristic of the tumor cells, or a tissue-specific marker, e.g., a cancer
marker.
In the context of adenovirus, a "heterologous polynucleotide" or "heterologous
gene" or "transgene" is any polynucleotide or gene that is not present in wild-
type
adenovirus. Preferably, the transgene will also not be expressed or present in
the target cell
prior to introduction by the adenovirus vector. Examples of preferred
transgenes are
provided below.
In the context of adenovirus, a "heterologous" promoter or enhancer is one
which is
not associated with or derived from an adenovirus gene.
CA 02404060 2002-09-23
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In the context of adenovirus, an "endogenous" promoter, enhancer, or THE is
native
to or derived from adenovirus. In the context of promoter, an "inactivation"
means that
there is a mutation of or deletion in part or all of the of the endogenous
promoter, ie, a
modification or alteration of the endogenous promoter, such as, for example, a
point
mutation or insertion, which disables the function of the promoter.
In the context of a target cell-specific TRE, a "heterologous" promoter or
enhancer
is one which is derived from a gene other than the gene from which a reference
target cell-
specific THE is derived.
A "host cell" includes an individual cell or cell culture which can be or has
been a
recipient of an adenoviral vectors) of this invention. Host cells include
progeny of a single
host cell, and the progeny may not necessarily be completely identical (in
morphology or ina
total DNA complement) to the original parent cell due to natural, accidental,
or deliberate
mutation and/or change. A host cell includes cells transfected or infected i~
vivo or in vitro
with an adenoviral vector of this invention.
"Replication" and "propagation" are used interchangeably and refer to the
ability of
an adenovirus vector of the invention to reproduce or proliferate. These terms
are well
understood in the art. For purposes of this invention, replication involves
production of
adenovirus proteins and is generally directed to reproduction of adenovirus.
Replication
can be measured using assays standard in the art and described herein, such as
a burst assay
or plaque assay. "Replication" and "propagation" include any activity directly
or indirectly
involved in the process of virus manufacture, including, but not limited to,
viral gene
expression; production of viral proteins, nucleic acids or other components;
packaging of
viral components into complete viruses; and cell lysis.
An "ADP coding sequence" is a polynucleotide that encodes ADP or a functional
fragment thereof. In the context of ADP, a "functional fragment" of ADP is one
that
exhibits cytotoxic activity, especially cell lysis, with respect to adenoviral
replication.
Ways to measure cytotoxic activity are known in the art and axe described
herein.
A polynucleotide that "encodes" an ADP polypeptide is one that can be
transcribed
and/or translated to produce an ADP polypeptide or a fragment thereof. The
anti-sense
strand of such a polynucleotide is also said to encode the sequence.
An "ADP polypeptide" is a polypeptide containing at least a portion, or
region, of
the amino acid sequence of an ADP and which displays a function associated
with ADP,
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particularly cytotoxicity, more particularly, cell lysis. As discussed herein,
these functions
can be measured using techniques known in the art. It is understood that
certain sequence
variations may be used, due to, for example, conservative amino acid
substitutions, which
may provide ADP polypeptides.
"Androgen receptor," or AR, as used herein refers to a protein whose function
is to
specifically bind to androgen and, as a consequence of the specific binding,
recognize and
bind to an androgen response element (ARE), following which the AR is capable
of
regulating transcriptional activity. The AR is a nuclear receptor that, when
activated, binds
to cellular androgen-responsive element(s). In normal cells the AR is
activated by
androgen, but in non-normal cells (including malignant cells) the AR may be
activated by
non-androgenic agents, including hormones other than androgens. Encompassed in
the
term "androgen receptor" are mutant forms of an androgen receptor, such as
those
characterized by amino acid additions, insertions, truncations and deletions,
as long as the
function is sufficiently preserved. Mutants include androgen receptors with
amino acid
additions, insertions, truncations and deletions, as long as the function is
sufficiently
preserved. In this context, a functional androgen receptor is one that binds
both androgen
and, upon androgen binding, an ARE.
A polynucleotide sequence that is "depicted in" a SEQ ID NO means that the
sequence is present as an identical contiguous sequence in the SEQ ID NO. The
term
encompasses portions, or regions of the SEQ ID NO as well as the entire
sequence
contained within the SEQ ID NO.
A "biological sample" encompasses a variety of sample types obtained from an
individual and can be used in a diagnostic or monitoring assay. The definition
encompasses blood and other liquid samples of biological origin, solid tissue
samples such
as a biopsy specimen or tissue cultures or cells derived therefrom, and the
progeny thereof.
The definition also includes samples that have been manipulated in any way
after their
procurement, such as by treatment with reagents, solubilization, or enrichment
for certain
components, such as proteins or polynucleotides. The term "biological sample"
encompasses a clinical sample, and also includes cells in culture, cell
supernatants, cell
lysates, serum, plasma, biological fluid, and tissue samples.
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An "individual" is a vertebrate, preferably a mammal, more preferably a human.
Mammals include, but are not limited to, farm animals, sport animals, rodents,
primates,
and pets.
An "effective amount" is an amount sufficient to effect beneficial or desired
results,
including clinical results. An effective amount can be administered in one or
more
administrations. For purposes of this invention, an effective amount of an
adenoviral
vector is an amount that is sufficient to palliate, ameliorate, stabilize,
reverse, slow or delay
the progression of the disease state.
A given THE is "derived from" a given gene if it is associated with that gene
in
nature.
"Expression" includes transcription and/or translation.
As used herein, the term "comprising" and its cognates are used in their
inclusive
sense; that is, equivalent to the term "including" and its corresponding
cognates.
"A," "an" and "the" include plural references unless the context clearly
dictates
I S otherwise.
Combination Adenoviral and Chemotherapeutic Therapy
Embodiments of the present invention include methods for the administration of
combinations of a target cell-specific adenoviral vector and at least one
antineoplastic
agents) to an individual with neoplasia. The antineoplastic agent includes
those listed in
Table 1. These include agents from each of the major classes of
chemotherapeutics,
including but not limited to: alkylating agents, alkaloids, antimetabolites,
anti-tumor
antibiotics, nitrosoureas, hormonal agonistslantagonists and analogs,
immunomodulators,
photosensitizers, enzymes and others. In some embodiments, the antineoplastic
is an
alkaloid, an antimetabolite, an antibiotic or an alkylating agent. In certain
embodiments the
antineoplastic agents include, for example, thiotepa, interferon alpha-2a, and
the M-VAC
combination (methotrexate-vinblastine, doxorubicin, cyclophosphamide).
Preferred
antineoplastic agents include, for example, 5-fluorouracil, cisplatin, 5-
azacytidine, and
gemcitabine. Particularly preferred embodiments include, but are not limited
to,
doxorubicin, estramustine, etoposide, mitoxantrone, docetaxel (TAXOTERE~),
paclitaxel
(TAXOL~), and mitomycin C.
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CA 02404060 2002-09-23
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Table 1: Antineoplastic Agents
ALKYLATING ANTIBIOTICS
AI:KAL(,IIDS.,tIGENTS:; ANALOGS ANTIMETABQhITESENZYMES
>IMMIJNOMODIn;ATORB
''
Docetaxel lkyl Sulfonatesclacinomycinsolic Acid -AsparaginaseInterferon-a
(TAXOTERETM) nalogs
toposide Busulfan ctinomycin Denopterin egasargaseInterferon-(3
Fl
Irinotecan Improsulfannthramycin datrexate Interferon-'y
aclitaxel iposulfan zaserine ethotrexate Interferon-a-2a
(TAXOLTM)
Teniposide leomycins iritrexim Interleukin-2
Topotecan ziridines Cactinomycineropterin entinan
Vinblastineenzodepa Carubicin Tomudex~ ropagermanium
incristine Carboquone CarzinophilinTrimetrexate PSI~
endesine Meturedepa Chromomycins Roquinimex
inorelbine Uredepa Dactinomycinurine Analogs Rituximab
DaunorubicinCladribine Sizofiran
thylenimines6-Diazo-5-oxo-L-Fludarabine Trastuzumab
and orleucine
ethylmedamines
ltretamine oxorubicin 6-Mercaptopurine enimex
Triethylenemelampirubicin Thiamiprine
ine
TriethylenephosphIdarubicin Thioguanine
oramide
Triethylenethiophenogaril
osphoramide
Mitomycins
itoxantroneyrimidine
nalogs
itrogen Mycophenoliccitabine
ustards Acid
Chlorambucilogalamycin 5-Azacytidine
ChlomaphazineOlivomycins6-Azauridine
Cyclophosphamideplomycin Carmofur
a
stramustineirarubicin Cytarabine
Ifosfamide Plicamycin,oxifluridine
echlorethamineorfiromycinmitefur
echlorethaminePuromycin nocitabine
Oxide
Hydrochloride
elphalan Streptonigrinloxuridine
ovembichin Streptozocinluorouracil
Valrubicin
erfosfamideTubercidin Gemcitabine
henesterineinostatin Tegafur
Prednimustineorubicin
Trofosfamide
Uracil Mustard
Carboplatin
Cisplatin
iboplatin
Oxaliplatin
Others
34
CA 02404060 2002-09-23
WO 01/72341 PCT/USO1/09042
,~ i' .ioxlcs , , ~..,
~; LAT~1G .v
LKALOIDS ' ANTIB ANTIMETABOLITESNZXMES jp~ylODULATORS:'
'AGENTS ANDANALi3GS.' : .
. -
_ acarbazine
annomustine
itobronitol
Mitolactol
iotepa
ipobroman
Temozolomide~
HQ~~..
AN~'AGONISTS/AGONISTSc
ITfiOSOUREAS'OTHERS ~.' PItOTOSENSTTIZER
ANALOGS:.
Carmustine AceglatoneDexamethasone Porfimer Sodium
ChlorozotocinAmsacrine Prednisone
Fotemustine Bisantrene
Lomustine DefosfamideAndrogens
Nimustine DemecolcineCalusterone
Ranimustine DiaziquoneDromostanolone
EflornithineEpitiostanol
ElliptiniumMepitiostane
Acetate
Etoglucid . Testolactone
Fenretinide
FinasterideAntiadrenals
Gallium Arninoglutethimide
Nitrate
HydroxyureaMitotane
LonidamineTrilostane
Miltefosine
MitoguazoneAntiandrogens
Mopidamol Bicalutamide
NitracrineFlutamide
PentostatinNilutamide
Phenamet
PodophyllinicAntiestrogens
Acid 2-
Ethylhydrazide
ProcarbazineDroloxifene
Razoxane Tamoxifen
SobuzoxaneToremifene
SpirogermaniuExemestane
m
Amsacrine Aromatase Inhibitors
Tretinoin Aminoglutethimide
TenuazonicAnastrozole
Acid
TriaziquoneFadrozole
2,2',2"- Foxtnestane
Triclorotriethyl
amine,
Urethan Letrozole
Topotecan
Estrogens
Fosfestrol
CA 02404060 2002-09-23
WO 01/72341 PCT/USO1/09042
HoBMONE..:
,
ITROSOIjREAsS'.QTBERS.: ANTAGONISTSIAGONISTSPHOTOSENSITiZER.
8t
''ANALOGS ~-,
- ~
Hexestrol
Polyestradiol
Phosphate
LHRH Analogs
Buserelin
Goserelin
Leuprolide
Triptorelin,
Progestogens
Chlormadinone
Acetate
Medroxyprogesterone
Megestrol Acetate
Melengestrol
This section provides exemplary non-inclusive vector and chemotherapeutic
combinations. The adenoviral vector used in the methods described herein is
generally a
replication-competent, target-cell specific adenoviral vector comprising an
adenovirus gene
essential for replication under transcriptional control of a TRE, embodiments
of which are
described infra. In some embodiments, the gene essential for replication in
the adenoviral
vector is an early gene, preferably ElA and/or E1B. In some embodiments the
ElA and
E 1 B genes are under transcriptional of identical TREs. In other embodiments
E 1 A and
E1B genes are under transcriptional control of non-identical (or heterologous)
TREs. In
some embodiments, the adenovirus vector comprises a transgene. In other
embodiments,
the adenovirus vector comprises ADP. In some embodiments, the adenovirus
vector
contains an E3 region.
In other embodiments, the adenovirus vectors comprise co-transcribed first and
second genes under transcriptional control of a heterologous, target cell-
specific
transcriptional regulatory element (TRE), wherein the second gene is under
translational
control of an internal ribosome entry site (IRES).
The choice of adenoviral vector is primarily determined by the identity of the
target
cells and therefore the type of cancer to be treated. As explained below in
detail, an
adenoviral vector comprising a PSA-TRE, PB-TRE, or hKLK2-THE would
preferentially
replicate in prostate cells; an adenoviral vector comprising a CEA-THE would
preferentially replicate in colorectal, gastric, pancreatic, breast and lung
cells; an AFP-THE
would preferentially replicate in hepatoma cells, or liver tumors; a
urothelial cell-specific
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THE (such as uroplakin) would preferentially replicate in bladder cells; a MUC-
THE would
preferentially replicate in breast cells; a melanocyte specific THE (such as
tyrosinase)
would preferentially replicate in melanoma cells.
Certain combinations of adenoviral vector and chemotherapeutic are
particularly
effective for the treatment of particular types of cancer using the methods
described above.
Based on our in vitro studies, not all combinations of target cell-specific
adenoviral vector
and chemotherapeutic result in synergy. As shown in Tables 5 and 6 in Examples
1 and 2,
gemcitabine used with CV790 (a liver-specific virus with ElA and E1B under
transcriptional control of two identical AFP-TREs) results in synergy.
However, when
gemcitabine is used with CV787 (a prostate-specific virus with ElA under
transcriptional
control of a PB-THE and E1B under transcriptional control of a PSE-TRE),
synergy is not
observed. 5-fluorouracil used with prostate-specific adenovirus..CV787 results
in synergy,
but when used with liver-specific adenovirus CV790, synergy is not observed.
In another
embodiment disclosed herein, CV884 used with doxorubicin provides synergistic
effect.
I 5 For example, with respect to treatment of prostate tumors, a replication-
competent
adenovirus in which a gene essential for replication, preferably one or more
early genes, is
under transcriptional control of a prostate specific TRE, as discussed below,
may be used in
conjunction with an antineoplastic agent that is in the alkaloid,
antimetabolite, antibiotic, or
alkylating agent class of antineoplastics. Preferred examples of
antineoplastic agents
include doxorubicin, mitoxantrone, paclitaxel, estramustine, etoposide and
docetaxel.
Additional examples of antineoplastic agents include, 5-fluorouracil or
cisplatin.
In some embodiments of the adenovirus vector, ElA is under transcriptional
control
of a prostate specific TRE. In other embodiments EIB is under transcriptional
control of a
prostate specific TRE. In yet other embodiments, both ElA and E1B are under
transcriptional control of prostate specific TREs, which may or may not be the
same
sequence. An example of a suitable prostate specific replication-competent
adenoviral
vector is one comprising probasin (PB)-THE controlling transcription of ElA,
and PSE-
TRE controlling transcription of E1B, such as CV787 as described in the
examples.
Particularly preferred embodiments include administration of the combination
of 5-
fluorouracil with a prostate specific adenoviral vector in which a PSA-THE
controls
transcription of ElA. An example of a suitable adenoviral vector is CV706.
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In some embodiments, a prostate specific adenoviral vector comprising ElA and
E1B under transcriptional control of two non-identical prostate specific TREs,
is
administered in conjunction with any of the following antineoplastic agents:
paclitaxel;
docetaxel; cisplatin; doxorubicin; estramustine; etoposide; mitoxantrone; and
5-
S fluorouracil. In some embodiments, the prostate specific THE controlling
transcription of
ElA and the prostate specific THE controlling transcription of E1B are
heterologous (i.e.,
of different sequence) with respect to each other. In some embodiments, the
prostate
specific THE controlling transcription of ElA is derived from probasin (PB)
and the
prostate specific THE controlling transcription of E1B is derived from
prostate specific
antigen (PSA). In other embodiments, the prostate specific THE controlling
transcription
of ElA is derived from PSA, and the prostate specific THE controlling
transcription of E1B
is derived from probasin. PSA-derived and PB-derived TREs are described
herein. In
some embodiments, the adenoviral vector is CV787. In some embodiments, an IRES
is
translationally linked to an adenovirus gene essential for replication, such
as E1B and in
preferred embodiments, E 1 B has its endogenous promoter deleted and the IRES
and E 1 B
are in frame. In other embodiments, the 19-kDa region of E1B is deleted.
Preferably, the prostate specific adenovirus vectors used in these methods
also
contains an E3 region, as described herein. For example, CV787 contains an E3
region.
With respect to liver tumors (hepatoma), any liver cell specific adenoviral
vector
may be used with the chemotherapeutic agents described herein. Preferably, the
THE is
derived from AFP. The liver specific adenovirus vectors may be used with
chemotherapeutic agents from any of the following classes: antimetabolites
(especially
DNA damaging agents); alkylating agents (especially platinum containing
agents);
antibiotics; alkaloids. Preferably, the chemotherapeutic agent is an
antibiotic such as
doxorubicin, mitoxantrone, or mitomycin-C. In some embodiments, the
chemotherapeutic
agent is paclitaxel , 5-azacytidine, gemcitabine, etoposide, or cisplatin. In
some
embodiments, ElA is under transcriptional control of an AFP-TRE. In other
embodiments,
E1B is under transcriptional control of an AFP-TRE. In yet other embodiments,
ElA and
E1B are under txanscriptional control of two AFP-TREs (which may be identical
or non-
identical). These vectors may or may not contain an E3 region. In some
embodiments,
E 1 A and E 1 B are co-transcribed and under transcriptional control of an AFP-
TRE, and
E1B is under translational control of an IRES (with EIB promoter preferably
deleted and
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preferably with the IRES and ElB in. frame). In other embodiments, the I9-kDa
region of
E1B is deleted.
An example of a suitable vector is CV790, in which ElA and E1B are each under
transcriptional control of identical AFP-TREs, and which further comprises an
E3 region.
Another example of a suitable vector is CV890, in which ElA and ElB are co-
transcribed
and under transcriptional control of an AFP-THE wherein E1B is under
translational
control of an IRES. Vectors such as these have displayed in vivo synergy in
conjunction
with doxorubicin. Accordingly, in some embodiments, the target cell-specific
adenoviral
vector has E 1 A under transcriptional control of an AFP-THE and E 1 B under
translational
control of an IRES, and further comprising an E3 region (such as CV890), and
the
antineoplastic is chosen from the antibiotic class of agents. Preferably, the
antineoplastic is
doxorubicin.
With respect to bladder tumors, any bladder cell specific adenoviral vector
may be
used with the chemotherapeutic agents described herein. Preferably, the THE is
derived
' from uroplakin. The bladder specific adenovirus vectors may be used with
chemotherapeutic agents from any of the following classes: antimetabolites
(especially
DNA damaging agents); alkylating agents (especially platinum containing
agents);
antibiotics; alkaloids, hormone antagonists/agonists and analogs and
immunomodulators.
Preferably, the chemotherapeutic agent is an antibiotic such as doxorubicin,
mitoxantrone,
~ bleomycin, valrubicin, or mitomycin C. In some embodiments, the
chemotherapeutic agent
is paclitaxel , etoposide, docetaxel, gemcitabine, 5-fluorouracil,
vinblastine, ifosfamide,
thiotepa, interferon alpha-2a, methotrexate, goserelin, leuprolide, gallium
nitrate,
cyclophosphamide, vincristine, carboplatin or cisplatin. Preferably the
chemotherapeutic
agent is cisplatin, thiotepa, mitomycin C, or interferon alpha-2a. In some
embodiments,
25. ElA is under transcriptional control of an uroplakin-TRE. In other
embodiments, E1B is
under transcriptional control of uroplakin-TRE. In yet other embodiments, ElA
and E1B
are under transcriptional control of uroplakin-TREs (which may be identical or
non-
identical). Examples of suitable vectors include CV829 and CV877, in which ElA
and
E1B are each under transcriptional control of heterologous uroplakin-derived
TREs, and
which further comprise an E3 region. These vectors may or may not contain an
E3 region.
In some embodiments of the vector, ElA and E1B are co-transcribed and under
transcriptional control of an uroplakin-TRE, and E1B is under translational
control of an
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IRES (with the E1B promoter preferably deleted and preferably IRES and E1B are
in
frame). In other embodiments, the 19-kDa region of E1B is deleted. These
vectors may or
may not contain an E3 region. Examples of vectors include CV874, CV87S and
CV876,
which comprise an E3 region. Another example includes CV884.
S With respect to colorectal or breast tumors, any colorectal or breast cell
specific
adenoviral vector may be used with the chemotherapeutic agents described
herein.
Preferably, the THE is derived from CEA. The colorectal or breast specific
adenovirus
vectors may be used with chemotherapeutic agents from any of the following
classes:
antimetabolites (especially DNA damaging agents); alkylating agents
(especially platinum
containing ); antibiotics; alkaloids; hormone antagonists/agonists and analogs
(especially
anti-estrogens). Preferably, the chemotherapeutic agent is an antibiotic such
as
doxorubicin, mitoxantrone, epirubicin, or mitomycin-C. In some embodiments,
the
chemotherapeutic agent is paclitaxel, S-fluorouracil, thiotepa, goserelin,
exemestane,
methotrexate, irinotecan, edatrexate, letrozole, leuprolide, cyclophosphamide,
vinblastine,
1 S prednisone, docetaxel, paclitaxel, or cisplatin. Preferably the
chemotherapeutic agent is a
hormone or hormone analog anti-estrogen such as tamoxifen, anastrozole,
exemestane or
letrozole. In some embodiments, ElA is under transcriptional control of an CEA-
TRE. In
other embodiments, E1B is under transcriptional control of an CEA-TRE. In yet
other
embodiments, ElA and E1B are each under transcriptional control of CEA-TREs
(which
may be identical or non-identical). These vectors may or may not contain an E3
region. In
some embodiments, ElA is co-transcribed with ElB and under transcriptional
control of an
CEA-TRE, and ElB is under translational control of an IRES (with the E1B
promoter
preferably deleted and preferably IRES and ElB are in frame). In other
embodiments, the
19-kDa region of ElB is deleted. These vectors may or may not contain an E3
region. An
2S example of a suitable vector is CV873, in which ElA is under
transcriptional control of a
CEA-THE and E 1 B is under translational control of an IRES, and which further
comprises
an E3 region.
With respect to melanoma, any melanoma specif c adenoviral vector may be used
with the chemotherapeutic agents described herein. Preferably, the THE is
derived from
tyrosinase. The melanoma specific adenovirus vectors may be used with
chemotherapeutic
agents from any of the following classes: antimetabolites (especially DNA
damaging
agents); alkylating agents (especially platinum containing agents);
antibiotics; alkaloids,
CA 02404060 2002-09-23
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hormone antagonistslagonists and analogs, nitrosoureas. In some embodiments,
the
chematherapeutic agent is 5-fluorouracil, gemcitabine, doxorubicin,
miroxantrone,
mitomycin, dacarbazine, carmustine, vinblastine, lomustine, tamoxifen,
docetaxel,
paclitaxel or cisplatin. In some embodiments, ElA is under transcriptional
control of a
tyrosinase-TRE. In other embodiments, ElB is under transcriptional control of
a
tyrosinase-TRE. In yet other embodiments, ElA and ElB are each under
transcriptional
control of a tyrosinase-TREs (which may be identical or non-identical). These
vectors may
or may not contain an E3 region. In some embodiments, ElA is co-transcribed
with E1B
and under transcriptional control of a tyrosinase-TRE, and E 1 B is under
translational
control of an IRES (with the ElB promoter preferably deleted and preferably
IRES and
E1B are in frame). In other embodiments, the 19-kDa region of E1B is deleted.
These
vectors may or may not contain an E3 region. An example is CV859, having ElA
co-
transcribed with E1B and under transcriptional control of a tyrosinase-THE and
E1B under
translational control of an IRES and an intact E3 region.
The specific choice of both the target cell-specific adenoviral vector and the
chemotherapeutic agents) is dependent upon, inter alia, the characteristics of
the disease to
be treated. These characteristics include, but are not limited to, the type of
cancer, location
of the tumor, identity of the target cell, stage of the disease and the
individual's response to
previous treatments, if any. It is well established that certain
antineoplastic agents are more
efficacious for certain types of cancer than others, for instance the use of
tamoxifen in the
treatment of breast cancer, the use of mitoxantrone or estramustine to treat
prostate tumors
or the use of doxorubicin and 5-fluorouracil to treat hepatoma.
In addition to the use of single antineoplastic agents in combination with a
particular adenoviral vector, the invention also includes the use of more than
one agent in
conjunction with an adenoviral vector. Table 2 lists non-limiting examples of
common
combinations of antineoplastic agents. These combinations of antineoplastics
when used to
treat neoplasia are often referred to as combination chemotherapy and are
often part of a
combined modality treatment which may also include surgery andlor radiation,
depending
on the characteristics of an individual's cancer. It is contemplated that the
combined
adenoviral/chemotherapy of the present invention can also be used as part of a
combined
modality treatment program. Preferred combinations of chemotherapeutic agents
include,
but are not limited to, doxorubicin and cisplatin; doxorubicin; and mitomycin
C;
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doxorubicin and mitoxantrone; and doxorubicin and paclitaxel (TAXOLTM). In
some
embodiments, these combinations are used with an adenovirus specific for AFP
producing
cells, such as liver cells. An example of a suitable vector is CV790.
In other embodiments, preferred combinations of chemotherapeutic agents
include,
but are not limited to, mitoxantrone and estramustine; paclitaxel (TAXOLTM)
and
estramustine; and docetaxel (TAXOTERETM) and estramustine. In some
embodiments,
these combinations are used with an adenovirus specific for prostate cells,
such as
adenoviruses containing PSA-TRE, hKLK-THE or PB-TRE. Examples of such
adenoviruses include CV787 and CV706.
In other embodiments, preferred combinations of chemotherapeutic agents
include,
but are not limited to M-VAC (methotrexate-vinblastine, doxorubicin,
cyclophosphamide),
CISCA (cyclophosphamide, doxorubicin, cisplatin), CMV (cisplatin,
methotrexate,
vinblastine), CAP (cyclophosphamide, doxorubicin, cisplatin), or MVMJ
(methotrexate,
vinblastine, mitoxantrone, carboplatin). In some embodiments, these
combinations are
used with an adenovirus specific for bladder cells, such as those containing a
uroplakin
TRE. Examples of such adenoviruses include vectors such as CV829, CV874,
CV875,
CV876, CV877, and CV884 described herein.
In other embodiments, preferred combinations include DBPT (dacarbazine,
cisplatin, carmustine, tamoxifen), VDD (vinblastine, dacarbazine, cisplatin).
In some
embodiments these combinations are used with adenovirus vectors specific for
melanoma,
such as those containing a tyrosinase-TREs. An example of a suitable vector is
CV859,
described herein.
In other embodiments preferred combinations include levamisole and 5-
fluorouracil
or leucovorin and fluorouracil. In particular embodiments these combinations
can be used
with colorectal specific adenoviral vectors, such as those containing a CEA-
TRE. An
example.of a vector is CV873, described herein .
In other embodiments preferred combinations include CAF (cyclophosphamide,
doxorubicin, 5-fluorouracil), CMF (cyclophosphamide, methotrexate, 5-
fluorouracil), CNF
(cyclophosphamide, mitoxantrone, 5-fluorouracil), FAC (5-fluorouracil,
doxorubicin,
cyclophosphamide), MF (methotrexate, 5-fluorouracil, leucovorin), MV
(mitomycin C,
vinblastine), CMFP (cyclophosphamide, methotrexate, 5-fluorouracil,
prednisone), VATH
(vinblastine, doxorubicin, thiotepa, fluoxymesterone). In particular
embodiments these
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combinations can be used with breast specific adenoviral vectors, such as
those containing
a CEA-TRE. An example of such a vector is CV873, described herein.
Listed below are selected acronyms for combination cancer chemotherapy
regimens comprising substances in The Merck Index.
Table 2: Cancer Combination Chemotherapy Drug Regimens
Acronym Drug regimens
AA cytarabine + doxorubicin
ABP doxorubicin + bleomycin + prednisone
ABVD doxorubicin + bleomycin + vinblastine + dacarbazine
AC doxorubicin + cyclophosphamide
ACVB doxorubicin + cyclophosphamide + vindesine
+ bleomycin
ADIC doxorubicin + dacarbazine
APO doxorubicin + prednisone + vincristine + 6-mercaptopurine
+
asparaginase + methotrexate
AV doxorubicin ~ vincristine
AVDP asparaginase + vincristine + daunorubicin
+ prednisone
BACOP bleomycin + doxorubicin + cyclophosphamide
+ vincristine +
prednisone
BAPP bleomycin + doxorubicin +, cisplatin + prednisone
B - CAVe bleomycin + lomustine + doxorubicin + vincristine
BCD methotrexate + doxorubicin + cisplatin
BCP carmustine + cyclophosphamide + prednisone
BCVPP carmustine + cyclophosphamide + ' vinblastine
+ procarbazine +
prednisone
B - DOPA bleomycin + dacarbazine + vincristine + prednisone
+ doxorubicin
BEP bleomycin + etoposide + cisplatin
BMP bleomycin + methotrexate + cisplatin
BOLD bleomycin + vincristine + lomustine + dacarbazine
CA cyclophosphamide + doxorubicin
CAF cyclophosphamide + doxorubicin + fluorouracil
CAME cyclophosphamide + doxorubicin + methotrexate
+ fluorouracil
CAP cyclophosphamide + doxorubicin + cisplatin
CAP-BOP cyclophosphamide + doxorubicin + procarbazine
+ bleomycin +
vincristine + prednisone
CAV cyclophosphamide + doxorubicin + vincristine
CAVE cyclophosphamide + doxorubicin + vincristine
+ etoposide
CAVEP cyclophosphamide + doxorubicin + vincristine
+ etoposide +
cisplatin
CBV cyclophosphamide + carmustine + etoposide
CC carboplatin + cyclophosphamide
CD cytarabine + duanorubicin
CFP cyclophosphamide + fluorouracil + prednisone
CFPMV cyclophosphamide + fluorouracil + prednisone
+ methotrexate +
vincristine
CFPT cyclophosphamide + fluorouracil + prednisone
+ tamoxifen
CHAD cyclophosphamide + hexamethylinelamine + doxorubicin
+ .
cisplatin
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Acronym Drug regimens
CHAMOCA cyclophosphamide + hydroxyurea + dactinomycin
+ methotrexate +
vincristine + doxorubicin
CHAP-5 cyclophosphamide + hexamethylmelamine + doxorubicin
+
cisplatin
CHF cyclophosphamide + hexamethylmelamine + fluorouracil
GhIVPP chlorambucil + vinblastine + procarbazine
+ prednisone
CHO cyclophosphamide + doxorubicin + vincristine
CHOP cyclophosphamide + doxorubicin + vincristine
+ prednisone
CHOP-B cyclophosphamide + doxorubicin + vincristine
+ prednisone +
bleomycin
CMF cyclophosphamide + methotrexate + fluorouracil
CMFP cyclophosphamide + methotrexate + fluorouracil
+ prednisone
CMFVP cyclophosphamide + methotrexate + fluorouracil
+ vincristine +
prednisone
C-MOPP cyclophosphamide + mechlorethamine + vincristine
+ procarbazine
+ prednisone
CMV cisplatin + methotrexate + vinblastine
COAP cyclophosphamide + vincristine + cytarabine
+ prednisolone
CODE cisplatin + vincristine + doxorubicin + etoposide
COMLA cyclophosphamide + vincristine + methotrexate
+ cytarabine
COMP cyclophosphamide + vincristine + methotrexate
+ prednisone
COP cyclophosphamide + vincristine + prednisone
COP-BLAM cyclophosphamide + vincristine + prednisone
+ bleomycin +
doxorubicin + procarbazine
COPP cyclophosphamide + vincristine + prednisone
+ procarbazine
CVF cyclophosphamide + vincristine + fluorouracil
CVP , cyclophosphamide + vincristine + prednisone
CVPP bleomycin + lomustine + doxorubicin + vinblastine
CYVADIC cyclophosphamide + vincristine + doxorubicin
+ dacarbazine
DCT daunorubicin + cytarabine + thioguanine
DICEP cycIophosphamide + etoposide + cisplatin
DVP duanorubicin + vincristine + prednisone
EAP etoposide + doxorubicin + cisplatin
EFP etoposide + fluorouracil + cisplatin
ELF etoposide + leucovorin + fluorouracil
EMA-CO etoposide + methotrexate + dactinomycin +
cyclophosphamide +
vincristine
ESHAP etoposide + methylprednisolone + cytarabine
+ cisplatin
FA fluorouracil + doxorubicin
FAC fluorouracil + doxorubicin + cyclophosphamide
FAM fluorouracil + doxorubicin + mitomycin C
FAMTX fluorouracil + doxorubicin + methotrexate
FAP fluorouracil + doxorubicin + cisplatin
FEB fluorouracil + epirubicin + carmustine
FUVAC fluorouracil + vinblastine + doxorubicin +
cyclophosphamide
HAD hexamethylmelamine + doxorubicin + cisplatin
H-CAP hexamethylmelamine + cyclophosphamide + doxorubicin
+
cisplatin
Hexa-CAF hexamethylmelamine + cyclophosphamide + methotrexate
+
fluorouracil
ICE ifosfamide + carboplatin + etoposide
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Acronym Drug regimens
IMVP -16 ifosfamide + methotrexate + etoposide
LOPP chlorambucil + vincristine + procarbazine
+ prednisone
LSAZI,Z cyclophosphamide + vincristine + prednisone
+ daunorubicin +
methotrexate + cytarabine +
thioguanine + colaspase + hydroxyurea
+ carmustine
M - 2 vincristine + carmustine + cyclophosphamide
+ melphalan +
prednisone
MAC methotrexate + dactinomycin + chlorambucil
MACC methotrexate + doxorubicin + cyclophosphamide
+ lomustine
MACOP-B methotrexate + doxorubicin + cyclophosphamide
+ vincristine +
prednisone + bleomycin
M-BACOD methotrexate + bleomycin + doxorubicin
+ cyclophosphamide +
vincristine + dexamethasone
MBD methotrexate + bleomycin + cisplatin
MC mitoxantrone + cytarabine
MCF mitoxantrone + cyclophosphamide + fluorouracil
MeGP methyl-CCNU + cyclophosphamide + prednisone
MINE mesna + ifosfamide + mitoxantrone + etoposide
MIP mitomycin + ifosfamide + cisplatin
MM mercaptopurine + methotrexate
MMM mitoxantrone + methotrexate + mitomycin
MOP mechlorethamine + vincristine + procarbazine
MOPP mechlorethamine + vincristine + procarbazine
+ prednisone
MP melphalan + prednisone
M-VAC methotrexate + vinblastine + doxorubicin
+ cisplatin
MV mitroxantrone + etoposide
MVP mitomycin + vindesine + cisplatin
MPPP mechlorethamine + vinblastine + procarbazine
+ prednisone
PAC cisplatin + doxorubicin + cyclophosphamide
PC cisplatin + cyclophosphamide
PCV procarbazine + lomustine + vincristine
PE cisplatin + etoposide
PEB cisplatin + etoposide + bleomycin
PF L - PAM and fluorouracil
PMF cisplatin + mitomycin C fluorouracil
ProMACE prednisone + methotrexate + doxorubicin
+ cyclophosphamide +
etoposide
ProMACE-CytaBOprednisone + methotrexate + doxorubicin
+ cyclophosphamide +
M etoposide + cytarabine + bleomycin + vincristine
+ methotrexate
ProMACE-MOPP prednisone + methotrexate + doxorubicin
+ cyclophosphamide +
etoposide + mechlorethamine + vincristine
+ procarbazine +
prednisone
PVP - 16B VP - 16 + bleomycin + cisplatin
PVB cisplatin + vinblastine + bleomycin
SMF streptozocin + mitomycin + fluorouracil
TC thioguanine + cytarabine
VAB-6 vinblastine + dactinomycin + bleomycin
+ cisplatin +
cyclophosphamide
VAG vincristine + dactinomycin + cyclophosphamide
VAD vincristine + doxorubicin + dexamethasone
VAMP vincristine + prednisone + methotrexate
+ 6-mercaptopurine
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Acronym Drug regimens
VAP-cyclo vincristine + doxorubicin + prednisolone + cyclophosphamide
VBAP vincristine + carmustine + dexamethasone + prednisone
VCAP vincristine + cyclophosphamide + doxorubicin + prednisone
VIP vindesine + ifosfamide + cisplatin
VMF etoposide + methotrexate + fluorouracil
VP vindesine + cisplatin
Administration and Assessment
There are a variety of delivery methods for the administration of
antineoplastic
agents, which are well known in the art, including oral and parenteral
methods. There are a
number of drawbacks to oral administration for a large number of
antineoplastic agents,
including low bioavailability, irritation of the digestive tract and the
necessity of
remembering to administer complicated combinations of drugs. The majority of
paxenteral
administration of antineoplastic agents is intravenously, as intramuscular and
subcutaneous
injection often Leads to irritation or damage to the tissue. Regional
variations of parenteral
injections include infra-arterial, intravesical, infra-tumor, intrathecal,
intrapleural,
intraperitoneal and intracavity injections.
Delivery methods for chemotherapeutic agents include intravenous,
intraparenteral
and introperitoneal methods as well as oral administration. Intravenous
methods also
15' include delivery through a vein of the extremities as well as including
more site specific
delivery, such as an intravenous drip into the portal vein of the Liver. Other
intraparenteral
methods of delivery include direct injections of an antineoplastic solution,
for example,
subcutaneously, intracavity or infra-tumor.
Delivery of adenoviral vectors is discussed infra and is generally
accomplished by
either site-specific injection or intravenously. Site-specific injections of
either vector or
antineoplastic agents) may include, for example, injections into the portal
vein of the liver
as well as intraperitoneal, intrapleural, intrathecal, infra-arterial, infra-
tumor injections or
topical application. These methods are easily accommodated in treatments using
the
combination of adenoviral vectors and chemotherapeutic agents.
The adenoviral vectors may be delivered to the target cell in a vaxiety of
ways,
including, but not limited to, liposomes, general transfection methods that
are well known
in the art (such as calcium phosphate precipitation or electroporation),
direct injection, and
intravenous infusion. The means of delivery will depend in large part on the
particular
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adenovixal vector (including its form) as well as the type and location of the
target cells
(i.e., whether the cells are in vitf-o or in vivo).
If used as a packaged adenovirus, adenovirus vectors may be administered in an
appropriate physiologically acceptable carrier at a dose of about 104 to about
1014. The
multiplicity of infection will generally be in the range of about 0.001 to
100. If
administered as a polynucleotide construct (i.e., not packaged as a virus)
about 0.01 pg to
about 1000 p,g of an adenoviral vector can be administered. The adenoviral
vectors) may
be administered one or more times, depending upon the intended use and the
immune
response potential of the host, and may also be administered as multiple,
simultaneous
injections. If an immune response is undesirable, the immune response may be
diminished
by employing a variety of imrnunosuppressants, so as to permit repetitive
administration,
without a strong immune response. If packaged as another viral form, such as
HSV, an
amount to be administered is based on standard knowledge about that particular
virus
(which is readily obtainable from, for example, published literature) and can
be determined
empirically.
Generally, the adenovirus and chemotherapeutic agent are administered as
compositions in a pharmaceutically acceptable excipient (and may or may not be
in the
same compositions), including, but not limited to, saline solutions, suitable
buffers,
preservatives, stabilizers, and may be administered in conjunction with
suitable agents such
as antiemetics. In some embodiments, an effective amount of an adenoviral
vector and an
effective amount of at least one antineoplastic agent are combined with a
suitable excipient
and/or buffer solutions and administered simultaneously from the same solution
by any of
the methods listed herein or those known in the art. This may be applicable
when the
antineoplastic agent does not compromise the viability and/or activity of the
adenoviral
vector itself. Where more than one antineoplastic agent is administered, the
agents may be
administered together in the same composition; sequentially in any order; or,
alternatively,
administered simultaneously in different compositions. If the agents are
administered
sequentially, administration may further comprise a time delay.
The chemotherapeutic agent and adenovirus may be administered simultaneously
or
sequentially, with various time intervals for sequential administration. In
some
embodiments, chemotherapeutic agents) and adenovirus vectors) are administered
simultaneously. As shown in the Examples, at least some antineoplastics do not
appear to
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compromise viral replication or specificity. The method of delivery will
depend upon both
the choice of the adenoviral vector and chemotherapeutic agents) and by the
characteristics
of the cancer under treatment.
In other embodiments, a chemotherapeutic agent and adenoviral vector can be
administered sequentially. This may be appropriate, for example, in instances
where the
antineoplastic agent is an alkylating agent, antimetabolite, nitrosourea or
other DNA
damaging agent which may compromise the viability and/or activity or the viral
vector, or
in instances in which it has been indicated that sequential administration
optimizes
effectiveness of the combination therapy. Sequential administration rnay be in
any order,
and accordingly encompasses the administration of an effective amount of an
adenoviral
vector first, followed by the administration of an effective amount of the
chemotherapeutic
agent. The interval between administration of adenovirus and chemotherapeutic
agent may
be in terms of at least (or, alternatively, less than) minutes, hours, or
days. Sequential
administration also encompasses administration of a chosen antineoplastic
agent followed
by the administration of the adenoviral vector. The interval between
administration may be
in terms of at least (or, alternatively, less than) minutes, hours, or days.
Administration
of the above-described methods may also include repeat doses or courses of
target-cell
specific adenovirus and chemotherapeutic agent depending, inter alia, upon the
individual's
response and the characteristics of the individual's disease. Repeat doses may
be
undertaken immediately following the first course of treatment (i.e., within
one day), or
after an interval of days, weeks or months to achieve and/or maintain
suppression of tumor
growth. A particular course of treatment according to the above-described
methods, for
example, combined adenoviral and chemotherapy, may later be followed by a
course of
combined radiation and adenoviral therapy.
Generally, an effective amount of adenovirus vector and chemotherapeutic
agents)
is administered, i.e., amounts sufficient to achieve the desired result, based
on general
empirical knowledge of a population's response to such amounts. Some
individuals are
refractory to these treatments, and it is understood that the methods
encompass
administration to these individuals. The amount to be given depends, inter
alia, on the type
of cancer, the condition of the individual, the extent of disease, the route
of administration,
how many doses will be administered, and the desired objective.
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A chemotherapeutic agents) is administered in a physiologically acceptable
carrier
appropriate to the method of delivery, as are known in the art and described
herein. The
amount of chemotherapeutic agents) administered is determined by the
characteristics of
the individual's disease, the method of delivery and the weight, age, general
health and
response of the individual. In some embodiments the amount of chemotherapeutic
agents)
administered will be the dosage known in the art to be effective given the
characteristics of
the individual and their disease. In other embodiments, due to the synergistic
effect of the
combination of adenoviral vector and chemotherapeutic agent, the amount of
chemotherapeutic agents) administered will be about 2x, about Sx, about 10x,
or about Sx
less than that known in the art to be effective for the particular individual
and
characteristics of the disease. In some embodiments, the amount of
chemotherapeutic
agents) administered will be about 20x, about 50x, about 100x or about 1000x
less than
that known in the art to be effective for the particular individual and
characteristics of the
disease. Dosages include courses of chemotherapy and repeat administrations of
the
chemotherapeutic agents) over the course of days, weeks or months and may
include an
increase or decrease in the interval between doses during administration of
the course of
chemotherapy, or increases or decreases in the actual amount of
chemotherapeutic agent
administered.
Examples of dosages known in the art for chemotherapeutic agents include, but
are
not limited to, doses of 60-75 mg/m2 for doxorubicin at 21 day intervals when
administered
as a single agent, and doxorubicin doses of 40-60 mg/ma when administered as a
component in a combination of chemotherapeutic agents. Typical doses known in
the art
for cisplatin are from 20 mg/m2 to 100 mg/ma; for etoposide 35-100 mg/m2; for
paclitaxel
135-175 mg/m2; for docetaxel 60-100 mg/m2; for mitomycin C 30-40 mg/m2;
gemcitabine
1000-1250 mg/m2; mitoxantrone 12-14 mg/m2 per cycle, 12-212 mg/m2 cumulative
over
course of treatment; thiotepa 0.3-0.8 mg/kg; 5-azacytidine 50-200 mg/m2/day; 5-
fluorouracil 7-12 mg/kg/day, not more than 800 mg/day. These dose may be
administered
on a variety of schedules known to those of skill in the art and depending on
the response
of the individual and the characteristic of the individual cancer.
Any of the methods described herein may further be used in conjunction with
combined modality treatment for suppressing tumor growth. Such combined
modality
treatment may or may not include surgery as a component of the treatment.
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Assessment may be determined by any of the techniques known in the art,
including
diagnostic methods such as imaging techniques, analysis of serum tumor markers
(which
may be measured, for example, by ELISA), biopsy (which could indicate the
presence of
killed tumor cells), and the presence, absence or amelioration of tumor
associated
symptoms.
Compositions ahd Kits ofAdehoviral Vectors and ChemotherapeuticAgehts
The invention also includes compositions comprising at least one
antineoplastic
agent, such as those listed in Table 1, and a target cell-specific adenoviral
vectors) as
described herein, where the stability, activity, and/or viability of the
adenoviral vector is
not compromised by the antineoplastic agents) (ie, the adenovirus vector
retains some to
all activity). These compositions can further comprise suitable pharmaceutical
such as,
saline solutions, suitable buffers, preservatives, stabilizers.
In some embodiments, the composition comprises a target cell-specific
adenoviral
vector comprising ElA under transcriptional control of a PB-TRE, E1B under
transcriptional control of a PSA-TRE, further comprising an E3 region (such as
CV787)
and the antineoplastic is 5-fluorouracil or cisplatin. In other embodiments,
the
antineoplastic is doxorubicin, estramustine, etoposide, mitoxantrone,
docetaxel
(TAXOTERETM) or paclitaxel (TAXOLTM). In other embodiments, the composition
comprises an adenovirus vector comprising ElA under transcriptional control of
a PSA-
THE (such as CV706) and the composition further comprises 5-fluorouracil or
cisplatin. In
other embodiments, the antineoplastic is doxorubicin, estramustine, etoposide,
mitoxantrone, docetaxel (TAXOTERETM) or paclitaxel (TAXOLTM). In other
embodiments, the composition comprises an adenoviral vector comprising an.
early gene
under transcriptional control of an AFP-THE (for example, E 1 A under
transcriptional
control of an AFP-TRE), E 1 B under transcriptional control of an AFP-TRE, an
intact E.3
region (such as CV790) and the composition further comprises 5-azacytidine,
cisplatin,
etoposide or gemcitabine, doxorubicin, mitomycin C, mitoxantrone, paclitaxel
or a
combination of antineoplastic agents such as, doxorubicin and cisplatin, or
doxorubicin and
mitomycin C or doxorubicin and mitoxantrone or doxorubicin and paclitaxel
(TAXOLTM).
In some embodiments, the adenovirus vector comprises co-transcribed first and
second genes, preferably adenovirus genes, under transcriptional control of a
heterologous,
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target cell-specific transcriptional regulatory element (TRE), wherein the
second gene is
under translational control of an internal ribosome entry site (IRES).
Fits comprising the combined antineoplastic agent(s), target cell-specific
adenoviral
vector, and suitable excipient, packaging, and labeling are also included in
the present
invention. The kit provides suitable dosages of each of the antineoplastic
agents) and
adenoviral vector. Embodiments include kits comprising, fox example, all of
the
compositions listed above. The kits preferably contain instructions for
administration to
individuals for appropriate cancer to effect suppression of tumor growth.
In some embodiments, the chemotherapeutic agent and adenoviral vector are
packaged separately in appropriate packaging. In other embodiments, the
chemotherapeutic agent and adenoviral vector are packaged together. Examples
of suitable
agents and adenoviral vectors have been discussed above and are described
herein.
Combination Adenoviral and Radiation Therapy
The invention also provides combination methods which employ the replication
competent target cell specific adenoviral vectors as described herein and
radiation. As
explained in more detail in Example 6, the combined treatment of neoplasia
with a target
cell-specific adenoviral vector and radiation results in a synergistic effect,
with earlier
eradication of the tumor compared to no treatment, radiation alone or virus
alone. When
used in combination with target cell-specific adenoviral vectors, the type of
radiation
treatment used is dependent upon the characteristics of the individual cancer
being treated.
The choice of suitable radiation therapy is well known by a person skilled in
the art and
decided on an individual basis. The choice of the target cell-specific
adenoviral vector is
largely governed by the identity of the target (neoplastic) cells and includes
X-rays, gamma
7G r~<.~ .,1~,1,., ,...".+;"10" 1.~+,. .......i.;,.t,.~ M..as_.._+:_._
:.._+_.,_.. ~t_..____ _____~______ _,__._.____ ___ ,
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a) administration of an effective amount of a replication-competent target
cell-
specific adenoviral vector to an individual with neoplasia; and
b) administration of an effective amount of an appropriate course of radiation
wherein radiation includes X-rays, gamma rays, alpha particles, beta
particles, electrons,
photons, neutrons, other ionizing radiation and radioactive isotopes.
In some embodiments, step (a) is performed before step (b). In other
embodiments,
step (b) is performed before step (a). In other embodiments, steps (a) and (b)
are performed
simultaneously.
The replication-competent target cell-specific adenoviral vector may be any of
the
I O replication-competent target cell-specific adenoviral vectors disclosed
herein, comprising a
gene essential for replication, preferably an early gene, under
transcriptional control of a
TRE. Preferably, the gene essential for replication is E1A or E1B or both.
Discussion of
exemplary embodiments of suitable adenoviral vectors in the previous section,
as well as
the section describing adenovirus vectors below, are applicable to these
methods.
In some embodiments, the gene essential for replication is ElA or E1B and in
some
embodiments, the vector comprises both ElA and E1B under transcriptional
control of a
cell-specific TRE. In some embodiments, the E 1 A and E 1 B genes are under
transcriptional
control of the same or similar TREs. The vectors may or may not include an E3
region. In
some embodiments, the adenovirus vector comprises co-transcribed first and
second genes,
preferably adenovirus genes, under transcriptional control of a heterologous,
target cell-
specific transcriptional regulatory element (TRE), wherein the second gene is
under
translational control of an internal ribosome entry site (IRES). In some
embodiments, the
first and second genes are E1A and E1B, respectively. In this embodiment it is
preferred
that E1B has its endogenous promoter deleted and in one embodiment; IRES and
E1B are
in frame.
In other embodiments, the adenovirus vector comprises ElA wherein the ElA
promoter is deleted and wherein the ElA gene is under transcriptional control
of a target
cell-specific TRE. In other embodiments, the adenovirus gene is E1B wherein
the E1B
promoter is deleted and wherein the E1B gene is under transcriptional control
of a target
cell-specific TRE. In other embodiments, the vector comprises ElA wherein the
ElA
promoter is deleted and E 1 B wherein the E 1 B promoter is deleted.
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In other embodiments, an enhancer element for the first andlor second
adenovirus
genes is deleted. In some embodiments, the EIA enhancer is deleted. In yet
other
embodiments, the EIA promoter is deleted and ElA enhancer I is deleted. In
further
embodiments, the THE has its endogenous silencer element deleted. In other
embodiments,
the adenovirus vector comprises E1B having a deletion in the 19-kDa region.
These
embodiments apply to any and all methods described herein.
Administration and Assessment
As is well-known in the art, radiation therapy includes treatment with X-rays
and
gamma-rays, as well as alpha and beta particles, photons, electrons, neutrons,
implants of
radioactive isotopes and other forms of ionizing radiation. Recent
experimental therapy
employs monoclonal antibodies specific to the malignant tumor to deliver
radioactive
isotopes directly to the site of the tumor, termed radioimmunotherapy. The
most common
type of radiation treatment is radiation directed to the body area containing
the neoplastic
tumor, which is known as regional or local radiation therapy.
The combined modality treatment of radiation and target cell-specific
adenoviral
therapy can be carried out in a number of ways, including delivery of the
adenoviral vector
followed by radiation therapy, or where vector delivery is followed by a time
delay of
seconds, minutes, hours or days and before radiation treatment. The combined
modality
treatment also incorporates administration of the radiation treatment followed
by the
adenoviral treatment, including but not necessarily requiring a time interval
between
radiation treatment and delivery of the adenovirus, of seconds, minutes, hours
or days.
Repeat dosages of adenoviral vector and/or radiation may be administered.
Administration of adenovirus vectors has been described above. Administration
of
radiation therapy can include methods well known in the art, such as internal
and external
radiation therapy. External therapy includes the administration of radiation
via high-energy
external beam radiation, administered either regionally (locally) to the tumor
site or whole
body irradiation. Examples of internal radiation (brachytherapy) include the
implantation
of radioactive isotopes in permanent, temporary, sealed, unsealed, intracavity
or interstitial
implants. The choice of implant is determined by the characteristics of the
neoplasia,
including the location and extent of the tumor. The choice between external or
internal
radiation treatment and type of external radiation treatment is also
determined by the
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characteristics of the neoplasia and can be determined by those skilled in the
art. An
additional type of radiation therapy is radioimmunotherapy in which
radioisotopes are
attached to monoclonal antibodies specific for the tumor cells.
The amount/course of radiation administered to the individual is determined by
the
characteristics of the individual's disease, the method of delivery and the
weight, age,
general health and response of the individual. For radiation therapy in
particular, the
location of the tumor is a determining factor in the administration of
radiation, as the
radiosensitivity of the tumor and surrounding tissue are variable according to
tissue type
(see Table 3), oxygen supply and other factors. In some embodiments the amount
of
radiation administered will be the dosage known in the art to be effective
given the
characteristics of the individual and the disease. In other embodiments, the
amount of
radiation administered will be about 2x, about Sx, about 10x, or about 15x
less than that
known in the art to be effective for the particular individual and
characteristics of the
disease. In some embodiments, the amount of radiation administered will be
about 20x,
about 50x, about 100x or about I OOOx less than that known in the art to be
effective for the
particular individual and characteristics of the disease.
Radiation treatment may also entail the administration of a radiosensitizing
agent or
radioprotectant to facilitate the txeatment. Recent evidence suggests that the
antineoplastic
agent TAXOLTM (paclitaxel) may function as a radiosensitizer. Liebmann et al.,
J.
National Cancer Inst. 86:441, 1994;. Similar evidence has been found fox
TAXOTERETI''i
(docetaxel). Creane et al., Int. J. Radiat. Biol. 75:731, 1999; Sikov et al.,
Front. Biosci.
May 1: 221, 1997. Other radiation sensitizers include E2F-1, anti-ras single
chain
antibody, p53, GM-CSF, and cytosine deaminase. A tumor specific adenovirus may
further
comprise a radiation sensitizer, such as p53 for example, or a chemo
sensitizer.
Repeat doses may be undertaken immediately following the first course of
treatment or after an interval of days, weeks or months to achieve suppression
of tumor
growth. A particular course of treatment according to the above-described
methods, fox
example, combined adenoviral and radiation therapy, may later be followed by a
course of ,
combined chemotherapy and adenoviral therapy.
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Table 3: Radiosensitivity of Various Tissues
Tumor or Tissue Type Relatue'Radiosensitivity.:.
lymphoma, leukemia, seminoma; high
dysgerminoma
squamous cell, cancer of the
oropharyngeal
glottis, bladder, skin & cervicalfairly high
epithelia
adenocarinomas of the alimentary
tract
vascular & connective tissue
(elements of
all tumors)
secondary neurovascularization,medium
astrocytomas
salivary gland tumors, hepatorna,
renal
cancer, pancreatic cancer, fairly low
chondrosarcoma, osteogenic
sarcomas
rhabdomyosarcoma, leiomyosarcomalow
&
ganglioneurofibrosarcoma
Assessment may be determined by any of the techniques known in the art,
including
diagnostic methods such as imaging techniques, analysis of serum tumor
markers, biopsy,
the presence, absence or amelioration of tumor associated symptoms.
Combination Treatment with Adenoviral, Chemotlaerapy and Radiation
Chemotherapy and radiation are commonly used as components of a combined
modality treatment, and the choice of chernotherapeutic agents) and type and
course of
radiation therapy is generally governed by the characteristics of the
individual cancer and
the response of the individual. While target cell-specific adenoviral vectors
can be used
with either radiation or chemotherapy, as separate courses of treatment, they
can also be
combined with both methods of treatment in the same course of therapy.
Accordingly, the
present invention encompasses combinations of the methods discussed above.
Accordingly, the invention includes methods for suppressing tumor growth in an
individual comprising the following steps, in any order:
a) administering to the individual an effective amount of a target cell-
specific
adenoviral vector and at least one antineoplastic agent; and
b) administering an effective amount of an appropriate course of radiation
therapy
to the individual.
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The method may further comprise the step of:
c) administering to the individual an additional dose of the
adenoviral/chemotherapeutic solution or radiation as necessary to treat the
individual's
neoplasia.
The method may further comprise time delays after any one of steps a), b) and
c).
A time delay interval may be days, weeks or months.
The antineoplastic may be chosen from the agents listed in Table 1 or a
combination
of agents may be chosen from the list in Table 2. Additional agents or
combinations of
agents known to those of skill in the art may also be used. The replication-
competent target
I O cell-specific adenoviral vector is chosen from the replication-competent
target cell-specific
adenoviral vectors disclosed herein.
In preferred embodiments the gene essential for replication in the adenoviral
vector
is an early gene. Even more preferably the gene essential for replication is
ElA or E1B or
both. In particularly preferred embodiments the EIA and E1B genes are under
transcriptional of the same or similar TREs. The vector may or may not contain
an E3
region.
In some embodiments, the adenovirus vector comprises co-transcribed first and
second genes under transcriptional control of a heterologous, target cell-
specific
transcriptional regulatory element (TRE), wherein the second gene is under
translational
control of an internal ribosome entry site (IRES). An adenovirus vector may
further
comprise E3.
In particular embodiments of the above described methods, the adenoviral
genes)
essential for replication is under the control of TRE(s) specific for target
cells such as, but
not limited to liver, prostate, bladder, colorectal, breast or melanoma cells.
In certain preferred embodiments of the above described methods, the
adenoviral
genes) essential for replication is under the control of a TRE(s) such as, but
not limited to
the PB-TRE, PSA-TRE, the MLTC-TRE, the AFP-TRE, the CEA-TRE, the hKLK2-TRE,
tyrosinase-TRE, and uroplakin-TRE, as described herein.
Illustrative embodiments of target cell-specific adenoviral vectors include
CV787,
CV790, CV890, CV706, CV829, CV859, CV873, CV874, CV875, CV876, CV877, and
CV884 as described herein.
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In a preferred embodiment, the adenoviral vector comprises a prostate specific
THE
or a liver specific THE and at least one of the chemotherapeutic agents is
from the alkaloid
class.
In another preferred embodiment, the adenoviral vector comprises a prostate
specific THE or a liver specific THE and at least one of the chemotherapeutic
agents is
paclitaxel (TAXOLTM) or docetaxel (TAXOTERETM) or a paclitaxel derivative.
In another preferred embodiment the adenoviral vector comprises a urothelial
specific THE and least one of the chemotherapeutic agents is paclitaxel
(TAXOLTM) or
docetaxel (TAXOTERETM) or a paclitaxel derivative.
Administration and Assessment
Administration of adenoviral vectors, chemotherapeutic agents and radiation
has
been described above. The choice of the adenoviral vector, chemotherapeutic
agents) and
radiation are dependent on the characteristics of the individual cancer and
the individual's
response to therapy. Such considerations are known to those skilled in the
art. The
invention encompasses embodiments which include the replication-competent
target cell-
specific adenoviral vectors discussed herein as well as those known to persons
of skill in
the art. The invention also encompasses embodiments which include the
combinations of
target cell-specific adenoviral vectors and chemotherapeutic agents discussed
herein which
can be further combined with radiation therapy.
The above-described methods include administration of the adenoviral vector,
radiation and chemotherapeutic(s) in any order and may include sequential
administration
or simultaneous administration of all or some of the components (i.e.
simultaneous
administration of chemotherapy and adenovirus followed sequentially by
radiation therapy
or sequential administration of adenovirus first, radiation second and
thirdly,
chemotherapy, etc.).
Repeat doses may be undertaken immediately following the first course of
treatment or after an interval of days, weeks or months to achieve suppression
of tumor
growth. Repeat doses of a particular component of the therapy may also be
administered
before the administration of the remaining components (i.e. administration of
multiple
doses of chemotherapeutic agents) followed by sequential administration of
radiation and
adenovirus or administration of multiple doses of radiation therapy followed
by
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simultaneous administration of chemotherapy and adenovirus, etc.). A
particular course of
treatment according to the above-described methods, for example, combined
adenoviral,
chemotherapeutic and radiation therapy, may later be followed by a course of
combined
chemotherapy and adenoviral therapy.
Any of the methods described herein may further be used in conjunction with
combined modality treatment for suppressing tumor growth. Such combined
modality
treatment may include surgery as a component of the treatment.
Assessment of the suppression of tumor growth may be determined by any of the
techniques known in the art, including diagnostic methods such as imaging
techniques,
analysis of serum tumor markers , biopsy, the presence, absence or
amelioration of tumor
associated symptoms.
Adenoviral Yectnrs
The adenoviral vectors used in the methods described herein are replication-
competent target-cell specific adenoviral vectors comprising an adenovirus
gene, preferably
a gene essential for replication under transcriptional control of a target
cell specific TRE.
The vector may or may not include an E3 region. In other embodiments, an
adenovirus
vector is a replication competent, target cell specific vector comprising E1B,
wherein E1B
has a deletion of part or all of the 19-kDa region.
In some embodiments the adenoviral gene essential for replication is an early
gene,
preferably E 1 A or E 1 B or both.
In some embodiments, the adenovirus vector comprises eo-transcribed first and
second genes under transcriptional control of a heterologous, target cell-
specific
transcriptional regulatory element (TRE), wherein the second gene is under
translational
control of an internal ribosome entry site (IRES). The adenovirus vector may
further
comprise E3.
The adenovirus vectors used in this invention replicate preferentially in TItE
functional cells referred to herein as target cells. This replication
preference is indicated by
comparing the level of replication (i.e., titer) in cells in which the THE is
active to the level
of replication in cells in which the THE is not active (i.e., a non-target
cell). The
replication preference is even more significant, as the adenovirus vectors
used in the
invention actually replicate at a significantly lower rate in THE non-
functional cells than
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wild type virus. Comparison of the adenovirus titer of a target cell to the
titer of a THE
inactive cell type provides a key indication that the overall replication
preference is
enhanced due to the replication in target cells as well as depressed
replication in non-target
cells. This is especially useful in the cancer context, in which targeted cell
killing is
desirable. The TRE's preferably control genes necessary for replication, where
the genes)
necessary for replication is an early genes) of the adenovirus, preferentially
the ElA or
E1B genes. Particularly preferred embodiments include where TRE's control both
the ElA
and E1B genes within the same viral construct. In another particularly
preferred
embodiment, the adenovirus vector comprises co-transcribed first and second
genes under
transcriptiona.l control of a heterologous, target cell-specific
transcriptional regulatory
element (TRE), wherein the second gene is under translational control of an
internal
ribosome entry site (IRES). In this embodiment, it is preferred that the
second gene has its
endogenous promoter mutated or deleted and in one embodiment, the IRES and
second
gene are in frame. In some embodiments, an adenovirus vector of the present
invention
further comprises E3.
Runaway infection is prevented due to the cell-specific requirements for viral
replication. Without wishing to be bound by any particular theory, production
of
adenovirus proteins can serve to activate and/or stimulate the immune system,
either
generally or specifically toward target cells producing adenoviral proteins
which can be an
. important consideration in the cancer context, where individuals are often
moderately to
severely immunocompromised.
In particular embodiments, the adenoviral vector may be a replication-
competent
target-cell specific adenoviral vector where the vector comprises an
adenoviral gene. In
one embodiment, the adenoviral gene is essential for replication and is under
transcriptional
control of a target cell-specific TRE.
In certain embodiments, the adenoviral vector may be a replication-competent
target-cell specific adenoviral vector wherein the gene essential for
replication is an early
gene. In other embodiments the gene essential for replication may be a late
gene.
In preferred embodiments the gene essential for replication is E 1 A or E 1 B.
In
particular embodiments, the adenovirus comprises both ElA and E1B. In further
embodiments, the gene essential for replication is E1B wherein E1B has a
deletion of part
or all of the 19-kDa region.
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In some embodiments, the adenovirus vector comprises co-transcribed first and
second genes under transcriptional control of a heterologous, target cell-
specific
transcriptional regulatory element (TRE), wherein the second gene is under
translational
control of an internal ribosome entry site (IRES). In this embodiment, it is
preferred that
the endogenous promoter of the second gene be mutated or deleted and in one
embodiment,
the IRES and second gene are in frame. .
In some embodiments of the adenovirus vector, ElA has a mutation in or
deletion
of its endogenous promoter. In some embodiments, E1B has°a mutation in
or a deletion of
its endogenous promoter. In some embodiments, ElA has a mutation in or
deletion of its
endogenous enhancer. In other embodiments, E1B has a deletion in part or all
of the 19-
kDa region.
In particular preferred embodiments, the target cell specific adenoviral
vector is
specific for target cells including bladder, liver, prostate, breast,
colorectal and melanoma
cells.
In certain preferred embodiments, the adenoviral genes) essential for
replication is
under the control of a TRE(s) such as, but not limited to PB-TRE, PSA-TRE, MUC-
TRE,
AFP-TRE, CEA-TRE, tyrosinase-TRE, hKLK2-TRE, and uroplakin-TRE, as described
herein.
Illustrative adenoviral vectors are summarized in Table 4.
In one aspect of the present invention, the adenovirus vectors comprise an
intergenic IRES elements) which links the translation of two or more genes,
thereby
removing any potential for homologous recombination based on the presence of
identical
TREs in the vector. Adenovirus vectors comprising an IRES are stable and in
some
embodiments provide better specificity than :vectors not containing an IRES.
Another
advantage of an adenovirus vector comprising an intergenic IRES is that the
use of an IRES
rather than a second THE may provide additional space in the vector for an
additional
genes) such as a therapeutic gene.
Thus, the adenovirus vectors comprising a second gene under control of an IRES
retain a high level of target cell specificity and remain stable in the target
cell.
Accordingly, in one aspect of the invention, the viral vectors disclosed
herein comprise at
least one IRES within a multicistronic transcript, wherein production of the
multicistronic
transcript is regulated by a heterologous, target cell-specific TRE. For
adenovirus vectors
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comprising a second gene under control of an IRES, it is preferred that the
endogenous
promoter of a gene under translational control of an IRES be deleted so that
the
endogenous promoter does not interfere with transcription of the second gene:
It is
preferred that the second gene be in frame with the IRES if the IRES contains
an initiation
codon. If an initiation codon, such as ATG, is present in the IRES, it is
preferred that the
initiation codon of the second gene is removed and that the IRES and the
second gene are
in frame. Alternatively, if the IRES does not contain an initiation codon or
if the initiation
codon is removed from the IRES, the initiation codon of the second gene is
used. In one
embodiment, the adenovirus vectors comprises the adenovirus essential genes,
ElA and
E 1 B genes, under the transcriptional control of a heterologous, cell-
specific TRE, and an
IRES introduced between ElA and E1B. Thus, both ElA and E1B are under common
transcriptional control, and translation of E I B coding region is obtained by
virtue of the
presence of the IRES. In one embodiment, ElA has its endogenous promoter
deleted. In
another embodiment, ElA has an endogenous enhancer deleted and in yet an
additional
embodiment, EIA has its endogenous promoter deleted and ElA enhancer I
deleted. In
another embodiment, ElB has its endogenous promoter deleted. In yet further
embodiments, E1B has a deletion of part or all of the 19-kDa region.
To provide cytotoxicity to target cells, one or more transgenes having a
cytotoxic
effect may be present in the vector. Additionally, or alternatively, an
adenovirus gene that
contributes to cytotoxicity and/or cell death, such as the adenovirus death
protein (ADP)
gene, can be included in the vector, optionally under the selective
transcriptional control of
a heterologous THE and optionally under the translational control of an IRES.
The subject vectors can be used for a wide~variety of purposes. The purpose
will
vary with the target cell. Suitable target cells are characterized by the
transcriptional
activation of the cell specific transcriptional response element in the
adenovirus vehicle.
The transcription initiation region will usually be activated in less than
about 5%, more
usually less than about 1%, and 'desirably by less than about 0.1% of the
cells in the host.
Transcriptional response elenzents (T.REs)
The adenovirus vectors of the invention comprise target cell specific TREs
which
direct preferential expression of an operatively linked gene (or genes) in a
particular target
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cell. A THE can be tissue-specific, tumor-specific, developmental stage-
specific, cell
status specific, etc., depending on the type of cell present in the tissue or
tumor.
Cell- and tissue-specific transcriptional regulatory elements, as well as
methods for
their identification, isolation, characterization, genetic manipulation and
use for regulation
of operatively linked coding sequences, are well known in the art. A THE can
be derived
from the transcriptional regulatory sequences of a single gene, or sequences
from different
genes can be combined to produce a functional TRE. A cell-specific THE is
preferentially
functional in a limited population (or type) of cells, e.g., prostate cells or
liver cells.
Accordingly, in some embodiments, the THE used is preferentially functional in
any of the
following cell types: prostate; liver; breast; urothelial cells (bladder);
colorectal; lung;
ovarian; pancreas; stomach; and uterine. In other embodiments, in accordance
with cell
status, the THE is functional in or during: low oxygen conditions (hypoxia);
certain stages
of cell cycle, such as S phase; elevated temperature; ionizing radiation.
As is known in the art, activity of TREs can be inducible. Inducible TREs
generally
exhibit low activity in the absence of inducer, and are up-regulated in the
presence of
inducer. Inducers include, for example, nucleic acids, polypeptides, small
molecules,
organic compounds and/or environmental conditions such as temperature,
pressure or
hypoxia. Inducible TREs may be preferred when expression is desired only at
certain times
or at certain locations, or when it is desirable to titrate the level of
expression using an
inducing agent. For example, transcriptional activity from the PSA-TRE, PB-THE
and
hKLK2-THE is inducible by androgen, as described herein and in
PCT/LTS98/04080.
Accordingly, in one embodiment of the present invention, an adenovirus vector
comprises
an inducible heterologous TRE.
THE multimers are also useful in the disclosed vectors. For example, a THE can
comprise a tandem series of at least two, at least three, at least four, or at
least five
promoter fragments. Alternatively, a THE can comprise one or more promoter
regions
along with one or more enhancer regions. THE multimers can also comprise
promoter
and/or enhancer sequences from different genes. The promoter and enhancer
components
of a THE can be in any orientation with respect to each other and can be in
any orientation
andlor any distance from the coding sequence of interest, as long as the
desired cell-
specific transcriptional activity is obtained.
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The disclosed vectors are designed such that replication is preferentially
enhanced
in target cells in which the TRE(s) is (are) functional. More than one THE can
be present
in a vector, as long as the TREs are functional in the same target cell.
However, it is
important to note that a given THE can be functional in more than one type of
target cell.
S For example, the CEA-THE functions in, among other cell types, gastric
cancer cells,
colorectal cancer cells, pancreatic cancer cells and lung cancer cells.
A THE for use in the present vectors may or may not comprise a silencer. The
presence of a silencer (i.e., a negative regulatory element known in the art)
can assist in
shutting off transcription (and thus replication) in non-target cells. Thus,
presence of a
silencer can confer enhanced cell-specific vector replication by more
effectively preventing
replication in non-target cells. Alternatively, lack of a silencer may
stimulate replication in
target cells, thus conferring enhanced target cell-specificity.
As is readily appreciated by one skilled in the art, a THE is a polynucleotide
sequence, and, as such, can exhibit function over a variety of sequence
permutations.
Methods of nucleotide substitution, addition, and deletion are known in the
art, and readily-
available functional assays (such as the CAT or luciferase reporter gene
assay) allow one of
ordinary skill to determine whether a sequence variant exhibits requisite cell-
specific
transcription regulatory function. Hence, functionally preserved variants of
TREs,
comprising nucleic acid substitutions, additions, and/or deletions, can be
used in the vectors
disclosed herein. Accordingly, variant TREs retain function in the target cell
but need not
exhibit maximal function. In fact, maximal transcriptional activation activity
of a TIDE
may not always be necessary to achieve a desired result, and the Level of
induction afforded
by a fragment of a THE may be sufFcient for certain applications. For example,
if used for
treatment or palliation of a disease state, less-than-maximal responsiveness
may be
sufficient if, for example, the target cells are not especially virulent
and/or the extent of
disease is relatively confined.
Certain base modifications may result in enhanced expression levels and/or
cell-
specificity. For example, nucleic acid sequence deletions or additions within
a THE can
move transcription regulatory protein binding sites closer or farther away
from each other
than they exist in their normal configuration, or rotate them so they are on
opposite sides of .
the DNA helix, thereby altering spatial relationship among TRE-bound
transcription
factors, resulting in a decrease or increase in transcription, as is known in
the art. Thus,
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while not wishing to be bound by theory, the present disclosure contemplates
the possibility
that certain modifications of a THE will result in modulated expression levels
as directed
by the TRE, including enhanced cell-specificity. Achievement of enhanced
expression
levels may be especially desirable in the case of more aggressive forms of
neoplastic
growth, and/or when a more rapid and/or aggressive pattern of cell killing is
warranted (for
example, in an immunocompromised individual).
Transcriptional activity directed by a THE (including both inhibition and
enhancement) can be measured in a number of ways known in the art (and
described in
more detail below), but is generally measured by detection and/or quantitation
of mRNA
and/or of a protein product encoded by the sequence under control of (i. e. ,
operably linked
to) a TRE.
As discussed herein, a THE can be of varying lengths, and of varying sequence
composition. The size of a heterologous THE will be determined in part by the
capacity of
the viral vector, which in turn depends upon the contemplated form of the
vector (see
infra). Generally minimal sizes are preferred for TREs, as this provides
potential room for
insertion of other sequences which may be desirable, such as transgenes
(discussed ih, fra)
and/or additional regulatory sequences. In a preferred embodiment, such an
additional
regulatory sequence is an IRES. However, if no additional sequences are
contemplated, or
if, for example, an adenoviral vector will be maintained and delivered free of
any viral
packaging constraints, larger THE sequences can be used as long as the
resultant adenoviral
vector remains replication-competent.
In a preferred embodiment, a viral vector is an adenoviral vector. An
adenoviral
vector can be packaged with extra sequences totaling up to about S% of the
genome size, or
approximately 1.8 kb, without requiring deletion of viral sequences. If non-
essential
sequences are removed from the adenovirus genome, an additional 4.6 kb of
insert can be
tolerated (i.e., for a total insertion capacity of about 6.4 kb). Examples of
non-essential
adenoviral sequences that can be deleted are E3, and E4 sequences other than
those which
encode E4 ORF6.
To minimize non-specific replication, endogenous (e. g., adenovirus) TREs are
preferably removed from the vector. Besides facilitating target cell-specific
replication,
removal of endogenous TREs also provides greater insert capacity in a vector,
which may
be of special concern if an adenoviral vector is to be packaged within a virus
particle. Even
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more importantly, deletion of endogenous TREs prevents the possibility of a
recombination
event whereby a heterologous THE is deleted and the endogenous THE assumes
transcriptional control of its respective adenovirus coding sequences (thus
allowing non-
specific replication). In one embodiment, an adenoviral vector is constructed
such that the
endogenous transcription control sequences of adenoviral genes are deleted and
replaced by
one or more heterologous TREs. However, endogenous TREs can be maintained in
the
adenovirus vector(s), provided that su~cient cell-specific replication
preference is
preserved. These embodiments are constructed by inserting heterologous TREs
between an
endogenous THE and a replication gene coding segment. Requisite cell-specific
replication
preference is determined by conducting assays that compare replication of the
adenovirus
vector in a cell which allows function of the heterologous TREs with
replication in a cell
which does not.
Generally, a THE will increase replication of a vector in a target cell by at
least
about 2-fold, preferably at least about 5-fold, preferably at least about 10-
fold more
preferably at least about 20-fold, more preferably at least about 50-fold,
more preferably at
least about 100-fold, more preferably at least about 200-fold, even more
preferably at least
about 400- to about 500- fold, even more preferably at least about 1000-fold,
compared to
basal levels of replication in the absence of a TRE. The acceptable
differential can be
determined empirically (by measurement of mRNA levels using, for example, RNA
blot
assays, RNase protection assays or other assays known in the art) and will
depend upon the
anticipated use of the vector and/or the desired result.
Replication-competent adenovirus vectors directed at specific target cells can
be
generated using TREs that are preferentially functional in a target cell. In
one embodiment
of the present invention, the target cell is a tumor cell. Non-limiting
examples of tumor
cell-specific heterologous TREs, and their respective target cells, include:
probasin (PB),
target cell, prostate cancer (PCT/LJS98/04132); a-fetoprotein (AFP), target
cell liver cancer
(PCT/L1S98/04084); mucin-like glycoprotein DF3 (MUCI ), target celh breast
carcinoma
(PCT/LTS98/04080); caxcinoembryonic antigen (CEA), target cells, colorectal,
gastric,
pancreatic, breast, and lung cancers (PCT/US98/04133); plasminogen activator
urokinase
(uPA) and its receptor gene, target cells, breast, colon, and liver cancers
(PCT/LTS98/04080); E2Fl (cell cycle S-phase specific promoter); target cell,
tumors with
disrupted retinoblastoma gene function, and HER-2/heu (c-erbB2/neu), target
cell, breast,
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ovarian, stomach, and lung cancers (PCTlLTS98/04080); tyrosinase, target cell,
melanoma
cells as described herein and uroplakins, target cell, bladder cells as
described herein.
Methods for identification, isolation, characterization and utilization of
additional target
cell-specific TREs are readily available to those of skill in the art.
In addition, tumor-specific TREs can be used in conjunction with tissue-
specific
TREs from the following exemplary genes (tissue in which the TREs are
specifically
functional are in parentheses): hypoxia responsive element, vascular
endothelial growth
factor receptor (endothelium), albumin (liver), factor VII (liver), fatty acid
synthase (liver),
Von Willebrand factor (brain endothelium), alpha-actin and myosin heavy chain
(both in
smooth muscle), synthetase I (small intestine) Na~~-K+-Cl- transporter
(kidney). Additional
tissue-specific TREs are known in the art.
In one embodiment of the present invention, a target cell-specific,
heterologous
THE is tumor cell-specific. A vector can comprise a single tumor cell-specific
THE or
multiple heterologous TREs which are tumor cell-specific and functional in the
same cell.
In another embodiment, a vector comprises one or more heterologous TREs which
are
tumor cell-specific and additionally comprises one or more heterologous TREs
which are
tissue specific, whereby all TREs are functional in the same cell.
Prostate-speeific TREs
In one embodiment, adenovirus vectors comprise heterologous TREs that are
prostate cell specific. For example, TREs that function preferentially in
prostate cells and
can be used to target adenovirus replication to prostate neoplasia, include,
but are not
limited to, TREs derived from the prostate-specific antigen gene (PSA-TRE)
(Henderson
U.S. Patent No. 5,698,443); the glandular kallikrein-1 gene (from the human
gene, hKLK2-
TRE) (PCT US98/16312), and the probasin gene (PB-TRE) (PCT/LTS98/04132). All
three
of these genes are preferentially expressed in prostate cells and their
expression is
androgen-inducible. Generally, expression of genes responsive to androgen
induction is
mediated by an androgen receptor (AR).
Prostate-specific Antigeh (PSA)
PSA is synthesized exclusively in prostatic epithelial cells and is
synthesized in
these cells whether they axe normal, hyperplastic, or malignant. This tissue-
specific
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expression of PSA has made it an excellent biomarker for benign prostatic
hyperplasia
(BPH) and prostatic carcinoma (CaP). Normal serum levels of PSA are typically
below 5
ng/ml, with elevated levels indicative of BPH or CaP. Lundwall et al. (1987)
FEBS Lett.
214:317; Lundwall (1989) Biochem. Biophys. Res. Comm. 161:1151; and Riegmann
et al.
(1991) Molec. Endocrin. 5:1921.
The region of the PSA gene that provides androgen-dependent cell specificity,
particularly in prostate cells, involves approximately 6.0 kilobases (kb).
Schuur et al.
(1996) J. Biol. Chem. 271:7043-7051. An enhancer region of approximately 1.5
kb in
humans is located between nt -5322 and nt -3739, relative to the transcription
start site of
the PSA gene. Within these enhancer sequences is an androgen response element
(ARE) a
sequence which binds androgen receptor. The sequence coordinates of the PSA
promoter
are from about nt -S40 to nt +8 relative to the transcription start site.
Juxtapositioning of
the enhancer and promoter yields a fully functional, minimal prostate-specific
THE
(PSA-TRE). Other portions of this approximately 6.0 kb region of the PSA gene
can be
used in the vectors described herein, as long as requisite functionality is
maintained.
Human glandular Kallikrein (hKLK2)
Human glandular kallikrein (hKLK2, encoding the hI~2 protein) is expressed
exclusively in the prostate and its expression is up-regulated by androgens,
primarily
through transcriptional activation. Wolf et al. (1992) Molec. Endocrihol.
6:753-762;
Morris (1989) Clin. Exp. Pharm. Physiol.16:345-35I; Qui et al. (I990) J.
Urol.144:1550-
1556; and Young et al. (1992) Biochem. 31:818-824. The levels of hK2 found in
various
tumors and in the serum of patients with prostate cancer indicate that hK2
antigen may be a
significant marker for prostate cancer. Charlesworth et al. (1997) Urology
49:487-493.
Expression of hI~2 has been detected in each of 257 radical prostatectomy
specimens
analyzed. Darson et al. (I997) Urology 49:857-862. The intensity and extent of
hK2
expression, detected using specific antibodies, was observed to increase from
benign
epithelium to high-grade prostatic intraepithelial neoplasia (PIN) and
adenocarcinoma.
The activity of the hKLK2 promoter has been described and a region up to nt -
2256
relative to the transcription start site was previously disclosed. Schedlich
et al. (1987) DNA
6:429-437. The hKLK2 promoter is androgen responsive and, in plasmid
constructs
wherein the promoter alone controls the expression of a reporter gene,
expression of the
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reporter gene is increased approximately 10-fold in the presence of androgen.
Martha et al.
(1993) Biochem. 32:6459-6464. hKLK2 enhancer activity is found within
a.polynucleotide
sequence approximately nt -12,014 to nt -2257 relative to the start of
transcription and,
when this sequence is operably linked to an hKLK2 promoter and a reporter
gene,
transcription of operably-linked sequences in prostate cells increases in the
presence of
androgen to levels approximately 30-fold to approximately I00-fold greater
than the level
of transcription in the absence of androgen. This induction is generally
independent of the
orientation and position of the enhancer sequences. Enhancer activity has also
been
demonstrated in the following regions (all relative to the transcription start
site): about
nt -3993 to about nt -3643, about nt -4814 to about nt -3643, about nt -5155
to about
nt -3387, about nt -6038 to about nt -2394.
Thus, a hKLK2 enhancer can be operably linked to an hKLK2 promoter or a
heterologous promoter to form a hKLK2 transcriptional regulatory element
(hKLK2-TRE).
A hKLK2-THE can then be operably linked to a heterologous polynucleotide to
confer
1 S hKLK2-TRE-specific transcriptional regulation on the linked gene, thus
increasing its
expression.
Probasin
The rat probasin (PB) gene encodes an androgen and zinc-regulated protein
first
characterized in the dorsolateral prostate of the rat. Dodd et al. (1983) J.
Biol. Chem.
258:10731-10737; Matusik et al. (1986) Biochem. Cell. Biol. 64:601-607; and
Sweetland
et al. (1988) Mol. Cell. Biochem. 84:3-15. The dorsolateral lobes of the
marine prostate are
considered the most homologous to the peripheral zone of the human prostate,
where
approximately 68% of human prostate cancers axe thought to originate.
A PB-THE has been shown to exist in an approximately 0.5 kb fragment of
sequence upstream of the probasin coding sequence, from about nt -426 to about
nt +28
relative to the transcription start site. This minimal promoter sequence from
the PB gene
appears to provide sufficient information to direct prostate-specific
developmental- and
hormone -regulated expression of an operably linked heterologous gene in
transgenic mice.
Greenberg et al. (1994) Mol. Endocrinol. 8:230-239.
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Alpha fetoprotei~
a-fetoprotein (AFP) is an oncofetal protein, the expression of which is
primarily
restricted to developing tissues of endodermal origin (yolk sac, fetal liver,
and gut),
although the level of its expression varies greatly depending on the tissue
and the
developmental stage. AFP is of clinical interest because the serum
concentration of AFP is
elevated in a majority of hepatoma patients, with high levels of AFP found in
patients with
advanced disease. High serum AFP levels in patients appear to be due to AFP
expression
in hepatocellular carcinoma (HCC), but not in surrounding normal liver. Thus,
expression
of the AFP gene appears to be characteristic of hepatoma cells. An AFP-THE is
described
in for example PCT/LTS98/04084.
According to published reports, the AFP-THE is responsive to cellular proteins
(transcription factors and/or co-factor(s)) associated with AFP-producing
cells, such as
AFP-binding protein (see, for example, IJ.S. Pat. No. 5,302,698) and comprises
at least a
portion of an AFP promoter and/or an AFP enhancer. Cell-specific TREs from the
AFP
gene have been identified. For example; the cloning and characterization of
human AFP-
specif c enhancer activity is described in Watanabe et al. (1987) J. Biol.
Chem. 262:4812-
4818. A 5' AFP regulatory region (containing the promoter, putative silencer,
and
enhancer) is contained within approximately 5 kb upstream from the
transcription start site.
Within the AFP regulatory region, a human AFP enhancer region is located
between about nt -3954 and about nt -3335, relative to the transcription start
site of the AFP
gene. The human AFP promoter encompasses a region from about nt -174 to about
nt +29.
Juxtapositioning of these two genetic elements, yields a fully functional AFP-
TRE. Ido et
al. (1995) Cancer Res. 55:3105-3109 describe a 259 by promoter fragment. (nt -
230 to
nt +29) that is specific for expression in HCC cells. The AFP enhancer,
located between
nt -3954 and nt -3335 relative to the transcription start site, contains two
regions, denoted A
and B. The promoter region contains typical TATA and CART boxes. Preferably,
the
AFP-THE contains at least one enhancer region. More preferably, the AFP-THE
contains
both enhancer regions.
Suitable target cells for vectors containing AFP-TREs are any cell type that
allow
an AFP-TItE to function. Preferred are cells that express or produce AFP,
including, but
not limited to, tumor cells expressing AFP. Examples of such cells are
hepatocellular
carcinoma (HCC) cells, gonadal and other germ cell tumors (especially
endodermal sinus
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tumors), brain tumor cells, ovarian tumor cells, acinar cell carcinoma of the
pancreas
(Kawarnoto et al. (1992) Hepatogastroente~ology 39:282-286), primary gall
bladder tumor
(Katsuragi et al. (1989) Rinsko Hoshasen 34:371-374), uterine endometrial
adenocarcinoma cells (Koyama et al. (1996) Jp~. J. Cahce~ Res. 87:612-617),
and any
metastases of the foregoing (which can occur in lung, adrenal gland, bone
marrow, and/or
spleen). In some cases, metastatic disease to the liver from certain
pancreatic and stomach
cancers produce AFP. Especially preferred as target cells for an AFP-THE are
hepatocellular carcinoma cells and any of their metastases.
AFP production can be measured (and hence AFP-producing cells can be
identified)
using immunoassays standard in the art, such as RIA, ELISA or protein
immunoblotting
(Western blots) to determine levels of AFP protein production; and/or RNA
blotting
(Northern blots) to determine AFP mRNA levels. Alternatively, such cells can
be
identified and/or characterized by their ability to activate transcriptionally
an AFP-THE
(i. e. , allow an AFP-THE to function).
See also co-owned PCT W098/39465 regarding AFP-TREs. As described in more
detail therein, an AFP-THE can comprise any number of configurations,
including, but not
limited to, an AFP promoter; an AFP enhancer; an AFP promoter and an AFP
enhancer; an
AFP promoter and a heterologous enhancer; a heterologous promoter and an AFP
enhancer; and multimers of the foregoing. The promoter and enhancer components
of an
AFP-THE can be in any orientation and/or distance from the coding sequence of
interest, as
long as the desired AFP cell-specific transcriptional activity is obtained. An
adenovirus
vector of the present invention can comprise an AFP-THE endogenous silencer
element or
the AFP-THE endogenous silencer element can be deleted.
Urolrinase Plasminogen Activator
The protein urokinase plasminogen activator (uPA) and its cell surface
receptor,
urokinase plasminogen activator receptor (uPAR), are expressed in many of the
most
frequently-occurring neoplasms and appear to represent important proteins in
cancer
metastasis. Both proteins are implicated in breast, colon, prostate, liver,
renal, lung and
ovarian cancer. Sequence elements that regulate uPA and uPAR transcription
have been
extensively studied. Riccio et al. (I985) Nucleic Acids Res. 13:2759-2771;
Cannio et al.
(1991) Nucleic Acids Res. 19:2303-2308.
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Carcihoembryonic ahtigeh (CEA)
CEA is a 180,000 Dalton, tumor-associated, glycoprotein antigen pxesent on
endodermally-derived neoplasms of the gastrointestinal tract, such as
colorectal, gastric
S (stomach) and pancreatic cancer, as well as other adenocarcinomas such as
breast and lung
cancers. CEA is of clinical interest because circulating CEA can be detected
in the great
majority of patients with CEA-positive tumors. In lung cancer, about 50% of
total cases
have circulating CEA, with high concentrations of CEA (greater than 20 ng/ml)
often
detected in adenocarcinomas. Approximately 50% of patients with gastric
carcinoma axe
serologically positive for CEA.
The 5'-flanking sequence of the CEA gene has been shown to confer cell-
specific
activity. The CEA promoter region, approximately the first 424 nucleotides
upstream of
the transcriptional start site in the 5' flanking region of the gene, was
shown to confer cell-
specific activity by virtue of providing higher promoter activity in CEA-
producing cells
than in non-producing HeLa cells. Schrewe et al. (1990) Mol. Cell. Biol.
10:2738-2748. In
addition, cell-specific enhancer regions have been found. See PCT/GB/02546 The
CEA
promoter, putative silencer, and enhancer elements appears to be contained
within a region
that extends approximately 14.5 kb upstream from the transcription start site.
Richards
et al. (1995); PCT/GB/02546. Further characterization of the 5'-flanking
region of the
CEA gene by Richards et al. (1995) supra indicated that two upstream regions
(one
between about -13.6 and about -10.7 kb, and the other between about -6.1 and
about-4.0 kb), when linked to the multimerized promoter, resulted in high-
level and
selective expression of a reporter construct iri CEA-producing LoVo and SW1463
cells.
Richards et al. (1995) supra also localized the promoter region between about
nt -90 and
about nt +69 relative to the transcriptional start site, with the region
between about nt -41
and about nt -18 being essential for expression. PCT/GB/02546 describes a
series of
5'-flanking CEA fragments which confer cell-specific activity, including
fragments
comprising the following sequences: about nt -299 to about nt +69; about nt -
90 to about
nt +69; nt -14,500 to nt -10,600; nt -13,600 to nt -10,600; and nt -6100 to nt
-3800, with all
coordinates being relative to the transcriptional start point. In addition,
cell-specific
transcription activity is conferred on an operably linked gene by the CEA
fragment from
nt -402 to nt +69.
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CEA-TREs fox use in the vectors disclosed herein are derived from mammalian
cells, including, but not limited to, human cells. Thus, any of the CEA-TREs
can be used
as long as the requisite desired functionality is displayed by the vector.
Mucin
The protein product of the MUCI gene (known as mucin, MUC1 protein; episialin;
poIymorphic epithelial mucin or PEM; EMA; DF3 antigen; NPGP; PAS-O; or CA15.3
antigen) is normally expressed mainly at the apical suxface of epithelial
cells lining the
glands or ducts of the stomach, pancreas, lungs, trachea, kidney, uterus,
salivary glands,
and mammary glands. Zotter et al. (1988) Cancer Rev. 11-12:55-101; and Girling
et al.
(1989) Iht. J. Cancer 43:1072-1076. However, mucin is overexpressed in 75-90%
of
human breast carcinomas. Kufe et al. (1984) Hybridoma 3:223 232. For reviews,
see
Hilkens (1988) Cancer Rev. 11-12:25-54; and Taylor-Papadimitriou, et al.
(1990) J. Nucl.
Med. Allied Sci. 34:144-150. Mucin protein expression correlates with the
degree of breast
tumor differentiation. Lundy et al. (1985) Breast Cahcer Res. Treat. 5:269-
276.
Ovexexpression of the MUCI gene in human breast carcinoma cells MCF-7 and
ZR-75-1 appears to occur at the transcxiptional level. Kufe et al. (1984)
supra; Kovarik
(1993) J. Biol. Chem. 268:9917-9926; and Abe et al. (1990) J. Cell. Physiol.
143:226-
23 I . The regulatory sequences of the MUCI gene have been cloned, including
the
approximately 0.9 kb upstream of the transcription start site which contains a
THE that
appears to be involved in cell-specific transcription. Abe et al. (1993) Proc.
Natl. Acad.
Sci. USA 90:282-286; Kovarik et al. (I993) supra; and Kovarik et al. (1996) J.
Biol.
Chem. 271:18140-18147.
MUCI-TREs are derived from mammalian cells, including but not limited to,
human cells. Preferably, the MUCI-THE is human. In one embodiment, the MUCl-
THE
contains the entire 0.9 kb 5' flanking sequence of the MUCI gene. In other
embodiments,
MUCI -TREs comprise the following sequences (relative to the transcription
start site of the
MUCI gene) operably-linked to a promoter: about nt -725 to about nt +31, about
nt -743 to
about nt +33, about nt -7S0 to about nt +33, and about nt -598 to about nt
+485.
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c-erbB2/HER-2/ueu
The c-e~bB2/neu gene (HER-2/neu or HER) is a transforming gene that encodes a
185 kD epidermal growth factor receptor-related transmembrane glycoprotein. In
humans,
the c-erbB2/neu protein is expressed during fetal development and, in adults,
the protein is
weakly detectable (by immunohistochemistry) in the epithelium of many normal
tissues.
Amplification and/or over-expression of the c-erbB2/neu gene has been
associated with
many human cancers, including breast, ovarian, uterine, prostate, stomach and
lung
cancers. The clinical consequences of overexpression of the c-erbB2/neu
protein have been
best studied in breast and ovarian cancer. c-erbB2/neu protein over-expression
occurs in 20
to 40% of intraductal carcinomas of the breast and 30% of ovarian cancers, and
is
associated with a poor prognosis in subcategories of both diseases.
Human, rat and mouse c-e~bB2lneu TREs have been identified and shown to confer
transcriptional activity specific to c-erbB2/neu-expressing cells. Tal et al.
(1987) Mol.
Cell. Biol. 7:2597 2601; Hudson et al. (1990) J. Biol. Chem. 265:4389-4393;
Grooteclaes et al. (1994) Cancer Res. 54:4193-4199; Ishii et al. (1987) Proc.
Natl. Acad.
Sci. USA 84:4374-4.378; and Scott et al. (1994) J. Biol. Chem. 269:19848-
19858.
Melarnocyte specific THE
It has been shown that some genes which encode melanoma proteins are
frequently
. expressed in melanoma/melanocytes, but silent in the majority of normal
tissues. A
variety of melanocyte-specific THE are known, are responsive to cellular
proteins
(transcription factors and/or co-factor(s)) associated with melanocytes, and
comprise at
least a portion of a melanocyte-specific promoter and/or a melanocyte-specific
enhancer.
Known transcription factors that control expression of one or more melanocyte-
specific
genes include the microphthalmia associated transcription factor MITF.
Yasumato et al.
(1997) J. Biol. Chem. 272:503-509. Other transcription factors that control
expression of
one or more melanocyte specific genes include MART-1/Melan-A, gp100, TRP-1 and
TRP-2
Methods are described herein for measuring the activity of a melanocyte-
specific
THE and thus for determining whether a given cell allows a melanocyte-specific
THE to
function.
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The melanocyte-specific TREs used in this invention are derived from mammalian
cells, including but not limited to, human, rat, and mouse. Any melanocyte-
specific TREs
may be used in the adenoviral vectors of the invention. Rodent and human 5'
flanking
sequences from genes expressed specifically or preferentially in melanoma
cells have been
described in the literature and are thus made available for practice of this
invention and
need not be described in detail herein. The following are some examples of
melanocyte-
specific TREs which can be used. A promoter and other control elements in the
human
tyrosinase gene 5' flanking region have been described and sequences have been
deposited
as GenBank Accession Nos. X16073 and D10751. Kikuchi et al. (1989) Biochim.
Biophys.
Acta 1009:283-286; and Shibata et al. (1992) J. Biol. Chem. 267:20584-20588. A
cis-
acting element has been defined that enhances melanocyte-specific expression
of human
tyrosinase gene. This element comprises a 20-by sequence known as tyrosinase
distal
element (TDE), contains a CATGTG motif, and lies at positions about -1874 to
about -
1835 relative to the human tyrosinase gene transcription start site. Yasumoto
et al. (1994)
Mol. Cell. Biol. 14:8058-8070. A promoter region comprising sequences from
about -209
to +61 of the human tyrosinase gene was found to direct melanocyte-specific
expression.
Shibata (1992). Similarly, the mouse tyrosinase 5' flanking region has been
analyzed and a
sequence deposited as GenBank Accession Nos. D00439 and X51743. Kliippel et
al.
(1991) Proc. Natl. Acad. Sci. USA 88:3777-3788. A minimal promoter has been
identified
for the mouse TRP-1 gene, and was reported to encompass nucleotides -44 to
+107 relative
to the transcription start site. Lowings et~al. (1992) Mol. Cell. Biol.
12:3653-3662. Two
regulatory regions required for melanocyte-specific expression of the human
TRP-2 gene
have been identified. Yokoyama et al. (1994) J. Biol. Chem. 269:27080-27087. A
human
MART-1 promoter region has been described and deposited as GenBank Accession
No.
U55231. Melanocyte-specific promoter activity was found in a 233-by fragment
of the
human MART-1 gene 5' flanking region. Butterfield et al. (1997) Gene 191:129-
134. A
basic-helix-loop-helix/leucine zipper-containing transcription factor, MITF
(microphthalmia associated transcription factor) was reported to be involved
in
transcriptional activation of tyrosinase and TRP-1 genes. Yasumoto et al.
(1997) J. Biol.
Chem.272:503-509.
In some embodiments, a melanocyte-specific THE comprises sequences derived
from the 5' flanking region of a human tyrosinase gene depicted in Table 14.
In some of
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these embodiments, the melanocyte-specific THE comprises tyrosinase
nucleotides from
about -231 to about +65 relative to the transcription start site (from about
nucleotide 244 to
about nucleotide 546 of SEQ ID NO:~ and may further comprise nucleotides from
about
-1956 to about -1716 relative to the human tyrosinase transcription start site
(from about
nucleotide 6 to about nucleotide -243 of SEQ ID NO:~. A tyrosinase THE can
comprise
nucleotides from about -231 to about + 65 juxtaposed to nucleotides from about
-1956 to
about -1716. It has been reported that nucleotides from about -1956 to about -
1716 relative
to the human tyrosinase transcription start site can confer melanocyte-
specific expression
of an operably linked reporter gene with either a homologous or a heterologous
promoter.
Accordingly, in some embodiments, a melanocyte-specific THE comprises
nucleotides
from about -1956 to about -1716 operably linked to a heterologous promoter.
A melanocyte-specific THE can also comprise multimers. For example, a
melanocyte-specific THE can comprise a tandem series of at least two, at least
three, at
least four, or at least five tyrosinase promoter fragments. Alternatively, a
melanocyte-
specific THE could have one or more tyrosinase promoter regions along with one
or more
tyrosinase enhancer regions. These multimers may also contain heterologous
promoter
and/or enhancer sequences.
Cell status-specific TREs
Cell status-specific TREs for use in the adenoviral vectors of the present
invention
can be derived from any species, preferably a mammal. A number of genes have
been
described which are expressed in response to, or in association with, a cell
status. Any of
these cell status-associated genes may be used to generate a cell status-
specific TRE.
An example of a cell status is cell cycle. An exemplary gene whose expression
is
associated with cell cycle is E2F-1, a ubiquitously expressed, growth-
regulated gene, which
exhibits peak transcriptional activity in S phase. Johnson et al. (1994) Genes
Dev. 8:1514-
1525. The RB protein, as well as other members of the RB family, form specific
complexes with E2F-1, thereby inhibiting its ability to activate
transcription. Thus, E2F-1-
responsive promoters are down-regulated by RB. Many tumor cells have disrupted
I~B
function, which can lead to de-repression of E2F-1-responsive promoters, and,
in turn, de-
regulated cell division.
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Accordingly, in one embodiment, the invention provides an E3-containing
adenoviral vector in which an adenoviral gene (preferably a gene necessary for
replication)
is under transcriptional control of a cell status-specific TRE, wherein the
cell status-specific
THE comprises a cell cycle-activated TRE. In one embodiment, the cell cycle-
activated
S THE is an E2F1 TRE.
Another group of genes that are regulated by cell status are those whose
expression
is increased in response to hypoxic conditions. Bunn and Poyton (1996)
Physiol. Rev.
76:839-885; Dachs and Stratford (1996) Br. J. Cancer 74:5126-5132; Guillemin
and
Krasnow (1997) Cell 89:9-12. Many tumors have insufficient blood supply, due
in part to
the fact that tumor cells typically grow faster than the endothelial cells
that make up the
blood vessels, resulting in areas of hypoxia in the tumor. Folkman (1989) J.
Natl. Cancer
Inst. 82:4-6; and I~allinowski (1996) The Cancer J. 9:37-40. An important
mediator. of
hypoxic responses is the transcriptional complex HIF-1, or hypoxia inducible
factor-1,
which interacts with a hypoxia-responsive element (HRE) in the regulatory
regions of
1 S several genes, including vascular endothelial growth factor, and several
genes encoding
glycolytic enzymes, including enolase-1. Murine HRE sequences have been
identified and
characterized. Firth et al. (1994) Proc. Natl. Acid. Sci. LISA 91:6496-6500.
An HRE from
a rat enolase-1 promoter is described in Jiang et al. (1997) Cancer Res.
S7:S328-S33S. An
HRE from a rat enolase-1 promoter is depicted in Table 14.
Accordingly, in one embodiment, an adenovirus vector comprises an adenovirus
gene, preferably an adenoviral gene essential for replication, under
transcriptional control
of a cell status-specific THE comprising an HRE. In one embodiment, the cell
status-
specific THE comprises the HRE depicted in Table 14
Other cell status-specific TREs include heat-inducible (i.e., heat shock)
promoters,
2S and promoters responsive to radiation exposure, including ionizing
radiation and UV
radiation. For example, the promoter region of the early growth response-1
(Egr-1) gene
contains an elements) inducible by ionizing radiation. Hallahan et al. (I995)
Nat. Med.
1:786-791; and Tsai-Morris et al. (1988) Nucl. Acids Res. 16:8835-8846. Heat-
inducible
promoters, including heat-inducible elements, have been described. See, for
example
Welsh (1990) in "Stress Proteins in Biology and Medicine", Morimoto, Tisseres,
and
Georgopoulos, eds. Cold Spring Harbor Laboratory Press; and Perisic et al.
(1989) Cell
59:797-806. Accordingly, in some embodiments, the cell status-specific THE
comprises an
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elements) responsive to ionizing radiation. In one embodiment, this THE
comprises a 5'
flanking sequence of an Egr-1 gene. In other embodiments, the cell status-
specific THE
comprises a heat shock responsive element.
The cell status-specific TREs listed above are provided as non-limiting
examples of
TREs that would function in the instant invention. Additional cell status-
specific TREs are
known in the art, as axe methods to identify and test cell status specificity
of suspected cell
status-specific TREs.
Urotlzelial cell specific TREs
Any urothelial cell-specific THE may be used in the adenoviral vectors of the
invention. A number of urothelial cell-specific proteins have been described,
among which
are the uroplakins. Uroplakins (LJP), including UPIa and UPIb (27 and 28 kDa,
respectively), UPII (15 kDa), and UPIII (47 kDa), are members of a group of
integral
membrane proteins that are major proteins of urothelial plaques. These plaques
cover a
~ large portion of the apical surface of mammalian urotheliurn and may play a
role as a
permeability barrier andlor as a physical stabilizer of the urothelial apical
surface. Wu et
al. (1994) J. Biol. Chem. 269:13716-13724. UPs are bladder-specific proteins,
and are
expressed on a significant proportion of urothelial-derived tumors, including
about 88% of
transitional cell carcinomas. Moll et al. (1995) Am. J. Pathol. 147:1383-1397;
and Wu et
al. (1998) Cancer Res. 58:1291-1297. The control of the expression of the
human UPII has
been studied, and a 3.6-kb region upstream of the mouse UPII gene has been
identified
which can confer urothelial-specific transcription on heterologous genes (Lin
et al. (1995)
Proc. Natl. Acad. Sci. USA 92:679-683).
Preferred urothelial cell-specific TREs include TREs derived from the
uroplakins
UPIa, UPIb, UPII, and UPIII, as well as urohingin. A uroplakin THE may be from
any
species, depending on the intended use of the adenovirus, as well as the
requisite
functionality is exhibited in the target or host cell. Significantly,
adenovirus constructs
comprising a urothelial cell-specific TREs have observed that such constructs
are capable
of selectively replicating in urothelial cells as opposed to smooth muscle
cells, which
adjoin urothelial cells in the bladder.
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Uroplakih
Urothelial-specific TREs derived from the hUPIl gene are described herein.
Accordingly, in some embodiments, an adenovirus vector of the invention
comprises an
adenovirus gene, preferably an adenoviral gene essential for replication,
under
transcriptional control of a urothelial cell-specific THE which comprises the
2.2 kb
sequence from the 5' flanking region of hUPII gene, as shown in Table 14. In
other
embodiments, an adenovirus vector of the invention comprises an adenovirus
gene,
preferably an adenoviral gene essential for replication, under transcriptional
control of a
urothelial cell-specific THE which comprises a 1.8 kb sequence from the 5'
flanking region
of hUPII gene, from nucleotides 430 to 2239 as shown in Table 14. In other
embodiments,
the urothelial cell-specific THE comprises a functional portion of the 2.2 kb
sequence
depicted in Table 14, or a functional portion of the 1.8 kb sequence of
nucleotides 430 to
2239 of the sequence depicted in Table 14, such as a fragment of 2000 by or
less, 1500 by
or less, or 1000 by or less, 600 by less, or at least 200 by which includes
the 200 by
fragment of the hUPII 5'-flanking region.
A 3.6 kb 5'-flanking sequence located from the mouse UPII (mUPII) gene which
confers urothelial cell-specific transcription on heterologous genes is one
urothelial cell-
specific THE useful in vectors of the instant invention (Table 14). Smaller
TREs (i.e., 3500
by or less, more preferably less than about 2000 bp, 1500 bp, or 1000 bp) are
preferred.
Smaller TREs derived from the mUPII 3.6 kb fragment are one group of preferred
urothelial cell-specific TREs. In particular, Inventors have identified an
approximately 600
by fragment from the 5' flanking DNA of the mUPII gene, which contains 540 by
of 5'
untransIated region (L1TR) of the mUPII gene, that confers urothelial cell-
specific
expression on heterologous genes.
Accordingly, in some embodiments, an adenovirus vector comprises an adenovirus
gene, preferably an adenoviral gene essential for replication, under
transcriptional control
of a urothelial cell-specific THE which comprises the 3.6 kb sequence from the
5' flanking
region of mouse UPII gene, as shown in Table 14. In other embodiments, the
urothelial
cell-specific THE comprises a functional portion of the 3.6 kb sequence
depicted in Table
14, such as a fragment of 3500 by or less, 2000 by or less, 1500 by or less,
or 1000 by or
less which includes the 540 by fragment of 5' UTR. The urothelial cell-
specific THE may
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also be a sequence which is substantially identical to the 3.6 kb mUPII 5'-
flanking region
or any of the described fragments thereof.
As an example of how urothelial cell-specific THE activity can be determined,
a
polynucleotide sequence or set of such sequences can be generated using
methods known
in the art, such as chemical synthesis, site-directed mutagenesis, PCR, and/or
recombinant
methods. The sequences) to be tested is inserted into a vector containing an
appropriate
reporter gene, including, but not limited to, chloramphenicol acetyl
transferase (CAT), (3-
galactosidase (encoded by the lacZ gene), luciferase (encoded by the luc
gene), a green
fluorescent protein, alkaline phosphatase, and horse radish peroxidase. Such
vectors and
assays are readily available, from, inter alia, commercial sources. Plasmids
thus
constructed are transfected into a suitable host cell to test for expression
of the reporter
gene as controlled by the putative target cell-specific THE using txansfection
methods
known in the art, such as calcium phosphate precipitation, electroporation,
Iiposornes
(lipofection) and DEAE dextran. Suitable host cells include any urothelial
cell type,
including but not limited to, KU-1, MYP3 (a non-tumorigenic rat urothelial
cell line), 8046
(rat bladder carcinoma cell line), cultured human urothelial cells (HUC), HCV-
29,
UM-UC-3, SW780, RT4, HL60, KG-I, and KG-lA. Non-urothelial cells, such as
LNCaP,
HBL-100, HLF, HLE, 3T3, Hep3B, HuH7, CADO-LC9, and HeLa are used as a control.
Results are obtained by measuring the level of expression of the reporter gene
using
standard assays. Comparison of expression between urothelial cells and control
indicates
presence or absence of transcriptional activation.
Comparisons betweemor among various urothelial cell-specific TREs can be
assessed by measuring and comparing levels of expression within a single
urothelial cell
line. It is understood that absolute transcriptional activity of a urothelial
cell-specific THE
will depend on several factors, such as the nature of the target cell,
delivery mode and form
of the urothelial cell-specific TRE, and the coding sequence that is to be
selectively
transcriptionally activated. To compensate for various plasmid sizes used,
activities can be
expressed as relative activity per mole of transfected plasmid. Alternatively,
the level of
transcription (i.e., mRNA) can be measured using standard Northern analysis
and
hybridization techniques. Levels of transfection (i.e., transfection
efficiencies) are
measured by co-transfecting a plasmid encoding a different reporter gene under
control of a
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different TRE, such as the CMV immediate early promoter. This analysis can
also indicate
negative regulatory regions, i.e., silencers.
Alternatively a putative urothelial cell-specific THE can be assessed for its
ability to
confer adenoviral replication preference for cells that allow a urothelial
cell-specific THE
to function. For this assay, constructs containing an adenovirus gene
essential to
replication operatively linked to a putative urothelial cell-specific THE are
transfected into
urothelial cells. Viral replication in those cells is compared, for example,
to viral
replication by wild type adenovirus in those cells and/or viral replication by
the construct in
non-urothelial cells.
THE configurations
A THE as used in the present invention can be present in a vaxiety of
configurations.
A THE can comprise multimers. For example, a THE can comprise a tandem series
of at
least two, at least three, at least four, or at least five target cell-
specific TREs. These
multimers may also contain heterologous promoter and/or enhancer sequences.
Optionally, a transcriptional terminator or transcriptional "silencer" can be
placed
upstream of the target cell-specific TRE, thus preventing unwanted read-
through
transcription of the coding segment under transcriptional control of the
target cell-specific
TRE. Also, optionally, the endogenous promoter of the coding segment to be
placed under
transcriptional control of the target cell-specific THE can be deleted.
A target cell-specific THE may or may not lack a silencer. The presence of a
silencer (i.e., a negative regulatory element) may assist in shutting off
transcription (and
thus replication) in non-permissive cells (i.e., a non-taxget cell). Thus,
presence of a
silencer may confer enhanced target cell-specific replication by more
effectively preventing
adenoviral vector replication in non-target cells. Alternatively, lack of a
silencer may assist
in effecting replication in target cells, thus conferring enhanced target cell-
specific
replication due to more effective replication in target cells.
It is also understood that the invention includes a target cell-specific THE
regulating
the transcription of a bicistronic mRNA in which translation of the second
mRNA is
associated by an IRES. An adenovirus vector may further include an additional
heterologous THE which may or may not be operably linked to the same genes) as
the
target cell-specific TRE. For example a THE (such as a cell type-specific or
cell status-
CA 02404060 2002-09-23
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specific TRE) may be juxtaposed to a second type of target-cell-specific TRE.
"Juxtaposed" means a target cell-specific THE and a second THE
transcriptionally control
the same gene. For these embodiments, the target cell-specific THE and the
second THE
may be in any of a number of configurations, including, but not limited to,
(a) next to each
S other (i.e., abutting); (b) both S' to the gene that is transcriptionally
controlled (i.e., may
have intervening sequences between them); (c) one THE S' and the other THE 3'
to the
gene.
As is readily appreciated by one skilled in the art, a target cell-specific
THE is a
polynucleotide sequence, and, as such, can exhibit function over a variety of
sequence
permutations. Methods of nucleotide substitution, addition, and deletion axe
known in the
art, and readily available functional assays (such as the CAT or Iuciferase
reporter gene
assay) allow one of ordinary skill to determine whether a sequence variant
exhibits
requisite target cell-specific transcription function. Hence, the invention
also includes
functionally-preserved variants of the THE nucleic acid sequences disclosed
herein, which
1.S include nucleic acid substitutions, additions, and/or deletions. The
variants of the
sequences disclosed herein may be 80%, 8S%, 90%, 9S%, 98%, 99% or more
identical, as
measured by, for example, ALIGN Plus (Scientific and Educational Software,
Pennsylvania), preferably using efault parameters, which are as follows:
mismatch = 2;
open gap = 0; extend gap = 2 to any of the urothelial cell-specific THE
sequences disclosed
herein. Variants of target cell-specific THE sequences may also hybridize at
high
stringency, that is at 68°C and O.1XSSC, to any of the target cell-
specific THE sequences
disclosed herein.
In terms of hybridization conditions, the higher the sequence identity
required, the
more stringent are the hybridization conditions if such sequences are
determined by their
ability to hybridize to a sequence of a THE disclosed herein. Accordingly, the
invention
also includes polynucleotides that are able to hybridize to a sequence
comprising at least
about 1S contiguous nucleotides (or more, such as about 2S, 3S, S0, 75 or 100
contiguous
nucleotides) of a sequence of a THE disclosed herein. The hybridization
conditions would
be stringent, i.e., 80°C (or higher temperature) and 6M SSC (or Iess
concentrated SSC).
Another set of stringent hybridization conditions is 68°C and 0.1 X
SSC. For discussion
regarding hybridization reactions, see below.
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Hybridization reactions can be perfornzed under conditions of different
"stringency". Conditions that increase stringency of a hybridization reaction
of widely
known and published in the art. See, for example, Sambrook et al. (1989) at
page 7.52.
Examples of relevant conditions include (in order of increasing stringency):
incubation
temperatures of 25°C, 37°C, 50°C and 68°C; buffer
concentrations of 10 X SSC, 6 X SSC,
1 X SSC, 0.1 X SSC (where SSC is 0.15 M NaCI and 15 mM citrate buffer) and
their
equivalents using other buffer systems; formamide concentrations of 0%, 25%,
50%, and
75%; incubation times from 5 minutes to 24 hours; 1, 2, or more washing steps;
wash
incubation times of 1, 2, or 15 minutes; and wash solutions of 6 X SSC, I X
SSC, 0.1 X
SSC, or deionized water. An exemplary set of stringent hybridization
conditions is 68°C
and 0.1 X SSC.
"Tm" is the temperature in degrees Celcius at which 50% of a polynucleotide
duplex
made of complementary strands hydrogen bonded in anti-parallel direction by
Watson-
Crick base pairing dissociates into single strands under conditions of the
experiment. Tm
may be predicted according to a standard formula, such as:
T,i, = 81.5 + 16.6 log(X+),+ 0.41 (%G/C) - 0.61 (%F) - 600/L
where [X~] is the cation concentration (usually sodium ion, Na~ in mol/L;
(%G/C) is the
number of G and C residues as a percentage of total residues in the duplex;
(%F) is the
percent formamide in solution (wt/vol); and L is the number of nucleotides in
each strand
of the duplex.
While not wishing to be bound by a single theory, the inventors note that it
is
possible that certain modifications will result in modulated resultant
expression levels,
including enhanced expression levels. Achievement of modulated resultant
expression
levels, preferably enhanced expression levels, may be especially desirable in
the case of
certain, more aggressive forms of cancer, or when a more rapid and/or
aggressive pattern of
cell killing is warranted (due to an immunocompromised condition of the
individual, for
example).
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Determination of THE activity
Activity of a THE can be determined, for example, as follows. A THE
polynucleotide sequence or set of such sequences can be generated using
methods known
in the art, such as chemical synthesis, site-directed mutagenesis, PCR, and/or
recombinant
methods. The sequences) to be tested can be inserted into a vector containing
a promoter
(if no promoter element is present in the TRE) and an appropriate reporter
gene encoding a
reporter protein, including, but not limited to, chloramphenicol acetyl
transferase (CAT), (3-
galactosidase (encoded by the lacZ gene), luciferase (encoded by the luc
gene), alkaline
phosphatase (AP), green fluorescent protein (GFP), and horseradish peroxidase
(HRP).
Such vectors and assays are readily available, from, inter alia, commercial
sources.
Plasmids thus constructed are transfected into a suitable host cell to test
for expression of
the reporter gene as controlled by the putative THE using transfection methods
known in
the art, such as calcium phosphate precipitation, electroporation, liposomes,
DEAE
dextran-mediated transfer, particle bombardment or direct injection. THE
activity is
measured by detection and/or quantitation of reporter gene-derived mRNA and/or
protein.
Reporter protein product can be detected directly (e.g., immunochemically) or
through its
enzymatic activity, if any, using an appropriate substrate. Generally, to
determine cell
specific activity of a TRE, a TRE-reporter gene construct is introduced into a
variety of cell
types. The amount of THE activity is determined in each cell type and compared
to that of
a reporter gene construct lacking the TRE. A THE is determined to be cell-
specific if it is
preferentially functional in one cell type, compared to a different type of
cell.
Internal Ribosome Entry Site (IRES)
IRES elements were first discovered in picornavirus mRNAs (Jackson RJ, Howell
MT, Kaminski A (1990) Trends Biochem Sci 15(12):477-83) and Jackson RJ and
Kaminski, A. (1995) RNA 1(10):985-1000). The present invention provides
improved
adenovirus vectors comprising co-transcribed first and second genes under
transcriptional
control of a heterologous, target cell-specific TRE, and wherein the second
gene (i.e.,
coding region) is under translational control of an internal ribosome entry
site (IRES). Any
IRES may be used in the adenovirus vectors of the invention, as long as they
exhibit
requisite function in the vectors. Example of IRES which can be used in the
present
invention include those provided in Table I and referenced in Table II.
Examples of IRES
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elements include the encephelomycarditis virus (EMCV) which is commercially
available
from Novagen (Duke et al. (1992) J. Virol 66(3):1602-9) the sequence for which
is
depicted in Table 1 (SEQ ID NO:1). Another example of an IRES element
disclosed herein
is the VEGF IRES (Huez et al. (1998) Mol Cell Biol 18(11):6178-90). This IRES
has a
short segment and the sequence is depicted in Table 1 (SEQ ID N0:2).
The IRES promotes direct internal ribosome entry to the initiation codon of a
downstream cistron, leading to cap-independent translation. Thus, the product
of a
downstream cistron can be expressed from a bicistronic (or multicistronic)
mRNA, without
requiring either cleavage of a polyprotein or generation of a monocistronic
mRNA.
Therefore, in one illustrative embodiment of the present invention, an
adenovirus vector
comprising E1B under txanslational control of an IRES allows translation of
E1B from a
bicistronic ElA-E1B mRNA under control of a target cell-specific TRE.
Internal ribosome entry sites are approximately 450 nucleotides in length and
are
characterized by moderate conservation of primary sequence and strong
conservation of
secondary structure. The most significant primary sequence feature of the IRES
is a
pyrimidine-rich site whose start is located approximately 25 nucleotides
upstream of the 3'
end of the IRES. See Jackson et al. (1990).
Three major classes of picornavirus IRES have been identified and
characterized:
(1) the cardio- and aphthovirus class (for example, the encephelomycarditis
virus, Jang et
al. (1990) Gene I~ev 4:1560-1572); (2) the entero- and rhinovirus class (for
example,
polioviruses, Borman et al. (1994) EMBO J. 13:314903157); and (3)the hepatitis
A virus
(HAV) class, Glass et al. (1993) Tlirol 193:842-852). For the frst two
classes, two general
principles apply. First, most of the 450-nucleotide sequence of the IRES
functions to
maintain particular secondary and tertiary structures conducive to ribosome
binding and
translational initiation. Second, the ribosome entry site is an AUG triplet
located at the 3'
end of the IRES, approximately 25 nucleotides downstream of a conserved
oligopyrimidine
tract. Translation initiation can occur either at the ribosome entry site
(cardioviruses) or at
the next downstream AUG (entero/rhinovirus class). Initiation occurs at both
sites in
aphthoviruses.
HCV and pestiviruses such as bovine viral diarrhea virus (BVDV) or classical
swine fever virus (CSFV) have 341 nt and 370 nt long 5'-UTR respectively.
These 5'-UTR
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fragments form similar RNA secondarystructures and can have moderately
efficient IRES
function (Tsukiyama-Kohara et al. (1992) J. Tlirol. 66:1476-1483; Frolov I et
al., (1998)
RNA 4:1418-1435). Table I depicts the 5'-UTR region from HCV genome sequence
(GenBank accession D14853).
Leishmania RNA virus 1 (LRV1) is a double-stranded RNA virus. Its 128 nt long
5'-UTR has IRES activity to facilitate the cap-independent translation, (Maga
et al. (1995)
Mol Cell Biol 15:4884-4889). This fragment also forms conserved stemloop
secondary
structure and at least the front part is essential.
Recent studies showed that both Friend-marine leukemia virus (MLV) 5'-UTR and
rat retrotransposon virus-like 30S (VL30) sequences contain IRES structure of
retroviral
origin (Torrent et al. (1996) Hum Gehe They 7:603-612). These fragments are
also
functional as packing signal when used in retroviruse derived vectors. Studies
of avian
reticuloendotheliosis virus type A (REV-A) show that its IRES maps downstream
of the
packaging/dimerization (E/DLS) sequence and the minimal IRES sequence appears
to be
within a 129 nt fragment (452-580) of the 5' leader, immediately upstream of
the gag AUG
codon (Lopez-Lastra et al. (1997) Hum Gene Ther 8:1855-I865).
In eukaryotic cells, translation is normally initiated by the ribosome
scanning from
the capped mRNA 5' end, under the control of initiation factors. However,
several cellular
mRNAs have been found to be with IRES structure to mediate the cap-independent
translation (van der Velde, et al. (I999) IntJBiochem Cell Biol. 31:87-106).
Examples are
immunoglobulin heavy-chain binding protein (BiP) (Macejak et al. (1991) Nature
353:90-94), antennapedia mRNA of Drosophilan (Oh et al. (1992) Gene and Dev
6:1643-1653), fibroblast growth factor-2 (FGF-2) (Vagner et al. (1995) Mol
Cell Biol
15:35-44), platelet-derived growth factor B (PDGF-B) (Bernstein et al. (1997)
JBiol Chem
272:9356-9362), insulin-like growth factor II (Teerink et al. (1995) Biochim
Biophys Acta
1264:403-408), and the translation initiation factor eIF4G (Gan et al. (1996)
JBiol Chem
271:623-626). Table 1 depicts the 5'-noncoding region for BiP and PDGF.
Recently,
'' vascular endothelial growth factor (VEGF) was also found to have IRES
element (Stein et
al. (1998) Mol Cell Biol 18:3112-3119; Huez et al. (1998) Mol Cell Biol
18:6178-6190).
Apart from the oligopyrimidine tract, nucleotide sequence per se does not
appear to
be important for IRES function. Without wishing to be bound by theory, a
possible
explanation for the function of an IRES is that it forms secondary and/or
tertiary structures
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which orient particular single-stranded regions of its sequence in a three-
dimensional
configuration that is conducive to interaction with a mammalian ribosome
(either ribosomal
protein and/or ribosomal RNA components) and/or initiation factors) and/or RNA
binding
proteins which interact with ribosomes and/or initiation factors. It is also
possible that the
three-dimensional structure of the IRES is determined or stabilized by one or
more RNA-
binding proteins. Thus it is possible to devise synthetic IRES sequences
having similar
single-stranded regions in a similar three-dimensional co~guration.
In certain cases, one or more t~ahs-acting cellular proteins may be required
for
IRES function. For example, the HAV and entero/rhinovirus IRESes function
inefficiently
in vitro in reticulocyte lysates. Supplementation of a reticulocyte Iysate
with a cytoplasmic
extract from HeLa, I~rebs II ascites, or L-cells restores activity of
entero/rhinovirus
IRESes. See, for example, Brown et al. (1979) Virology 97:396-405; and Dorner
et al.
(1984) J. Virol. 50:507-514. Activity of the HAV IRES in vitro is stimulated
by liver
cytoplasmic extracts. Glass et al. (1993) Virology 193:1047-1050. These
observations
indicate that cell-specific translational regulation can be achieved through
the use of a cell-
specific IRES. Furthermore, coordinated cell-specific transcriptional and
translational
regulatory elements can be included in a vector to further increase cell
specificity of viral
replication. For example, the combination of an AFP-THE and a HAV-IRES can be
used
to direct preferential replication of a vector in hepatic cells. Thus, in one
illustrative
embodiment, a vector comprises an AFP-THE regulating the transcription of a
bicistronic
E I A-E 1 B mRNA in which E I B translation is regulated by an ECMV IRES. In
another
illustrative embodiment, the vector comprises a probasin-THE regulating the
transcription
of a bicistronic ElA-ElB mRNA in which E1B translation is regulated by an ECMV
IRES.
In yet another illustrative embodiment, a vectox comprises a CMV-THE
regulating the
transcription of a bicistronic ElA-E1B mRNA in which E1B translation is
regulated by an
ECMV IRES. In examples disclosed herein, E1B has a deletion of the 19-kDa
region.
Examples of IRES which can be used in the present invention include those
provided in Table 12 and Table 13. In order to test for an IRES sequence which
may be
used in the present invention, a test vector is produced having a reporter
gene, such as
luciferase, for example, placed under translational control of an IRES to be
tested. A
desired cell type is transfected with the vector containing the desired IRES-
reporter gene
and an assay is performed to detect the presence of the reporter gene. In one
illustrative
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example, the test vector comprises a co-transcribed chloramphenicol
transferase (CAT) and
luciferase encoding gene transcriptionally driven by a CMV promoter wherein
the
luciferase encoding gene is translationally driven by an IRES to be tested.
Host cells are
transiently transfected with the test vector by means known to those of skill
in the art and
assayed for the presence of luciferase.
IRES may be prepared using standard recombinant and synthetic methods known in
the art, and as described in the Examples. For cloning convenience,
restriction sites may be
engineered into the ends of the IRES fragments to be used.
Adenovirus early genes
The adenovirus vectors of the invention comprise adenovirus genes under the
control of a target cell-specific TRE. Preferably an adenovirus gene essential
for
replication. Any gene that is essential for adenovirus replication, such as
ElA, E1B, E2,
E4 or any of the late genes, is useful. The adenovirus may also comprise E3.
In addition,
one or more of the genes can be a transgene or heterologous gene. Any of the
various
adenovirus serotypes can be used, such as, for example, Ad2, AdS, Adl2 and
Ad40. For
purposes of illustration, the Ad5 serotype is exemplified herein.
The ElA gene is expressed immediately (between 0 and 2 hours) after viral
infection, before any other viral genes. EIA protein is a traps-acting
positive
transcriptional regulatory factor, and is required for the expression of the
other early viral
genes E1B, E2, E3, E4, and the promoter-proximal major late genes. Despite the
nomenclature, the promoter proximal genes driven by the major late promoter
are also
expressed during early times after Ad5 infection. Flint (1982) Biochem.
Biophys. Acta
651:175-208; Flint (1986) Advances Virus Research 31:169-228; and Grand (1987)
Biochem. J. 241:25-38. In the absence of a functional ElA gene, viral
infection does not
proceed, because the gene products necessary for viral DNA replication are not
produced.
Nevins (1989) Adv. Virus Res. 31:35-81. The transcription start site of Ad5
ElA is at
coordinate 498 and the ATG start site of the ElA protein is at coordinate 560
in the virus
genome.
The E1B protein is necessary in traps for transport of late mRNA from the
nucleus
to the cytoplasm. Defects in ElB expression result in poor expression of Iate
viral proteins
and an inability to shut off host cell protein synthesis. The promoter of ElB
has been
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implicated as the defining element of difference in the host range of Ad40 and
AdS:
clinically Ad40 is an enterovirus, whereas Ad5 causes acute conjunctivitis.
Bailey et al.
(1993) Virology 193:631; Bailey et al. (1994) Virology 202:695-706. The ElB
promoter
of Ad5 consists of a single high-affinity recognition site for Spl and a TATA
box, and
extends from Ad5 nt 1636 to 1701.
Adenovirus E1B 19-kDa (19K) protein is a potent inhibitor of apoptosis and
cooperates with ElA to produce oncogenic transformation of primary cells (Rao,
et al.,
1992, Cell Biology, 89:7742-7746). During productive adenovirus infection, ElA
stimulates host cell DNA synthesis, thereby causing cells to aberrantly go
through the cell
cycle. In response to cell cycle deregulation, the host cell undergoes
apoptosis. As a
defense mechanism, the E1B 19-kDa protein inhibits this ElA-induced apoptosis
and
allows assembly of viral progeny to be completed before the cell commits
suicide. ElB 19-
kDa conducts anti-apoptotic function by multiple mechanisms. E1B 19-kDa
inhibits the
apoptosis of multiple stimuli, including Ela, p53 and TNF, for example.
According to
wild-type Ad5 , the E1B 19-kDa region is located between nucleotide 1714 and
nucleotide
2244. The ElB 19-kDa region has been described in, for example, Rao et al.,
P~oc. Natl.
Acad. Sci. USA, 89:7742-7746.
In a preferred embodiment, expression of the ElA and E1B regions of the Ad
genome is facilitated in a cell-specific fashion by placing a cell-specific
THE upstream of
ElA and a internal ribosome entry site between ElA and E1B.
The E2 region of adenovirus encodes proteins related to replication of the
adenoviral genome, including the 72 kD DNA-binding protein, the 80 kD
precursor
terminal protein and the viral DNA polymerase. The E2 region of Ad5 is
transcribed in a
rightward orientation from two. promoters, termed E2 early and E2 late,
mapping at 76.0
and 72.0 map units, respectively. While the E2 late promoter is transiently
active during
late stages of infection and is independent of the ElA transactivator protein,
the E2 early
promoter is crucial during the early phases of viral replication.
The E2 early promoter of Ad5 is located between nucleotides 27,050 and 27,150,
and consists of a major and a minor transcription initiation site (the latter
accounting for
about 5% of E2 transcripts), two non-canonical TATA boxes, two E2F
transcription factor
binding sites and an ATF transcription factor binding site. For a detailed
review of E2
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promoter architecture see Swaminathan et al. (1995) Curr. Topics in Micro. and
Imm. 199
part 3:177-194.
The E2 late promoter overlaps with the coding sequences of a gene encoded by
the
counterstrand and is therefore not amenable for genetic manipulation. However,
the E2
early promoter overlaps by only a few base pairs with sequences on the
counterstrand
which encode a 33 kD protein. Notably, an SpeI restriction site (Ad5 position
27,082) is
part of the stop codon for the above mentioned 33 kD protein and conveniently
separates
the major E2 early transcription initiation site and TATA box from the
upstream E2F and
ATF binding sites. Therefore, insertion of a heterologous THE having SpeI ends
into the
SpeI site disrupts the endogenous E2 early promoter of Ad5 and allows TRE-
regulated
expression of E2 transcripts.
An E3 region refers to the region of the adenoviral genome that encodes the E3
products. The E3 region has been described in various publications, including,
for
example, Wold et al. (1995) Curr. Topics Microbiol. Immunol. 199:237-274.
Generally,
the E3 region is located between about 28583 and about 30470 of the adenoviral
genome.
An E3 region for use in the present invention may be from any adenovirus
serotype. An E3
sequence is a polynucleotide sequence that contains a sequence from an E3
region. In
some embodiments, the sequence encodes ADP. In other embodiments, the sequence
encodes other than ADP and excludes a sequence encoding only ADP. As is well
known in
the art, the ADP coding region is located in the E3 region within the
adenoviral genome
from about 29468 by to about 29773 bp; including the Y leader, the location of
ADP is
from about 28375 by to about 29773 by for AdS. Other ADP regions for other
serotypes
are known in the art. An E3 sequence includes, but is not limited to,
deletions; insertions;
fusions; and substitutions. An E3 sequence may also comprise an E3 region or a
portion of
the E3 region. It is understood that, as an "E3 sequence" is not limited to an
"E3 region",
alternative references herein to an "E3 region" or "E3 sequence" do not
indicate that these
terms are interchangeable. Assays for determining a functional E3 sequence for
purposes
of this invention are described herein.
The E4 gene has a number of transcription products and encodes two
polypeptides
(the products of open reading frames (ORFs) 3 and 6) which are responsible for
stimulating
the replication of viral genomic DNA and stimulating late gene expression,
through
interaction with heterodimers of cellular transcription factors E2F-l and DP-
1. The ORF 6
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protein requires interaction with the E1B 55 kD protein for activity while the
ORF 3 protein
does not. In the absence of functional ORF 3- and ORF 6-encoded proteins,
efficiency of
plaque formation is less than 10-6 that of wild type virus.
To further increase cell-specificity of replication, it is possible to take
advantage of
the interaction between the E4 ORF 6 gene product and the E1B 55 kD protein.
For
example, if E4 ORFs I-3 are deleted, viral DNA replication and late gene
synthesis
becomes dependent on E4 ORF6 protein. By generating such a deletion in a
vector in
which the E1B region is regulated by a cell-specific TRE, a virus is obtained
in which both
E1B and E4 functions are dependent on the cell-specific THE which regulates
E1B.
I O Late genes relevant to the disclosed vectors are L 1, L2 and L3, which
encode
proteins of the virion. All of these genes (typically coding for structural
proteins) are
probably required for adenoviral replication. All late genes are under the
control of the
major late promoter (MLP), which is located in Ad5 between nucleotides 5986
and 6048.
In one embodiment, an adenovirus early gene is under transcriptional control
of a
cell specific, heterologous TRE. In additional embodiments, the early gene is
selected from
the group including ElA, ElB, E2, E3, E4. In another embodiment, an adenovirus
late
gene is under transcriptional control of a cell specific, heterologous TRE. In
further
embodiments, two or more early genes are under the control of heterologous
TREs that
function in the same target cell. The heterologous TREs can be the same or
different, or
one can be a variant of the other. In additional embodiments, two or more late
genes are
under the control of heterologous TREs that function in the same target cell.
The
heterologous TREs can be the same or different, or one can be a variant of the
other. In yet
another embodiment, one or more early genes) and one or more late genes) are
under
transcriptional control of the same or different heterologous TREs~ wherein
the TREs
function in the same target cell.
In some embodiments of the present invention, the adenovirus vector comprises
the
essential gene E1A and the ElA promoter is deleted. In other embodiments, the
adenovirus
vector comprises the essential gene ElA and the ElA enhancer I is deleted. In
yet other
embodiments, the ElA promoter is deleted and ElA enhancer I is deleted. In
other
embodiments, an internal ribosome entry site (IRES) is inserted upstream of
E1B (so that
E1B is translationally linked), and a target cell-specific THE is operably
linked to ElA. In
still other embodiments, an (IRES) is inserted upstream of ElB (so that EIB is
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translationally linked), and target cell-specific THE is operably linked to
ElA, which may
or may not maintain the ElA promoter andlor enhancer I (i.e., the ElA promoter
and/or
enhancer I may be, but not necessarily be, deleted). In other embodiments, the
19-kDa
region of E1B is deleted. For adenovirus vectors comprising a second gene
under control
of an IRES, it is preferred that the endogenous promoter of a gene under
translational
control of an IRES be deleted so that the endogenous promoter does not
interfere with
transcription of the second gene. It is preferred that the second gene be in
frame with the
TRES if the IRES contains an initiation codon. If an initiation codon, such as
ATG, is
present in the IRES, it is preferred that the initiation codon of the second
gene is removed
and that the IRES and second gene are in frame. Alternatively, if the IRES
does not
contain an initiation codon or if the initiation codon is removed from the
IRES, the
initiation codon of the second gene is used.
Adenovirus death protein (ADP) gene and gene product
In the construction of adenovirus vectors, the E3 region is often deleted to
facilitate
insertion of one or more TREs and/or transgenes. In some embodiments, however,
the
adenovirus death protein (ADP), encoded within the E3 region, is retained in
an adenovirus
vector. The ADP gene, under control of the major late promoter (MLP), appears
to code
for a protein (ADP) that is important in expediting host cell lysis. Tollefson
et al. (1992) J.
Virol. 66:3633; and Tollefson et al. (1996) J. hirol. 70:2296. Thus, inclusion
of an ADP
gene in a viral vector can render the vector more potent, making possible more
effective
treatment andlor a lower dosage requirement.
An ADP coding sequence is obtained preferably from Ad2 (since this is the
strain in
which the ADP has been most fully characterized) using techniques known in the
art, such
as PCR. Preferably, the Y leader (which is an important sequence for
correct.expression of
late genes) is also obtained and placed in operative linkage to the ADP coding
sequence.
The ADP coding sequence (with or without the Y leader) is then introduced into
an
adenoviral genome, for example, in the E3 region, where expression of the ADP
coding
sequence will be driven by the MLP. The ADP coding sequence can, of course,
also be
inserted in other locations of the adenovirus genome, such as the E4 region.
Alternatively,
the ADP coding sequence can be operably linked to a heterologous TRE,
including, but not
limited to, another viral THE or a target cell-specific THE (see infra). In
another
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embodiment, the ADP gene is present in a viral genome such that it is
transcribed as part of
a mufti-cistronic mRNA in which its translation is associated with an IRES.
E3-containing target cell-specific adenoviral vectors
In some embodiments, the adenovirus vectors contain an E3 region, or a portion
of
S an E3 region. Inclusion of the E3 region of adenovirus can enhance
cytotoxicity of the
target cell-specific adenoviral vectors of the present invention. Adenoviral
vectors
containing an E3 region may maintain their high level of specificity and can
be (a)
significantly more cytotoxic; (b) produce higher virus yield including
extracellular virus
yield; (c) form larger plaques; (d) produce rapid cell death; and (e) kill
tumors more
efficiently in vivo than vectors lacking the E3 region. The adenoviral vectors
of this
invention may contain the E3 region or a portion of the E3 region. It is
understood that, as
inclusion of E3 confers observable and measurable functionality on the
adenoviral vectors,
for example, increased replication and production, functionally equivalent (in
which
functionality is essentially maintained, preserved, or even enhanced or
diminished) variants
1 S of E3 may be constructed. Fox example, portions of E3 may be used. A
portion may be,
non-inclusively, either of the following: (a) deletion, preferably at the 3'
end; (b) inclusion
of one or more various open reading frames of E3. Five proteins which are
encoded by the
Ad-E3 region have been identified and characterized: (1) a 19-kDa glycoprotein
(gpl9k) is
one of the most abundant adenovirus early proteins, and is known to inhibit
transport of the
major histocompatibility complex class I molecules to the cell surface, thus
impairing both
peptide recognition and clearance of Ad-infected cells by cytotoxic T
lymphocytes (CTLs);
(2) E3 14.7k protein and the E3 10.4k/l4.Sk complex of proteins inhibit the
cytotoxic and
inflammatory responses mediated by tumor necrosis factor (TNF); (3) E3
10.4k/l4.Sk
protein complex down regulates the epidermal growth factor receptor, which may
inhibit
2S inflammation and activate quiescent infected cells for efficient virus
replication; (4) E3
11.6k protein (adenoviral death protein, ADP) from adenovirus 2 and 5 appears
to promote
cell death and release of virus from infected cells. The functions of three E3-
encoded
proteins -- 3.6k, 6.7k and l2.Sk -- axe unknown. A ninth protein having a
molecular weight
of 7.S kDa has been postulated to exist, but has not been detected in cells
infected with
wild-type adenovirus. Wold et al. (1995) Curr. Topics Microbiol. Immunol.
199:237-274.
The E3 region is schematically depicted in FIG. 6. These intact, portions, or
variants of E3
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may be readily constructed using standard knowledge and techniques in the art.
Preferably,
an intact E3 region is used.
In the adenovirus vectors of the present invention, E3 may or may not be under
transcriptional control of native adenoviral transcriptional control
element(s). The E3
promoter is located within the coding sequence for virion protein VIII, an
essential protein
which is highly conserved among adenovirus serotypes. In some embodiments, E3
is under
transcriptional control of a heterologous TRE, including, but not limited to,
a target cell-
specific TRE. Accordingly, in one embodiment, the invention provides an
adenoviral
vector, preferably replication competent, that comprises E3 region (or a
portion of E3)
under transcriptional control of a target cell-specific TRE. In other
embodiments, the E3
region is under transcriptional control of a native adenoviral TRE, and the
vector further
comprises an adenoviral gene essential for replication under transcriptional
control of a
target cell-specific TRE. In other embodiments, the E3 region is under
transcriptional
control of a target cell-specific TRE, and the vector further comprises an
adenoviral gene
essential for replication under transcriptional control of a target cell-
specific TRE.
Transgenes under transcriptional control of a target cell specific THE
Various other replication-competent adenovirus vectors can be made according
to
the present invention in which, in addition to having a single or multiple
adenovirus genes)
under control of a target cell-specific TRE, a transgene(s) is/are also under
control of a
target cell-specific THE and optionally under translational control of an
IRES. Transgenes
include, but are not limited to, therapeutic transgenes and reporter genes.
Transgenes can
be inserted into the adenoviral vector to produce, for example, certain
chemotherapeutic
agents, cheinoprotectants, chemosensitizers, radioprotectants and
radiosensitizers.
Examples of such genes include, for example, genes encoding, p53, Adenovirus
ElA,
HSV-TK, Cytosine dearninase (CDA), Cytochrome p450, TAXOLTM or others.
Reporter genes
For example, a target cell-specific THE can be introduced into an adenovirus
vector
immediately upstream of and operably linked to an early gene such as ElA or
E1B, and
this construct may further comprise a second co-transcribed gene under
translational
control of an IRES. The second gene may be a reporter gene. The reporter gene
can
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encode a reporter protein, including, but not limited to, chloramphenicol
acetyl transferase
(CAT), (3-galactosidase (encoded by the lacZ gene), luciferase, alkaline
phosphatase, a
green fluorescent protein, and horse radish peroxidase. For detection of a
putative cancer
cells) in a biological sample, the biological sample may be treated with
modified
adenoviruses in which a reporter gene (e.g., luciferase) is under control of a
target cell-
specific TRE. The target cell-specific THE will be transcriptionally active in
cells that
allow the target cell-specific THE to function, and luciferase will be
produced. This
production will allow detection of target cells, including cancer cells in,
for example, a
human host or a biological sample. Alternatively, an adenovirus can be
constructed in
which a gene encoding a product conditionally required for survival (e.g., an
antibiotic
resistance marker) is under transcriptional control of a target cell-specific
TRE. When this
adenovirus is introduced into a biological sample, the target cells will
become antibiotic
resistant. An antibiotic can then be introduced into the medium to kill the
non-cancerous
cells.
Therapeutic trahsgehes
Transgenes also include genes which may confer a therapeutic effect, such as
enhancing cytotoxicity so as to eliminate unwanted target cells. In this way,
various
genetic capabilities may be intxod.uced into target cells, particularly cancer
cells. For
example, in certain instances, it may be desirable to enhance the degree
and/or rate of
cytotoxic activity, due to, for example, the relatively refractory nature or
particular
aggressiveness of the cancerous target cell. This could be accomplished by
coupling the
target cell-specific cytotoxic activity with cell-specific expression of, for
example, HSV-tk
and/or cytosine deaminase (cd), which renders cells capable of metabolizing 5-
fluorocytosine (5-FC) to the chemotherapeutic agent 5-fluorouracil (5-FLT).
Using these
types of transgenes may also confer a bystander effect.
Other desirable transgenes that may be introduced via an adenovirus vectors)
include genes encoding cytotoxic proteins, such as the A chains of diphtheria
toxin, ricin or
abrin (Palmiter et al. (1987) Cell 50: 435; Maxwell et al. (1987) Mol. Cell.
Biol. 7: 1576;
Behringer et al. (1988) Ge~zes Dev. 2: 453; Messing et al. (1992) Neuron 8:
507; Piatak et
al. (1988) J. Biol. Chem. 263: 4937; Lamb et al. (1985) Eu~. J. Biochem. 148:
265; Frankel
et al. (1989) Mol. Cell. Biol. 9: 415), genes encoding a factor capable of
initiating
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apoptosis, sequences encoding antisense transcripts or ribozymes, which among
other
capabilities may be directed to mRNAs encoding proteins essential for
proliferation, such
as structural proteins, or transcription factors; viral or other pathogenic
proteins, where the
pathogen proliferates intracellularly; genes that encode an engineered
cytoplasmic variant
of a nuclease (e.g. RNase A) or protease (e.g. awsin, papain, proteinase I~,
carboxypeptidase, etc.), or encode the Fas gene, and the like. Other genes of
interest
include cytokines, antigens, transmembrane proteins, and the like, such as IL-
l, -2, -6, -12,
GM-CSF, G-CSF, M-CSF, IFN-a, -(3, -x, TNF-a, -~3, TGF-a, -(3, NGF, and the
like. The
positive effector genes could be used in an earlier phase, followed by
cytotoxic activity due
to replication.
Preparation of tlae adehovirus vectors
The adenovirus vectors of this invention can be prepared using recombinant
techniques that are standard in the art. Generally, a target cell-specific THE
is inserted 5'
to the adenoviral gene of interest, preferably an adenoviral replication gene,
more
preferably one or more early replication genes (although late genes) can be
used). A target
cell-specific THE can be prepared using oligonucleotide synthesis (if the
sequence is
known) or recombinant methods (such as PCR and/or restriction enzymes).
Convenient
restriction sites, either in the natural adeno-DNA sequence or introduced by
methods such
as PCR or site-directed mutagenesis, provide an insertion site for a target
cell-specific TRE.
Accordingly, convenient restriction sites for annealing (i.e., inserting) a
target cell-specific
THE can be engineered onto the 5' and 3' ends of a UP-THE using standard
recombinant
methods, such as PCR.
Polynucleotides used for making adenoviral vectors of this invention may be
obtained using standard methods in the art, such as chemical synthesis,
recombinant
methods and/or obtained from biological sources.
Adenoviral vectors containing all replication-essential elements, with the
desired
elements (e.g., ElA) under control of a target cell-specific TRE, are
conveniently prepared
by homologous recombination or in vitro ligation of two plasmids, one
providing the left-
hand portion of adenovirus and the other plasmid providing the right-hand
region, one or
more of which contains at least one adenovirus gene under control of a target
cell-specific
TRE. If homologous recombination is used, the two plasmids should share at
least about
CA 02404060 2002-09-23
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S00 by of sequence overlap. Each plasmid, as desired, may be independently
manipulated,
followed by cotransfection in a competent host, providing complementing genes
as
appropriate, or the appropriate transcription factors for initiation of
transcription from a
target cell-specific THE for propagation of the adenovirus. Plasmids are
generally
introduced into a suitable host cell such as 293 cells using appropriate means
of
transduction, such as cationic liposomes. Alternatively, in vitro ligation of
the right and
left-hand portions of the adenovirus genome can also be used to construct
recombinant
adenovirus derivative containing all the replication-essential portions of
adenovirus
genome. Berkner et al. (1983) Nucleic Acid Research 11: 6003-6020; Bridge et
al. (1989)
J. Virol. 63 : 631-63 8.
Fox convenience, plasmids are available that provide the necessary portions of
adenovirus. Plasmid pXC.l (McKinnon (1982) Gene 19:33-42) contains the wild-
type left-
hand end of AdS. pBHGlO (Belt et al. (1994); Microbix Biosystems Inc.,
Toronto)
provides the right-hand end of AdS, with a deletion in E3. The deletion in E3
provides
room in the virus to insert a 3 kb target cell-specific THE without deleting
the endogenous
enhancer/promoter. The gene for E3 is located on the opposite strand from E4
(r-strand).
pBHGl 1 provides an even laxger E3 deletion (an additional 0.3 kb is deleted).
Bett et al.
(1994). Alternatively, the use of pBHGE3 (Microbix Biosystems, Inc.) provides
the right
hand end of AdS, with a full-length of E3.
For manipulation of the early genes, the transcription start site of Ad5 E 1 A
is at 498
and the ATG start site of the ElA coding segment is at 560 in the virus
genome. This
region can be used for insertion of a taxget cell-specific TRE. A restriction
site may be
introduced by employing polymerase chain reaction (PCR), where the primer that
is
employed may be limited to the Ad5 genome, or may involve a portion of the
plasmid
carrying the Ad5 genomic DNA. For example, where pBR322 is used, the primers
may use
the EcoRI site in the pBR322 backbone and the XbaI site at nt 1339 of AdS. By
carrying
out the PCR in two steps, where overlapping primers at the center of the
region introduce a
nucleotide sequence change resulting in a unique restriction site, one can
provide fox
insertion of target cell-specific THE at that site.
A similar strategy may also be used for insertion of a target cell-specific
THE
element to regulate E1B. The E1B promoter of Ad5 consists of a single high-
affinity
recognition site for SpI and a TATA box. This region extends from Ad5 nt 1636
to 1701.
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By insertion of a target cell-specific THE in this region, one can provide for
cell-specific
transcription of the EIB gene. By employing the left-hand region modified with
the cell-
specific response element regulating EIA, as the template for introducing a
target cell-
specific THE to regulate E1B, the resulting adenovirus vector will be
dependent upon the
cell-specific transcription factors for expression of both ElA and ElB. In
some
embodiments, part or all of the 19-kDa region of E1B is deleted.
Similarly, a target cell-specific THE can be inserted upstream of the E2 gene
to
make its expression cell-specific. The E2 early promoter, mapping in Ad5 from
27050-
27150, consists of a major and a minor transcription initiation site, the
latter accounting for
about 5% of the E2 transcripts, two non-canonical TATA boxes, two E2F
transcription
factor binding sites and an ATF transcription factor binding site (for a
detailed review of
the E2 promoter architecture see Swaminathan .et al., Cur. Topics ih Micro.
and Immunol.
(I995) 199(part 3):177-194.
The E2 late promoter overlaps with the coding sequences of a gene encoded by
the
counterstrand and is therefore not amenable for genetic manipulation. However,
the E2
early promoter overlaps only for a few base pairs with sequences coding for a
33 kD
protein on the counterstrand. Notably, the SpeI restriction site (Ad5 position
27082) is part
of the stop codon for the above mentioned 33 kD protein and conveniently
separates the
major EZ early transcription initiation site and TATA-binding protein site
from the
upstream transcription factor binding sites E2F and ATF. Therefore, insertion
of a target
cell-specific THE having SpeI ends into the SpeI site in the 1-strand would
disrupt the
endogenous E2 early promoter of Ad5 and should allow target cell-restricted
expression of
E2 transcripts.
For E4, one must use the right hand portion of the adenovirus genome. The E4
transcription start site is predominantly at about nt 35605, the TATA box at
about nt 35631
and the first AUG/CUG of ORF I is at about nt 35532. Virtanen et al. (1984) J.
Virol. 51:
822-831. Using any of the above strategies for the other genes, a UP-THE may
be
introduced upstream from the transcription start site. For the construction of
full-length
adenovirus with a target cell-specific THE inserted in the E4 region, the co-
transfection and
homologous recombination are performed in W162 cells (Weinberg et al. (1983)
Proc.
Natl. Acad. Sci. 80:5383-5386) which provide E4 proteins in traps to
complement defects
in synthesis of these proteins.
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Adenoviral constructs containing an E3 region can be generated wherein
homologous recombination between an E3-containing adenoviral plasmid, for
example,
BHGE3 (Microbix Biosystems Inc., Toronto) and a non-E3-containing adenoviral
plasmid,
is carried out.
Alternatively, an adenoviral vector comprising an E3 region can be introduced
into
cells, for example 293 cells, along with an adenoviral construct or an
adenoviral plasmid
construct, where they can undergo homologous recombination to yield adenovirus
containing an E3 region. In this case, the E3-containing adenoviral vector and
the
adenoviral construct or plasmid construct contain complementary regions of
adenovirus,
for example, one contains the left-hand and the other contains the right-hand
region, with
sufficient sequence overlap as to allow homologous recombination.
Alternatively, an E3-containing adenoviral vector of the invention can be
constructed using other conventional methods including standard recombinant
methods .
(e.g., using restriction nucleases and/or PCR), chemical synthesis, or a
combination of any
of these. Further, deletions of portions of the E3 region can be created using
standard
techniques of molecular biology.
Insertion of an IRES into a vector is accomplished by methods and techniques
that
are known in the art and described herein supra, including but not limited to,
restriction
enzyme digestion, ligation, and PCR. A DNA copy of an IRES can be obtained by
chemical synthesis, or by making a eDNA copy of, for example, a picornavirus
IRES. See,
for example, Duke et al. (1995) J. Vvirol. 66(3):1602-9) for a description of
the EMCV
IRES and Huez et a1. (1998), Mol. Cell. Biol. 18(11):6178-90) for a
description of the
VEGF IRES. The internal translation initiation sequence is inserted into a
vector genome
at a site such that it lies upstream of a S'-distal coding region in a
multicistronic mRNA.
For example, in a preferred embodiment of an adenovirus vector in which
production of a
bicistronic ElA-E1B mRNA is under the control of a target cell-specific TRE,
the E1B
promoter is deleted or inactivated, and an IRES sequence is placed between ElA
and E1B.
In other embodiments, part or all of the 19-kDa region of ElB is deleted. IRES
sequences
of cardioviruses and certain aphthoviruses contain an AUG codon at the 3' end
of the IRES
that serves as both a ribosome entry site and as a translation initiation
site. Accordingly,
this type of IRES is introduced into a vector so as to replace the translation
initiation codon
of the protein whose translation it regulates. However, in an IRES of the
entero/rhinovirus
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class, the AUG at the 3' end of the IRES is used for ribosome entry only, and
translation is
initiated at the next downstream AUG codon. Accordingly, if an
entero/rhinovirus IRES is
used in a vector for translational regulation of a downstream coding region,
the AUG (or
other translation initiation codon) of the downstream gene is retained in the
vector
construct.
Methods of packaging polynucleotides into adenovirus particles are known in
the
art and are also described in co-owned PCT PCT/US98/04080.
The following examples are offered by way of illustration and should not be
considered as limiting the scope of the invention. The specific examples
exemplify the
adenovirus 5 serotype, however, persons skilled in the art will realize these
techniques may
be applied to other adenoviral serotypes.
EXAMPLES
Table 4 summarizes descriptions of the various .replication-competent target-
cell
specific adenoviral constructs used in these studies, and described previously
herein.
Preparation of these aderioviral vectors (including their components) employ
standard
techniques in the art. See also PCT/LTS99/03117, PCT/LTS98/16312,
PCT/US98/04133,
PCT/US98/04132, PCT/LJS98/04084, PCT/US98/04080, PCT/US97/13888,
PCT/LJS96/10838, PCT/LJS95/00845. In these publications, a CV designation is
also
denoted as CN. For example, CV706 is also denoted as CN706.
Table 4: Summary Description of Adenoviral Constructs
AbEIVOVIRA~. 'TARGETE1A TltE E1B THE E3 EIA 'E1B
L YEGTOR . ' : PROMOTER '
: CELL ~-/- j PROMOTER
TYPE
CV706 Prostate PSE N/A - + +
CV787 Prostate PB PSE + + +
CV790 Liver AFP AFP + + +
(0.827kb) (0.827kb)
CV829 Bladder hUPII mUPII + - +
CV859 Melanoma tyrosinaseIRES + -
CV873 ColorectalCEA IRES + -
Breast
CV874 Bladder mUPII(2kb)IRES + - -
CV875 Bladder hUPII(lkb)IRES + - -
CV876 Bladder hUPII(2kb)LRES + -
CV877 Bladder mUPII(lkb)hUPII(lkb)+ - -
CV890 Liver AFP IRES + - -
CV884 Bladder hUPII TRES + - -
(l.8kb)
For all constructs the ElA enhancer is present.
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PSA, prostate specific enhancer/promoter; PB, rat probasin promoter; AFP, a-
fetoprotein promoter; mUPII, mouse uroplakin II promoter; hUPII, human
uroplakin II
promoter; tyrosinase, melanocyte specific TRE; IRES, internal ribosome entry
site.
Example 1: Treatment of in vitro Tumor Cells with Combined Prostate Cell
Specific
Adenoviral Vector CV787 and Chemotherapy and In vivo assessment.
In vitro assessment.
CV787 is a prostate-specific, replication competent adenovirus vector that
preferentially replicates in prostate cancer cells. In this vector, ElA is
under transcriptional
control of a 452bp PB TRE, and E1B is under transcriptional control of l.6kb
PSA-TRE.
CV787 alone can, in a single intratumoral dose (1x108 particles per mm3 of
tumor) or a
single intravenous dose (1x1011 particles per animal) eliminate established
tumors within 6
weeks in nude mouse xenografts. The data below demonstrate that CV787-
mediated,
replication-dependent oncolytic cytotoxicity can be enhanced in conjunction
with standard
chemotherapeutic agents including paclitaxel (TAXOLTM), doxorubicin,
mitoxantrone and
docetaxel (TAXOTERETM), while the specificity of CV787-based cytopathogenicity
remains specific to prostate cancer cells. These data suggest that the
combination of
CV787 with chemotherapy is more effective than chemotherapy treatment alone or
virus
treatment alone.
Cell lines and culture
The human LNCaP (prostate carcinoma), HBL-100 (breast epithelia), and OVCAR-
3 (ovarian carcinoma) were obtained from the American Type Culture Collection
(Rockville, MD). The human embryonic kidney cell line, 293, which expresses
the
adenoviral EIA and EIB gene products serves as a production cell line, and was
purchased
from Microbix: Biosystem, Inc. (Toronto, Canada). Cells were maintained at 37
C with 5%
C02 in RPMI 1640 (Life Technologies, Gaithersburg, MD) supplemented with 100
units/ml
penicillin and 100 ~,glml of streptomycin (Life Technologies, Gaithersburg,
MD).
Chemotherapeutic agents and virus
Paclitaxel (TAXOLTM, Bristol-Myers Squibb, Princeton, N~, docetaxel
(TAXOTERETM, Rhone-Poulenc Rorer Pharmaceuticals, Inc., Collegeville, PA), and
the
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chemotherapeutic agents listed in Table 5, were purchased from the Stanford
University
Hospital pharmacy (Palo Alto, CA). These agents were diluted with medium
without fetal
bovine serum (FBS) just before use for in vitro studies and with 0.9% NaCI for
iva vivo
studies.
CV787 is a prostate-specific replication-competent adenovirus. Yu et al.
(1999)
Cancer Res. 59:4200. Two prostate-specific transcription response elements
(TRE), the rat
probasin promoter and the human prostate-specific antigen (PSE)
promoter/enhancer, were
inserted upstream of the ElA and E1B encoding regions in the viral genome,
respectively,
using methods known in the art. The expression of the E 1 A gene and the E 1 B
gene are
then controlled by these TREs.
Combination study of CV787 with paclitaxel (TAXOLTM), docetaxel (TAXOTERE~) or
other chemotherapeutic agents in vitro
In preliminary experiments, we examined the chemosensitivity to different
agents,
as well as the oncolytic effect of CV787 in the prostate carcinoma LNCaP
cells. Cells were
plated in 96-well plates at a density of 20,000 cells per well. Twenty-four
hours later, the
cells were infected with CV787 at various multiplicities of infection (MOI).
Subsequently,
medium (50 ~,1) containing 10% heat-inactivated serum and various
concentrations of
chemotherapeutic agents were added to the appropriate wells. Cells were
incubated at 37°C
in 5% C02 for an additional two days. Cell viability was measured using the
MTT assay.
Mosmann et al., (1983). Briefly, 50 ~,1 of 1 mg/ml MTT vital dye (Sigma, St.
Louis, MO)
was added to each well and allowed to incubate for 3 h at 37°C and 5%
CO~. Then, plates
were drained to remove untransformed MTT and blot. 100 ~,l of isopropanol was
added to
each well, the plate was incubated for I S minutes and vigorously shaken
(Microshaker II,
Dynatech) in order to ensure solubilization of the blue formazan. The optical
density of
each well was quantitated using an automatic plate reader (Molecular Devices,
Sunnyvale,
CA) with a 560 nm test wavelength and 690 nm reference wavelength. Cell
viability was
defined as the ratio of the mean absorbance of 9 treatment wells minus the
blank to the
mean absorbance of 6 untreated matched controls minus the blank. Blank is
defined as the
mean absorbance of six wells containing medium alone. Each experiment was
performed
at least twice.
Other chemotherapeutics were tested, using the protocols described above.
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Results of iu vitro experiments
To study potential synergy or enhancement in treatment when administering
CV787
and chemotherapy i~ vitro, the effectiveness of the combined treatment at
several
concentrations of paclitaxel, ranging from 0-62.5 nM, or docetaxel, ranging
from 125-250
nM, with CV787 at various MOIs, ranging from 0-10 MOI, was evaluated in the
prostate
carcinoma LNCaP cells. Cells were treated with CV787 and paclitaxel or
docetaxel and
the cell viability was determined at various time points after treatment by
an.MTT assay, as
shown in Figures 2-4. Figure 2 presents data for treatment with a combination
of CV787
(MOI 0.01) and paclitaxel (6.25 nM), showing the synergistic cytotoxicity of
the
combination treatment compared to virus alone or chemotherapy alone. An
enhanced
cytotoxicity was observed in the combination treatment between CV787 and
paclitaxel.
For example, CV787 at an MOI of 0.01 produced 85% cell survival 6 days after
virus
infection and paclitaxel at 6.25 nM showed 100% survival in LNCaP. When CV787
and
paclitaxel were combined at these concentrations, cell survival dropped to
18%,
demonstrating a greater effect than just an additive effect. To determine
whether the timing
of administration for the testing articles affected the combined oncolytic
effect, LNCaP
cells were treated with paclitaxel for 24 hours before or after infection with
CV787.
Results showed that there were no significant differences in oncolytic
activity between cells
treated with paclitaxel before or after infection with CV787.
Cytotoxicity was also measured for the combination treatment of CV787 and
docetaxel, Figs. 3 and 4, and synergistic effects were observed. LNCaP cells
were infected
with CV787 at an MOI of 0.01 after a 24 hour incubation with docetaxel at 3.12
nM and
cell viability was determined by MTT, as shown in Figure 3. The cell survival
was 25% of
the control at day 7 post treatment, whereas CV787 alone produced 95% cell
survival and
docetaxel alone showed 95% cell survival in prostate carcinoma LNCaP cells. No
significant difference in the effectiveness of the combined therapy of
docetaxel and CV787
infection was observed by varying the time of virus administration. As
presented in Figure
3, LNCaP cells treated with docetaxel for 24 hours, then infected with CV787
produced
similar cell viability to the treatment of which the LNCaP cells were infected
with CV787
24 hours prior to docetaxel Figure 4.
The protocols described above were used to screen a number of different
chemotherapeutic agents from various classes of chemotherapeutics. The results
are
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presented in Figures 5-9 and are summarized in Table 5, below. The summarized
results
are for experiments in which drug was added 24 hours before the introduction
of the virus,
except in the case of doxorubicin, in which the virus was added 24 hours prior
to the
administration of the drug. Figures 3-4 compare the order of administration
for a
combination of docetaxel and CV787. CV787 was administered at MOI of either
0.1 or
0.01 as indicated in Figures 5-9. Chemotherapeutics were administered in the
following
amounts: paclitaxel (6.25 nM); docetaxel (3.12 nM); mitoxantrone (100 nM);
etoposide
(500 ng/ml); doxorubicin (50 ng/ml); cisplatin (8.25 ~M); 5-fluorouracil (35
~,M);
estramustine (Smg/mI); gemcitabine (50 ng/ml); flutamide (l5ng/ml); goserelin
(SO~,g/~,g);
leuprolide (SnM); and vinblastine (80mg/ml).
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Table 5: Synergistic Effects of CV787/Chemotherapeutic Combinations
TARGETl CHEMOTHERAPEUTIZ'
VIRUS CELL LINE (GENT : CLASS OFAGENT '' - SYNERGY
~
CV787 Prostate 5-FluorouracilAntimetabolites Yes
cancer/ (acting as
LNCaP (5-FU) pseudosubstrate for
essential
enzymatic reactions)
CV787 Prostate Cisplatin Alkylating agent (Plantinum-Yes
cancer/
LNCaP containing agents -
Causing single-
and double-strand break
in DNA)
CV787 Prostate Doxorubicin Antibiotics (anticycline;Yes
cancer/
LNCaP interrupting DNA replication
and
transcription, causing
strand break)
CV787 Prostate Estramustine Alkylating agent Yes
cancer/
LNCaP
CV787 LNCaP Etoposide Plant alkaloid (inhibitingYes
the
assembly of microtubules
and
disrupting mitosis
CV787 Prostate Mitoxantrone Antibiotics (anticycline)Yes
cancer/
LNCap
CV787 Prostate TAXOTERE11"' plant alkaloids Yes
cancer/
LNCaP (docetaxel)
CV787 Prostate TAXOLI'"1 plant alkaloids Yes
cancer/
LNCaP (paclitaxel)
CV787 Prostate Gemcitabine Antimetabolite No
cancer/
LNCaP
CV787 Prostate Flutamide Anti-androgen No
cancer/
LNCaP
CV787 Prostate ZOLADEXI'"1 Hormonal analog No
cancer/
LNCaP (goserelin)
CV787 Prostate LUPRONI'"1 Testosterone analog No
cancer/
LNCaP (leuprolide)
CV787 prostate Vinblastine Plant alkaloids No
cancer/
LNCaP
The following experiments were designed to test the specificity and viability
of the
replication-competent target cell-specific adenoviral vectors described herein
in the
presence of antineoplastic (chemotherapeutic) agents.
Virus yield
Virus yield was determined to characterize the specificity of combination
treatment
of CV787 and paclitaxel or docetaxel. 5 x 105 293, LNCaP, HBL-100 and OVCAR-3
cells
were plated in duplicate into six-well .plates. Twenty-four hours later,
medium was
aspirated and replaced with 1.0 ml of serum-free RPMI 1640 containing CV787 at
a MOI
of 1 PFU (plaque-forming unit) per cell. After a 4 hour incubation at
37°C with 5% C02,
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cells were washed twice with pre-warmed phosphate buffered saline (PBS), and 2
ml of
complete RPMI 1640 containing the indicated chemotherapeutic agents were added
into
each well in concentrations and amounts as indicated below. After an
additional 72 hours,
the cells were scraped into the culture medium, and the cells were lysed by
three freeze-
s thaw cycles. The supernatant of each duplicate point was tested for virus
production by
triplicate plaque assay for 12 days under semisolid agarose on 293 cells. Yu
et al. Cancer
Research (1999) 59:1698.
Paclitaxel does hot ihlaibit CV787 replication
Paclitaxel (TAXOLTM) and docetaxel (TAXOTERE~) are antineoplastic agents
belonging to the taxoid family. They are novel antimicrotubule agents that
promote the
assembly of microtubules from tubulin dimers and stabilize microtubules by
preventing
depolymerization. This stability results in the inhibition of the normal
dynamic
reorganization of the micxotubule network that is essential for vital
interphase and mitotic
cellular functions. In addition, they induce abnormal arrays or "bundles" of
microtubules
throughout the cell cycle and multiple asters of microtubules during mitosis.
To examine the effect of paclitaxel on the virus replication, we ran a virus
yield
assay. LNCaP cells were infected with CV787 at a MOI of 0.1 for 4 hours,
followed by
incubation in RPMI 1640 containing paclitaxel at a final concentration of 6.25
nM. Cells
were harvested 6 days post-infection and the number of infectious virus
particles were
determined on 293 cells by a standard plaque assay. As shown in the Figures,
cells treated
with CV787 and paclitaxel produced 7,000 pfu per cell, while the cells
infected with
CV787 alone generated about 4,600 pfu per cell, suggesting that paclitaxel
does not inhibit
CV787 replication.
In addition, the chemotherapeutics mitoxantrone, doxorubicin and etoposide
were
also tested with CV787 according to the above protocol. None of these
chemotherapeutics,
from different classes of agents, showed a reduction in viral yield compared
to CV787
without chemotherapeutic agent.
Paclitaxel does hot alter CV78Ts specificity
In order to evaluate whether addition of paclitaxel could change the
specificity of
CV787's oncolytic activity, we tested viral replication efficiency in four
cell lines including
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a permissive cell line LNCaP, and two non-permissive cell lines, HBL-100
(breast
epithelia) and OVCAR-3 (ovarian carcinoma). These three cell lines were
infected with
either CV787 at an MOI of 0.1 or CV787 and paclitaxel, with a final paclitaxel
concentration of 6.25 nM in the medium. Progeny virus yield was determined 48
hours
after infection by plaque assay on 293 cells. Results presented in Figure 12
show that
prostate cancer (LNCaP) treated with CV787 and paclitaxel produced a similar
burst size to
the cells infected with CV787 alone, which produced about 800 pfu per cell.
CV787
replicated poorly in the non-prostate cancer cells tested (HBL-100 and OVCAR-
3),
producing 1000 to 10,000-fold lower virus yield compared to the burst size in
LNCaP cells.
Interestingly, the burst size in the LNCaP cells treated with CV787 and
paclitaxel is similar
to that in the cells infected by CV787 alone. These data indicate that CV787
in the presence
of paclitaxel replicates efficiently in prostate cancer cells, but is still
attenuated in non-
prostate cancer cells. Combination treatment does not change CV787 replication
efficiency
in the non-prostate cells and retains a high selectivity. Similar results were
obtained for
combinations of CV787 and mitoxantxone (MXT) and doxorubicin (DOXO).
To further assess the specificity of the combination treatment of CV787 and
paclitaxel, the viability of various infected cells was estimated using the
MTT assay to
measure mitochondria) activity. HEIR-293, LNCaP, HBL-100 and OVCAR-3 cells
were
infected with CV787 at an MOI of 0.1 in the presence or absence of paclitaxel.
The
percentage of cell viability in the combination treatment group versus
paclitaxel treatment
group was plotted in Figure 13. Combination of CV787 and paclitaxel was toxic
to 293, a
permissive production cell line, and LNCaP cells, prostate cancer cells, but
not to HBL-
100, normal breast epithelial cells, and OVCAR-3 cells, ovarian cancer cells.
There were
no surviving LNCaP cells 9 days after infection. In contrast, the viability of
HBL-100 and
OVCAR-3 cells treated with CV787 and paclitaxel was similar to that of cells
treated with
paclitaxel (ratio of cell survival between combination group and paclitaxel
group was
approximately 1 ). The results suggest that the presence of paclitaxel does
not alter the
cytotoxic effect of CV787.
Similar results were observed using the above protocols and a combination of
CV787 and mitoxantrone Figure 14.
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In vivo assesment
Using the PSA+ LNCaP xenograft model of prostate cancer, a single i.v. dose of
1x10 particles CV787 and docetaxel in combination eliminates large pre-
existent distant
tumors. Toxicity studies do not show a synergistic increase of toxicity of
CV787 and
taxane. These experiments demonstrate a synergistic antitumor efficacy for
CV787 when
combined with taxane, and demonstrate an in vivo single-does curative
therapeutic index
for CV787 of over 1000:1.
Cell viability
MTT assays were performed by seeding LNCaP, HBL-100, OVCAR-3, HepG2,
and 293 cells at 5000 cells per well in a 96 well plate (Falcon) 48 hr prior
to infection as
previously described (Denizot, 2000, Jlmmuhol. Methods 89:271-7.) with
modifications.
Cells were either infected with CV787 at.an MOI of 2 PFU/cell or treated with
the
indicated chemotherapeutic agents (Paclitaxel at 6.25 nM and Docetaxel at 3.12
nM). Cell
viability was measured at the times indicated by removing the media and
replacing it with
50 ~,1 of a 1 mg/ml solution of MTT (3-(4,5-Dimethylthiazol-2-yly 2,5-diphenyl-
2H
tetrazolium bromide) (Sigma, St. Louis, MO) and incubating for 3hrs at 3hrs
at. 37°C.
After removing the MTT solution, the crystals remaining in the wells were
solubilized by
the addition of 50 ~l of isopropanol followed by vigorous shaking. The
absorbency was
determined using a microplate reader (Molecular Dynamics) at 560 nm (test
wavelength)
and 690 nm (reference wavelength). The percentage of surviving cells was
estimated by
dividing the ODsso - OD6so of virus infected cells by the ODsso - OD6so of
mock infected
cells. 12 replica samples were taken for each time point and each experiment
was repeated
at least three times.
Statistical analysis
The dose-response interactions between taxane and CV787 at the point of ICso
were
evaluated by the isobologram method of Steel and Peckham (Steel, 1993, lit. J.
Rad. Ohc.
Biol. Phys. 5:85.) as modified by Aoe et al. (Aoe, K. et al. 1999, Ahtica~ce~
Res. 19:291-
299.) The ICso was defined as the concentration of drug that produced 50% cell
growth
inhibition, i.e. 50% reduction in absorbance. Cells were exposed to drugs
sequentially for
24 h and cell viability was determined by the MTT assay after 6 days. The dose-
response
curves were plotted with CurveExpert (Version 1.34) on a semilog scale as a
percentage of
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the control, the absorbance of which was obtained from the samples not exposed
to the
drugs. ICSO value of CV787 and taxane in LNCaP was then determined. Based upon
the
dose-response curves of CV787 alone and taxane alone, isobolograms (three
isoeffect
curves, model 1 and model 2 lines) were computed. The envelope of additivity,
surrounded
S by model 1 and model 2 isobologram lines, was constructed from the dose
response curves
of CV787 alone and taxane alone. The observed data were compared with the
predicted
maximum and minimum data for presence of synergism, additivity, or antagonism
by a
statistical analysis using the Stat View 4.01 software program (Abacus
Concepts, Berkeley,
California). When the data points of the drug combination fall within the area
surrounded
by model I and/or model 2 lines (i.e. within the envelope of additivity), the
combination is
described as additive. A combination that gives data points to the left of the
envelope of
additivity can be described as supraadditive (synergism) and a combination
that gives data
points to the right of the envelope of additivity, can be described as
subadditive
(antagonistic) (Keno, Y. et al. 1998, Cancer Chemo. Pharm. 42:91-98.)
Fractional tumor
volume (FTV) relative to untreated controls was determined as described
previously
(Yokoyama, Y. et al., 2000, Cancer Res. 60:2190-2196.).
One-step growth curve and Yirus yield
One-step growth curves of CV787 in the presence or absence of docetaxel were
performed in LNCaP cells to determine burst size. Monalayers of LNCaP cells
were
infected at a multiplicity of 2 PFU/cell with CV787. After a 4 hour incubation
at 37°C with
5% C02, cells were washed twice with pre-warmed PBS, and 2 ml of complete RPMI
1640
containing docetaxel at a concentration of either 0 nM or 3.12 nM was added
into each
well. At the indicated times thereafter, duplicate cell samples were harvested
and lysed by
three cycles of freeze-thawing. Virus was titered in triplicate (Yu, D.-C. et
al., 1999,
Cancer Res. 59:1498-1504.).
Virus yield was used to determine if CV787 retained specificity in the
combination
treatment of CV787 and taxane. 5 x 105 cells of 293, LNCaP, HBL-100, HepG2 and
OVCAR-3 were plated in duplicate into six-well plates. Twenty-four hours
later, medium
was aspirated and replaced with 1.0 mi of serum-free RPMI 1640 containing
CV787 at an
MOI of 1 PFU (plaque-forming unit) per cell. After a 4 hour incubation at
37°C with 5%
C02, cells were washed twice with pre-warmed PBS, and 2 ml of complete RPMI
1640
containing the indicated taxane was added to each well. After an additional 72
hours, cells
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were scraped into the culture medium, and lysed by three freeze-thaw cycles.
Virus
production was monitored by triplicate plaque assay (Yu, D:-C., et al., 1999,
Cancer Res.
59:1498-1504.).
Immunoblots
~ LNCaP cells treated with CV787, taxane, or both CV787 and taxane, were
incubated for the indicated times. Cells were washed with cold PBS, and lysed
for 30 min
on ice in SOmM Tris, pH8.0, 150mM NaCl, 1% IGEPAL CA360 (NP40 equivalent from
Sigma), 0.5% sodium deoxycholate, and protease inhibitor cocktail (Roche, Palo
Alto,
California). After 30 min centrifugation at 4°C, the supernatant was
removed and protein
concentration was determined by the ESL protein assay kit (Roche). Fifty
micrograms of
protein/lane were separated on 8-16% SDS-PAGE and electroblotted onto Hybond
ECL
membranes (Amersham Pharmacia, Buckinghamshire, England). The membrane were
blocked overnight in PBST (PBS with 0.1 % Tween-20) supplemented with 5%
nonfat dry
milk: Primary antibody incubation was done at room temperature for 2-3 hrs
with
PBST/1 % nonfat dry milk diluted antibody, followed by wash and lhr incubation
with
diluted horseradish peroxidase-conjugated secondary antibody. Enhanced
chemiluminescence (ECL; Amersham Pharmacia) was used for detection. Antibodies
for
p53 and poly-ADP-ribose-polymerase were from Roche. Antibodies against Fas/Fas-
L,
caspase 7, Bcl-2, Bcl-XL, Bax and secondary antibodies were purchased from
Santa Cruz
Biotechnology Inc. (Santa Cruz, California). All antibodies were used
according
manufacturer's instruction. For quantifying the bands, the gels were scanned
and bands
were analyzed by Multi-Analyst software (Blo-Rad).
In vivo antitumor efficacy
Six to eight week old athymic Balb/c nulnu mice were obtained from Simonson
Laboratories (Gilroy, CA) and acclimatized to laboratory conditions one week
prior to
tumor implantation. Xenografts were established by injecting 1 x 106 LNCaP
cells,
suspended in 100 ~,d of RPM1 1640 and 100 ~d of matrigel, subcutaneously near
the small
of the back. When tumors reached between 400 mm3 and 600 mm3, mice were
randomized
into groups of four. The first group received 1 x 101° particles of
CV787 at day 1 via the
tail vein intravenously (i.v.). CV787 was diluted in 0.1 ml lyophilized buffer
(5% sucrose,
1% glycine, 1 mM MgCl2, 0.05% Tween-80 in 10 mM Tris buffer) and injected into
the
tail vein using a 28-gauge needle. The second group was given taxane only.
Paclitaxel was
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intraperitoneally administered at a dose of 20 mg/kg, daily for 4 days
starting at day 2.
Docetaxel was intravenously administered at a dose of 5 or 12.5 mg/kg at day
2, 5 and 8.
The third group was given CV787 (i.v.) at day l and taxane at the same doses
and schedule
as the second group. As a control, a fourth group was treated with 0.1 ml of
normal saline
(i.e. control) i.v. at day 1 and then i.p. or i.v. for 4 days. The dose and
route of
administration of paclitaxel were selected according to studies in nude mice
(Riondel, J. et
al., 1986, Cancer Chemother Pharmacol. 17:137-42.) (Chahinian, A.P. et
aL,1998, J. Surg.
Onc. 67:104- 111.). For docetaxel, the dose was selected based on the human
clinical
dose.(RPR Pharm. Inc., Collegeville, PA) and determined by a dose-range
fording study in
nude mice. Tumors were measured weekly in two dimensions by external caliper
and
volume was estimated by the formula [length (mm) x width (mm)21/2 (7). Animals
were
humanely killed when their tumor burden became excessive. Serum was harvested
weekly
by retro-orbital bleed. The difference in mean tumor volume between treatment
groups
was compared for statistical significance using the unpaired, two-tailed, t-
test. Blood
samples were collected at various time points for determining prostate-
specific antigen.
Federal and institutional guidelines for animal care were followed.
Immuhohistochemistry
Four groups of mice (n=6) were treated with vehicle, CV787 (1 x101°
particles per
animal), paclitaxel (15 rng/kg) or a combination of CV787 and paclitaxel at
these identical
doses. Half the animals were sacrificed on day 9 and the other half on day 16.
Tumors
were fixed in 10% neutral buffered formalin~ embedded in paraffin and
sectioned using
standard procedures. For detecting adenovirus, tissue sections were blocked
with ready-to-
use normal rabbit serum (Biogenex, San Carlos, CA) for 20 min and incubated
with goat
anti-Ad antibody (Biodesign International, I~ennebunkport, ME) diluted 1:200
in PBS for
30 min. Alkaline phosphatase staining was performed using Super SensitiveTM
streptaviden-biotin alkaline phosphatase reagents and Fast RedTM chromogen
(Biogenex) as
suggested by the manufacturer. Sections were counterstained with Gill's
hematoxylin and
mounted with Gel MountTM (Biomedia, Foster City, CA).
Apoptotic cells were detected using M30 monoclonal antibody with reagents from
the M30 CytoDEATHTM kit (Roche Molecular Biochemicals, Indianapolis, III as
suggested by the manufacturer. Paraffin-embedded tumor sections were heated in
citric
acid buffer for 15 min to retrieve antigen, hybridized with M30 antibody, then
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counterstained with Harries hematoxilin (Roche Molecular Biochemicals). The
stained
sections were analyzed under a light microscope and pictures of representative
sections
taken.
Isobolograms were also generated to show the synergy between CV787 and
docetaxel. Dose-response curve analysis indicated that the ICSO at day 5 in
LNCaP cells for
CV787 and docetaxel was 0.368 MOI and 8.14 nM, respectively. The combined data
points fell to the left of the envelope of additivity, or restated the IC50 in
LNCaP cells of
CV787 in combination with docetaxel occurred at smaller doses than that
predicted from
the use of CV787 or docetaxel alone. Thus, sequential exposure to CV787
followed by
docetaxel produced synergistic effects.
To determine whether the timing of administration for the tested compounds
affected the combined cytotoxic effect, LNCaP cells were treated with
paclitaxel for 24
hours before or after infection with CV787. There were no significant
differences in
cytotoxic activity between cells treated with paclitaxel before infection,
after infection, or
I S simultaneously with CV787. Similar results were obtained for docetaxel.
Taxane Increases Ch787 burst size in LNCaP calls
Paclitaxel and docetaxel are antineoplastic agents belonging to the taxane
family.
They are novel antimicrotubule agents that promote the assembly of
microtubulas from
tubulin dimers and stabilize microtubules by preventing depolymerization. This
stability
results in the inhibition of the normal dynamic reorganization of the
microtubule network
that is essential for vital interphase and mitotic cellular functions
(Blagosklonny, M.V. et
al., 2000, J. U~ol. 163:1022-6.). In addition, the taxanes induce abnormal
arrays or
"bundles" of microtubules throughout the cell cycle and multiple asters of
rnicrotubules
during mitosis. One possible explanation for the synergy seen with taxane and
CV787 is
that taxane may augment the ability of CV787 to replicate in LNCaP cells.
To examine the effect of paclitaxel and docetaxel on virus replication, we
performed the one-step growth curve. LNCaP cells were infected with CV787 at
an MOI
of 1 for 4 hrs, followed by incubation in RPMI 1640 containing docetaxel at a
final
concentration of 3.12 nM. Cells were harvested at various times post-infection
and the
number of infectious virus particles was determined on 293 cells by standard
plaque assay
(Yu, D.-C. et al., 1999, Cancer Res. 59:4200-4203.). Although the initial rate
of increase
of CV7137 in cells treated with CV787 and docetaxel was similar to that of
cells treated
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with CV787 alone, a plateau was reached for CV787 at approximately 72 post-
infection
and at approximately 96 hours post-infection for CV787 and docetaxel. Cells
treated with
CV787 and docetaxel produced 30,000 PFU per cell, while the cells infected
with CV787
alone generated about 15,000 PFU per cell. Thus, docetaxel does not inhibit
CV787
replication, but actually increases virus replication efficiency. A similar
results was
obtained in a parallel study with paclitaxel.
Combination of taxane and CY787Increases the p53 expression
To address the synergistic mechanism behind combination treatment, LNCaP cells
were treated with various agents and the expression of apoptotic related
protein markers
were compared by Western blot. The treatments for LNCaP cells were grouped as
(1)
docetaxel alone at 6.0 nM, (2) CV787 alone at, MOI 0.5, and (3) CV787
(MOI=0.5) and
docetaxel (6.0 nM) together. For each treatment group, cells were collected at
different
. time points and subjected to various antibodies by Western blot. Under these
experimental
conditions, in the first 48 hours after treatment, the combination of CV787
and taxane
increased p53 expression up to 2 to 8-fold compared to virus alone or drug
alone at 24 or
48 hours.
In contrast, the apoptotic indicators caspase-7 and poly-ADP-ribose-polymerase
did
not show a significant change. In addition, the combination of CV787 and
taxane did not
change Fas/Fas-L or Bcl-2, Bcl-XL, and Bax expression compared to the single
agent
group. Previously, it was suggested that paclitaxel-induced apoptosis was not
mediated by
Bcl-2 family change. In the current study, we did not observe a significant
change of BcI-
2 expression in the cells treated with docetaxel alone, CV787 alone, or
docetaxel and
CV787. Liu and Stein has reported that paclitaxel treated LNCaP cells
experienced
alteration In bcl-XL and Bak expression. However, under our condition of low
concentration of docetaxel, there was no dramatic change detected. From the
increased p53
expression, p53-dependent apoptosis may play a major role in the synergy of
CV787
and taxane.
Synergistic efficacy of taxane with CY787 in vivo
The ih vivo antitumor efficacy of CV767 in combination with taxane was
assessed
in the LNCaP mouse xenograft model. We have shown previously that a single
intravenous administration of CV787 at 1x1011 particles per animal can
eliminate
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subcutaneous xenograft tumors in 6 weeks (Yu, D.-C. et al. (1999) supra. This
data was
extended using studies up through 10 weeks. Established human prostate tumors
(LNCaP
cells) were treated with either vehicle, CV787 (1x10i° particles per
animal), paclitaxel (20
mg/kg), or both CV787 and paclitaxel. For the combination treatment, animals
were
intravenously injected with either CV787 or vehicle, and twenty-four hours
later, paclitaxel
was administered intraperitoneally (i.p.) daily for four days. The tumor
volume data shows
that there was a significant decrease in tumor volume between control and all
treatment
groups. In this study, single doses of CV787 or 4 doses of paclitaxel over
four days were
effective in producing partial tumor regression 7 weeks or 2 weeks after
treatment, whereas
the combination produced a near complete regression within 2 weeks. Four weeks
after
treatment, relative tumor volume decreased to 3% of baseline (from 418 mm3 to
14 mm3)
for the combination treatment group and 31 % of baseline for the paclitaxel
group, but
increased to 216% of baseline for the vehicle-treated group and 162% of
baseline for the
CV787 group. These changes were statistically significant by Students t-test
(p<0.05) for
the comparison of the combination treatment of CV787 with paclitaxel to any of
the
vehicles, CV787 or paclitaxel, alone. Additionally, serum PSA levels in mice
injected with
vehicle increased, whereas the levels in mice injected with CV787 and
paclitaxel decreased
to ~2% of their staffing values within 4 weeks.
Combination therapy showed more than additive effect (e.g. synergy) on tumor
growth inhibition. On day 21, there was 4.4-fold improvement in anti-tumor
activity in the
combination group when compared with the expected additive effect. At this
time point,
CV787 alone or paclitaxel alone inhibited tumor growth by 20% or 70%,
respectively
(fractional tumor volume, 0.815 mm3 and 0.287 mm3 respectively) when compared
with
the control group. With time, there was a progressive improvement in anti-
tumor activity.
On day 42, CV787 and paclitaxel combination group showed a 9.2-fold higher
inhibition of
tumor growth over additive effect (expected fractional tumor volume). These
data
demonstrated a synergistic efficacy between CV787 and paclitaxel in LNCaP
xenografts.
A synergistic effect was also observed in the combination treatment of
xenograft
tumors with CV787 and docetaxel. Results from LNCaP prostate tumor xenografts
treated
with CV787 and docetaxel, both administered intravenously: in the combination
treatment
group, animals were intravenously injected with docetaxel (5.0 mg/kg) on day
2, day 5 and
day 8, following a single intravenous injection of CV787 (1x101°
particles per animal) on
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day 1. Both CV787 and docetaxel appear to be effective in producing
stabilization of
tumor'growth in the LNCaP mouse model, whereas a combination of the two
produce a
complete regression within 5 weeks (Figure 613). Analysis on fractional tumor
volume,
indicated a synergistic effect between CV787 and docetaxel in LNCaP
xenografts. For
example, on day 42, CV787 and docetaxel combination group showed a 6.4-fold
higher
inhibition of tumor growth over an additive effect.
To fixrther investigate the dose range for CV787 treatment in combination with
docetaxel, we fixed the dose of docetaxel at I2.5 rng/kg and varied the dose
of CV787 from
1x108, 1x109, to 1x101° particles per animal. Treatment with CV787
alone or docetaxel
alone resulted in tumor growth inhibition. However, the combination of CV787
and
docetaxel had the greatest effect of the treatments tested. Complete
regression was
achieved in the animals treated with docetaxel and CV787 at a dose of either
1x101°, 1x109,
or 1x108 particles. Synergy of anti-tumor activity was also evident using
lxl0~ particles
per animal but complete regression was not observed. These changes were
statistically
significant by the Student's t-test for the comparison of combination
treatment of CV787
and docetaxel to any of the vehicle, CV787 alone, or docetaxel alone
treatments, with no
statistical difference between the three combination treatment groups. Recall
the complete
response dose of CV787 alone is 1x1011 particles per animal (Yu, D.-C. et al.,
1999, Supra.
Thus, the combination of CV787 and docetaxel produces a complete response with
1000-
- fold less virus, compared to the use of CV787 alone.
Virus replication within LNCaP tumors was documented by immunohistochemical
staining of tumor sections using polyclonal antibodies to Adenovirus type 5
(Chen, Y. et
al., 2000, Hum. Gone Then. 1-1:1153-1567.) No evidence of virus replication
was found in
the tumors treated with either vehicle or paclitaxel, whereas evidence of
necrosis and
multifocal inflammation was observed in a small portion of tumors treated with
paclitaxel.
In the CV787-txeated tumors, while positively stained cells were visible
throughout the
tumors, infected cells were predominantly located near the tumor vasculature.
The most
intriguing phenomena were in the samples treated with both the virus and
paclitaxel. While
few virus-infected cells were detected, mast of the cells in the sections were
empty and
virtually devoid of cellular content. The remaining cells were much smaller
and appeared
to have undergone a morphological change.
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Tumor cells were also tested for apoptosis using the M30 CytoDEATHTM detection
kit, which recognizes a specific caspase cleavage site within cytokeratin 18
in early events
of apoptosis. Three tumors from each group, CV787 alone, paclitaxel, or both
CV787 and
paclitaxel, were analyzed 9 days after the start of dosing. Few apoptotic
cells were
detected in the paclitaxel-treated tumor, while a significant amount of
apoptotic cells along
the blood vessel were present in the CV787-infected tumors. However,
combination
treatment produced more apoptotic cells than in the any of the other samples.
In
conclusion, the immunohistochemical analysis of CV787 treated tumors suggests
that both
virus replication-dependent cytolysis and apoptosis contribute to the
antitumor effect of
CV787 and taxane.
Finally, and of clinical significance are two other results. First, healthier
animals,
characterized by body weight, were observed in the combination treatment group
as
compared to groups treated with either agent alone. Of particular interest is
the transient
weight loss using docetaxel alone, from which animals are protected from by
the use of
CV787 in combination with docetaxel. Indeed, animals treated with both CV787
and
taxotere gain 24% more weight than untreated control animals (Table 2).
Second, formal
toxicology studies in Balb/C mice failed to show synergistic toxicity from the
combined
use of docetaxel and CV787
Example 2: In vitro Treatment of HepG2 and Hep3B Tumor Cells with Replication
Competent Target Cell-Specific Adenoviral Vector CV790 and
Chemotherapy
Regimen for in vitro study of adenoviral vector and chemotherapeutic agent
A preliminary experiment was performed to compare three different protocols:
Adding virus first, drug first or virus and drug together (Figures 15-17).
HepG2 and
Hep3B cells were treated with 10 ng/ml doxorubicin and 0.01 MOI of CV790.
Figure 15
shows a synergistic effect in the panel of virus infection first. Virus first
indicates
administration of the virus about 10-14 hrs before drug application. Drug
first
indicates administration of the chemotherapeutic agent 10-14 hrs before virus
infection
Figure 16. The results of administration of adenovirus vector and drug
together are shown
in Figure 17. For the combination of CV790 and doxorubicin, virus first
administration
resulted in the greatest killing of liver cancer cells. This order of
administration was not
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the most effective for CV787 combined with paclitaxel (TAXOLTM) or docetaxel
(TAXOTERETM).
In order to study the killing effect of virus and drug on liver cancer cells,
an in vitro
cell viability study (MTT assay) was carried out using chemotherapeutic agents
and the
CV790 adenovirus on HepG2 and Hep3B hepatoma cells. Protocols for cell growth
and
MTT assay were as described as in Example 1. CV790 was constructed according
to
methods known in the art with the ElA and E1B genes under the control of the a-
fetoprotein promoter (approximately 0.8 kb), with an intact E3 region. The
structure of
CV790 can be summarized as AFP/ElA, AFP/E1B, E2, E3, E4. The hepatoma cells
were
grown in well plates, then treated with CV790 and various chemotherapeutic
agents, as
shown in Figures 15-22. After treatment, cells were incubated with MTT and
cell viability
at different time points from days 2-10 were compared. The MTT assay
determines the
number of cancerous cells still viable after treatment with the
CV790/chemotherapy
combination. Dead cells are equal to 1-the percentage of viable cells.
The following is the list of chemotherapeutic agents (drugs) and the sources
for the
drugs used in this study.
1. 5-Fluorouracil, (Sigma, St. Louis, MO) catalog number F-6627
2. Doxorubicin hydrochloride, (Sigma, St. Louis, MO) catalog number D-1 515
3. Cis-platinum (if)-diammine dichloride (cisplatin), (Sigma, St. Louis, MO)
catalog
number P-4394
4. 5-azacytidine, (Sigma, St. Louis, MO) catalog number A-2385
5. Mitomycin C, (Sigma, St. Louis, MO) catalog number M-0505
6. TAXOLTM, (Mead Johnson oncology products, New Jersey) catalog number C 0015-
3475-30
7. Gemcitabine, (Lilly, Indiana) catalog number nC 0002-7501-O1
8. Etoposide, (Bristol Laboratories, New Jersey) catalog number nC 0015-3095-
20
9. Mitoxantrone, (Immunex Corp., Seattle, WA), catalog number NDC 58406-640-03
Screening of chemotherapeutic agents for synergistic effects with CY790
The cytotoxicity of different chemotherapeutic agents combined with CV790 in
Hep3B and HepG2 hepatoma cells were tested using the methodology described in
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Example 1 and above, with virus added before treatment with chemotherapeutic
agent. The
results are shown in Figures 18-22 and summarized in Table 6, below. These
results
correspond to the virus first regimen described above. Doxorubicin, mitomycin
C,
mitoxantrone, cisplatin, gerncitabine, 5-azacytidine, etoposide and TAXOLTM
displayed
synergistic effects of cytotoxicity when combined with CV790 compared to the
cytotoxicity of the drug or virus alone. 5-Fluorouracil did not show
synergistic effects with
respect to virus and chemotherapy alone. For the experiments summarized in
Table 6 the
administered dose of CV790 was either MOI 0.1 or 0.01, as shown in the
Figures. The
chemotherapeutic agents were administered in the following amounts:
doxorubicin (50
ng/ml); cisplatin (10 p,g/ml); taxol (6.5 ng/ml); 5-fluorouracil (100 pg/ml);
mitoxantrone
(100nM); mitomycin C (I0 ~,g/ml); 5-azacytidine (10~.g/ml); etoposide (I
~,g/ml); and
gemcitabine (SOng/ml).
20
Table 6: Synergistic Effects of CV790/Chemotherapeutic Combinations
Chemotherapeutic
'Virus Cell.lin'e.agent. . CIa'ss of'agent SocivERGY:
. . '
CV790 HepG2, Hep3B5-FluorouracilAntimetabolites No
CV790 HepG2, Hep3B5-AzacytidineAntimetabolite Yes
(DNA
damaging agent)
CV790 HepG2, Hep3BCisplatin Alkylating agent Yes
(Plantinum-containing
agents)
CV790 HepG2, Hep3BDoxorubicin Antibiotics (anticycline)Yes
CV790 HepG2, Hep3BTAXOL""' Plant alkaloids Yes
(paclitaxel) '-
CV790 HepG2, Hep3BEtoposide Plant allcaloids Yes
CV790 HepG2, Hep3BGemcitabine Antimetabolite Yes
(DNA
damaging agent)
CV790 HepG2, Hep3BMitomycin Antibiotics Yes
C
CV790 HepG2, Hep3BMitoxantroneAntibiotics (anticycline)Yes
Example 3: In vitro Treatment of HepG2 and Hep3B Tumor Cells with Replication-
Competent AFP-Producing Cell-Specific Adenoviral Vector CV790 and
Combination Chemotherapy
In addition to screening single chemotherapeutic agents co-administered with
replication-competent target cell-specific adenoviral vectors, a screen was
completed of a
number of combination chemotherapy regimens which were co-administered with
CV790,
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a hepatoma specific adenoviral vector. Examples of such combination or
multiple drug
chemotherapy regimens can be found in Table 2. The protocols for the
administration of
the drugs and virus were as described in Examples l and 2, as was the
monitoring of cell
viability by MTT assay. The regimen followed was the virus first regimen. A
range of
drug concentrations were tested.
Treatment of hepatoma cells (Hep3B and HepG2) with a combination of multiple
chemotherapy drugs plus CV790 showed a synergistic enhancement of cytotoxicity
toward .
the hepatoma cells compared to the treatment of the hepatoma cells with either
the virus
alone or the multiple drug combination alone. Results are summarized in Table
7 below.
Table 7: Synergistic effects of CV790lCombination Chemotherapeutics
~:C~TE~OT~IERAk'EU'~'~C
~' VIRUS~ CELL LINEAGENT ' CLASS OF AGENT SYNERGrY
, w
CV790 HepG2, Hep3BDoxorubicin Anticycline antibioticsYes
&
Cisplatin & Plantinum-containing
agents
CV790 HepG2, Hep3BDoxorubicin Anticycline antibioticsYes
&
Mitomycin C
CV790 HepG2, Hep3BDoxorubicin Anticycline antibioticsYes
&
Mitoxantrone
CV790 HepG2, Hep3BDoxorubicin Anticycline antibioticsYes
&
TAXOL~ & Plant alkaloids
Example 4: Treatment of Prostate Tumor Xenografts with CV787 and
Chemotherapeutic Agents
After a synergistic effect was observed in vitro for the suppression of tumor
cell
growth with combinations of CV787 and a numberof chemotherapeutic agents, a
subset of
these agents were examined for evidence of synergistic results in suppressing
tumor growth
in vivo. In vivo studies indicated that the combination of CV787 with
paclitaxel or
docetaxel could eliminate tumors within 2-4 weeks with ten-fold less virus
(1x10 particles
per mm3 for intratumoral administration, 1x101° particle per animal for
intravenous
administration) compared to a previously effective dose for virus alone. Yu et
al. (1999)
Cancer Res. 59:4200. Below are described detailed examples for CV787 and
paclitaxel
and CV787 and docetaxel.
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Six to eight week old athymic Balb/c nu/nu mice were obtained from Simonson
Laboratories (Gilroy, CA) and acclimatized to laboratory conditions one week
prior to
tumor implantation. Xenografts were established by injecting 1x106 LNCaP cells
subcutaneously near the small of the back suspended in 100 ~.1 of RPMI 1640
and 100 ~,1 of
maltrigel (Collaborative Biochemical Products). When tumors reached between
400 mm3
and 600 mm3, mice were randomized in groups of four each to receive either
1x10°
particles of CV787 at day 1 via the tail vein or paclitaxel, 20 mg/kg
intraperitoneally (i.p.)
daily for 4 days starting at day 2, versus controls treated with normal saline
0.1 ml i.v. at
day 1 and then i.p. for 4 days. In addition, another group of mice received
the combination
of CV787 and paclitaxel at the same doses and schedule as above. CV787 was
diluted in
lyophilized buffer and injected into tail vein in a volume of 0.1 ml using a
28-gauge needle.
The dose and route of administration of paclitaxel were selected according to
studies in
nude mice by Riondel et al., (1986) Cancer Chemother Pharmacol.17:137. These
authors
conducted acute toxicity studies of paclitaxel in nude mice and selected the
unit dose of
12.5 mg/kg daily, being 1/20th of the LD50 dose (lethal dose for 50% of
animals). Tumors
were measured weekly in two dimensions by external caliper and volume was
estimated by
the formula [length (mm) x width (mm)a)/2. Animals were humanely killed when
their
tumor burden became excessive. Serum was harvested weekly by retro-orbital
bleed. The
difference in mean tumor volume between treatment groups was compared for
statistical
significance using the unpaired, two-tailed, t-test. Blood samples were
collected at various
time points for determining prostate-specific antigen (PSA). The level of PSA
is directly
related to tumor size, and tumor regression is accompanied by a fall in PSA
levels.
Ahti-tumor efficacy of the combined therapy of iutratumorally administered
CV787 with
paclitaxel
The in vivo antitumor efficacy of intratumorally administered CV787 and the
interaction of CV787 in the combination with paclitaxel was assessed in the
LNCaP mouse
xenograft model as described above. The following treatments were administered
(n=6 per
treatment group):
Vehicle (negative control).
GV787 (active control) at a dose of 1 x 10' particles per mm3 of tumor, at day
I .
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Paclitaxel at a dose of 15 mg/kg of animal weight, starting at day 2, daily
for four
days.
CV787 (1 x 10' particles per mm) and paclitaxel (15 mg/kg), scheduled as
above.
All treatment groups received identical injections of the active agent or
vehicle
control into both the tumor and peritoneum. Tumor volume was measured just
before the
injection of test articles and once a week for 6 weeks thereafter.
The following changes in average tumor volumes were measured 6 weeks after
treatment. Average tumor volume increased in vehicle-treated animals from 425
mm3 to
983 mm3 (231 % of baseline) 6 weeks after treatment and in the paclitaxel
group from 405
mm3 to 630 mm3(166% of baseline) Figure 23. Tumor volumes in the CV787 1x10
particles/mm3 group dropped from 419 to 379 mm3 (90% of baseline) whereas the
average
tumor volume in the combination treatment group of CV787 with paclitaxel
decreased from
413 mm3 to 45 mm3 ( 11 % of baseline) within six weeks after treatment. These
changes
were statistically significant by Student's t-test for the comparison of
combination
treatment of CV787 with paclitaxel to any of the vehicles, CV787 alone or
paclitaxel alone
treatment. It is suggested that the combination of CV787 with paclitaxel
produces a
synergistically enhanced anti-tumor efficacy, more effective than virus
treatment alone or
paclitaxel treatment alone.
Anti-tumor efficacy of the combined therapy of intravenously administered
CV787
with paclitaxel
In vivo studies of intravenously administered CV787 in conjunction with
paclitaxel
or docetaxel were performed in the same mouse xenograft model as used for the
intratumoral injection study. All test articles were administered via tail
vein except that
paclitaxel was injected intraperitoneally into animals.
The efficacy of intravenously administered CV787 and paclitaxel was assessed
as
described above. The following treatments were administered in this study:
Vehicle (negative control).
CV787 (active control) at a dose of 1 x 101° particles per animal at
day 1.
Paclitaxel at a dose of 20 mg/lcg of animal weight, starting at day 2, daily
for 4 days.
CV787 (1x101° particles/animal) and paclitaxel (20 mg/kg), scheduled as
above.
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Tumor volumes were measured just before the injection of test articles and
once a
week for 10 weeks thereafter.
In this study, single doses of CV787 and paclitaxel both appeared to be
effective in
producing tumor regression in the LNCaP mouse model, whereas the combination
produced a complete regression in 4 weeks Figure 25. Four weeks after
treatment, relative
tumor volume decreased to 3%. of baseline (from 418 mm3 to 14 mm3) for the
combination
treatment group and 216% of baseline for the vehicle-treated group, 31 % of
baseline for
the paclitaxel group and 162% of baseline for the CV787 group. Ten weeks after
treatment, 100% of the animals in the combination therapy group were tumor
free, and
animals were followed for 90 days without tumor regrowth. Relative tumor
volume in the
CV787-treated group decreased to 28% of baseline, while the tumors in the
paclitaxel-
treated group progressively grew back to 149% of baseline. This result
indicated that
paclitaxel alone could not cure cancers in this xenograft model. CV787
appeared to be
highly effective and virus alone took.a relatively long period of time to cure
cancer at this
dose level. However, the combination of CV787 and paclitaxel effectively
eliminated
tumors within 4 weeks after administration. In summary, the combination of
paclitaxel
with intravenously administered CV787 was far more effective than chemotherapy
or virus
treatment alone
Figure 24 depicts the change in tumor growth upon varying does of paclitaxel
(TAXOL~) and CV787 combined with paclitaxel (TAXOLTM). paclitaxel.(TAXOLTM) at
10 mg/kg has approximately the same efficacy over a 5 week period as does
paclitaxel
(TAXOLTM) at 2 mg/kg when combined with CV787 (1 x 101° particles).
Each of these
treatments merely arrests tumor growth while not actually causing and
regression of the
tumor. A dose of 2 mg/kg of paclitaxel (TAXOL~1'IVl) alone, however, leads to
progressive
enlargement of the tumor. A combination of paclitaxel (TAXOLTM) at 10 mg/kg
combined
with a 1 x 101° particle dose of CV787, however, leads to complete
suppression of tumor
growth by the third week of the in vivo trial.
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Ahti-tumor efficacy of the combined therapy of intravenously administered
CV787
and docetaxel
The efficacy of intravenously administered CV787 and docetaxel was also
assessed
as described above. All test articles were administered into animals via tail
vein. The
following treatments were administered in this study:
Vehicle (negative control).
CV787 (active control) at a dose of 1x101° particles per animal at
day 1.
Docetaxel at a dose of 10 mg/kg of animal weight, starting at day 2, daily for
4
days.
CV787 (1x101° particles/animal) and paclitaxel (10 mg/kg), scheduled as
above.
Tumor.volumes were measured just before the injection of test articles and
once a
week for 6 weeks thereafter.
In this study, single dose of CV787 and docetaxel both appeared to be
effective in
producing tumor regression in the LNCaP mouse model, whereas the combination
produced a complete regression within 4 weeks. Four weeks after treatment,
relative tumor
volume decreased to 2% of baseline for the combination treatment group and
226% of
baseline for the vehicle-treated group, 49% of baseline for the docetaxel
group and 132%
of baseline for the CV787 group. These changes were statistically significant
by the
Student's t-test for the comparison of combination treatment of CV787 and
docetaxel to
any of the vehicle, CV787 alone or docetaxel alone treatment. It is suggested
that the
combination of CV787 and docetaxel produces an enhanced anti-tumor efficacy,
much
better than virus alone or docetaxel alone.
An alternate presentation of these data are found in Figure 28 in which the
data are
reported as tumor volumes.
Following the successful treatment of the LNCaP xenografts with the above-
described method, the dosage of docetaxel was decreased by 50% to 5 mg/kg and
the
CV787 dose was maintained at 1 x 10r° particles. As shown in Figure 29
significant
regression of the tumor is observed for the CV787/docetaxel combination
therapy by week
3. At week 4 the tumor volume is less than the tumor volume which remains
steady for the
remainder of the experiment. The minimum tumor volume for docetaxel alone,
approximately 50% of the original tumor volume, is reached by week 1, however,
in the
following weeks tumor growth resumes and by week 6 has reached the starting
tumor
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volume. Treatment with a tenfold higher dose of CV787 (1 x 1011 particles) is
significantly
more effective than the lower dose of CV787 (1 x 101° particles) or
docetaxel alone, but is
slower to regress tumor growth and even at week 6 does not equal the reduction
in tumor
volume as the combination of CV787 (1 x 101° particles) and docetaxel
(5 mg/kg).
In summary, in vivo studies showed that direct intratumoral or intravenous
injection
of CV787 in conjunction with paclitaxel or docetaxel has an enhanced anti-
tumor eff cacy,
resulting in a significantly lower tumor burden observed in the combination
treatment. The
virus dose in the combination treatment was 10-fold lower than our previously
effective
dose for virus treatment alone, 1 x 1011 particles and a hundred percent of
treated animals
had complete tumor regression within 4 weeks in the intravenous administration
regimen.
These data provide supportive evidence for the potential development of a
combination
clinical regimen of CV787 with paclitaxel or docetaxel for clinical treatment
of prostate
cancer.
A number of other chemotherapeutic agents were screened for synergistic effect
when combined with CV787 for the i~ vivo treatment of cancer. Results are
summarized in
Table 8, below, and representative' data shown in Figures 23, 25-27. Table 8
also includes
data for the CN706 adenoviral vector, a replication-competent prostate cell-
specific
adenoviral construct (see Table 4). CV787 was administered in amounts ranging
between 1
x 10' particles/mm3, and 1 x 1011 particles, as indicated in the Figures.
Chemotherapeutic
agents were administered in the following amounts: paclitaxel (TAXOLTM; 2
mglkg to 20
mg/kg as shown); docetaxel (TAXOTERETM; 5-10 mg/kg, as shown); mitoxantrone (3
mg/kg); estramustine (14 mg/kg daily at days 2-5, 7-11, 13-17, and 20-
24);cisplatin
(4mg/kg); and 5-fluorouracil (30mg/kg).
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Table 8: Synergistic effects of Adenovirus/Chemotherapeutic
Combinations ih vivo
:. Chexnotherapeat~c:.. 'v'...
Virus. CeII:Iirie ~ agent Class-of.agerit.;Syqei~gy::
.
CV706 Prostate 5-Fluorouracil AntimetabolitesYes
cancer
xenografts
CV787 Prostate Cisplatin Alkylating Yes
cancer Agent
xenografts (Plantinum-
containing
agents)
CV787 Prostate Estramustine Alkylating Yes
cancer agent
xenografts
CV787 Prostate Mitoxantrone Antibiotics No
cancer
xenografts (anticycline)
CV787 Prostate TAXOTERE't"1 plant AlkaloidsYes
cancer
xenografts (docetaxel)
CV787 Prostate TAXOL""1 ~ Plant alkaloidsYes
cancer
xenografts (paclitaxel)
Example 5: Treatment of Hep3B Tumor Xenografts with Replication-Competent
Hepatoma Specific CV790 and Doxorubicin and Hepatoma Specific
CV890 and Doxorubicin
CV790 is an AFP producing hepatocellular carcinoma specific adenovirus, with
EIA and E1B under the control of an identical AFP promoter (827bp) and
enhancer with an
E3 region. The CV890 adenovirus construct is also a hepatoma or liver-specific
adenoviral
mutant with the E 1 A and E 1 B genes under transcriptional control of 827bp
AFP promoter,
wherein ElB is under translational control of EMCV IRES and having an intact
E3 region.
The structure of CV890 therefore reads as AFP/ElA, IRES/E1B, E2, E3, E4. Ih
vivo
studies of the efficacy of combinations of CV790 and doxorubicin and CV890 and
doxorubicin were performed according to the protocols described in detail in
Example 4,
with minor alterations which axe described below.
Xenografts in the study of CV790 and CV890 combined with chemotherapeutic
agents utilized liver carcinoma Hep3B cells, instead of LNCaP prostate
carcinoma. Virus,
CV790 or CV890, was administered by a single intravenous injection of 1 x 1011
particles
through the tail veins of the nude mice. One day after virus delivery, a
single dose of
doxorubicin was given to each animal by i.p. injection. The doxorubicin dose
was 10
mg/kg for both doxorubicin alone and doxorubicin combined with virus
treatments. Tumor
volume was measured once a week for six weeks according to the protocol in
Example 4.
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Both CV790/doxorubicin and CV890/doxorubicin treatment of the hepatoma
showed synergistic results. Four weeks after treatment with either
CV790/doxorubicin or
CV890/doxorubicin the relative tumor volume was less than 10%. Unlike mice
treated
with either virus alone or doxorubicin alone, after week 4, the relative tumor
volume did
not increase for either the either CV790/doxorubicin or CV890/doxorubicin
treated mice.
At week 6 in the control mice, the relative tumor volume was approximately
1000% in the
CV790 study and approximately 600% in the CV890 study 4 weeks after treatment.
The
relative tumor volumes of mice treated with virus alone were 250% (CV790) and
520%
(CV890) while the relative tumor volumes for mice treated with doxorubicin
alone were
450% with 280% in the CV790 study and 500% in the CV890 study. These results
are
shown in Figure 30 (CV790/doxorubicin) and Figure 31 (CV890/doxorubicin).
Example 6,: In vitro Treatment of Tumor Cells with Combined Target Cell-
Specific
Adenoviral Vector CV787 and Radiation Therapy
LNCaP prostate carcinoma cells were pre-seeded in 96 well plates in the RPMI
medium at 10,000 cells per well. After infection with CV787 (0.01 MOI)
according to
above described protocols (Example 1), the cells were incubated at 37°C
with 5% C02 for
24 hours, and then irradiated as monolayers using Cesium 137 gamma rays (used
for all
radiation studies) at a dose of 2 Gy. An MTT assay as described in Example 1
was
performed to determine cell viability (1- % of viable cells = dead cells). The
results axe
shown in Figures 10; 32; 33; 34; 35;and 36. The results show that the combined
adenoviral/radiation treatment is synergistically enhanced over treatment with
either virus
or radiation alone.
Figure 33 summarizes the results of a comparison of the treatment of LNCaP
prostate carcinoma cells with 6 Gy radiation combined with CV787 (MOI 0.1), 6
Gy
radiation alone, CV787 treatment alone, or no treatment. Protocols for the
treatment are as
described above for CV787 and 2 Gy radiation. Synergistic results were
observed for the
combined treatment of adenovirus and radiation compared to either treatment
alone.
In Figure 32 the same procedure was followed as those described above with the
treatment consisting of CV787 (MOI 0.1) and 6 Gy radiation, except that the
virus was
added 24 hours after LNCaP cells were irradiated. The results indicate that
virus treatment
either before or after irradiation leads to synergy in terms of cell killing.
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To establish a dose response curve, LNCaP cells were prepared and treated as
described above, with CV787 (MOI 0.01) administered first, followed after 24
hours with
varying doses of radiation. Sepaxate cell cultures were irradiated with an
increasing dose of
radiation starting at 0 Gy, up to 8 Gy (CV787 was kept at the same level of
multiplicity of
S infection of 0.01), then 6 days after irradiation, the cells were subjected
to a MTT assay as
described above in Example 1. Figure 36 shows the resulting dose response
curve, with
nearly 100% cell death at day 6 for an 8 Gy dose of radiation.
To determine the effect of radiation on the viability of the replication-
competent
target cell-specific adenoviral vectors, virus yield was measured according to
the protocol
described in Example 8. LNCaP prostate carcinoma cells were seeded in well
plates as
described in Example l and above, then treated with either radiation (6 Gy)
followed by
administration of CV787 (MOI 0.1) 24 hours later or treated with CV787 (MOI
0.1)
followed 24 hours later by irradiation (6 Gy), as described above in this
example. These
results were compared to the virus yield determined in LNCaP cells treated
with CV787
(MOI 0.1) alone. In both cases, with either radiation administered first
Figure 34 or virus
administered first Figure 35, the virus yield over time is comparable to the
virus yield in
LNCap cells which are not treated with radiation. These results indicate that
the
combination treatment produces more virus than virus alone.
Example 7: Construction of a Replication-Competent Adenovirus Vector
Comprising
an AFP-THE and an EMCV IRES
The encephalomyocarditis virus (ECMV) IRES as depicted in Table 12 was
introduced between the ElA and E1B regions of a replication-competent
adenovirus vector
specific for cells expressing AFP as follows. Table 12 shows the 519 base
.pair IRES
segment which was PCR amplified from Novagen's pCITE vector by primers A/B as
listed
in Table 9. A 98 base pair deletion in the ElA promoter region was created in
PXC.1, a
plasmid which contains the left-most 16 mu of AdS. Plasmid pXC.l (McI~innon
(1982)
Gene 19:33-42) contains the wild-type left-hand end of AdS, from Adenovirus 5
nt 22 to
5790 including the inverted terminal repeat, the packaging sequence, and the E
1 a and E 1 b
genes in vector pBR322. pBHGlO (Bett. et al. (1994) Proc. Natl. Acad. Sci. USA
91:8802-
8806; Microbix Biosystems Inc., Toronto) provides the right-hand end of AdS,
with a
deletion in E3. The resultant plasmid, CP306 (PCT/US98/I6312), was used as the
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backbone in overlap PCR to generate CP624. To place a Sall site between Ela
and Elb,
primers C/D, E/F (Table 9) were used to amplify CP306, plasmid derived from
pXC.l and
lacking the Ela promoter. After first round PCR using CP306 as template and
primers
C/D, E/F, the resultant two DNA fragments were mixed together for another
round of
overlapping PCR with primers C/F. The overlap PCR product was cloned by blunt
end
ligation to vector. The resultant plasmid, CP624 (Table 10), contains 100 by
deletion in
Ela/Elb intergenic region and introduces Sall site into the junction. On this
plasmid, the
endogenous Ela promoter is deleted, and the Ela polyadenylation signal and the
Elb
promoter are replaced by the Sall site. Next, the Sal l fragment of CP625 was
cloned into
the Sall site in CP624 to generate CP627 (Table 10). CP627 has an EMCV IRES
connecting adenovirus essential genes Ela and Elb. In CP627, a series of
different tumor-
specific promoters can be placed at the Pi~Al site in front of Ela to achieve
transcriptional
control 'on E 1 expression.
Table 9
PrimerSequence Note
A. S'-GACGTCGACTAATTCCGGTTATTTTCCA For PCR EMCV IRES, GTCGAC
is a SalI site.
B. S'-GACGTCGACATCGTGTTTTTCAAAGGAA For PCR EMCV IRES, GTCGAC
is a SalI site.
C. S'-CCTGAGACGCCCGACATCACCTGTG AdS sequence to 1314 to 1338.
D. S'-GTCGACCATTCAGCAAACAAAGGCGTTAACAntisense of AdS sequence
1572 to 1586.
GTCGAC is a SalI site. Underline
region
overlaps with E.
E. S'-TGCTGAATGGTCGACATGGAGGCTTGGGAGAdS sequence 1714 to 1728.
GTCGAC is a
SaII site. Underline region
overlaps with D.
F. S'-CACAAACCGCTCTCCACAGATGCATG Antisense of AdS sequence
2070 to 2094.
For generating a liver cancer-specific virus, an about 0.8kb AFP promoter
fragment
as shown in Table 14 was placed into the Pi~AI site of CP627 thereby yielding
plasmid
CP686. Full-length viral genomes were obtained by recombination between CP686
and a
plasmid containing a right arm of an adenovirus genome. The right arms used in
vinrs
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recombination were pBHGE3 (Microbix Biosystems Inc.), containing an intact E3
region,
and pBHGl l or pBHGlO (Bett et al. (1994) containing a deletion in the E3
region.
The virus obtained by recombination of CP686 with a right arm containing an
intact
E3 region was named CV890. The virus obtained by recombination of CP686 with a
right
arm containing a deleted E3 region (pBHG 10) was named CV840. The structure of
all
viral genomes was confirmed by conducting PCR amplifications that were
diagnostic for
the corresponding specific regions.
Therefore, adenovirus vector designated CV890 comprises 0.8 kb AFP promoter,
ElA, a deletion of the ElA promoter, EMCV IRES, E1B a deletion of the E1B
promoter
and an intact E3 region. Adenovirus vector CV840 comprises AFP promoter, ElA,
a
deletion of the ElA promoter, EMCV IRES, E1B, a deletion of the ElB promoter
and a
deleted E3 region.
Table 10
Plasmid Brief description
designation
CP306 An ElA promoter deleted plasmid derived from pXC.l
CP624 Overlap PCR product from CP306 to generate 100 by deletion and
introduce a S al l site at E 1 A and E 1 B j unction; E 1 A and E l B promoter
deleted in E 1 A/E 1 B intergenic region.
CP625 ~ EMCV IRES element ligated to PCR-blunt vector (Invitrogen pCR~ blunt
vector).
CP627 IRES element derived from CP625 by Sall digestion and ligated to CP624
Sall site placing IRES upstream from E1B.
CP628 Probasin promoter derived from CP251 by PinAl digestion and cloned into
PinAl site on CP627.
CP629 HCMV IE promoter amplified from pCMV beta (Clontech) with PirzAl at
5' and 3' ends ligated into CP627 Pi~AI site.
CP630 A 163 by long VEGF IRES fragment (Table 1) cloned into the Sall site on
CP628.
CP686 AFP promoter from CP219 digested with PinAl and cloned into Pi~AI site
on CP627.
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Example 8: Construction of a Replication-Competent Adenovirus Vector with a
Probasin THE and an EMCV IRES
The probasin promoter as shown in Table 14 was inserted at the PinAI site of
plasmid CP627 (see Example 8) to generate CP628, which contains a probasin
promoter
upstream of ElA and an EMCV IRES between ElA and E1B. Full-length viral
genomes
were obtained by recombination between CP628 and a plasrnid containing a right
arm of'an
adenovirus genome. The right arms used in virus recombination were pBHGE3,
containing
an intact E3 region, and pBHGl 1 or pBHGlO containing a deletion in the E3
region. The
structure of all viral genomes was confirmed by conducting PCR amplifications
that were
diagnostic for the corresponding specific regions.
Therefore, adenovirus designated CV 834 comprises probasin promoter, ElA, a .
deletion of the ElA promoter, EMCV IRES, E1B, a deletion of the E1B endogenous
promoter and a deleted E3 region.
Example 9: Construction of a Replication-Competent Adenovirus Vector with a
bCMV-THE and an EMCV IRES
The hCMV immediate early gene (IE) promoter from plasmid CP629, originally
derived from pCMVBeta (Clonetech, Palo Alto) was inserted at the PinAI site of
plasmid
CP627 (see Example 8) to generate CP629, containing a CMV IE promoter upstream
of
ElA and an IRES between ElA and E1B. Full-length viral genomes were obtained
by
recombination between CP629 and a plasmid containing a right arm of an
adenovirus
genome. The right aims used in virus recombination were pBHGE3, containing an
intact
E3 region, and pBHGl1 or pBHGlO containing a deletion in the E3 region. The
structure
of all viral genomes was confirmed by conducting PCR amplifications that were
diagnostic
for the corresponding specific regions.
Therefore, adenovirus vector designated CV835 comprises hCMV-IE promoter,
ElA, a deletion of the ElA promoter, EMCV IRES, E1B a deletion in the E1B
endogenous
promoter and a deleted E3 region. CV835 lacks the hCMV enhancer and is
therefore not
tissue specific. By adding the hCMV IE enhancer sequence to CV835, the vector
is made
tissue specific.
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Example 10: Comparison of Dual THE Vectors with Single TRE/IRES-Containing
Vectors
Two liver cancer-specific adenovirus vectors, CV790 and CV733 (also designated
CN790 and CN733, respectively), were generated and characterized. See
PCT/LTS98/04084. These viruses contain two AFP TREs, one upstream of EIA and
one
upstream of E1B. They differ in that CV790 contains an intact E3 region, while
the E3
region is deleted in CV733. Replication of these two viruses was compared with
that of the
newly generated IRES-containing viruses, CV890 and CV840 (see Example I).
Virus replication was compared, in different cell types, using a virus yield
assay as
described in Example 4. Cells were infected with each type of virus and, 72
hrs after
infection, virus yield was determined by a plaque assay. The results indicate
that vectors
containing an IRES between ElA and E1B (CV890 and CV840), in which E1B
translation
is regulated by the IRES, replicate to similar extents as normal adenovirus
and viruses with
dual AFP TREs, in AFP-producing cells such as 293 cells and hepatoma cells. In
SK-Hep-1 (liver cells), PA-1 (ovarian carcinoma) and LNCaP cells (prostate
cells) the
IRES-containing viruses do not replicate as well as dual THE or wild-type
adenoviruses,
indicating that the IRES-containing viruses have higher specificity for
hepatoma cells.
Based on these results, it is concluded that IRES-containing vectors have
unaltered
replication levels, but are more stable and have better target cell
specificity, compared to
dual-THE vectors.
Example 11: Uroplakin adenoviral constructs containing an EMCV IRES
A number of E3-containing viral constructs were prepared which contained
uroplakin II sequences (mouse and/or human) as well as an EMCV internal
ribosome entry
site (IRES). The viral constructs are summarized in Table 11. All of these
vectors lacked
an E 1 A promoter and retained the E 1 A enhancer.
The 519 base pair EMCV IRES segment was PCR amplified from Novagen's
pCITE vector by primers AB:
primer A: 5'-GACGTCGACTAATTCCGGTTATTTTCCA
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primer B 5'-GACGTCGACATCGTGTTTTTCAAAGGAA (GTCGAC is a SaII
site).
The EMCV IRES element was ligated to PCR blunt vector (Invitrogen pCR~ blunt
vector).
CP1066
The 1.9kb-(-1885 to +1) fragment of mouse UPII from CP620 was digested with
AflIII (blunted) and HindIII and inserted into pGL3-Basic from CP620 which had
been
digested with XhoI (blunted) and HindIII to generate CP1066.
CP1086
The 1.9kb mouse UPII insert was digested with PinAI and ligated with CP269
(CMV driving E 1 A and IRES driving E 1 B with the deletions of E 1 A/E 1 B
endogenous
promoter) which was similarly cut by PinAI.
CP1087
The lkb (-1128 to +1) human UPII was digested with PinAI from CP665 and
inserted into CP629 which had been cut by PinA.I and purified (to elute CMV).
CPX088
The 2.2kb (-2225 to +1) human UPII was amplified from CP657 with primer
127.2.1 (5'-AGGACCGGTCACTATAGGGCACGCGTGGT-3') PLUS 127.2.2 (5'-
AGGACCGGTGGGATGCTGGGCTGGGAGGTGG-3') and digested with PinAI and
ligated with CP629 cut with PinAI.
CP627 is an Ad5 plasmid with an internal ribosome entry site (IRES) from
encephelomycarditis virus (EMCV) at the junction of ElA and E1B. First, CP306
(Yu et
al., 1999) was amplified with primer pairs 96.74.3/96.74.6 and
96.74.4/96.74.5.
The two PCR products were mixed and amplified with primer pairs 96.74.3 and
96.74.5. The resultant PCR product contains a 100bp deletion in E1A-E1B
intergenic
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region and a new SaII site at the junction. EMCV IRES fragment was amplified
from
pCITE-3a(+) (Novagen) using primers 96.74.1 and 96.74.2. The SaTI fragment
containing
IRES was placed into SaII site to generate CP627 with the bicistronic E1A-IRES-
E1B
cassette. CP629 is a plasmid with CMV promoter amplified from pCMVbeta
(Clontech)
with primer 99.120.1 and 99.120.2 and cloned into PinAI site of CP627.
CP657 is a plasmid with 2.2kb 5' flanking region of human UP II gene in pGL3-
Basic (Promega). The 2.2kb hUPII was amplified by PCR from GenomeWalker
product
with primer 100.113.1 and 100.113.2 and TA-cloned into pGEM-T to generate
CP655.
The 2.Zkb insert digested from SacII (blunt-ended) and KpnI was cloned info
pGL3-Basic at HindIII (blunted) and KpnI to create CP657.
CP1089
The 1kb (-965 to +1) mouse UPII was digested by PinAI from CP263 and inserted
into CN422 (PSE driving E 1 A and GKE driving E 1 B with the deletions of E 1
A/E 1 B
endogenous promoter) cut by PinAI and purified and further digested with EagI
and ligated
with lkb (-1128 to +1) human UPII cut from CP669 with EagI.
CP1129
The 1.8kb hUPII fragment with PinAI site was amplified from CP657 with primer
127.50.1 and 127.2.2 and cloned into PinAI site of CP629.
CP1131
CP686 was constructed by replacing the CMV promoter in CP629 with an AFP
fragment from CP219. A 1.4kb DNA fragment was released from CP686 by digesting
it
with BssHII, filling with Klenow, then digesting with BgIII. This DNA fragment
was
then cloned into a similarly cut CP686 to generate CP1199. In CP1199, most of
the ElB
19-KDa region was deleted. The 1.8kb hUPII fragment with PinAI site was
amplified
from CP657 by PCR with primer 127.50.1 and 127.2.2 and inserted into similarly
digested CP 1199 to create CP 1131.
The plasmids above were all co-transfected with pBHGE3 to generate CV874 (from
CP1086), CV875 (from CP1087), CV876 (from 1088) and CV877 (from CP1089),
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CV 882 (from CP 1129) and CV 884 (from CP 1131 ). CP 108 8, CP 1129 and CP
1131 were
cotransfected with pBHGE3 for construction of CV876, CV892 and CV884,
respectively
by lipofectAMINE (GibcoBRL) for 11-14 days. pBHGE3 was purchased from
Microbix, Inc., and was described previously. The cells were lysed by three
freeze-thaw
cycles and plagued on 293 cells for a week. The single plaques were picked and
amplified by infection in 293 cells for 3-5 days. The viral DNAs were isolated
from the
lysates and the constructs were confirmed by PCR with primer 31.166.1/ 51.176
for
CV876 and primer 127.50.1/51.176 for CV882 and CV884 at El region and primer
32.32.1/2 for all three viruses at E3 region.
TABLE 11
Name Vector Ad 5 VectorElA THE E1B THE E3
CV874 CP1086 pBHGE3 1.9 kb mUPIIIRES intact
CV875 CP1087 pBHGE3 1.0 kb hUPIIIRES intact
CV CP 1088 pBHGE3 2.2 kb hUPIIIRES intact
876
CV877 CP1089 pBHGE3 1.0 kb mUPII1.0 kb hUPII (E1Bintact
promoter deleted)
CV882 CP1129 pBHGE3 1.8 kb hUPIIIRES intact
CV884 CP1131 pBHGE3 1.8 kb hUPiiIRES (ElB 19-kDa intact
deleted)
Viruses
are tested
and characterized
as described
above.
Primer sequences:
96.74.1 GACGTCGACATCGTGTTTTTCAAAGGAA
96.74.2 GACGTCGACTAATTCCGGTTATTTTCCA
96.74.3 CCTGAGACGCCCGACATCACCTGTG
96.74.4 TGCTGAATGGTCGACATGGAGGCTTGGGAG
96.74.5 CACAACCGCTCTCCACAGATGCATG
_ GTCGACCATTCAGCAAACAAAGGCGTTAAC
96.74.6
100.113.1 AGGGGTACCCACTATAGGGCACGCGTGGT
100. Z ACCCAAGCTTGGGATGCTGGGCTGGGAGGTGG
13.2
127.2.2 AGGACCGGTGGGATGCTGGGCTGGGAGGTGG
127.50.1 AGGACCGGTCAGGCTTCACCCCAGACCCAC
31.166.1 TGCGCCGGTGTACACAGGAAGTGA
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32.32.1 GAGTTTGTGCCATCGGTCTAC
32.32.2 AATCAATCCTTAGTCCTCCTG
51.176 GCAGAAAAATCTTCCAAACACTCCC
99.120.1 ACGTACACCGGTCGTTACATAACTTAC
99.120.2 CTAGCAACCGGTCGGTTCACTAAACG
Example 12: Construction of a Replication-Competent Adenovirus Vector with a
Tyrosinase THE and EMCV IRES
CP621 is a plasmid containing a human tyrosinase enhancer and promoter
elements
in a PinAl fragment. This fragment is ligated to the PinAl site on CP627 to
generate
CP1078. CP1078 is combined with pBHGE3 to generate a new melanoma specific
virus,
CV859. Table 14 depicts the polynucleotide sequence of the PinAl fragment
which
contains a tyrosinase promoter and enhancer.
Example 13: Construction of a Replication-Competent Adenovirus Vector with a
Probasin-THE and a VEGF IRES
Using a strategy similar to that described in Example 8, the IRES fragment
from the
mouse vascular endothelial growth factor (VEGF) gene is amplified and cloned
into CP628
at the SalI site. Table 12 depicts the IRES fragment obtainable from vascular
endothelial
growth factor (VEGF) mRNA. In order to clone this fragment into the Ela/Elb
intergenic
region, two pieces of long oligonucleotide are synthesized. The sense
oligonucleotide is
shown in the Table , whereas the second piece is the corresponding antisense
one. After
annealing the two together to create a duplex, the duplex is subjected to SaII
digestion and
the resulting fragment is cloned into the SaII site on CP628. The resulting
plasmid, CP630,
has a probasin promoter in front of E1 a and an VEGF IRES element in front of
E 1 b. This
plasmid is used to construct a prostate cancer-specific virus comprising the
VEGF IRES
element.
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Example 14: Construction of a Replication-Competent Adenovirus Vector with an
AFP-THE and a VEGF IRES
Using a strategy similar to Example 8, a PinAI fragment which contains AFP THE
can be obtained. This AFP THE is cloned into the PinA.l site in front of ElA
on CP628
yielding plasmid CP1077. This plasmid has the AFP THE for E1 transcriptional
control and
the VEGF IRES element before Elb. CP1077 can be recombined with pBHGE3 to
generate a liver-specific adenovirus, designated as CV858.
Example 15: Construction of a Replication-Competent AdenovirusVector with a
hKLK2-THE and a EMCV IRES
Using a strategy similar to Example 1, the THE fragment from human glandular
kallikrein II as shown in Table 14 was cloned into the PinAI site in CP627.
The resultant
plasmid, CP1079, is cotransfected with pBHGE3 to create CV860.
Example 16: Construction of a Replication-Competent Adenovirus Vector with a
CEA-THE and a EMCV IRES
Using a strategy similar to Example 1, the THE fragment from Carcinembryonic
antigen (CEA)(Table 14, SEQ ID NO:~ is used to construct virus designated
CV873. A
PinAI fragment containing the CEA-THE was cloned into the PinAI site in front
of ElA of
CP627 for the transcriptional control. The resultant plasmid CP1080 is used
together with
pBHGE3 to generate CV873.
Example 17: Adenovirus Vectors with Urothelial Cell-Specific TREs
A number of plasmid constructs were generated as intermediates for adenovirus
type 5 (Ad 5) vector constructs. The plasmid constructs were based on plasmid
CP321 (Yu
et al., 1999, Cancer Res. 59:4200-4203), which contains a prostate-specific
enhancer
inserted at a PinAI site upstream of the E1A gene and at a EagI site upstream
of the ElB
gene. Constructs were created by inserting various UPII-derived 5'-flanking
DNA
sequences into the PinAI and EagI sites and removing the prostate-specific
enhancer.
Characteristics of the plasmid CP669 are ElA THE l.Okb hUPII and E1B THE l.Okb
mUPI
and lacked the E 1 A promoter and which contained the E 1 A enhancer.
Infectious
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recombinant adenoviral vectors was produced by co-transfecting 293 cells with
the UPII
5'-flanking DNA/E1 constructs and an Ad 5 backbone vector (pBHGlO or pBHGE3,
Microbix, Inc.) as described in Yu et al. (id.) to produce CV829,which has an
intact E3
region.
Example 18: In vitro Characterization of Melanocyte-Specif c TRE-Containing
Adenoviral Constructs
An especially useful objective in the development of melanocyte cell-
specific adenoviral vectors is to treat patients with melanoma. Methods are
described
below for measuring the activity of a melanocyte-specific THE and thus for
determining
whether a given cell allows a melanocyte-specific THE to function.
Cells and Culture Methoals
Host cells such as, HepG2 (liver); Lovo (colon); LNCaP (prostate); PMEL
(melanoma); SKMeI (melanoma); 6361 (melanoma) and MeWo cells are obtained at
passage 9 from the American Type Culture Collection (Rockville, MD). MeWo
cells are
maintained in RPMI 1640 medium (RPMI) supplemented with 10% fetal bovine serum
(FBS; Intergen Corp.), 100 units/mL of penicillin, and 100 units/mL
streptomycin. MeWo
cells being assayed for luciferase expression are maintained in 10% strip-
serum
(charcoal/dextran treated fetal bovine serum to remove T3, T4, and steroids;
Gemini
Bioproduct, Inc., Calabasas, CA) RPMI.
Trausfectiohs of MeWo Cells
For transfections, MeWo cells are plated out at a cell density of 5 x 105
cells per 6-
cm culture dish (Falcon, N~ in complete RPMI. DNAs are introduced into MeWo
cells
after being complexed with a 1:1 molar lipid mixture of N-jl-(2,3-
dioleyloxy)propyl-
N,N,N trimethylammonium chloride (DOTAPTM; Avanti Polar Lipids, AL) and
dioleoyl-
phosphatidylethanolamine (DOPETM; Avanti Polar Lipids, AL); DNAllipid
complexes are
prepared in serxm-free RPMI at a 2:1 molar ratio. Typically, 8 ~.g (24.2
nmole) of DNA is
diluted into 200 ~,L of incomplete RPMI and added dropwise to 50 nmole of
transfecting,
lipids in 200 ~,L of RPMI with gentle vortexing to insure homogenous mixing of
components. The DNA/lipid complexes are allowed to anneal at room temperature
for 15
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minutes prior to their addition to MeWo cells. Medium is removed from MeWo
cells and
replaced with 1 mL of serum-free RPMI followed by the dropwise addition of
DNA/lipid
complexes. Cells are incubated with complexes for 4-5 hours at 37°C, 5%
COZ. Medium
was removed and cells washed once with PBS. The cells were then trypsinized
and
resuspended in 10% strip-serum RPMI (phenol red free). Cells were replated
into an
opaque 96-well tissue culture plate (Falcon, NJ) at a cell density of 40,000
cells/well per
100 ~.L media and assayed.
Plaque assays
To determine whether the adenoviral constructs described above replicate
preferentially in melanocytes, plaque assays are performed. Plaguing
efficiency is
evaluated in the following cell types: melanoma cells (MeWo), prostate tumor
cell lines
(LNCaP), breast normal cell line (HBL-100), ovarian tumor cell line (OVCAR-3,
SK-OV-
3), and human embryonic kidney cells (293). 293 cells serve as a positive
control for
plaguing efficiency, since this cell line expresses Ad5 ElA and E1B proteins.
For
analyzing constructs comprising a melanocyte-specific TRE, cells that allow a
melanocyte-
specific THE to function, such as the cell lines provided above and cells that
do not allow
such function, such as HuH7, HeLa, PA-1, or 6361, are used. The plaque assay
is
performed as follows: Confluent cell monolayers are seeded in 6-well dishes
eighteen
hours before infection. The monolayers are infected with 10-fold serial
dilutions of each
virus. After infecting monolayers for four hours in serum-free media (MEM),
the medium
is removed and replaced with a solution of 0.75% low melting point agarose and
tissue
culture media. Plaques are scored two weeks after infection.
Example 19: Iu vitro and Ih vivo assays of anti-tumor activity
An especially useful objective in the development of urothelial cell-specific
adenoviral vectors is to treat patients with bladder cancer. An initial
indicator of the
feasibility is to test the vectors) for cytotoxic activity against cell lines
and tumor
xenografts grown subcutaneously in Balb/c nu/nu mice.
Ih vitro characterization of CV876
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Virus yield assay for CV876
X 105293, RT-4, SW780, PA-1, 6361, MKNI, HBL-100, Fibroblast (from lung)
and Smooth muscle cells (from bladder) were plated into each well of six-well
plates.
Twenty-four hours later, medium was aspirated and replaced with lml of serum-
free
5 RPMI 1640 containing CV802 (wt.AdS with E3) or CV876 at a MOI of 2 pfu/cell.
After a
4-h incubation at 37°C, cells were washed with prewarmed PBS, and 2m1
of complete
RPMI 1640 were added to each well. After an additional 72h at 37°C, the
cells were
scraped into medium and lysed by three freeze-thaw cycles. The lysates were
tested for
virus production by triplicate plaque assay for 8-10 days under semisolid
agarose .on
293 cells.
Unlike wt. AdS, CV802 which grows well in all of the cells tested, CV876
replicates much better in permissive cells (293, RT-4 and SW780) than in non-
permissive
cells (PA-l, 6361, MKN1, HBL-100 and primary cells) by about 100-10000 fold.
Noticeably, the replication in SW780 for CV876 is about 100 fold less than
CV802, which
indicates the limitation of this virus in efficacy.
Growth curve experiment for CV876
5 X 105 RT-4, PA-l, Smooth muscle and Fibroblast cells were plated into each
well
of six-well plates. Twenty-four hours later, medium was aspirated and replaced
with lml
of serum-free RPMI 1640 containing CV802 (wt.AdS with 133) or CV876 at a MOI
of
2 pfu/cell. After a 4-h incubation at 37°C, cells were washed with
prewarmed PBS, and
2m1 of complete RPMI 1640 were added to each well. At different time points of
0, 12, 24,
36, 48, 72, 96 and 120h, the cells were scraped into medium and lysed by three
freeze-thaw
cycles. The lysates were tested for virus production by triplicate plaque
assay for 8-10 days
under semisolid agarose on 293 cells.
Very similar as in virus yield assay, CV876 replicates well only in RT-4 but
not in
primary cells and PA-1 over a 120h period of time. However, CV876 does show a
delay of
replication in RT-4 compared to CV802.
Cytopathic effect assay for CV876
5 X 105293, RT-4, SW780, PA-l, MKN1 and LNCap were plated into each well of
six-well plates. Twenty-four hours later, medium was aspirated and replaced
with lml of
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serum-free RPMI 1640 containing CV802 (wt.AdS with E3) or CV876 at increasing
MOI
from 0.001 to 10 (the data shown was at MOI 1). After a 4-h incubation at
37°C, medium
was replaced with 3m1 of complete RPMI 1640 and incubated at 37°C for 6-
8 days when
cytopathic effect was observed for CV802 at MOI 0.01.
CV802 shows efficacy in all the cells tested while CV876 only kills the
permissive
cells (293, RT-4 and SW780) but not the non-permissive cells (PA-l, MIEN-l and
LNCap).
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MTT assay for CV876
2 X 104293, RT-4, SW780, MI~N1, PA-l, HBL-100, Smooth muscle cells (from
bladder) and Fibroblast (from Lung) were plated into each well of 96-well
plates. Twenty-
four hours later, the cells were infected with CV802 and CV876 at increasing
MOI from
0.001 to 10 in complete RPMI 1640. A rapid colorimetric assay for cell growth
and
survival was run at different time point of day 1, 3,5,7 and 10. The medium
was replaced
by SOuI of MTT at lmg/ml solution, which is converted to an insoluble purple
formazan by
dehydrogenase enzymes present in active mitochondria of live cells. After 3-4h
-incubation
at 37°C, the solution was replaced by isopropanol and the plates were
incubated at 30°C for
1h and read at S60nm test wavelength and 690nm reference wavelength.
Similar as the results in CPE assay, CV876 shows efficacy only in permissive
cells
but not in non-permissive cells. Again, in RT-4 and SW780, CV876 kills the
cells much
slower than CV802.
Ih vitro characterization of CV882
1S Virus yield assay for CV882
5 X 105293, RT-4, SW780, 6361, LNCap, HBL-100, MI~Nl, PA-1, Fibroblast and
Smooth muscle cells were plated into each well of six-well plates. Twenty-four
hours later,
medium was aspirated and replaced with lml of serum-free RPMI 1640 containing
CV802
(wt.AdS with E3) or CV882 at a MOI of 2 pfu/cell. After a 4-h incubation at
37°C, cells
were washed with prewarmed PBS, and 2m1 of complete RPMI 1640 were added to
each
well. After an additional 72h at 37°C, the cells were scraped into
medium and lysed by
three freeze-thaw cycles. The Iysates were tested for virus production by
triplicate plaque
assay for 8-10 days under semisolid agarose on 293 cells.
The replication of CV882 in permissive cells (293, RT-4 and SW780) is
comparable to CV802 (the difference is less than 100 fold) while it shows over
1000-
1000000 fold difference in non-permissive cells (G361, LNCap, HBL-100, MI~Nl,
PA-1
and primary cells).
Growth curve experiment for CV882
S X l OSRT-4, PA-l, and Fibroblast cells were plated into each well of six-
well
plates. Twenty-four hours later, medium was aspirated and replaced with lml of
serum-
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free RPMI 1640 containing CV802 (wt.AdS with E3) or CV882 at a MOI of 2
pfu/cell.
i
After a 4-h incubation at 37°C, cells were washed with prewarmed PBS,
and 2m1 of
complete RPMI 1640 were added to each well. At different time points of 0, 12,
24, 36, 48,
72, 96 and 120h, the cells were scraped into medium and lysed by three, freeze-
thaw
cycles. The lysates were tested for virus production by triplicate plaque
assay for 8-10 days
under semisolid agarose on 293 cells.
Very similar as in virus yield assay, CV882 replicates well only in RT-4 but
not in
primary cells and PA-1 over a 120h period of time. Additionally, CV882 shows
better
replication in RT-4 compared to CV876.
Cytopathic effect assay for CV882
5 X 105293, RT-4, SW780, HBL-100, 6361, PA-1 and Fibroblast cells were plated
into each well of six-well plates. Twenty-four hours later, medium was
aspirated and
replaced with lml of serum-free RPNI 1640 containing CV802 (wt.AdS with E3) or
CV882
at increasing MOI from 0.001 to 10 (the data shown was at MOI 1). After a 4-h
incubation
at 37°C, medium was replaced with 3m1 of complete RPMI 1640 and
incubated at 37°C for
6-8 days when cytopathic effect was observed for CV802 at MOI 0.01.
CV802 shows efficacy in all the cells tested while CV882 only kills the
permissive
cells (293, RT-4 and SW780) but not the non-permissive cells (HBL-100, 6361,
PA-l and
Fibroblast cells).
MTT assay for CV882
2 X 104RT-4, SW780, PA-1, HBL-100, U118 and Fibroblast were plated into each
well of 96-well plates. Twenty-four hours later, the cells were infected with
CV802 and
CV882 at increasing MOI from 0.001 to 10 in complete RPMI 1640. A rapid
colorimetric
assay for cell growth and survival was run at different time points of day 1,
3, 5, 7 and 10.
The medium was replaced by SOuI of MTT at lmg/ml solution, which is converted
to an
insoluble purple formazan by dehydrogenase enzymes present in active
mitochondria of
live cells. After 3-4h incubation at 37°C, the solution was replaced by
isopropanol and the
plates were incubated at 30°C for 1h and read at 560nm test wavelength
and 694nm
reference wavelength.
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Similar as the results in CPE assay, CV882 shows efFcacy only in permissive
cells
but not in non-permissive cells.
In Vitro Characterization of CV884
Virus yield assay for CV884
5 X 105293, RT-4, SW780, 6361, LNCap, HBL-100, MI~Nl, PA-1, Fibroblast and
Smooth muscle cells were plated into each well of six-well plates. Twenty-four
hours later,
medium was aspirated and replaced with lml of serum-free RPMI 1640 containing
CV802
(wt.AdS with E3) or CV984 at a MOI of 2 pfu/cell. After a 4-h incubation at
37°C, cells
were washed with prewarmed PBS, and 2m1 of complete RPMI 1640 were added to
each
well. After an additional 72h at 37°C, the cells were scraped into
medium and lysed by
three freeze-thaw cycles. The lysates were tested for virus production by
triplicate plaque
assay for 8-10 days under semisolid agarose on 293 cells.
The replication of CV884 is very similar as CV802 in permissive cells (293, RT-
4
and SW780) but shows over 1000 fold difference with CV802 in non-permissive
cells
(G361, LNCap, HBL-100, MKN1, PA-1 and primary cells). CV884 shows better
efficacy
than CV876 and CV882 without losing much specificity.
Growth curve experiment for CV884
5 X 105RT-4, PA-1, Smooth muscle and Fibroblast cells were plated into each
well
of six-well plates. Twenty-four hours later, medium was aspirated and replaced
with lml
of serum-free RPMI 1640 containing CV802 (wt.AdS with E3) or CV884 at a MOI of
2 pfu/cell. After a 4-h incubation at 37°C, cells were washed with
prewarmed PBS, and
2m1 of complete RPMI 1640 were added to each well. At different time points of
0, 12, 24,
36, 48, 72, 96 and 120h, the cells were scraped into medium and lysed by three
freeze-thaw
cycles. The lysates were tested for virus production by triplicate plaque
assay for 8-10 days
under semisolid agarose on 293 cells.
Very similar as in virus yield assay, CV884 replicates very well only in RT-4
(similar as CV802) but not in primary cells and PA-1. Again, the replication
of CV884 is
better than CV882 and CV876.
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Cytopathic effect assay for CV884
S X 105293, RT-4, SW780, 6361, PA-1 and Fibroblast cells were plated into each
well of six-well plates. Twenty-four hours later, medium was aspirated and
replaced with
lml of serum-free RPMI 1640 containing CV802 (wt.AdS with E3) or CV884 at
increasing
S MOI from 0.001 to 10 (the data shown was at MOI 1). After a 4-h incubation
at 37°C,
medium was replaced with 3m1 of complete RPMI 1640 and incubated at
37°C for 6-8 days
when cytopathic effect was observed for CV802 at MOI 0.01.
CV802 shows efficacy in all the cells tested while CV884 only kills the
permissive
cells (293, RT-4 and SW7$0) but not the non-permissive cells (G361, PA-I and
Fibroblast
cells).
MTT assay for CV884
2 X 104293, RT-4, SW780, Ul 18, Fibroblast and Smooth muscle cells were plated
into each well of 96-well plates. Twenty-four hours later, the cells were
infected with
CV802 and CV884 at increasing MOI from 0.001 to 10 in complete RPMI 1640. A
rapid
1S colorimetric assay for cell growth and survival was run at different time
points of day 1, 3,
S, 7 and 10. The medium was replaced by SOuI of MTT at lmg/ml solution which
is
converted to an insoluble purple formazan by dehydrogenase enzymes present in
active
mitochondria of live cells. After 3-4h incubation at 37°C, the solution
was replaced by
isopropanol and the plates were incubated at 30°C for 1h and read at
S60nm test wavelength
and 690nm reference wavelength.
Similar as the results in CPE assay, CV884 shows strong efficacy (similar as
wt. AdS) only in permissive cells but not in non-permissive cells.
In vivo activity of CV808
Mice were given subcutaneous (SC) injections of 1 x 106 sW780 cells. When
2S tumors grew to about S00 mm3, CV808 was introduced into the mice (S X 10'
PFU of virus
in 0.1 ml PBS and 10% glycerol) intratumorally. Control mice received vehicle
alone.
Tumor sizes were measured weekly. The data indicate that CV808 was effective
at
suppressing tumor growth.
While it is highly possible that a therapeutic based on the viruses described
here
would be given intralesionally (i.e., direct injection), it would also be
desirable to
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determine if intravenous (IV) administration of adenovirus vector can affect
tumor growth.
If so, then it is conceivable that the virus could be used to treat metastatic
tumor deposits
inaccessible to direct injection. For this experiment, groups of mice bearing
bladder
epithelial tumors are inoculated with 108 to 101° PFU of an adenoviral
vector by tail vein
injection, or with buffer used to carry the virus as a negative control. The
effect of IV
injection of the adenovixal vector on tumor size is compared to vehicle
treatment.
Example 20: Synergistic Effect of CV 890 with Chemotherapeutics
Materials and Methods
Cells
Hepatocellular carcinoma cell lines HepG2, Hep3B, PLC/PRF/5, SNU449, and
Sk-Hep-1, Chang liver cell (human normal liver cells), as well as other tumor
cell lines
PA-1 (ovarian carcinoma), UM-UC-3 (bladder carcinoma), SW 780 (bladder
carcinoma),
HBL100 (breast epithelia), Colo 201 (Colon adenocarcinoma), U 118 MG
(glioblastoma)
and LNCaP (prostate carcinoma) were obtained from the American Type Culture
Collection. HuH-7 (liver carcinoma) was a generous gift of Dr. Patricia Marion
(Stanford
University). 293 cells (human embryonic kidney containing the E1 region of
Adenovirus)
were purchased from Microbix, Inc. (Toronto, Canada). The primary cells nBdSMC
(normal human bladder smooth muscle cells), nHLFC (normal human lung
fibroblast
cells), and nHMEC (normal human mammary epithelial cells) were purchased from
Clonetics (San Diego, California). All tumor cell lines were maintained in
RPMI 1640
(BioWhittaker, Inc.) supplemented with 10% fetal bovine serum (Irvine
Scientific), 100
U/ml penicillin and 100 ug/ml streptomycin. Primary cells were maintained in
accordance
with vendor instructions (Clonetics, San Diego). Cells were tested for the
expression of
AFP by immunoassay (Genzyme Diagnostics, San Carlos, CA).
Virus yield and one-step growth curves
Six well dishes (Falcon) were seeded with 5x105 cells per well of calls of
interest 24
hrs prior to infection. Cells were infected at an multiplicity of infection
(MOI) of 2
PFU/cell for three hours in serum-free media. After 3 hours, the virus
containing media
was removed, monolayers were washed three times with PBS, and 4 ml of complete
media
(RPMI1640 + 10% FBS) was added to each well. 72 hours post infection, cells
were
scraped into the culture medium and lysed by three cycles of freeze-thaw.
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The one-step growth curves time points were harvested at various time points
after
infection. Two independent infections of each virus cell-combination were
titered in
duplicate on 293 cells (Yu et al., 1999, Cancer Research, 59:1498-1504.
Northern blot analysis
Hep3B or HBL100 cells were infected at an MOI of 20 PFU/cell (plaque forming
unit per cell) with either CV802 or CV890 and harvested 24 hours post
infection. Total
cell RNA was purified using the RNeasy protocol (Qiagen). The Northern blot
was
conducted using NorthernMax Plus reagents (Ambion, Austin, Texas). Sug of RNA
was
fractionated on a 1% agarose, formaldehyde-based denaturing gel and
transferred to a
BrightStax-Plus (Ambion) positively charged membrane by capillary transfer.
The
antisense RNA probes for ElA (adenovirus genome SOlbp to 1141bp) or E1B
(1540bp-3910bp) were PCR products cloned in pGEM-T easy (Promega) and
transcription
labeled with [a 32P] UTP. Blots were hybridized at 68°C for 14 hours
with ZipHyb
solution and washed using standard methods (Ambion). Membranes were exposed to
BioMax film (Kodak).
Western blot ana~sis
Hep3B or HBL100 cells were infected at MOI of 20 PFU/cell with either CV802 or
CV890 and harvested 24 hours post infection. Cells were washed with cold PBS
and lysed
for 30 min on ice in (SOmM Tris, pH8.0, 150 mM NaCI, 1% IGEPAL CA360 a NP40
equivalent (Sigma), 0.5% sodium deoxycholate, and protease inhibitor cocktail
from
(Roche, Palo Alto, California). After 30 min centrifugation at 4C, the
supernatant was
harvested and the protein concentration determined with protein assay ESL kit
(Roche).
Fifty micrograms of protein per lane were separated on 816% SDS-PAGE and
electroblotted onto Hybond ECL membrane (Amersham Pharmacia, Piscataway, New
Jersey). The membrane was blocked overnight in PBST (PBS with 0.1% Tween-20)
supplemented with 5% nonfat dry milk. Primary antibody incubation was done at
room
temperature for 2-3 hrs with PBST/1 % milk diluted antibody, followed by wash
and 1 hr
incubation with diluted horseradish peroxidase-conjugated secondary antibody
(Santa Cruz
Biotechnology Inc., Santa Cruz, California). Enhanced chemiluminescence (ECL;
Amersham Pharmacia) was used for the detection. ElA antibody (clone M58) was
from
NeoMarkers (Fremont, California), E1B-21 kD antibody was from Oncogene
(Cambridge,
Massachusetts). All antibodies were used according manufacturer's instruction.
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Cell viability assay and statistical analysis
To determine the cell killing effect of virus and chemotherapeutic agent in
combination treatment, a cell viability assay was conducted as previously
described with
modifications (Denizot, 1986, Journal Immunology. Methods, 89:271-277). On 96
well
S plates, cells of interest were seeded at 10,000 calls per well 48 hr prior
to infection. Cells
were then treated with virus alone, drug alone, or in combination. Cell
viability was
measured at different time points by removing the media, adding SO p1 of
lmg/ml solution
of MTT (3-(4,S-Dimethylthiazol-2-yl)-2,S-diphenyl-2H-tetrazolium bromide)
(Sigma, St.
Louis, MO) and incubating for 3 hrs at 37°C. After removing the MTT
solution, the
crystals remaining in the wells were solubilized by the addition of SO ~Cl of
isopropanol
followed by 30C incubation for O.S hr. The absorbency was determined on a
microplate
reader (Moleculax Dynamics) at S60 nm (test wavelength) and 690 nm (reference
wavelength). The percentage of surviving cells was estimated by dividing the
ODsso-OD6so
of virus or drug treated cells by the ODsso-OD6so of control cells. 6 replica
samples were
1 S taken for each time point and each experiment was repeated at least three
times.
For statistical analysis, CurveExpert (shareware by Daniel Hyams, version
1.34)
was used to plot the dose-response curves for virus and drugs. Based upon the
dose-
response curves, the isobolograms were made according to the original theory
of Steel and
Peckham (1993, Int. J. Rc~d. Onc. Biol. Phys., 5:85) and method described in
Aoe et al.
(1999, Anticancer Res. 19:291-299).
Animal studies
Six to eight week old athymic BALB/C nulnu mice were obtained from Simonson
Laboratories (Gilroy, California) and acclimated to laboratory conditions one
week prior to
tumor implantation. Xenografts were established by injecting 1x106 Hep3B,
HepG2 or
2S LNCaP cells suspended in 100~I of RPMI 1640 media subcutaneously. When
tumors
reached between 200 mm3 and 300 mm3, mice were randomized and dosed with 100
p1 of
test article via intratumoral or the tail vein injection. Tumors were measured
in two
dimensions by external caliper and volume was estimated by the formula [length
(mm) x
width (mm)2]/2. Animals were humanely killed when their tumor burden became
excessive. Serum was harvested weekly by retro-orbital bleed. The level of AFP
in the
serum was determined by AFP Immunoassay kit (Genzyme Diagnostics, San Carlos,
CA).
The difference in mean tumor volume and mean serum AFP concentration between
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treatment groups was compared for statistical significance using the unpaired,
two-tailed, t-
test.
Transcription and Translation of ElA/E1B Bicistronic Cassette of CV890 in
Different Cells
In wild type adenovirus infection, ElA and E1B genes produce a family of
alternatively spliced products. It has been found that there are five ElA
mRNAs, among
them 12S (880 nucleotides, nts) and 13S (1018 nts) mRNAs are the dominant ones
that are
expressed both early and late after infection. The I2S and I3S mRNAs encode
the gene
product of 243 amino acids (243R) and 289 amino acids (289R) respectively
(reviewed by
Shenk, 1996). The two major E1B transcripts that code for l9kD and SSkD
proteins are
12S (1031 nts) and 22S (2287 nts) mRNAs. E18 12S mRNA only codes the l9kD
product,
whereas the 22S mRNA codes for both l9kD and SSkD products due to different
initiation
sites during translation. In the current study, the generation of ElA-IRES-ElB
bicistronic
cassette was expected to change the pattern of ElA and E1B transcripts in
viral infection.
1 S Therefore, Northern blot analysis was conducted to evaluate the steady-
state Level of E 1 A
and E1B transcripts. First, CV802 or CV890 were infected to Hep3B (AFP) or
HBL100
(AFP) cells for 24 hours. The total RNA samples were separated on agarose gels
and
processed for Northern blot by hybridizing to antisense RNA probes. The
Northern blot
with ElA probe visualized the 12S and 13S mRNAs in both wild type CV802
infected
cells. For CV890, ElA transcripts can only be seen in Hep3B cells, indicating
the
conditional transcription of ElA. It is of interest to find that in CV890,
there is only one
large transcript (about 3.SlKb), whereas the I2S and 13S mRNAs are no longer
present.
This large transcript indicates the continuous transcription of ElA-IRES-E1B
bicistronic
cassette, suggesting an alteration of viral EIA splicing pattern in CV890.
Transcription of
E1B from CV890 also appeaxs to be AFP-dependent. It is clear that both 12S and
22S
mRNAs of E1B were present in wild type CV802 samples, whereas the 128 mRNA and
an
enlaxged 22S mRNA (3.SKb) appeared in CV890 infected cells. Obviously, the
identity of
this enlaxged mRNA is the same 3.SKb transcript as visualized in ElA blot,
which is from
the transcription of EIA/E1B bicistronic cassette. Therefore, the E1B mRNA is
tagged
after ElA mRNA in this large transcript. This laxge transcript contains all
the coding
information for E 1 A, E I B 19kD and E I B SSkD. The mRNA splice pattern that
appears in
CV802 is not valid in CV890, thel2S mRNA with EIB probe disappeared.
Meanwhile, in
the ElB Northern blot, due to the selection of our E1B probe (1540bp-3910bp),
mRNA of
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the Adenovirus gene IX (3580bp-4070bp), the hexon-associated protein, was also
detected.
In CV890 infected Hep3B cells, gene IX expression is equivalent to that of
CV802,
whereas in CV890 infected HBL100, its expression was also completely shut
down. This
result further demonstrated that the AFP controlled E 1 A/E 1 B expression is
the key for late
gene expression as well as viral replication.
Results of the same samples in the Western blot also indicate that CV890 has
AFP
dependent expression of E 1 A and E 1 B. Under our experimental conditions, E
1 A
expression level of CV890 in Hep3B cells is similar to that of CV802. However,
when
E1B l9kD protein was detected, it was found that the expression level was much
lower
than CV802 ElA. Previously, it has been addressed that IRES-mediated second
gene has
less expression (Mizuguchi et al., 2000, Mol. Ther. 1:376-382). Taken
together, CV890
infection in permissive Hep3B cells can produce normal amounts of ElA and
lesser
amounts of E1B proteins capable of initiating a normal productive infection.
In AFP- cells,
however, this process was attenuated due to a lack of ElA and E1B gene
transcription and
translation. These data demonstrated that the expression of both ElA and ElB
genes are
under the control of AFP THE and the artificial ElA/E1B bicistronic cassette
is functioning
properly in CV890.
In Vitro Replication Specificity of CV890 in Tumor Cells and Primary Cells
From i~ vitro comparison of virus yield, CV890 has a better specificity
profile than
CV732 (CV732 is an AFP-producing, cell-specific adenovirus variant in which
the ElA
gene is under control of AFP-TRE). In order to gain further insights of using
CV890 in
liver cancer therapy, more tumor cell lines and primary cells were tested to
characterize in .
vitro virus replication. First, all cells in the study were analyzed for their
AFP status by
AFP immune assay. Based on AFP produced in the cells and media, all the cells
were
divided into three groups, high (>2.5 ~,g/106 cells/10 days), low (<0.6
~.g/106 cells/10 days)
and none (undetectable in our study) (Table 15). It was confirmed that
replication of
CV890 in different cell lines correlates well with the AFP status of the host
cell. Among
the group of liver cell lines, CV890 only replicates well in AFP+ cells,
including Hep3B,
HepG2, Huh7, SNU449 and PLC/PRF/5. The amount of AFP required for the promoter
activity seems very low as one of the hepatoma cell lines, SNU449, a previous
reported
AFP- cell (Park et al., 1995, I~t. J. Cancer 62:276-282), produces very low
AFP (about 60
ng/106 cells/10 days) compared to other cells. Nevertheless, even with very
low amount of
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AFP, SNU449 cells can still support CV890 replication to the extent comparable
to cells
producing significantly higher levels of AFP such as HepG2. Compared to CV802,
CV890
is attenuated 5,000 to 100,000 fold in cells that do not produce AFP,
including the
hepatoma cell Sk-Hepl and Chang liver cell, other tumor cells and primary
cells. Taken
together the results indicate that CV890 has shown a good specificity profile
from a broad
spectrum of tumor cells. Among them, only the AFP+ liver cells, AFP production
level
from high to low, are permissive for CV890.
In another experiment, CV890 was compared to CV802 for their single step
growth
curves on different cells. Results demonstrated that CV890 has a similar
growth kinetics to-
wild-type CV802 in AFP+ cells except that virus yields are slightly lower (2-8
fold) in low
AFP producing cells. In consideration of experimental error, there is no
dramatic
difference in the replication of CV890 and CV802 in AFP+ hepatoma cells.
However, the
growth curves of CV890 in AFP- cells showed clear attenuation. During a 5 day
experiment, CV890 failed to replicate in AFP- cells including hepatoma cell
(Change liver)
and primary cells (nHLFC). From all the in vitro virus replication studies, it
is clear that
replication of CV890 is under the tight control of AFP-THE and this adenovirus
variant has
an excellent specificity profile of preferentially targeting AFP producing
hepatocellular
carcinoma cells.
In Yivo Specificity and Efficacy of CV890
CV890 specificity was also evaluated in animals bearing prostate cancer LNCaP
xenografts. In this in vivo test, nude mice with prostate xenograft were
intravenously
injected with either CV890 or CV787, a prostate cancer specific adenovirus
variant (Yu et
al., 1999, Cancer Research, 59:4200-4203). Tumor volumes were documented and
indicated that only CV787 had a significant antitumor efficacy in LNCaP
xenografts, while
tumors in the animals treated with CV890 grew, from 400 mm3 to approximately
1200
mm3 in six weeks, similar to the group treated with vehicle. This study
indicates that
CV890 does not attack LNCaP xenograft and keeps the good specificity profile
under in
vivo conditions.
To evaluate ih vivo antitumor efficacy of CV890, different studies were
carried out
in the nude mouse model harboring human hepatoma xenografts. First, BALB/c
nu/nu
mice with HepG2 or Hep3B xenografts were established, animals were fiu~ther
challenged
with single dose or multiple doses of CV890 into the tumor mass (intratumoral
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administration, IT) or via their tail vein (intravenous administration, IV).
Tumor volume
and the level of serum AFP were monitored weekly after the start of treatment,
and hence
the efficacy of the treatment was determined. The ih vitro cytotoxicity study
has
demonstrated that CV890 has a better cytolytic effect than CV732. In order to
further
examine their antitumor activity, we first conducted animal study to compare
CV890 to
CV732. Animals harboring 300 mm3 Hep3B xenograft were grouped (n=6) and
injected
with vehicle alone (control group), CV890 (1x1011 particlesldose, CV890
group), or
CV732 (1x1011 particles/dose, CV732 group). The Hep3B xenograft is a very
aggressive
tumor model and tumors grow very fast. Most animals can not survive long
because of
excessive tumor burden. During a six week study, single intravenous
administration of
CV890 have shown significant tumor growth inhibition, whereas control mice had
over 10
fold tumor growth at week 5. In the group treated with CV732, single dose IV
injection
also reduced the tumor growth as compared to control group, however, it was
much less
effective compared to CV890. For example, the average tumor volume of the
CV890
treated group dropped from 312 mm3 to 219 mm3, while tumor volume increased
from 308
mm3 to 1542 mrn3 5 weeks after treatment in control. Both control group and
the CV732
group were terminated at week 5 because excessive tumor size. Previously,
CV732 has
been demonstrated to restrict the hepatoma tumor from growth after 5 doses of
intravenous
administration. Similar efficacy can be achieved with just a single
intravenous
administration of CV890, indicating that under in vivo conditions, CV890 has
better
efficacy than CV732 in hepatoma xenografts. In this experiment, 4 out of five
CV890
treated mice were tumor free three weeks after treatment. However, in GV732
group,
xenografts in two mice stopped growing but none of treated animals were tumor
free
through the six-week experiment. There was no tumor reduction in this group or
the
control group of animals. By statistical analysis, the differences in mean
relative tumor
volumes and serum AFP concentrations between CV890 treated and CV732 treated
or
vehicle treated tumors are significant (p<0.01)). Taken together, these
studies suggest that
CV890 has a significant antitumor activity and its oncolytic efficacy is
better than CV732,
an adenovirus variant similar to AvEla04I, in which the AFP THE was applied to
control
ElA alone (Hallenback et al, 1999, Hum. Gene. Ther., I0:172I-1733).
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Synergistic Antitumor Efficacy of CV890 in Combination with Chemotherapeutic
A_ gents,
In this example, different chemotherapeutic agents were tested in combination
with
CV890 for their ih vitro killing effect in Hep3B or HepG2 cells. Drug
concentrations were
optimized to the extent that they would not generate extensive cytotoxic
effect on their
own. Under such conditions, some agents had shown higher cell killing effect
in
combination with CV890. Among them, doxorubicin, a drug currently used in
treatment of
HCC showed synergistic cytotoxicity with CV890. In experiments using
doxorubicin
together with CV890 on Hep3B cells, doxorubicin at lOng/ml did not generate
cytotoxicity,
whereas CV890 at an MOI of 0.01 (pfu/cell) only had about 35% of cell killed
at day 9.
However, when both were applied together, 90% cells were killed 9 days after
treatment.
In order to determine the potential synergistic effect from the combination
treatment, the
MTT cell viability data were subjected to further statistical analysis. Figure
38 shows a
representative ICso isobologram of doxorubicin and CV890 on Hep3B cells at day
5. First,.
the dose-response curves of doxorubicin alone or CV890 alone were made. Based
on the
original theory of Steel and Peckham (I993) and method by Aoe et al. (1999),
three
isoeffect curves (mode I and mode 2a, 2b) were constructed. From this
isobologram,
several data points were in the synergy or additive area, indicating that
combination of
CV890 and doxorubicin provides synergistic effect on killing of Hep3B cells.
Although CV890 alone has good antitumor activity, we applied combination
therapy with doxorubicin for in vivo evaluation of synergy. Animals harboring
300 mm3
Hep3B xenografts were grouped (n=6) and injected with vehicle alone (control
group),
CV890 alone (1x101 particles/dose, CV890 group) doxorubicin alone (l0mg/kg,
doxorubicin group), or CV890 in combination with doxorubicin (combination
group).
Figure 38 shows weekly change of the relative tumor size normalized to 100% at
day 1. In
this experiment, by week six, all animals in the control group had excessive
tumor which
has increased by 700% of baseline, whereas in CV890 group and combination
group,
animals had either tumor free or tumor reduction. Of the eight Hep3B
xenografts, treated
with CV890, three animals (37.5%) had no palpable tumor at week S, another
three animals
had tumor regressed by more than 60%. In combination group, four out of eight
animals
were tumor free from week 5, another four animals had tumor reduction about
90%. All
the animals in the CV890 and combination group were alive and tumor was
suppressed
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even ten weeks following treatment whereas the control animals were sacrificed
for
excessive tumor burden after week 6. Furthermore, CV890 also caused a drop in
the serum
AFP concentration in these mice. Statistical analysis shows that differences
in mean
relative tumor volumes and serum AFP concentrations between CV890 and vehicle
treated
group or combination and doxorubicin treated group are significant at
different times
(p<0.005).
The strong efficacy in the combination treatment shows that single IV
injection of
CV890 in combination of doxorubicin can eradicate aggressive Hep3B xenografts
in most
of the animals.
Table 15. AFP production in different tumor cells
AFP
CELLS (ng/106cells/lOdays)
Hep3B 2645
HepG2 3140 High
HuH7 45 85
SNU449 60
Low
PLC/PRF/5 600
Chang 0
SK-Hep 1 0
HBL 100 0 None
PA-1 0
LoVo 0
Example 21: CV706 in combination with Irradiation produces synergy
Materials and Methods
CeII culture and virus
The human LNCaP (prostate carcinoma), OVCAR-3 (ovary carcinoma) and HBL-
100 (breast epithelia) cell lines were obtained from the American Type Culture
Collection
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(ATCC, Rockville, MD). The human embryonic kidney cell line, 293, which
expresses the
Adenoviral ElA and E1B gene products, was purchased from Microbix Biosystem,
Inc.
(Toronto, Canada). Cells were maintained at 37°C with 5% C02 in RPMI
1640
supplemented with 10% fetal bovine serum (FBS, Hyclone, Utah), 100 units/ml
penicillin
and 100 ~g/ml of streptomycin (Life Technologies, Gaithersburg, MD).
CV706 is a prostate-specific replication competent Adenoviruswariant. One
prostate-specific transcription response element (TRE), the human prostate-
specific antigen
promoter and enhancer (PSE), was inserted upstream of the ElA encoding region
in the
viral genome (Rodriguez et al., 1997, Cancer Research, 57: 2559-2563).
Similarly, CV787
is also a prostate-specific replication competent Adenovirus variant, which
contain two
prostate-specific TREs, the probasin promoter and PSE, inserted upstream of
the ElA and
ElB encoding regions in the viral genome, respectively (Yu et al., 1999,
supra).. Both
CV706 and CV787 are currently in clinical trials for organ-confined prostate
cancer and
metastatic hormone refractory prostate cancer (DeWeese et al., 2001).
Cell Viability and Irradiation
MTT assays were performed to measure cell viability as described by (Yu et al,
1999, supra). Briefly, HBL-100, OVCAR-3 and LNCaP cells (2x104 cells/well, 96
well
plate) were either infected with CV706 or CV787 at various MOI (from 0.0001 to
1) or
treated with irradiation at the indicated dosages. Cells were incubated in
growth medium
for 24 hr to allow for viral replication. After 24 hr, cells were exposed to a
single dose of
y-irradiation (0 ~ 40 Gy) (Mark 1 Research Irradiator Model #1608A, Caesium
137
source). Cell viability was measured at the times indicated by removing the
media and
replacing it with 50 ~,1 of lmg/ml solution of MTT (3-(4,5-Dimethylthiazol-2-
yl)-2,5-
diphenyl-2H tetrazolium bromide) (Sigma, St. Louis, MO) and incubating for 3
hrs at
37°C. After removing the MIT solution, the crystals remaining in the
wells were
solubilized by the addition of 50 g1 of isopropanol and placed in a
30°C incubator for 30
min for the crystals to dissolve. Plates were vibrated for 10 sec prior to
reading. The
absorbency was determined on a microplate reader (Molecular Dynamics) at 560
nm (test
wavelength) and 690 nm (reference wavelength). At least, 8 replica samples
were taken for
each time point and the percentage of surviving cells was estimated by
dividing the ODSSO -
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OD69o of virus infected cells by the OD56o - OD69o of mock infected cells.
Statistical Analysis
The dose-response interactions between CV706 and irradiation at the point of
ICso
were evaluated by the isobologram method of Steel and Peckham as modified by
Aoe et al.
(Aoe et al. 1999, Anticancer Res. 19:291-299). The ICso defined as the
concentration of
drug that produced 50% cell growth inhibition, i.e. 50% reduction in
absorbance.
Isobolograms (three isoefFect curves, model 1 and model 2) were computed as
described
previously (Yu et aL, 2001). Fractional tumor volume (FTV) relative to
untreated controls
was determined based on the method described previously (Yokoyama et al.,
2000; Yu et
al., 2001, Cancer Research).
On Step Growth Curve and Virus Yield
One-step growth curve of CV706 in the presence and absence of irradiation were
performed in LNCaP cells to determine burst size. Monolayers of LNCaP cells
were
infected with CV706 at MOIs 0.01, 0.1 and 1. After 24 hour incubation at
37°C with 5%
C02, cells were exposed to a single dose of y-irradiation at 10 Gy. At the
indicated times
thereafter, duplicate cell samples were harvested and lysed by three cycles of
freeze-
thawing. Virus yield was determined by plaque assay as described in (Yu et
al., 1999,
Cancer Research, 59:1498-1504).
In Vivo Antitumor Efficacy
Six to eight week old athymic Balb/c nu/nu mice were obtained from Simonson
Laboratories (Gilroy, CA) and acclimatized to laboratory conditions one week
prior to
tumor implantation. Xenografts were established either by injecting 1 x 106
LNCaP cells
subcutaneously near the small of the back suspended in 100 p1 of RPMI 1640 and
100 p1 of
matrigel (back tumor) or by injecting cells into the right gastrocnemius
muscle (i.m.) (leg
tumor). When tumors reached between 300 mm3 and 500 mm3, mice were randomized
into
groups of four. The first group received CV706 at day 0 via intratumoral
(i.t.)
administration. CV706 was diluted by PBS containing 10% glycerol and injected
into
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tumor as 0.4 ~l of diluted virus (Ixl O~ particles) per mm3 of tumor using a
28-gauge
needle. The second group was given irradiation only. For irradiation mice were
immobilized in Lucite chambers and their whole body was shielded with lead
except for the
tumor bearing sites on their back or tumor-bearing hind leg. This tumor-
bearing site in
back or leg was irradiated with a Mark 1 Research Irradiator (Model #1 608A,
J.H.
Shepherd Associates) at various doses (0, 5, 10 and 20 Gy) 1 day after CV706
injection or
mock injection. The third group was given CV706 (i.t.) at day 0 and irradiated
at the same
doses at day 1. As a control, a fourth group was treated with virus dilution
buffer (i.e.
control) i.t. at day 0. Tumors were measured weekly in two dimensions by
external caliper
and volume for back tumors was estimated by the formula [length (mm) x width
(mm)a~/2
(Yu et al., 1999b). Volumes of i.m. leg tumors were determined using the
following
formula (Alfieri and Hahn, I978, Cancer Research, 38:3006-3011): volume (cm3)
= d'3-
(0.6)2d', where d' is the average diameter of the tumor-bearing Ieg (cm), and
the product
(0.6)2d' is the correction factor for normal leg volume. Animals were humanely
killed
when their tumor burden became excessive. The difference in relative tumor
volumes
between treatment groups was compared fox statistical significance using the
type 2 (two-
sample equal variance), two-tailed, t-test. Blood samples were collected at
various time
points by retro-orbital bleed for determining prostate-specific antigen.
Federal and
institutional guidelines for animal care were followed.
Histochemistry Analysis
Four groups of mice (n=6) were treated with vehicle, CV706 (1x10' particles
per
mm3 of tumor), irradiation (10 Gy) or a combination of CV706 and irradiation.
Half the
animals were sacrificed on day 7 and the other half on day 14. The tumor
samples were
embedded in paraffin blocks and 4-~.irn sections were cut and stained with
Hematoxylin and
Eosin (H&E). Histology methods for detecting Adenovirus antigens were as
described (Yu
et al., 1999, Cancer Reseaxch, 59:4200-4203). The necrotic cells were scored
on coded
slides at Iight microscopy at x 400 magnification. The number of necrosis was
based on
scoring 500 points per section as either necrotic or nonnecrotic. The average
necrosis score
was calculated based on counting in 10 fields distributed evenly across the
area of tumor
section. The light-microscopic features used to identify necrosis included
cell size,
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indistinct cell border, eosinophilic cytoplasm, loss or condensation of the
nucleus, and
associated inflammation (Milross et aL, 2000). To assess the effect of CV706,
irradiation
or the combination treatment on tumor vascularization, the number of blood
vessels was
counted at a magnification of x 400 and the average blood vessels were
calculated from 10
S fields distributed evenly across the area of whole tumor section. Apoptotic
cells were
detected using TUNEL assay (Ruche Molecular Biochemicals, Indianapolis, IN) as
suggested by the manufacturer. The morphological features used to identify
apoptosis in
the tumor sections have been previously described, associated with positive
terminal
deoxynucleotidyl transferase-mediated nick end labeling staining (Milross, et
al., 2000).
The apoptotic cells were scored on coded slides at x 400 magnification and
average. score
of apoptotic cells was calculated from 10 fields of nonnecrotic areas selected
randomly
across each tumor section.
RESULTS
CV706 in Combination with Irradiation Produce Synergistic Cytotoxicity fn
Prostate
1S Carcinoma LNCaP Cells
To study the potential interaction between a prostate-specific Adenovirus
variant
CV706 and radiation i~ vitro, the effectiveness of combined treatment of
several
combinations of CV706 and irradiation at various doses was evaluated in the
PSA-
producing prostate carcinoma LNCaP cell line. LNCaP cells were either mock-
infected, or
infected with CV706. One day later, cells received a single dose of y-
irradiation (0, SGy,
l OGy and 20Gy) and the cell viability was then determined at various time
points by the
MTT assay. Several viral MOIs and radiation doses were tested to determine the
dose-
response curves in LNCaP cells, such that the selected dose shows greater
combined
efficacy with radiation or virus, but minimal cell killing when treated with
the same dose of
virus alone or radiation alone. Infecting LNCaP cells with CV706 at an MOI of
0.01
resulting in 80% cell survival S days after infection, while irradiation at a
dose of 10 Gy
resulted in 78% survival S days after treatment. However, when CV787 and
radiation were
combined at these doses, cell survival dropped to 20% S days after treatment.
Cell viability
dropped further to 8% 9 days after combination treatment, while cells treated
with virus at
MOI 0.01 alone or radiation 10 Gy alone retained 70% or 60% cell viability,
respectively.
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Isobolograms were generated from the models to determine the presence of
synergy, additivity, or antagonism between CV706 and irradiation. The results
indicate
that sequential exposure to CV706 followed by irradiation produced synergistic
cytotoxicity. The enhanced cytotoxicity was also observed in LNCaP cells when
CV787, a
second prostate-specific Adenovirus variant, was combined with radiation Taken
together,
our ih vitro data demonstrate that prostate-specif c Adenovirus variants in
combination
with irradiation produce synergistic cell cytotoxicity in prostate carcinoma
LNCaP cells.
Irradiation Increases CY706 Burst Size in LNCaP CeIIs
Irradiation kills mammalian cells in the reproductive (also known as
clonogenic)
death pathway. DNA is the target, and double-stranded breaks in the DNA are
regarded as
the specific lesions that initiate this lethal response. Most radiation
induced DNA double-
stranded breaks are rapidly repaired by constitutively expressed DNA repair
mechanisms.
Residual unrepaired or misrepaired breaks lead to genetic instability and to
increased
frequency of mutations and chromosomal aberrations (Garzotto et al., 1999).
Because of
its small target size, the adenoviral genome (36kb) is far less likely to
sustain radiation-
induced damage as it is 1 OS-fold smaller than that of human cells (3x106 kb).
To examine the effect of irradiation on virus replication, we performed a one-
step
growth curve. LNCaP cells were infected with CV706 at an MOI of 0.1 for 24
hrs,
followed by irradiation at a dose of 10 Gy. Cells were harvested at various
times post-
infection and the number of infectious virus particles was determined on 293
cells by
standard plaque assay (Yu et al., 1999, supra). Although the initial rate of
increase of
CV706 in cells treated with CV706 and irradiation was similar to that of cells
treated with
CV706 alone, cells treated with CV706 and irradiation produced a larger burst
size than
CV706 alone. Fox example, cells treated with CV706 and irradiation produced
8,000 PFU
per cell 9 days post-infection, while the cells infected with CV706 alone
generated about
500 PFU per cell 9 days after virus infection. A bigger virus burst size was
also observed
in the combination treatment of irradiation and CV706 at MOIs 0.01 or 1. Cells
treated
with CV706 at MOI of 0.01., and 1 produced 15 and 3500 PFU per cell, whereas
cells
treated with CV706 at MOI of 0.01 and 1 combined with irradiation, produced
4750, and
8700 PFU per cell respectively, at 9 days after virus infection. Thus,
irradiation does not
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inhibit CV706 replication, but significantly increases virus propagation.
Cytotoxicity of CV706 in Combination with Irradiation Remains to Be Specific
to
Prostate Cancer Cells
In order to evaluate whether the addition of radiation could change the
specificity of
CV706's cytotoxic activity, we assess the specificity of the combination
treatment of
CV706 and radiation by measuring viability of various infected cell lines
using the MTT
assay. LNCaP, HBL-100 and OVCAR-3 cells were infected with CV706 at an MOI of
0.01 for 24 hrs, followed by a single dose of radiation at 10 Gy. The
percentage of cell
viability versus time post treatment was plotted. The combination of CV706 and
radiation
was toxic to LNCaP cells, but not to HBL-100 and OVCAR-3 cells. There were few
surviving LNCaP cells 9 days after infection. In contrast, the viability of
HBL-100 and
OVCAR-3 cells treated with CV706 and radiation was more than 90% throughout
the
course of the experiment, similar to that of cells treated with radiation
alone. This data
suggests that combination with irradiation does not alter CV706's specificity.
Synergistic Efficacy of CV706 in Combination with Irradiation Ih T~ivo
The in vivo antitumor efficacy of CV706 in combination with irradiation was
assessed in the LNCaP mouse xenograft model. We have shown previously that a
single
intratumoral administration of CV706 at SxlOg particles per mm3 of tumor can
eliminate
subcutaneous xenograft tumors in 6 weeks (Rodriguez et al., 1997,supra)
Established
human prostate cancer xenografts (LNCaP cells) were treated with either
vehicle, CV706
(1x10' particles/mrn3), irradiation (10 Gy), or both CV706 and irradiation.
Fox the
combination treatment, animals were intratumorally injected with either CV706
or vehicle,
and 24 hours later, animals received a single dose of irradiation. In this
study, a single dose
of l OGy was used because it caused a tumor growth delay in a previous pilot
study. The
dose of 1x10 particles per mm3 of tumor was selected based on our previous
studies on its
antitumor efficacy (Yu et al., 1999, supra.
The tumor volume data shows that there was a significant decrease in tumor
volume
between control and all treatment groups. In all cases although single doses
of CV706 or
irradiation were effective in producing tumor growth inhibition, the
combination of the two
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showed significant tumor regression. For example, tumor volume of the group
treated with
irradiation (10 Gy) was 119.76% of baseline 6 weeks after treatment, while the
tumor
volume of the group treated with CV706 was 97.39% of baseline 6 weeks after
administration. However, when CV706 was combined with irradiation at similar
doses, a
statistically significant drop in the relative tumor volume (4% of baseline )
was observed
(p<0.01). Additionally, relative PSA level in serum of mice was also monitored
for anti-
tumor efficacy. Relative PSA level in mice increased to 370% of baseline 6
weeks after
receiving vehicle treatment, increased to 139% after receiving irradiation
alone, reduced to
84% of baseline after being treated with CV706 alone, whereas the PSA levels
in mice
treated with CV706 and irradiation decreased to less than 1 % of their
starting values within
6 weeks.
After 7 days, combination treatment showed more than additive effect on tumor
growth inhibition at all the time points studied. On day 21, there was more
than 2-fold
improvement in anti-tumor activity in the combination group when compared with
the
expected additive effect. At this time point, both CV706 and irradiation (10
Gy) per se
inhibited tumor growth by 26% and 34%, respectively (fractional tumor volume,
0.7419
mm3 and 0.6645 mm3, respectively) when compared with the control group. This
anti-
tumor activity further improved with time. On day 42, the group treated with
the
combination of CV706 and irradiation showed a 6.69-fold higher inhibition of
tumor
growth over the expected fractional tumor volume. These observation further
strengthen the
idea of synergy between CV706 and irradiation in the eradication of LNCaP
xenografts.
Enhanced antitumor efficacy was also observed in the animal model in which the
prostate cancer tumors are implanted in hind limb of mice. In this study,
tumors were
produced by inoculation of 1x106 cells into limb muscle. Those tumors which
were
attained a volume of 200 mm3 to 300 mm3 were randomized into four groups and
treated as
described above for back tumors. As before the weekly tumor volume
measurements
showed that combination treatment of CV706 and irradiation led to significant
antitumor
activity in comparison to either CV706 or irradiation. For example, tumor
volume of the
group treated with irradiation (20 Gy) was 70% of baseline 4 weeks after
treatment, while
the tumor volume of the group treated with CV706 (5x10' particle per mm3 of
tumor) was
75% of baseline 4 weeks after administration. However, when CV706 was combined
with
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irradiation at these dose levels, the tumor volume dropped to 8% of baseline.
A series of experiments were then designed to examine the effects of various
factors, including the sequencing of the agents, timing of irradiation
following virus
administration and irradiation fractionation. The effect of order of
administration for the
tested agents was examined in an in vivo study using back tumor xenograft
model. LNCaP
xenografts were irradiated 24 hr before or after CV706 administration. Weekly
measured
tumor volume indicated that treatment with CV706 prior to irradiation was
significantly
superior to irradiation followed by CV706.
The second study was designed to evaluate the timing of irradiation following
virus
administration. Tumors were treated with CV706 at day 0 and followed by
irradiation at
various periods of time. The results of average tumor volume indicated that
similar
antitumor efficacy was achieved when tumors treated with CV706 at day 0
following by
irradiation 1 day or 4 days after virus administration, both eliminated tumors
within 6
weeks after treatment. However, the antitumor activity was decreased when the
tumors
were treated with irradiation 7 days after CV706 administration.
The third study was designed to assess the effect of radiation fractionation
on
antitumor efficacy. Animals with human prostate cancer tumors on their backs
were
randomized into five groups. Two of which were treated with either CV706 at
day 0
followed by a single dose of radiation at 10 Gy on day l, or CV706 at day 0
followed with
four fractional doses of radiation at 2.5 Gy on day 1, 2, 6 and 8. Weekly
measured tumor
volume data indicated that both treatments eliminated the pre-existing tumors
6 weeks after
treatment and produced an synergistic antitumor activity when compared to
either agent
alone. However, no significant difference in antitumor efficacy was observed
between
these two combination groups as long as the total doses of irradiation was the
same.
Synergistic antitumor efficacy of CV706 in combination irradiation was further
documented by tumor histological analysis. First of all, more necrotic cells
were observed
in the tumors treated with CV706 plus irradiation compared with either agent
alone. The
amount of necrosis in tumors treated with CV706 alone was higher than control
tumor or
tumor treated with radiation. Evidence of necrosis and multifocal inflammation
was
observed in a small portion of tumors treated with radiation. In the tumor
treated with both
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the virus and radiation, a few virus-infected cells were detected. Most of the
cells in the
sections were empty and virtually devoid of cellular content. Significantly
increases in the
extent of necrosis was a dominant histological feature, which makes up about
95% of the
tumor mass in this treatment group. The average necrosis scores in a x 400
magnification
for the tumors treated with vehicle, radiation, CV706 and both were 5.42.17,
6748.24,
258.2 80.76 and 46I .637.87, respectively. The presence of mass necrosis in
the tumors
treated with CV706 or CV706 plus radiation suggests that the induction of
necrosis greatly
attributes CV706 or CV706 plus radiation's anti-tumor efficacy in vivo.
Student T test
showed that tumor cell necrosis caused by CV706 in combination with radiation
was
significantly greater than by CV706 (p<0.03) and irradiation perse (p<0.0001).
This
observation is in agreement with the number of apoptotic cells observed in the
treated
tumors. The number of apoptotic cells, detected using TUNEL assay (Milross et
al., 2000)
in the tumors treated with CV706 and irradiation is 16-fold higher than
vehicle, 8.8-fold
higher than irradiation and 3.2-fold higher than CV706.
Secondly, a significant reduction in blood vessel numbers was observed in the
tumors treated with CV706 in combination with irradiation. Average number of
blood
vessel observed at a magnification of 400x in tumors treated with vehicle,
CV706, radiation
or the combination of CV706 and radiation were 87.56.3, 27.58.9, 58.53.1 and
4.51.9,
respectively. Significantly reduced numbers of blood vessels in the tumors
treated with
combination in comparison to CV706 alone or irradiation alone (p<0.01) suggest
that the
reduction of tumor vascularization may contribute to enhanced tumor
regression. It is
unclear at this time as to the precise mechanism by which this reduction in
blood vessel
number is achieved. The possibility for such an eventuality through direct
damage of
endothelial cells or indirectly through the destruction of tumor vasculature
by extensive
necrosis seems highly possible. CD31 is expressed constitutively on the
surface of adult
and embryonic endothelial cells and has been used as a marker to detect
angiogenesis
(Giatromanolaki et al., 1997, Clin. Can. Res. 3 (l2pt 1): 2485-92).
Immunohistochemical
staining was performed to examine the effect of treatment on tumor angigenesis
by using
monoclonal antibody against CD31 (Horak et al., 1992). Tumors treated with
CV706
followed by irradiation showed a significantly lower level of CD31 positive
vessel when
compared to radiation (p=0.003) or CV706 alone (p=0.03). When compared to
untreated
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mice, CV706/radiation treated mice exhibited significantly lower (4-fold) CD31
positive
blood vessel counts (p<0.0001), whereas, radiation treated or CV706 treated
mice
displayed 1.6-fold (p=0.03) or 2.1-fold (p=0.004) lower CD3l positive blood
vessel counts.
These observations suggest that CV706 in combination with radiation may be
inhibiting
tumor angiogenesis to a significant extent.
Finally, treatment employing the combination seems to have a beneficial effect
on
the general health of the treated animals in comparison to the individual
treatment. The
quality of life of the treated animals seems to be greatly improved as
evidenced by the
general appearance and significant gain in the body weight. Indeed, animals
treated with
both CV706 and irradiation gain 38% more weight than untreated control
animals, 22%
more than CV706 treated animals and 25% more weight than irradiation treated
animals.
The combination treatment seems to protect the animals from the transient
weight loss
observed in the case of animals treated with irradiation alone.
1f
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Table 12: IRES Sequences
SEQ ID NO:- A S 19 base pair IRES obtainable from
encephelomycarditis
virus
(EMCV).
S 1 GACGTCGACrAATTCCGGTTATTTTCCACCATATTGCCGTCTTTTGGCAA
SalT
51 TGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGG
101 GTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAG
151 GAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTGTAGCGAC
1O 201 CCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCC
251 AAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTGC
301 CACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAG
351 CGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGG
401 GATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTTAGTCGAGG
IS 451 TTAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGA
SalI
502 AAAACACGATGTCGACGTC
SEQ ID NO:~ An LRES obtainable from vascular endothelial growth
20 factor (VEGF).
1 ACGTAGTCGACAGCGCAGAGGCTTGGGGCAGCCGAGCGGCAGCCAGGCCC
Sall
51 CGGCCCGGGCCTCGGTTCCAGAAGGGAGAGGAGCCCGCCAAGGCGCGCAA
101 GAGAGCGGGCTGCCTCGCAGTCCGAGCCGGAGAGGGAGCGCGAGCCGCGC
2S 151 CGGCCCCGGACGGCCTCCGAAACCATGGTCGACACGTA
Sall
SEQ ID NO:- A S'UTR region of HCV.
1 GCCAGCCCCCTGATGGGGGCGACACTCCGCCATGAATCACTCCCCTGTGAGGAACTACTG
3O 61 TCTTCACGCAGAAAGCGTCTAGCCATGGCGTTAGTATGAGTGTCGTGCAGCCTCCAGGAC
121 CCCCCCTCCCGGGAGAGCCATAGTGGTCTGCGGAACCGGTGAGTACACCGGAATTGCCAG
187. GACGACCGGGTCCTTTCTTGGATTAACCCGCTCAATGCCTGGAGATTTGGGCGTGCCCCC
241 GCAAGACTGCTAGCCGAGTAGTGTTGGGTCGCGAAAGGCCTTGTGGTACTGCCTGATAGG
301 GTGCTTGCGAGTGCCCCGGGAGGTCTCGTAGACCGTGCACC (341)
3S
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SEQ ID NO:- A S'UTR region of BiP SEQ ID N0:4
1 CCCGGGGTCACTCCTGCTGGACCTACTCCGACCCCCTAGGCCGGGAGTGAAGGCGGGACT
61 TGTGCGGTTACCAGCGGAAATGCCTCGGGGTCAGAAGTCGCAGGAGAGATAGACAGCTGC
121 TGAACCAATGGGACCAGCGGATGGGGCGGATGTTATCTACCATTGGTGAACGTTAGAAAC
S 181 GAATAGCAGCCAATGAATCAGCTGGGGGGGCGGAGCAGTGACGTTTATTGCGGAGGGGGC
241 CGCTTCGAATCGGCGGCGGCCAGCTTGGTGGCCTGGGCCAATGAACGGCCTCCAACGAGC
301 AGGGCCTTCACCAATCGGCGGCCTCCACGACGGGGCTGGGGGAGGGTATATAAGCCGAGT
361 AGGCGACGGTGAGGTCGACGCCGGCCAAGACAGCACAGACAGATTGACCTATTGGGGTGT
421 TTCGCGAGTGTGAGAGGGAAGCGCCGCGGCCTGTATTTCTAGACCTGCCCTTCGCCTGGT
IO 481 TCGTGGCGCC'T'TGTGACCCCGGGCCCCTGCCGCCTGCAAGTCGAAATTGCGCTGTGCTCC
541 TGTGCTACGGCCTGTGGCTGGACTGCCTGCTGCTGCCCAACTGGCTGGCAAGATG
(595)
SEQ ID NO:- A S'UTR of PDGF SEQ ID NO:S
1 GTTTGCACCTCTCCCTGCCCGGGTGCTCGAGCTGCCGTTGCAAAGCCAACTTTGGAAAAA
IS 61 GTTTTTTGGGGGAGACTTGGGCCTTGAGGTGCCCAGCTCCGCGCTTTCCGATTTTGGGGG
121 CTTTCCAGAAAATGTTGCAAAAAAGCTAAGCCGGCGGGCAGAGGAAAACGCCTGTAGCCG
181 GCGAGTGAAGACGAACCATCGACTGCCGTGTTCCTTTTCCTCTTGGAGGTTGGAGTCCCC
241 TGGGCGCCCCCACACCCCTAGACGCCTCGGCTGGTTCGCGACGCAGCCCCCCGGCCGTGG
301 ATGCTGCACTCGGGCTCGGGATCCGCCCAGGTAGCCGGCCTCGGACCCAGGTCCTGCGCC
ZO 361 CAGGTCCTCCCCTGCCCCCCAGCGACGGAGCCGGGGCCGGGGGCGGCGGCGCCGGGGGCA
421 TGCGGGTGAGCCGCGGCTGCAGAGGCCTGAGCGCCTGATCGCCGCGGACCTGAGCCGAGC
481 CCACCCCCCTCCCCAGCCCCCCACCCTGGCCGCGGGGGCGGCGCGCTCGATCTACGCGTC
541 CGGGGCCCCGCGGGGCCGGGCCCGGAGTCGGCATG (575)
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Table 13: Literature References For IRES
IRES Host Example Reference
PicornavirusHAV Glass et al., 1993. Virol 193:842-852
EMCV Jang ~ Wimmer, 1990. Gene Dev 4:1560-1572
Poliovirus Borman et al., 1994. EMBO J 13:3149-3157
HCV and HCV Tsukiyama-Kohara et al., 1992. J
Virol 66:1476-
pestivirus 1483
BVDV Frolov I et al., 1998. RNA. 4:1418-1435
LeishmaniaLRV-1 Maga et al., 1995. Mol Cell Biol
15:4884-4889
virus
RetrovirusesMoMLV Torrent et al., 1996. Hum Gene Ther
7:603-612
VL30 (Harvey
marine sarcoma
virus)
REV Lopez-Lastra et al., 1997. Hum Gene
Ther
8:1855-1865
EukaryoticBiP Macejak & Sarnow, 1991. Nature 353:90-94
mRNA
antennapedia Oh et al., 1992: Gene & Dev 6:1643-1653
mRNA
FGF-2 Vagner et al., 1995. Mol Cell Biol
15:35-44
PDGF-B Bernstein et al., 1997. J Biol Chem
272:9356-
9362
TGFII Teerink et al., 1995. Biochim Biophys
Acta
1264:403-408
eIF4G Gan & Rhoads, 1996. J Biol Chem
271:623-626
VEGF Stein et al., 1998. Mol Cell Biol
18:3112-3119;
Huez et al., 1998. Mol Cell Biol
18:6178-6190
165
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Table 14: THE Sequences
Nucleotide sequence of a human uroplakin II S' flanking region. Position +1
(the
translational start site) is denoted with an asterisk. SEQ ID NO:_ (number 1
of SEQ ID
NO:- corresponds to position -2239 with respect to the translational start
site).
S
TCGATAGGTA CCCACTATAG GGCACGCGTG GTCGACGGCC CGGGCTGGTC
1 50
TGGCAACTTC AAGTGTGGGC CTTTCAGACC GGCATCATCA GTGTTACGGG
51 100
GAAGTCACTA GGAATGCAGA ATTGATTGAG CACGGTGGCT CACACCTGTA
101 150
IS ATCCCAACAC TCTGGGAGGC CAAGGCAGGT GGATCACTTG TGGTCAGGAG
151 200
TTTGAGACCA GCCTGGCCAA CATGGTGAAA. CCTCATCTCT ACTAAAAATA
201 250
CAAAAATTAG CTGGGAATGG TGGCACATGC CTATAATCCC AGTTACTCAG
251 300
GAGGCTGAGG CAGGAGAATC ATTTGAACCT GGGAGGCAGA GGTTGCAGTG
2S 301 350
AGCCGAGATC ACGCCACTGC ACTCCAGCCT GGGTGACACA GCGAGACTCT
351 ~ 400
3O GTCTCAAAAA F~AAA.AAAATG CAGAATTTCA GGCTTCACCC CAGACCCACT
401 450
GCATGACTGC ATGAGAAGCT GCATCTTAAC AAGATCCCTG GTAATTCATA
451 500
3S
CGCATATTAA ATTTGGAGAT GCACTGGCGT AAGACCCTCC TACTCTCTGC
501 550
TTAGGCCCAT GAGTTCTTCC TTTACTGTCA TTCTCCACTC ACCCCAAACT
40 551 600
TTGAGCCTAC CCTTCCCACC TTGGCGGTAA GGACACAACC TCCCTCACAT
601 650
4S TCCTACCAGG ACCCTAAGCT TCCCTGGGAC TGAGGAAGAT AGAATAGTTC
651 700
GTGGAGCAAA CAGATATACA GCAACAGTCT CTGTACAGCT CTCAGGCTTC
166
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701 750
.TGGAAGTTCT ACAGCCTCTC CCGACAAAGT ATTCCACTTT CCACAAGTAA
751 800
CTCTATGTGT CTGAGTCTCA GTTTCCACTT TTCTCTCTCT CTCTCTCTCT
801 850
CAACTTTCTG AGACAGAGTT TCACTTAGTC GCCCAGGCTG GAGTGCAGGG
857. 900
GCACAATCTC GGCTCACTGC AACCTCCACC TCCTGGGTTC AAGTGTTTCT
901 950
CCTGTCTCAG CCTCCCGAGT AGCTGGGATT ACAGGCACAC ACCACCGCGT
951 1000
. TAGTTTTTGT ATTTTTGGTA GAGATGGTGT TTCGCCATAT TGGCCAGGCT
1001 1050
GATCTCGAAC TCCTGACCTC AGGTGATCCG CCCACCTCGG CCTCCCAAAG
1051 1100
TGCTGGGATT ACAGGCATGA GCCACCACGC CCGGCTGATC TCTTTTCTAT
2S 1101 1150
TTTAATAGAG ATCAAACTCT CTGTGTTGCC TAGGCTGGTC TTGAACTCCT
1151 1200
3O GGCCTCGAGT GATCCTCCCA CCTTGGCCTC CCAAAGTGTT GAGATTACAG
1201 1250
GCATGAGCCA CTGTGCCTGG CCTCAGTTCT ACTACAAAAG GAAGCCAGTA
1251 1300
35
CCAGCTACCA CCCAGGGTGG CTGTAGGGCT ACAATGGAGC ACACAGAACC
1301 1350
CCTACCCAGG GCCCGGAAGA AGCCCCGACT CCTCTCCCCT CCCTCTGCCC
40 1351 1400
AGAACTCCTC CGCTTCTTTC TGATGTAGCC CAGGGCCGGA GGAGGCAGTC
1401 1450
45 AGGGAAGTTC TGTCTCTTTT TCATGTTATC TTACGAGGTC TCTTTTCTCC
1451 1500
ATTCTCAGTC CAACAAATGG TTGCTGCCCA AGGCTGACTG TGCCCACCCC
1501 1550
S0
167
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CAACCCCTGC TGGCCAGGGT CAATGTCTGT CTCTCTGGTC TCTCCAGAAG
1551 1600
TCTTCCATGG CCACCTTCGT CCCCACCCTC CAGAGGAATC TGAAACCGCA
S 1601 1650
TGTGCTCCCT GGCCCCCACA GCCCCTGCCT CTCCCAGAGC AGCAGTACCT
1651 1700
IO AAGCCTCAGT GCACTCCAAG AATTGAAACC CTCAGTCTGC TGCCCCTCCC
1701 1750
CACCAGAATG TTTCTCTCCC ATTCTTACCC ACTCAAGGCC CTTTCAGTAG
1751 1800
1S
CCCCTTGGAG TATTCTCTTC CTACATATCA GGGCAACTTC CAAACTCATC
1801 1850
ACCCTTCTGA GGGGTGGGGG AAAGACCCCC ACCACATCGG GGGAGCAGTC
20 1851 1900
CTCCAAGGAC TGGCCAGTCT CCAGATGCCC GTGCACACAG GAACACTGCC
1901 1950
2S TTATGCACGG GAGTCCCAGA AGAAGGGGTG ATTTCTTTCC CCACCTTAGT
1951 2000
TACACCATCA AGACCCAGCC AGGGCATCCC CCCTCCTGGC CTGAGGGCCA
2001 2050
GCTCCCCATC CTGAAAAACC TGTCTGCTCT CCCCACCCCT TTGAGGCTAT
2051 2100
AGGGCCCAAG GGGCAGGTTG GACTGGATTC CCCTCCAGCC CCTCCCGCCC
3S 2101 2150
CCAGGACAAA ATCAGCCACC CCAGGGGCAG GGCCTCACTT GCCTCAGGAA
2151 2200
4O CCCCAGCCTG CCAGCACCTA TTCCACCTCC CAGCCCAGCA
2201 2239
Nucleotide sequence of a mouse uroplakin II S' flanking region. The
translational
start site is denoted with an asterisk. SEQ ID NO:- (number I of SEQ ID
4S N0:6 corresponds to position -3592 with respect to the translational start
site).
CTCGAGGATCTCGGCCCTCTTTCTGCATCCTTGTCCTAAATCATTTTCAT
168
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1 50
ATCTTGCTAGACCTCAGTTTGAGAGAAACGAACCTTCTCATTTTCAAGTT
51 100
G GAGGTTCAAAGTGGCTCACTCAAAGTTACAAGCCAACAC
101 150
TCACCACTACGAGTACAATGGCCACCATTAGTGCTGGCATGCCCCAGGAG
151 200
ACAGGCATGCATATTATTCTAGATGACTGGGAGGCAGAGGGGTGGCCTAG
201 250
TGAGGTCAGACTGTGGACAGATCAGGCAGATGTGGGTTCTGATCCCAATT
251 300
CCTCAGGCCGCAGAACTACTGTGGTTCAAGAAGGGGACAAAAGGACTGCA
301 350
GTCCGGAACAGGAGGTCCATTTGAGAGCTGACTGAGCAGAAGAGGAAAGT
351 400
GAAGAACTTCTGGGGCAAGAGCTTACCCTACTTTACAGCTTTGTTGTCTT
401 450
CTTTACTCCAGGGGCGTCCCTGGTACTCAGTAAATGTCTGTTGGCTTGAG
451 500
GAACATATGTGTAAGGAGGAAGGAGAGGGAACTTGAGGGAGTTAAGACTC
501 550
AAG.AATCAATCAAGGAGAGGACAGCAGAGAAGACAGGGTTTGGGAGAGAG
551 600
40
ACTCCAGACATTGGCCCTGGTTCCCTTCTTGGCCACTGTGAAACCCTCCA
601 650
GAGGAACTGAGTGCTGTGGCTTTAAATGATCTCAGCACTGTCAGTGAAGC
651 700
GCTCTGCTCAAAGAGTTATCCTCTTGCTCCTGTGCCGGGGCCTCCCCCTC
701 750
CTCTCAGCTCCCAAACCCTTCTCAGCCACTGTGATGGCATAATTAGATGC
751 800
GAGAGCTCAGACCGTCAGGTCTGCTCCAGGAACCACCCATTTTCCCCAAC
801 850
CCCAGAGAAAGGTCCTAGTGGAAA.AGTGGGGGCCACTGAAGGGCTGATGG
851 900
GGTTCTGTCCTTTCCCCCATGCTGGGTGGACTTAAAGTCTGCGATGTGTG
900 950
169
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TAGGGGGTAGAAGACAACAGAACCTGGGGGCTCCGGCTGGGAGCAGGAGG
951 1000
AACTCTCACCAGACGATCTCCAAATTTACTGTGCAATGGACGATCAGGAA
1001 1050
ACTGGTTCAGATGTAGCTTCTGATACAGTGGGTCTGAGGTAAAACCCGAA
1051 1100
ACTTAATTTCTTTCAAAAATTTAAAGTTGCATTTATTATTTTATATGTGT
1101 1150
GCCCATATGTGTGCCACAGTGTCTATGTGGAGGTCAGAGGGCAAGTTGTG
1151 1200
GGCATTGGCTCTCTCCTTTCATAATGTGGCTTCTGGGGACCAAAATGTCA
1201 1250
GGCATGGTGGCAAGAGCTTTTACCTGTTGAGCCATCTCATGGTTTCGTAA
2252 1300
AACTTCCTATGACGCTTACAGGTAACGCAGAGACACAGACTCACATTTGG
1301 1350
AGTTAGCAGATGCTGTATTGGTGTAAACACTCATACACAGACACACACAC
1351' 1400
ATACTCATACACACACACACACACTTATCACATGCACACACATACTCGTA
1401 1450
TACACACAGACACACACACATGCACTCTCACATTCACATATTCATACACA
1451 1500
TCCACACACACACTCATCCACACACACAGACACACATACTCATCCACACA
1501 1550
CACACACACACATACTCATACACACACACAGACACACATACTCATACACA
1551 1600
CACACAGACACACACATATAATCATACATACACAGACACACTCATACATG
1601 1650
TGCACACACACACTCATCCACACACACACACTCATACACACACACACTCA
1651 1700
TACACACACACACTCATACACACACACACGAGGTTTTTCTCAGGCTGCCT
1701 1750
TTGGGTGGAGACTGGAACTGATTTCTGTTTTTCAGCTCCTTGGCTTTTTG
1751 1800
TCCCTTTAGATGAGATCTCCTCCTCACTTTACACACAGAAAGATCACACA
1801 1850
CGAGGGAGAACTGGCGGTGCGGAAGAGGGCTACACGGTAGGGTGTCAGGG
170
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1851 1900
TCAGGAGATCTTCCTGGCAAGTCTCAAACCTCCACATAGCACAGTGTTTA
1901 1950
CGTGAGGATTTAGGAGGAATCAGGAAGAGGATTGGTTTACTGCAGAGCAG
1952 2000
ACCATATAGGTCCACTCCTAAGCCCCATTTGAAATTAGAAGTGAGACAGT
2001 2050
GTGGGATAAAAAGAGCAGATCTCTGGTCACATTTTTAAAGGGATATGAGG
2051 3000
GTCCTGTGCCTTTAAGCCTTCCCATCTCCCTCCAATCCCCCCTCACCTTC
2101 2150
CCCACCCTAACCCTCCCCAGGTTTCTGGAGGAGCAGAGTTGCGTCTTCTC
2151 2200
CCTGCCCTGCCGAGCTGCTCACTGGCTGCTCTAGAGGCTGTGCTTTGCGG
2201 2250
TCTCCATGGAAACCATTAGTTGCTAAGCAACTGGAGCATCATCTGTGCTG
2251 2300
AGCTCAGGTCCTATCGAGTTCACCTAGCTGAGACACCCACGCCCCTGCAG
2301 2350
CCACTTTGCAGTGACAAGCCTGAGTCTCAGGTTCTGCATCTATAAAAACG
2351 2400
AGTAGCCTTTCAGGAGGGCATGCAGAGCCCCCTGGCCAGCGTCTAGAGGA
2401 2450
GAGGTGACTGAGTGGGGCCATGTCACTCGTCCATGGCTGGAGAACCTCCA
2451 2500
TCAGTCTCCCAGTTAGCCTGGGGCAGGAGAGAACCAGAGGAGCTGTGGCT
2501 2550
GCTGATTGGATGATTTACGTACCCAATCTGTTGTCCCAGGCATCGAACCC
2551 2600
CAGAGCGACCTGCACACATGCCACCGCTGCCCCGCCCTCCACCTCCTCTG
2601 2650
CTCCTGGTTACAGGATTGTTTTGTCTTGAAGGGTTTTGTTGTTGCTACTT
2651 2700
TTTGCTTTGTTTTTTCTTTTTTAACATAAGGTTTCTCTGTGTAGCCCTAG
2701 2750
CTGTCCTGGAACTCACTCTGTAGACCAGGCTGGCCTCAAACTCAGAAATC
2751 2800
171
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CACCTTCCTCCCAAGTGCTGGGATTAAAGGCATTCGCACCATCGCCCAGC
2801 2850
CCCCGGTCTTGTTTCCTAAGGTTTTCCTGCTTTACTCGCTACCCGTTGCA
2851 2900
CAACCGCTTGCTGTCCAAGTCTGTTTGTATCTACTCCACCGCCCACTAGC
2901 2950
CTTGCTGGACTGGACCTACGTTTACCTGGAAGCCTTCACTAACTTCCCTT
2951 3000
GTCTCCACCTTCTGGAGAAATCTGAAGGCTCACACTGATACCCTCCGCTT
3001 3050.
CTCCCAGAGTCGCAGTTTCTTAGGCCTCAGTTAAATACCAGAATTGGATC
3051 3100
TCAGGCTCTGCTATCCCCACCCTACCTAACCAACCCCCTCCTCTCCCATC
3101 3150
CTTACTAGCCAAAGCCCTTTCAACCCTTGGGGCTTTTCCTACACCTACAC
3151 3200
ACCAGGGCAATTTTAGAACTCATGGCTCTCCTAGAAA.ACGCCTACCTCCT
3201 3250
TGGAGACTGACCCTCTACAGTCCAGGAGGCAGACACTCAGACAGAGGAAC
3251 3300
TCTGTCCTTCAGTCGCGGGAGTTCCAGAAAGAGCCATACTCCCCTGCAGA
3301 3350
GCTAACTAAGCTGCCAGGACCCAGCCAGAGCATCCCCCTTTAGCCGAGGG
3351 3400
CCAGCTCCCCAGAATGAAAAACCTGTCTGGGGCCCCTCCCTGAGGCTACA
3401 3450
GTCGCCAAGGGGCAAGTTGGACTGGATTCCCAGCAGCCCCTCCCACTCCG
3451 3500
AGACAAAATCAGCTACCCTGGGGCAGGCCTCATTGGCCCCAGGAAACCCC
3501 3550
AGCCTGTCAGCACCTGTTCCAGGATCCAGTCCCAGCGCAGTA
3551
3592
AFP-TRE. SEQ ID NO:-
1 GCATTGCTGTGAACTCTGTACTTAGGACTAAACTTTGAGCAATAACACACATAGATTGAG
61 GATTGTTTGCTGTTAGCATACAAACTCTGGTTCAAAGCTCCTCTTTATTGCTTGTCTTGG
172
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l21 AAAATTTGCTGTTCTTCATGGTTTCTCTTTTCACTGCTATCTATTTTTCTCAACCACTCA
181 CATGGCTACAATAACTGTCTGCAAGCTTATGATTCCCAAATATCTATCTCTAGCCTCAAT
S 241 CTTGTTCCAGAAGATAAAAAGTAGTATTCAAATGCACATCAACGTCTCCACTTGGAGGGC
301 TTAAAGACGTTTCAACATACAAACCGGGGAGTTTTGCCTGGAATGTTTCCTAAAATGTGT
361 CCTGTAGCACATAGGGTCCTCTTGTTCCTTAAAATCTAATTACTTTTAGCCCAGTGCTCA
421 TCCCACCTATGGGGAGATGAGAGTGAAAAGGGAGCCTGATTAATAATTACACTAAGTCAA
481 TAGGCATAGAGCCAGGACTGTTTGGGTAAACTGGTCACTTTATCTTAAACTAAATATATC
IS 541 CAAAACTGAACATGTACTTAGTTACTAAGTCTTTGACTTTATCTCATTCATACCACTCAG
601 CTTTATCCAGGCCACTTATGAGCTCTGTGTCCTTGAACATAAAATACAAATAACCGCTAT
661 GCTGTTAATTATTGGCAAATGTCCCATTTTCAACCTAAGGAAATACCATAAAGTAACAGA
721 TATACCAACAAAAGGTTACTAGTTAACAGGCATTGCCTGAAAAGAGTATAAAAGAATTTC
781 AGCATGATTTTCCATATTGTGCTTCCACCACTGCCAATAACA (822)
2S
Probasin -THE SEQ ID NO:-
-426
5'-AAGCTTCCACAAGTGCATTTAGCCTCTCCAGTATTGCTGATGAATCCACAGT
TCAGGTTCAATGGCGTTCAAAACTTGATCAAAAATGACCAGACTTTATATTTA
3S CACCAACATCTATCTGATTGGAGGAATGGATAATAGTCATCATGTTTAAACAT
CTACCATTCCAGTTAAGAAAATATGATAGCATCTTGTTCTTAGTCTTTTTCTTA
ARE-1
4O ATAGGGACATAAAGCCCACAAATAAA.AATATGCCTGAAGAATGGGACAGGC
ATTGGGCATTGTCCATGCCTAGTAAAGTACTCCAAGAACCTATTTGTATACTA
ARE-2
4S GATGACACAATGTCAATGTCTGTGTACAACTGCCAACTGGGATGCAAGACAC
TGCCCATGCCAATCATCCTGAAAAGCAGCTATAAAAAGCAGGAAGCTACTCT
CART box TATAA box
S0 +1 +28
GCAC_CTTGTCAGTAGGTCCAGATACCTACAG-3'
Transcription site
SS
173
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Tyrosinase-THE SEQ ID NO:
PinAl end
1 CCGG AAAATGATAAGTTGAATTCTGTCTTCGAGAACATAGAAAAGAA
51 TTATGAAATGCCAACATGTGGTTACAAGTAATGCAGACCCAAGGCTCCCC
S I01 AGGGACAAGAAGTCTTGTGTTAACTCTTTGTGGCTCTGAAAGAAAGAGAG
151 AGAGAAAAGATTAAGCCTCCTTGTGGAGATCATGTGATGACTTCCTGATT
201 CCAGCCAGAGCGAGCATTTCCATGGAAACTTCTCTTCCTCTT TCGA BOG
3) TTACTAACCTTATTGTTAATATTCTAACCATAAGAATTAAACTATTAA.T
301 GGTGAATAGAGTTTTTCACTTTAACATAGGCCTATCCCACTGGTGGGATA
1O 351 CGAGCCAATTCGAAAGAAAAAGTCAGTCATGTGCTTTTCAGAGGATGAAA
401 GCTTAAGATAA.AGACTAAAAGTGTTTGATGCTGGAGGTGGGAGTGGTATT
451 ATATAGGTCTCAGCCAAGACATGTGATAATCACTGTAGTAGTAGCTGGAA
501 AGAGAAATCTGTGACTCCAATTAGCCAGTTCCTGCAGACCTTGTG'~ ~~0
PinAI end
15 Human glandular kallikrein-THE SEQ ID NO:-
gaattcagaa ataggggaag gttgaggaag gacactgaac tcaaagggga tacagtgatt 60
ggtttatttg tcttctcttc acaacattgg tgctggagga attcccaccc tgaggttatg 120
aagatgtctg aacacccaac acatagcact ggagatatga gctcgacaag agtttctcag 180
ccacagagat tcacagccta gggcaggagg acactgtacg ccaggcagaa tgacatggga 240
2S attgcgctca cgattggctt gaagaagcaa ggactgtggg aggtgggctt tgtagtaaca 300
agagggcagg gtgaactctg attcccatgg gggaatgtga tggtcctgtt acaaattttt 360
caagctggca gggaataaaa cccattacgg tgaggacctg tggagggcgg ctgccccaac 420
tgataaagga aatagccagg tgggggcctt tcccattgta ggggggacat atctggcaat 480
agaagccttt gagacccttt agggtacaag tactgaggca gcaaataaaa tgaaatctta 540
3$ tttttcaact ttatactgca tgggtgtgaa gatatatttg tttctgtaca gggggtgagg 600
gaaaggaggg gaggaggaaa gttcctgcag gtctggtttg gtcttgtgat ccagggggtc 660
ttggaactat ttaaattaaa ttaaattaaa acaagcgact gttttaaatt aaattaaatt 720
aaattaaatt ttactttatt ttatcttaag ttctgggcta catgtgcagg acgtgcagct 780
ttgttacata ggtaaacgtg tgccatggtg gtttgctgta cctatcaacc catcacctag 840
4S gtattaagcc cagcatgcat tagctgtttt tcctgacgct ctccctctcc ctgactccca 900
caacaggccc cagtgtgtgt tgttcccctc cctgtgtcca tgtgttctca ttgttcagct 960
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cccacttata agtgagaaca tgtggtgttt ggttttctgt ttctgtgtta gtttgctgag 1020
gataatggct tccacctcca tccatgttcc tgcaaaggac gtgatcttat tcttttttat 1080
ggttgcatag aaattgtttt tacaaatcca attgatattg tatttaatta caagttaatc 1140
taattagcat actagaagag attacagaag atattaggta cattgaatga ggaaatatat 1200
aaaataggac gaaggtgaaa tattaggtag gaaaagtata atagttgaaa gaagtaaaaa 1260
aaaatatgca tgagtagcag aatgtaaaag aggtgaagaa cgtaatagtg actttttaga 1320
ccagattgaa ggacagagac agaaaaattt taaggaattg ctaaaccatg tgagtgttag 1380
IS aagtacagtc aataacatta aagcctcagg aggagaaaag aataggaaag gaggaaatat 1440
gtgaataaat agtagagaca tgtttgatgg attttaaaat atttgaaaga cctcacatca 1500
aaggattcat accgtgccat tgaagaggaa gatggaaaag ccaagaagcc agatgaaagt 1560
a
tagaaatatt attggcaaag cttaaatgtt aaaagtccta gagagaaagg atggcagaaa 1620
tattggcggg aaagaatgca gaacctagaa tataaattca tcccaacagt ttggtagtgt 1680
gcagctgtag ccttttctag ataatacact attgtcatac atcgcttaag cgagtgtaaa 1740
atggtctcct cactttattt atttatatat ttatttagtt ttgagatgga gcctcgctct 1800
gtctcctagg ctggagtgca atagtgcgat accactcact gcaacctctg cctcctctgt 1860
tcaagtgatt ttcttacetc agcctcccga gtagctggga ttacaggtgc gtgccaccac 1920
acccggctaa tttttgtatt ttttgtagag acggggtttt gccatgttgg ccaggctggt 1980
cttgaactcc tgacatcagg tgatccacct gccttggcct cctaaagtgc tgggattaca 2040
ggcatgagcc accgtgccca accactttat ttatttttta tttttatttt taaatttcag 2100
cttctatttg aaatacaggg ggcacatata taggattgtt acatgggtat attgaactca 2160
ggtagtgatc atactaccca acaggtaggt tttcaaccca ctccccctct tttcctcccc 2220
attctagtag tgtgcagtgt ctattgttct catgtttatg tctatgtgtg ctccaggttt 2280
4S agctcccacc tgtaagtgag aacgtgtggt atttgatttt ctgtccctgt gttaattcac 2340
ttaggattat ggcttccagc tccattcata ttgctgtaaa ggatatgatt catttttcat 2400
ggccatgcag tattccatat tgcgtataga tcacattttc tttctttttt ttttttgaga 2460
SO
cggagtcttg ctttgctgcc taggctggag tgcagtagca cgatctcggc tcactgcaag 2520
cttcacctcc ggggttcacg tcattcttct gtctcagctt cccaagtagc tgggactaca 2580
55 ggcgcccgcc accacgtccg gctaattttt ttgtgtgttt ttagtagaga tgggggtttc 2640
actgtgttag ccaggatggt cttgatctcc tgaccttgtg gtccacctgc ctcggtctcc 2700
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caaagtgctg ggattacagg ggtgagccac tgcgcccggc ccatatatac cacattttct 2760
ttaaccaatc caccattgat gggcaactag gtagattcca tggattccac agttttgcta 2820
S ttgtgtgcag tgtggcagta gacatatgaa tgaatgtgtc tttttggtat aatgatttgc 2880
attcctttgg gtatacagtc attaatagga gtgctgggtt gaacggtggc tctgtttaaa 2940
attctttgag aattttccaa actgtttgcc atagagagca aactaattta catttccacg 3000
aacagtatat aagcattccc ttttctccac agctttgtca tcatggtttt tttttttctt 3060
tattttaaaa aagaatatgt tgttgttttc ccagggtaca tgtgcaggat gtgcaggttt 3120
gttacatagg tagtaaacgt gagccatggt ggtttgctgc acctgtcaac ccattacctg 3180
ggtatgaagc cctgcctgca ttagctcttt tccctaatgc tctcactact gccccaccct 3240
caccctgaca gggcaaacag acaacctaca gaatgggagg aaatttttgc aatctattca 3300
w
tctgacaaag gtcaagaata tccagaatct acaaggaact taagcaaatt tttacttttt 3360
aataatagcc actctgactg gcgtgaaatg gtatctcatt gtggttttca tttgaatttc 3420
2S tctgatgatc agtgacgatg agcatttttt catatttgtt ggctgcttgt acgtcttttg.3480
agaagtgtct cttcatgcct tttggccact ttaatgggat tattttttgc tttttagttt 3540
aagttcctta tagattctgg atattagact tcttattgga tgcatagttt gtgaatactc 3600
tcttccattc tgtaggttgt ctgtttactc tattgatggc ttcttttgct gtgccgaagc 3660
atcttagttt aattagaaac cacctgccaa tttttgtttt tgttgcaatt gcttttgggg 3720
3S acttagtcat aaactctttg ccaaggtctg ggtcaagaag agtatttcct aggttttctt 3780
ctagaatttt gaaagtctga atgtaaacat ttgcattttt aatgcatctt gagttagttt 3840
ttgtatatgt gaaaggtcta ctctcatttt ctttccctct ttctttcttt ctttcttttc 3900
tttCtttCtt tCtttCtttC tttCtttCtt tCtttCtttC tttCtttttg tCCttCtttC 3960
tttctttctt tctctttctt tctctctttc tttttttttt ttgatggagt attgctctgt 4020
4S tgcccaggct gcagtgcagc ggcacgatct cggctcactg caacctctgc ctcctgggtt 4080
SO
caactgattc tcctgcatca gccttccaag tagctgggat tataggcgcc cgccaccacg 4140
cccgactaat ttttgtattt ttagtagaga cggggttgtg ccatgttggc caggctggtt 4200
tgaaactcct gacctcaaac gatctgcctg ccttggcctc ccaaagtgct gggattacag 4260
gtgtgagcca ctgtgcccag ccaagaatgt cattttctaa gaggtccaag aacctcaaga 4320
5S tattttggga ccttgagaag agaggaattc atacaggtat tacaagcaca gcctaatggc 4380
aaatctttgg catggcttgg cttcaagact ttaggctctt aaaagtcgaa tccaaaaatt 4440
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tttataaaag ctccagctaa gctaccttaa aaggggcctg tatggctgat cactcttctt 4500
gctatacttt acacaaataa acaggccaaa tataatgagg ccaaaattta ttttgcaaat 4560
aaattggtcc tgctatgatt tactcttggt aagaacaggg aaaatagaga aaaatttaga 4620
ttgcatctga cctttttttc tgaattttta tatgtgccta caatttgagc taaatcctga 4680
attattttct ggttgcaaaa actctctaaa gaagaacttg gttttcattg tcttcgtgac 4740
acatttatct ggctctttac tagaacagct ttcttgtttt.tggtgttcta gcttgtgtgc 4800
cttacagttc tactcttcaa attattgtta tgtgtatctc atagttttcc ttcttttgag 4860
aaaactgaag ccatggtatt ctgaggacta gagatgactc aacagagctg gtgaatctcc 4920
tcatatgcaa tccactgggc tcgatctgct tcaaattgct gatgcactgc tgctaaagct 4980
atacatttaa aaccctcact aaaggatcag ggaccatcat ggaagaggag gaaacatgaa 5040
attgtaagag ccagattcgg ggggtagagt gtggaggtca gagcaactec accttgaata 5100
agaaggtaaa gcaacctatc ctgaaagcta acctgccatg gtggcttctg attaacctct 5160
~,S gttctaggaa gactgacagt ttgggtctgt gtcattgccc aaatctcatg ttaaattgta 5220
atccccagtg ttcggaggtg ggacttggtg gtaggtgatt cggtcatggg agtagatttt 5280
cttctttgtg gtgttacagt gatagtgagt gagttctcgt gagatctggt catttaaaag 5340
tgtgtggccc ctcccctccc tctcttggtc ctcctactgc catgtaagat acctgctcct 5400
gctttgcctt ctaccataag taaaagcccc ctgaggcctc cccagaagca gatgccacca 5460
tgcttcctgt acagcctgca gaaccatcag ccaattaaac ctcttttctg tataaattac 5520
cagtcttgag tatctcttta cagcagtgtg agaacggact aatacaaggg tctccaaaat 5580
tccaagttta tgtattcttt cttgccaaat agcaggtatt taccataaat cctgtcctta 5640
ggtcaaacaa ccttgatggc atcgtacttc aattgtctta cacattcctt ctgaatgact 5700
cctcccctat ggcatataag ccctgggtct tgggggataa tggcagaggg gtccaccatc 5760
ttgtctggct gccacctgag,acacggacat ggcttctgtt ggtaagtctc tattaaatgt 5820
ttctttctaa gaaactggat ttgtcagctt gtttctttgg cctctcagct tcctcagact 5880
ttggggtagg ttgcacaacc ctgcccacca cgaaacaaat gtttaatatg ataaatatgg 5940
atagatataa tccacataaa taaaagctct tggagggccc tcaataattg ttaagagtgt 6000
aaatgtgtcc aaagatggaa aatgtttgag aactactgtc ccagagattt tcctgagttc 6060
SS tagagtgtgg gaatatagaa cctggagctt ggcttcttca gcctagaatc aggagtatgg 6120
ggctgaagtc tgaagcttgg cttcagcagt ttggggttgg cttccggagc acatatttga 6180
177
CA 02404060 2002-09-23
WO 01/72341 PCT/USO1/09042
catgttgcga ctgtgatttg gggtttggta tttgctctga atcctaatgt ctgtccttga 6240
ggcatctaga atctgaaatc tgtggtcaga attctattat cttgagtagg acatctccag 6300
tcctggttct gccttctagg gctggagtct gtagtcagtg acccggtctg gcatttcaac 6360
ttcatataca gtgggctatc ttttggtcca tgtttcaacc aaacaaccga ataaaccatt 6420
agaacctttc cccacttccc tagctgcaat gttaaaccta ggatttctgt ttaataggtt 6480
catatgaata atttcagcct gatccaactt tacattcctt ctaccgttat tctacaccca 6540
ccttaaaaat gcattcccaa tatattccct ggattctacc tatatatggt aatcctggct 6600
1S ttgccagttt ctagtgcatt aacatacctg atttacattc ttttacttta aagtggaaat 6660
aagagtccct ctgcagagtt caggagttct caagatggcc cttacttctg acatcaattg 6720
agatttcaag ggagtcgcca agatcatcct caggttcagt gattgctggt agccctcata 6780
taactcaatg aaagctgtta tgctcatggc tatggtttat tacagcaaaa gaatagagat 6840
gaaaatctag caagggaaga gttgcatggg gcaaagacaa ggagagctcc aagtgcagag 6900
attcctgttg ttttctccca gtggtgtcat ggaaagcagt atcttctcca tacaatgatg 6960
tgtgataata ttcagtgtat tgccaatcag ggaactcaac tgagccttga ttatattgga 7020
gcttggttgc acagacatgt cgaccacctt catggctgaa ctttagtact tagcccctcc 7080
agacgtctac agctgatagg ctgtaaccca acattgtcac cataaatcac attgttagac 7140
tatccagtgt ggcccaagct cccgtgtaaa cacaggcact ctaaacaggc aggatatttc 7200
aaaagcttag agatgacctc ccaggagctg aatgcaaaga cctggcctct ttgggcaagg 7260
agaatccttt accgcacact ctccttcaca gggttattgt gaggatcaaa tgtggtcatg 7320
tgtgtgagac accagcacat gtctggctgt ggagagtgac ttctatgtgt gctaacattg 7380
ctgagtgcta agaaagtatt aggcatggct ttcagcactc acagatgctc atctaatcct 7440
cacaacatgg ctacagggtg ggcactacta gcctcatttg acagaggaaa ggactgtgga 7500
a
4S taagaagggg gtgaccaata ggtcagagtc attctggatg caaggggctc cagaggacca 7560
SO
tgattagaca ttgtctgcag agaaattatg gctggatgtc tctgccccgg aaagggggat 7620
gcactttcct tgacccccta tctcagatct tgactttgag gttatctcag acttcctcta 7680
tgataccagg agcccatcat aatctctctg tgtcctctcc ccttcctcag tcttactgcc 7740
cactcttccc agctccatct ccagctggcc aggtgtagcc acagtaccta actctttgca 7800
55 gagaactata aatgtgtatc ctacagggga gaaaaaaaaa aagaactctg aaagagctga 7860
cattttaccg acttgcaaac acataagcta acctgccagt tttgtgctgg tagaactcat 7920
178
CA 02404060 2002-09-23
WO 01/72341 PCT/USO1/09042
gagactcctg ggtcagaggc aaaagatttt attacccaca gctaaggagg cagcatgaac 7980
tttgtgttca catttgttca ctttgccccc caattcatat gggatgatca gagcagttca 8040
S ggtggatgga cacaggggtt tgtggcaaag gtgagcaacc taggcttaga aatcctcaat 8100
cttataagaa ggtactagca aacttgtcca gtctttgtat ctgacggaga tattatcttt 8160
ataattgggt tgaaagcaga cctactctgg aggaacatat tgtatttatt gtcctgaaca 8220
gtaaacaaat ctgctgtaaa atagacgtta actttattat ctaaggcagt aagcaaacct 8280
agatctgaag gcgataccat cttgcaaggc tatctgctgt acaaatatgc ttgaaaagat 8340
ggtccagaaa agaaaacggt attattgcct ttgctcagaa gacacacaga aacataagag 8400
aaccatggaa aattgtctcc caacactgtt cacccagagc cttccactct tgtctgcagg 8460
acagtcttaa catcccatca ttagtgtgtc taccacatct ggcttcaccg tgcctaacca 8520
agatttctag gtccagttcc ccaccatgtt tggcagtgcc ccactgccaa ccccagaata 8580
agggagtgct cagaattccg aggggacatg ggtggggatc agaacttctg ggcttgagtg 8640
2S cagagggggc ccatactcct'tggttccgaa ggaggaagag gctggaggtg aatgtccttg 8700
gaggggagga atgtgggttc tgaactctta aatccccaag ggaggagact ggtaaggtcc 8760
cagcttccga ggtactgacg tgggaatggc ctgagaggtc taagaatccc gtatcctcgg 8820
gaaggagggg ctgaaattgt gaggggttga gttgcagggg tttgttagct tgagactcct 8880
tggtgggtcc ctgggaagca aggactggaa ccattggctc cagggtttgg tgtgaaggta 8940
atgggatctc ctgattctca aagggtcaga ggactgagag ttgcccatgc tttgatcttt 9000
ccatctactc cttactccac ttgagggtaa tcacctactc ttctagttcc acaagagtgc 9060
gcctgcgcga gtataatctg cacatgtgcc atgtcccgag gcctggggca tcatccactc 9120
atcattcagc atctgcgcta tgcgggcgag gccggcgcca tgacgtcatg tagctgcgac 9180
tatccctgca gcgcgcctct cccgtcacgt cccaaccatg gagctgtgga cgtgcgtccc 9240
ctggtggatg tggcctgcgt ggtgccaggc cggggcctgg tgtccgataa agatcctaga 9300
accacaggaa accaggactg aaaggtgcta gagaatggcc atatgtcgct gtccatgaaa 9360
tctcaaggac ttctgggtgg agggcacagg agcctgaact tacgggtttg ccccagtcca 9420
ctgtcctccc aagtgagtct cccagatacg aggcactgtg ccagcatcag cttcatctgt 9480
accacatctt gtaacaggga ctacccagga ccctgatgaa caccatggtg tgtgcaggaa 9540
SS gagggggtga aggcatggac tcctgtgtgg tcagagccca gagggggcca tgacgggtgg 9600
ggaggaggct gtggactggc tcgagaagtg ggatgtggtt gtgtttgatt tcctttggcc 9660
179
CA 02404060 2002-09-23
WO 01/72341 PCT/USO1/09042
agataaagtg ctggatatag cattgaaaac ggagtatgaa gaccagttag aatggagggt 9720
caggttggag ttgagttaca gatggggtaa aattctgctt cggatgagtt tggggattgg 9780
caatctaaag gtggtttggg atggcatggc tttgggatgg aaataggttt gtttttatgt 9840
tggctgggaa gggtgtgggg attgaattgg ggatgaagta ggtttagttt tggagataga 9900
atacatggag ctggctattg catgcgagga tgtgcattag tttggtttga tctttaaata 9960
aaggaggcta ttagggttgt cttgaattag attaagttgt gttgggttga tgggttgggc 10020
ttgtgggtga tgtggttgga ttgggctgtg ttaaattggt ttgggtcagg ttttggttga 10080
ggttatcatg gggatgagga tatgcttggg acatggattc aggtggttct cattcaagct 10140
gaggcaaatt tcctttcaga cggtcattcc agggaacgag tggttgtgtg ggggaaatca 10200
ggccactggc tgtgaatatc cctctatcct ggtcttgaat tgtgattatc tatgtccatt 10260
ctgtctcctt cactgtactt ggaattgatc tggtcattca gctggaaatg ggggaagatt 10320
ttgtcaaatt cttgagacac agctgggtct ggatcagcgt aagccttcct tctggtttta 10380
ttgaacagat gaaatcacat tttttttttc aaaatcacag aaatcttata gagttaacag 10440
tggactctta taataagagt taacaccagg actcttattc ttgattcttt tctgagacac 10500
caaaatgaga tttctcaatg ccaccctaat tctttttttt tttttttttt tttttgagac 10560
acagtctggg tcttttgctc tgtcactcag gctggagcgc agtggtgtga tcatagctca 10620
ctgaaccctt gacctcctgg acttaaggga tcctcctgct tcagcctcct gagtagatgg 10680
3$ ggctacaggt gcttgccacc acacctggct aattaaattt tttttttttt tttgtagaga 10740
aagggtctca ctttgttgcc ctggctgatc ttgaacttct gacttcaagt gattcttcag 10800
ccttggactc ccaaagcact gggattgctg gcatgagcca ctcaccgtgc ctggcttgca 10860
gcttaatctt ggagtgtata aacctggctc ctgatagcta gacatttcag tgagaaggag 10920
gcattggatt ttgcatgagg acaattctga cctaggaggg caggtcaaca ggaatccccg 10980
4S ctgtacctgt acgttgtaca ggcatggaga atgaggagtg aggaggccgt accggaaccc 11040
catattgttt agtggacatt ggattttgaa ataataggga acttggtctg ggagagtcat 11100
atttctggat tggacaatat gtggtatcac aaggttttat gatgagggag aaatgtatgt 11160
ggggaaccat tttctgagtg tggaagtgca agaatcagag agtagctgaa tgccaacgct 11220
tctatttcag gaacatggta agttggaggt ccagctctcg ggctcagacg ggtataggga 11280
SS ccaggaagtc tcacaatccg atcattctga tatttcaggg catattaggt ttggggtgca 11340
aaggaagtac ttgggactta ggcacatgag actttgtatt gaaaatcaat gattggggct,11400
180
CA 02404060 2002-09-23
WO 01/72341 PCT/USO1/09042
ggccgtggtg ctcacgcctg taatctcatc actttgggag accgaagtgg gaggatggct 11460
tgatctcaag agttggacac cagcctaggc aacatggcca gaccctctct ctacaaaaaa 11520
S attaaaaatt agctggatgt ggtggtgcat gcttgtggtc tcagctatcc tggaggctga 11580
gacaggagaa tcggttgagt ctgggagttc aaggctacag ggagctgcga tcacgccgct 11640
gcactccagc ctgggaaaca gagtgagact gtctcagaat ttttttaaaa aagaatcagt 21700
gatcatccca acccctgttg ctgttcatcc tgagcctgcc ttctctggct ttgttcccta 11760
gatcacatct ccatgatcca taggccctgc ccaatctgac ctcacaccgt gggaatgcct 11820
1S ccagactgat ctagtatgtg tggaacagca agtgctggct ctccctcccc ttccacagct 11880
ctgggtgtgg gagggggttg tccagcctcc agcagcatgg ggagggcctt ggtcagcatc 11940
taggtgccaa cagggcaagg gcggggtcct ggagaatgaa ggctttatag ggctcctcag 12000
25
ggaggccccc cagccccaaa ctgcaccacc tggccgtgga caccggt 12047
HRE-THE SEQ ID NO:-
ccccgagg cagtgcat gaggctcagg gcgtgcgt gagtcgcagcgagaccccg gggtgcag gccgga
PSA-THE SEQ ID NO:-
aagcttctag ttttcttttc ccggtgacat cgtggaaagc actagcatct ctaagcaatg 60
atctgtgaca atattcacag tgtaatgcca tccagggaac tcaactgagc cttgatgtcc 120
3$ agagattttt gtgttttttt ctgagactga gtctcgctct gtgccaggct ggagtgcagt 180
ggtgcaacct tggctcactg caagctccgc ctcctgggtt cacgccattc tcctgcctca 240
gcctcctgag tagctgggac tacaggcacc cgccaccacg cctggctaat ttttttgtat 300
ttttagtaga gatggggttt cactgtgtta gccaggatgg tctcagtctc ctgacctcgt 360
gatctgccca ccttggcctc ccaaagtgct gggatgacag gcgtgagcca ccgcgcctgg 420
4J ccgatatcca gagatttttt ggggggctcc atcacacaga catgttgact gtcttcatgg 480
S0
ttgactttta gtatccagcc cctctagaaa tctagctgat atagtgtggc tcaaaacctt 540
cagcacaaat cacaccgtta gactatctgg tgtggcccaa accttcaggt gaacaaaggg 600
actctaatct ggcaggatac tccaaagcat tagagatgac ctcttgcaaa gaaaaagaaa 660
tggaaaagaa aaagaaagaa aggaaaaaaa aaaaaaaaaa gagatgacct ctcaggctct 720
SS gaggggaaac gcctgaggtc tttgagcaag gtcagtcctc tgttgcacag tctccctcac 780
181
CA 02404060 2002-09-23
S
WO 01/72341 PCT/USO1/09042
agggtcattg tgacgatcaa atgtggtcac gtgtatgagg caccagcaca tgcctggctc 840
tggggagtgc cgtgtaagtg tatgcttgca ctgctgaatg gctgggatgt gtcagggatt 900
atcttcagca cttacagatg ctcatctcat cctcacagca tcactatggg atgggtatta 960
ctggcctcat ttgatggaga aagtggctgt ggctcagaaa ggggggacca ctagaccagg 1020
1~ gacactctgg atgctgggga ctccagagac catgaccact caccaactgc agagaaatta 1080
attgtggcct gatgtccctg tcctggagag ggtggaggtg gaccttcact aacctcctac 1140
cttgaccctc tcttttaggg ctctttctga cctccaccat ggtactagga ccccattgta 1200
ttctgtaccc tcttgactct atgaccccca ccgcccactg catccagctg ggtcccctcc 1260
tatctctatt cccagctggc cagtgcagtc tcagtgccca cctgtttgtc agtaactctg 1320
2~ aaggggctga cattttactg acttgcaaac aaataagcta actttccaga gttttgtgaa 1380
2S
tgctggcaga gtccatgaga ctcctgagtc agaggcaaag gcttttactg ctcacagctt 1440
agcagacagc atgaggttca tgttcacatt agtacacctt gcccccccca aatcttgtag 1500
ggtgaccaga gcagtctagg tggatgctgt gcagaagggg tttgtgccac tggtgagaaa 1560
cctgagatta ggaatcctca atcttatact gggacaactt gcaaacctgc tcagcctttg 1620
30 tctctgatga agatattatc ttcatgatct tggattgaaa acagacctac tctggaggaa 1680
catattgtat cgattgtcct tgacagtaaa caaatctgtt gtaagagaca ttatctttat 1740
tatctaggac agtaagcaag cctggatctg agagagatat catcttgcaa ggatgcctgc 1800
tttacaaaca tccttgaaac aacaatccag aaaaaaaaag gtgttactgt ctttgctcag 1860
aagacacaca gatacgtgac agaaccatgg agaattgcct cccaacgctg ttcagccaga 1920
4~ gccttccacc ctttctgcag gacagtctca acgttccacc attaaatact tcttctatca 1980
catcccgctt ctttatgcct aaccaaggtt ctaggtcccg atcgactgtg tctggcagca 2040
ctccactgcc aaacccagaa taaggcagcg ctcaggatcc cgaaggggca tggctgggga 2100
tcagaacttc tgggtttgag tgaggagtgg gtccaccctc ttgaatttca aaggaggaag 2160
aggctggatg tgaaggtact gggggaggga aagtgtcagt tccgaactct taggtcaatg 2220
S~ agggaggaga ctggtaaggt cccagctccc gaggtactga tgtgggaatg gcctaagaat 2280
SS
ctcatatcct caggaagaag gtgctggaat cctgaggggt agagttctgg gtatatttgt 2340
ggcttaaggc tctttggccc ctgaaggcag aggctggaac cattaggtcc agggtttggg 2400
gtgatagtaa tgggatctct tgattcctca agagtctgag gatcgagggt tgcccattct 2460
tccatcttgc cacctaatcc ttactccact tgagggtatc accagccctt ctagctccat 2520
182
CA 02404060 2002-09-23
WO 01/72341 PCT/USO1/09042
gaaggtcccc tgggcaagca caatctgagc atgaaagatg ccccagaggc cttgggtgtc 2580
atccactcat catccagcat cacactctga gggtgtggcc agcaccatga cgtcatgttg 2640
ctgtgactat ccctgcagcg tgcctctcca gccacctgcc aaccgtagag ctgcccatcc 2700
tcctctggtg ggagtggcct gcatggtgcc aggctgaggc ctagtgtcag acagggagcc 2760
tggaatcata gggatccagg actcaaaagt gctagagaat ggccatatgt caccatccat 2820
gaaatctcaa gggcttctgg gtggagggca cagggacctg aacttatggt ttcccaagtc 2880
1S
tattgctctc ccaagtgagt ctcccagata cgaggcactg tgccagcatc agccttatct 2940
ccaccacatc ttgtaaaagg actacccagg gccctgatga acaccatggt gtgtacagga 3000
gtagggggtg gaggcacgga ctcctgtgag gtcacagcca agggagcatc atcatgggtg 3060
2~ gggaggaggc aatggacagg cttgagaacg gggatgtggt tgtatttggt tttctttggt 3120
tagataaagt gctgggtata ggattgagag tggagtatga agaccagtta ggatggagga 3180
tcagattgga gttgggttag ataaagtgct gggtatagga ttgagagtgg agtatgaaga 3240
ccagttagga tggaggatca gattggagtt gggttagaga tggggtaaaa ttgtgctccg 3300
gatgagtttg ggattgacac tgtggaggtg gtttgggatg gcatggcttt gggatggaaa 330
tagatttgtt ttgatgttgg ctcagacatc cttggggatt gaactgggga tgaagctggg 3420
tttgattttg gaggtagaag acgtggaagt agctgtcaga tttgacagtg gccatgagtt 3480
ttgtttgatg gggaatcaaa caatggggga agacataagg gttggcttgt taggttaagt 3540
tgcgttgggt tgatggggtc ggggctgtgt ataatgcagt tggattggtt tgtattaaat 3600
tgggttgggt caggttttgg ttgaggatga gttgaggata tgcttgggga caccggatcc 3660
atgaggttct cactggagtg gagacaaact tcctttccag gatgaatcca gggaagcctt 3720
aattcacgtg taggggaggt caggccactg gctaagtata tccttccact ccagctctaa 3780
gatggtctta aattgtgatt atctatatcc acttctgtct ccctcactgt gcttggagtt 3840
tacctgatca ctcaactaga aacaggggaa gattttatca aattcttttt tttttttttt 3900
tttttttgag acagagtctc actctgttgc ccaggctgga gtgcagtggc gcagtctcgg 3960
ctcactgcaa cctctgcctc ccaggttcaa gtgattctcc tgcctcagcc tcctgagttg 4020
ctgggattac aggcatgcag caccatgccc agctaatttt tgtattttta gtagagatgg 4080
ggtttcacca atgtttgcca ggctggcctc gaactcctga cctggtgatc cacctgcctc 4140
SS
agcctcccaa agtgctggga ttacaggcgt cagccaccgc gcccagccac ttttgtcaaa 4200
ttcttgagac acagctcggg ctggatcaag tgagctactc tggttttatt gaacagctga 4260
183
CA 02404060 2002-09-23
WO 01/72341 PCT/USO1/09042
aataaccaac tttttggaaa ttgatgaaat cttacggagt taacagtgga ggtaccaggg 4320
ctcttaagag ttcccgattc tcttctgaga ctacaaattg tgattttgca tgccacctta 4380
atcttttttt tttttttttt aaatcgaggt ttcagtctca ttctatttcc caggctggag 4440
ttcaatagcg tgatcacagc tcactgtagc cttgaactcc tggccttaag agattctcct 4500
1~ gcttcggtct cccaatagct aagactacag tagtccacca ccatatccag ataattttta 4560
aattttttgg ggggccgggc acagtggctc acgcctgtaa tcccaacacc atgggaggct 4620
gagatgggtg gatcacgagg tcaggagttt gagaccagcc tgaccaacat ggtgaaactc 4680
tgtctctact aaaaaaaaaa aaaatagaaa aattagccgg gcgtggtggc acacggcacc 4740
tgtaatccca gctactgagg aggctgaggc aggagaatca cttgaaccca gaaggcagag 4800
gttgcaatga gccgagattg cgccactgca ctccagcctg ggtgacagag tgagactctg 4860
tctcaaaaaa aaaaaatttt tttttttttt ttgtagagat ggatcttgct ttgtttctct 4920
ggttggcctt gaactcctgg cttcaagtga tcctcctacc ttggcctcgg aaagtgttgg 4980
gattacaggc gtgagccacc atgactgacc tgtcgttaat cttgaggtac ataaacctgg 5040
ctcctaaagg ctaaaggcta aatatttgtt ggagaagggg cattggattt tgcatgagga 5100
3~ tgattctgac ctgggagggc aggtcagcag gcatctctgt tgcacagata gagtgtacag 5160
gtctggagaa caaggagtgg ggggttattg gaattccaca ttgtttgctg cacgttggat 5220
tttgaaatgc tagggaactt tgggagactc atatttctgg gctagaggat ctgtggacca 5280
caagatcttt ttatgatgac agtagcaatg tatctgtgga gctggattct gggttgggag 5340
tgcaaggaaa agaatgtact aaatgccaag acatctattt caggagcatg aggaataaaa 5400
4~ gttctagttt ctggtctcag agtggtgcat ggatcaggga gtctcacaat ctcctgagtg 5460
ctggtgtctt agggcacact gggtcttgga gtgcaaagga tctaggcacg tgaggctttg 5520
tatgaagaat cggggatcgt acccaccccc tgtttctgtt tcatcctggg catgtctcct 5580
ctgcctttgt cccctagatg aagtctccat gagctacaag ggcctggtgc atccagggtg 5640
atctagtaat tgcagaacag caagtgctag ctctccctcc ccttccacag ctctgggtgt 5700
SD gggagggggt tgtccagcct ccagcagcat ggggagggcc ttggtcagcc tctgggtgcc 5760
agcagggcag gggcggagtc ctggggaatg aaggttttat agggctcctg ggggaggctc 5820
cccagcccca agctt 5835
184
CA 02404060 2002-09-23
WO 01/72341 PCT/USO1/09042
CEA THE SEQ ID NO:
aagcttttta gtgctttaga cagtgagctg gtctgtctaa cccaagtgac ctaggctcca60
tactcagccc cagaagtgaa gggtgaagct gggtggagcc aaacca.ggca 120
agcctaccct
cagggctccc agtggcctga gaaccattgg accc~ggacc cattacttct agggtaagga'180
aggtacaaac accagatcca accatggtct ggggggacag ctgtcaaatg cctaaaaata240
tacctgggag aggagcaggc aaactatcac tgccccaggt tctctgaaca gaaacagagg300
ggcaacccaa agtccaaatc caggtgagca ggtgcaccaa atgcccagag atatgacgag360
gcaagaagtg aaggaaccac ccctgcatca aatgttttgc atgggaagga gaagggggtt420
gctcatgttc ccaatccagg agaatgcatt tgggatctgc cttcttcrca ctccttggtt480
agcaagacta agcaaccagg actctggatt tggggaaaga cgttta~t~g tggaggccag540
tgatgacaat cccacgaggg cctaggtgaa gagggcagga aggctcgaga cactggggac600
-
tgagtgaaaa ccacacccat gatctgcacc acccatggat gctcct~c~t tgctcacctt660
tctgttgata tcagatggcc ccattttctg taccttcaca gaaggacaca ggct~gggtc720
tgtgcatggc cttcatcccc ggggccatgt gaggacagca ggtgggaaag atcatgggt.c780
ctcctgggtc ctgcagggcc agaacattca tcacccatac tgacctcc~a gatgggaatg840
gcttccctgg ggctgggcca acggggcctg ggcaggggag aaaggacgtc aggggacagg900
gaggaagggt catcgagacc cagcctggaa ggttcttgtc tctgacrztc caggatttac960
ttccctgcat ctacctttgg tcattttccc tcagcaatga ccagctctgc ttcctgatct1020
cagcctccca ccctggacac agca.ccccag tccctggccc ggctgcatcc 1080
acccaatacc
ctgataaccc aggacccatt acttctaggg taaggagggt cca.ggagaca 1140
gaaactgagg
aaaggtctga agaagtcaca tctgtcctgg ccagagggga aaaaccatca gatgctgaacI~00
caggagaatg ttgacccagg aaagggaccg aggacccaag aaaggagtca gaccaccagg1260
gtttgcctga gaggaaggat caaggccccg agggaaagca, gggctggctg 1320
catgtg'cagg
acactggtgg ggcatatgtg tcttagattc tccctgaatt cagtgtccct gccatggcca1380
gactctctac tcaggcctgg acatgctgaa ataggaca~t ggccttgtcc tctctcccca1440
ccatttggca agagacataa aggacattcc aggacatgcc ttcctgggag gtccaggttc1500
tctgtctcac acctcaggga ctgtagttac tgcatcagcc atggtaggtg ctgatctcac1560
ccagcctgtc caggcccttc cactctccac tttgtgacca tgtccaggac cacccctcag1620
atcctgagcc tgcaaatacc cccttgctgg gtgggtggat tcagtaaaca gtoagctcct1680
185
CA 02404060 2002-09-23
WO 01/72341 PCT/USO1/09042
atccagcecc-cagagcca.cc tctgtcaccttCctgctggqcatcatcccaccttcacaag1740
cactaaagagcatggqgagacctggctagctgggtttctgcatcacaaagaaaataatcc1800
cccaggttcggattcccagggctctgtatgtggagctgacagacctgaggccaggagata1860
gcagaggtcagccctagggagggtgggtcatccacccaggggacaqgggtgcaccagcct1924'
tgctactgaaagggcctccccaggacagcgccatcagccctgcctgagagctttgctaaa1980
cagcagtcagaggaggccatggcagtggctgagctcctgctccaggccccaacagaccag2040
accaacagcacaatgcagtccttccccaacgtcacaggtccca.aagggaaactgaggtg2100
a
ctacctaaccttagagccatcaggggagataacagcccaatttcccaaacaggccagttt2160
.
caatcccatg.acaatgacctctctgctctcattcttcccaaaataggacgctgattctcc2220
cccaccatggatttctcccttgtcccgggagccttttctgccccctatga-tctgggcact 2280
cctgacacacacctcctctctggtgacatatcagggtccctcactgtcaagcagtccaga2340
aaggaca.gaaccttggac~gcgcccatctcagcttcacccttcctccttcacagggttca2400
gggcaaagaataaatggcagaggccagtgagcccagagatggtgacaggcagtgacccag2460
gggcagatgectggagcaggagctggcggggccacagggagaaggtgatg~caggaaggga2520
aacccagaaatgggcaggaaaggaggacacaggctctgtg~gggctgcagcccagggttgg2580
acta~gagtgtgaagccatctcagcaagtaaggccaggtcccatgaacaa-gagtgggagc2640
acgtggcttcctgctctgtatatggggtgggggattccatgccccatagaaccagatggc2700
cggggttcagatggagaaggagcaggacag~gggatccccaggataggaggacacCagtgt2760
ccccacccaggcaggtgactgatgaatgggcatgcagggtcctcctgggctgggctctcc2820
ctttgtccctcaggattccttgaaggaacatccggaagccgaccacatctacctggtggg2880
ttctggggagtccatgtaaagccaggagcttgtgttgctaggaggcjgtcatggcatgtgc2940
tggaggcaccaaagagagaacctgagggcaggcaggacctggtctgaggaggcatggga.3000
a
gcccagatggggagatggatgtcaggaaaggctgccccatcagggagggtgatagcaatg3060
gggggtctgtgggagtgggcacgtgggattccctgggctctgccaagttccctcccatag3120
tcacaacctggggacactgcccatgaaggggcgcctttgcccagccagatgctgctggtt3180
ctgcccatccactaccctctctgctccagccactctgggtctttctccagatgccctgga3240
caac~ctggcctgggcctgtccctgagaggtgttgggagaagctgagtctctggggaca3300
c
ctct~atcagagtctgaaaggcacatcaggaaacatccctggtctccaqgactaggcaat33ou
186
CA 02404060 2002-09-23
WO 01/72341 PCT/USO1/09042
gaggaaagggccccagctcctccctttgccactgagagggtcgaccctgggtggccaca.g3420
tgacttctgcgtctgtccca,gtcaccctgaaaccacaacaaaaccccagccccagaccct 3480
gcaggtacaatacatgtggggacagtctgtacccaggggaagccagttctctcttcctag 3540
gagaccgggcctcagggctgtgcccggggcaggcgggggcagcacgtgcctgtccttgag 3600
aactcgggaccttaagggtctctgctctgtgaggcacagcaaggatccttctgtccagag 3669
atgaaagcagctcctgcccctcctctgacctcttcctccttcccaaatctcaaccaacaa 3720
ataggtgtttcaaatctcatcatcaaatcttcatccatccacatgagaaa.gcttaaaacc 3?80
caatggattgacaacatcaa..gagttggaacagtggacat ggagatgttacttgtggaaa _
a 3840
tttagatgtgttcagctatcgggcaggagaatctgtgtcaaattccagcatggttcagaa 3900
gaatcaaaaagtgtcacagtccaaatgtgcaacagtgcaggggataaaactgtggtgcat~3960
tcaaactgagggatattttggaacatgagaaaggaagggattgctgctgcacagaacatg 4020
gatgatctcacacatagagttgaaaga~aggagtcaatcgcagaatagaaaatgatcac't4080
aattccacctctataaagtttccaagaggaaaacccaattctgctgctagagatcagaat 4140
ggaggtgacctgtgccttgcaatggctg~gagggtcacgggagtgtcacttagtgcaggc 4200
aatgtgccgtatcttaatctgggcagggctttcatgagcacataggaatgagacattac 4260
c
tgctgtgttcattttacttc.accggaaaag aagaataaaatcagccgggcgcggtggctc 4320
acgcctgtaatcccagcactttagaaggct.gaggtgggcagattacttgagtcaggagt 4380
g
tcaagaccaccctggccaatatggtgaaaccccggctctactaaaaatacaaaaattagc 4440
tgggca.tggtggtgcgcgcctgtaatcccagctactcgggaggctgaggctggaca.attg4500
cttggacccaggaagcagaggttgcagtgagccaagattgtgccactgcactccagcttg 4560
'
ggcaacagagccagactctgtaaaaaa~aa aaaaaaaaaaaaaaaaagaaagaaagaaaa 462_0
agaaaagaaagtataaaatctctttgggtt aacaaaaaaagatccacaaaacaaacacca 4680
gctcttatcaaacttacacaactctgcc~g agaacaggaaacacaaatactcattaactc 4
? 40
acttttgtggcaataaaaccttcatgtcsaaaggagaccaggacacaatgaggaagtaaa 4800
actgcaggccctacttgggtgcagagagggaaaatccacaaataaaacattaccagaagg 4860
agctaagatttactgcattgagttcattccccaggtatgcaaggtgattttaacacctga 4920
aaatcaatcattgcctttactacatagacagattagctagaaaaaaattacaactagcag 4980
aacagaagcaatttggccttcctaaaat~ccacz~catatcatcatgatggagacagtgc 5040
agacgccaatgacaataaaaagagggacctccgtcacccggtaaacatgtccacacagct 5100
187
CA 02404060 2002-09-23
WO 01/72341 PCT/USO1/09042
ccagcaagca-~ccgtcttcc cagtgaatca ccctttaatcagccccaggc 5160-
ctctaacctc
aaggctgcct gcgatggccacacaggctccaacccgtgggcctcaacctcccgcagaqgc 5220
tctcctttgg ccaccccatggggagagcatgaggacaqggcagagccctctgatgccca.c5280
acatggcagg agctgacgccagagccatgggggctggagagcagagctgc.tggggtcaga 5346.
gcttcctgag gacacccaggcct'aagggaaggcagctccctggatgggggcaaccaggct 5400
ccgggctcca acctcagagcccgcatgggaggagccagcactctaggcctttcctagggt 5460
gactctgagg ggaccctgacacgacaggatcgctgaatgcacccgagatgaaggggccac 5520
cacgggaccc tgctctcgtggcagatcagg~ag~gagtgggacaccatgcGaggcccccat 5580
ggcatggctg cgactgacccaggccactcccctgcatgcatcagcctcggtaagtcacat .5640.
gaccaagccc aggaccaatgtggaaggaaggaaacagcatcccctttagtgatggaaccc 5700
aaggtcagtg caaagagaggccatgagcagttaggaagggtggtccaacctacagcacaa 5760
accatcatct atcataagtagaagccctgctccatgacccctgcatttaaataaacgttt 5820
gttaaatgag tcaaattccctcaccatgagagctcaGctgtgtgtaggcccatcacacac.5880
acaaacacac acacacacacacacacacacacacacaczcacagggaaagtgcaggatcc 5940
tggacagcac caggcaggcttcacaggcag~gcaaacagcgtgaatgacccatgcagtgc 6000
cctgggccccatcagctca.g tggggctaggcaggggagag 6060
agaccctatg
agggctgaga
acttagagagggtggggcctccagggagggggcLgcagggagctgggtactgccctcca.g6120
ggagggggctgcagggagctgggtactgccctccagggagggggctgcagggagctgggt 6180
actgccctccagggagggggctgcagggagctgggtactglccctccagggagggggctgc 6240
agggagctgggtactgccctccagggaggcaggagca.ctgttcccaacagagagcacatc 6300
ttcctgcagcagctgcacagacacaggagcccccatgactgccctgggccagggtgtgga ~
63s0
ttccaaatttcgtgccccattgggtgggaGggaggttgaccgtgacatccaaggggcatc 6420
tgtgattccaaacttaaactactgtgcctacaaaataggaaataaccctactttttctac 6480
tatctcaaattccctaagcacaagctagcaccctttaaatcaggaagttcagtcactcct 6540
ggggtcctcccatgcccccagtctgacttgczgqtgcacagggtggctgacatctgtcct.6600
tgctcctcctcttggctcaactgccgcccctcctgggggtgactgatggtcaggacaagg 6660
gatcctagagctggccccatgattgacaggaaggcaggac 6720
:.tggcctcca
ttctgaagac
taggggtgtc 6780
aagagagctg
ggcatcccac
aga;ctgcac
~agatgacgc
ggacagaggg
188
CA 02404060 2002-09-23
WO 01/72341 PCT/USO1/09042
tgacacagggctcagggcttcagacgggtcgggaggctcagctgagagttcagagacaga 6840
cctgaggagcctcagtgggaaaagaagcactgaagtgggaagttctggaatgttctggac 6900
aagcctgagtgctctaaggaaatgctcccaccccgatgtagcctgcagcactggacggtc 6960
tgtgtacctccccgctgcccatcctctcacagcccccgcctctagggacacaactcctgc 7020
cctaacatgcatctttcctgtctcattccacacaaaagggcctctggggtccctgttctg 7080
cattgcaagg agtggaggtcacgttcccacagaccacccagcaacagggtcctatggagg 7140
tgcggtcagg aggatcacacgtccccccatgcccaggggactgactctgggggtgatgga 7200
ttggcctgga ggccactggtcccctctgtccctgaggggaatctgcaccctggaggctgc 7260
cacatccctc ctgattctttcagctgagggcccttcttgaaatcccagggaggactcaac 7320
ccccactqgg aaaggcccagtgtggacggttccacagcagcccagctaaggcccttggac 7380
acagatcctg agtgagagaacctttagggacacaggtgcacggccatgtcccc~gtgccc 7440
acaczgagca ggggcatctggaccctgagtgtgtagctcccgcgactgaacccagccctt 7500
ccccaatgac gtgacccctggggtggctccaggtctccagtccatgccaccaaaatctcc 7560
agattgaggg tcctcccttgagtccctgatgcctgtccaggagctgccccctgagcaaat 7620
ctagagtgca gagggctgggattgtggcagtaaaagcagccacatttgtctcagqaagga 768'0
aagggaggac atgagctccaggaagggcgatggcgtcctctagtgggcgcctcctgttaa ?740
tgagcaaaaa ggggccaggagagttgagagatcagggctggccttggactaaggctcaga 7800
tggagaggac tgaggtgcaaagagggggctgaagtaggggagtggtcgggagagatggga 7860
ggagcaggtaaggggaagcc tacagcagag 7920
ccagggaggc ctctccactc
cgggggaggg
ctcagcattgacatttggggtggtcgtgct agtggggttctgtaagttgtagggtgttca.7980
gcaccatctggggactctacccacGaaatg ccagcaggactccctccccaagctctaaca 8040
accaacaatgtctccagactttccaaatgt cccctggagagcaaaattgcttctggcaga 8100
atcactgatctacgtcagtctctaaaagtg actcatcagcgaaatccttcacctcttggg 8160
agaagaatcacaagtgtgagaggggtagaaactgcagacttcaaaatctttccaaaagag 8220
ttttacttaatcagcagtttgatgtcccagqagaagatacatttagagtgtttagagttg 8280
atgccacatggctgcctgtacctcacagcaagagcagagtgggttttccaagggcctgta 8340
accacaactggaatgacactcactgggttacattacaaag'tggaatgtggggaattctgt 8400
agactttgggaagggaaatgtatgacgtaagcccacagcctaaggcagtggacagtccac 8460
tttgaggctctcaccatctaggagacatctcagccatgaacatagccacatctgtcatta 820
189
CA 02404060 2002-09-23
WO 01/72341 PCT/USO1/09042
gaaaacatgh--tttattaaga ggaaaaatctaggctagaagtgctttatgctcttttttct 8580
ctttatgttcaaattcatatacttttagatcattcct~aaagaagaatctatccccctaa 8640
gtaaatgttatcactgactggatagtgttggtgtctcactcccaa~ccctgtgtggtgac 8?00
agtgccctgcttccccagccctgggccctctctgattcctgagag~tttgggtgctcctt 8?fi8>
cattaggaggaagagaggaagggtgtttttaatattctcaccattcacccatccaCctct 8820
.taga~.actgggaagaatcagttgcccactcttggatttgatcctccaattaatgacctct 8880
atttctgtcc'cttgtccatt tcaacaatgtgacaggcctaagaggtgccttctccatgtg 89.40
atttttgaggagaaggttctcaagataagttttctcacacctctttgaattacctccacc 9000
tgtgtccccatcaccattaccagcagcatttggaccctttttcta~tagtcagatgcttt 9060'
ccacctcttgagggtgtatactgtatgctctctacacaggaatatgcagaggaaatagaa 9120
aaagggaaatcgcattactattcagagagaagaagacctttatgtgaatgaatgagagtc 9180
taaaatcctaagagagcccatataaaattattaccagtgctaaaactacaaaagttacac 9240
taacagtaaactagaataataaaacatgcatcacagttgctggtaaagctaaatcagata 9300
tttttttcttagaaaaagcattccatgtgtgttgcagtgatgaczggagtgcccttcagt 9360
caatatgctgcctgtaatttttgttccctggcagaat~attgtcttttctccctttaaa 9420
tcttaaatgcaaaactaaaggcagctcctgggccccctccccaaactcagctgcctgcaa 9480
cca.gccccacgaagagcagaggcctgagcttccctggtcaaaatagggggctagggagct9540
taaccttgctcgataaagctgtgttcccagaatgtcgctcctgttcccaggggcaccagc9600
ctggagggtggtgagcctcactggtggcctgatgcttaccttgtgccctcaczccagtgg9660
tcactggaaccttgaacacttggctgtcgcccggatctgcagatg~caagaacttctgga9?20
agtcaaattactgcccacttctccagggcagatacctgtgaacatccaaaaccatgccac9?80
agaaccctgcctggggtctacaacacatatggactgtgagcaccaagtccagccctgaat9840
ctgtgaccacctgccaagatgcccctaactgggatccaccaatcactgcacatggcaggc9900
agcgaggcttggaggtgcttcgccacaaggcagccccaatttgctgggagtttcttggca9960
cctggtagtggtgaggagccttgggaccctcaggattactccccttaagcatagtgggga10020
cccttctgcatccccagcaggtgccccgctcttcagagcctctctctctgaggtttaccc10080
agacccctgcaccaatgagaccatgctgaagcctcagagagagagatggagctttgacca10140
ggagccgctcttccttgagggccaggg.:agggaaagcac~gaggcagcaccaggagtggga- 10200
190
CA 02404060 2002-09-23
WO 01/72341 PCT/USO1/09042
acaccagtgt~ctaagcccct gatgagaacagaqtggtctctcccatatgcccataccagg10260
cctgtgaacagaatcctccttctgcagtgacaatgtctgagaggacgacatgtttcccag10320
cctaacgtgcagccatgcccatctacccactgcctactgcaggacagcaccaacccagga10380
gctgggaagctgggagaagacatggaatacceatggcttctcaccttcctccagtccagt1b4d0
gggca.ccatttatgcctaggacacccacctgccggccccaggctcttaagagttaggtca1050
cctaggtgcctctgggaggccgaggeaggagaattgcttgaacccgggaggcagaggttg10560
cagtgagccgagatcacaccactgcactccagcctgggtqacagaatgagactctgtctc10620
aaaaaaaaagagaaagatagcatcagtggctaccaagggctaggggcaggggaaggtgga10680
gagttaatgattaatagtatgaagtttctatgtgagatgatgaaaatgttctggaaaaaa10740
aaatatagtggtgaggatgtag2atattgtgaatataattaacggcatttaattgtacac10800
ttaacatgat taatgtggca tattttatct tatgtatttc actacatcca agaaacactg 10860
ggagagggaa agcccaccat gtaaaataca cccaccctaa tCagatagtc ctcattgtac 10920
ccaggtacag gcccctcatg acctgcacag gaataaetaa ggatttaagg acatgaggct 10980
tcccagccaa ctgcaggtgc acaacataaa tgtatctgca aacagactga gagtaaagct 11040
gggggcacaa acctcagcac tgccaggaca cacacccttc tcgtggattc tgactttatc 11100
tgacccggcc ca.ctgtccag atcttgttgt gggattggga caagggaggt cataaagcct 11160
gtccccaggg cactctgtgt gagcacacga gacctcccca cccccccacc gttaggtctc 11220
cacacataga tctgaccatt aggcattgtg aggaggactc tagcgcgggc tca.gggatca 11280
caccagagaa tcaggtacag agaggaagac gggqctcgag gagctgatgg atgacacaga 11340
gcagggttcc tgcagtccac aggtccagct caccctggtg taggtgcccc atccccctga 11400
tccaggcatc cctgacacag ctccct~cccg'gagcctcctc ccaggtqaca catcagggtc 11460
cctcactcaa gctgtccaga gagggcagca ccttggacag cgcccacccc acttcactct 11520
tcctccctca cagggctcag ggctcagggc tcaagtctca gaacaaatgg cagaggccag 11580
tgagcccagagatggtgacaggqcaatgatccaggggcagctgcctgaaacgggagcagg11640
tgaagccacagatgggagaagatggttcagga~gaaaaatccaggaatgggcaggagagg11700
agaggaggacacaggctctgtggggctgcagcccaggatgggactaagtgtgaagacatc11760
tcagcaggtgaggccaggtcccatgaacagagaagcagctcccacctcccctgatgcacg11820
gacacacagagtgtgtggtgc~gtgcccccagagtcgggctctcctgttctggtccccag11880
ggagtgagaagtgaggttgacttgtccctgctcctctctgctaccccaacattcaccttc11940
191
CA 02404060 2002-09-23
WO 01/72341 PCT/USO1/09042
tcctcatgcc-cctctctctc aaatatgatt Ct~gatctatg tccccgccca aatctcatgt 120b0
caaattgtaa accccaatgt tggagqtggg gccttgtgag aagtgattgg ataatgcggg 120 60
tggattttct gctttgatgc tgtttctgtg atagagatct cacatgatct ggttgtttaa 12120
aagtgtgtag cacctctccc ctctctctct ctctctctta ctcatgctct gccatgtaag 1218D
acgttcctgt ttccccttca ccgtccagaa tgattgtaag ttttctgagg cctccccagg 12240
agcagaagcc actatgcttc ctgtacaact gcagaatgat gagcgaatta aacctctttt 12300
ctttataaat tacccagtct caggtatttc tttatagcaa tgcgaggaca.gactaataca 12360
atcttctact cccagatccc cgcacacgct tagccccaga catcactgcc cctgggagca 12420
tgcacagcgc agcctcctgc cgacaaaagc aaagtcacaa aaggtgacaa aaatctgcat 12480
ttggggacat ctgattgtga aagagggagg acagtacact tgtagccaca gagactgggg 12540
ctcaccgagc tgaaacctgg tagcactttg gcataacatg tgcatgaccc gtgttcaatg 12600
tctacagatc agtgttgat3t aaaacagcct ggtctggggc cgctgctgtc cccacttccc 12660
tcctgtccac cagagggcgg cagagttcct cccaccctgg agcctcccca ggggctgctg 12720
acctccctca gccgggccca cagcccagca gggtccaccc tcacccgggt cacctcggcc 12780
czcgtcctcc tcgccctccg agctcctcac acggactctg tcagctcctc cctgcagcct 12840
atcgg~cgcc cacctgaggc ttgtcggccg cccacttgag gcctgtcggc tgccctctgc 12900
aggcagct~c tgtcccctac accccctcct tccccgggct cagctgaaag ggcgtctccc 12960
agggcagctc cctgtgatct ccaggaczgc tcagtctctc acaggctccg acgcccccta 13020
tgctg~.cacc tcacagccct gtcattacca ttaactcctc~agtcccatga agttcactga 13080
gcgcctgtct cccggttaca ggaaaactct gtgacaggga ccacgtctgt cctgctctct 13140
gtggaatccc agggcccagc ccagtgcctg acacggaaca gatgctceat aaatactggt 13200
taaatctgtg ggagatctct aaaaagaagc ztatcacctc cgtgtggccc ccagcagtca 13260
gagtc~gttc catgtggaca caggggcact ggcaccagca tgggaggagg ccagcaagtg 13320
cccgcggctg ccccaggaat gaggcctcaa cccccagagc ttcagaaggg aggacagagg 13380
cctgcaggga atagatcctc cggcctgacc ctgcagccta atccagagtt cagggtcagc 13440
tcacaccacg tcgaccctgg tcagcatccc tagggcagtt ccagacaagg ccggaggtct 13500
cctcttgccc tccagggggt gacattgcac acagacatca ctcaggaaac ggattcccct 13560
ggacacgaac ctggctttgc taaggaagtg- gaggtggagc ctggtttcca tcccttgctc 13620
192
CA 02404060 2002-09-23
WO 01/72341 PCT/USO1/09042
caacagaccc ttctgatctc tcccacatac ctgctctgtt cctttctcgg tcctatgagg 13680
accctgttct gccaggggtc cctgtgcaac tccagactcc ctcctggtac caccatgggg 13740
aaggtggggt gatcacagga cagtcagcct cgcagagaca cagaccaccc aggactgtca 13800
gggagaacat ggacaggccc tgagccgcag ctcagccaac agacacggag agggagggtc .13860
cccctggagc cttccccaag gacagcagag cccagagtca cccacctccc tccaccacag 13920
tcctctcttt ccaggacaca caagacacct ccccctccac atgcaggatc tggggactcc 13980
tgagacctct gggcctgggt ctccatccct gggtcagtgg cggggttggt ggtactggag 14040
acagagggct ggtccctccc cagccaccac ccagtgagcc tttttctagc ccccagagcc 14100
acctctgtca~ccttcctgtt gggcatcatc ccaccttccc ~gagccctgg agagcatggg 14160
gagacccggg accctgctgg gtttctctgt cacaaaggaa aataatcccc ctggtgtgac 14220
agacccaagg acagaacaca gcagaggtca gcactgggga agacaggttg tcctcccagg 14280
ggatgggggt ccatccacct tgccgaaaag atttgtctga ggaactga~a atagaaggga 14340
aaaaagagga gggacaaaag aggcagaaat gagaggggag gagacagacg acacctgaat 14400
aaagaccaca_cccatgaccc acgtgatgct gagaagtact cctgccctag gaagagactc 14460
~ transcription start site
agggcaqagg gaggaaggac agcagaccag acagtcacag cagccttgac aaaacgttcc 14520
tggaactcaa gctcttctcc acagaqgagg acagagcaga cagcagagsc catgoagtct 14580
ccctcggccc ctccccacag atggtgcatc ccctggcaga ggctcctgct cacaggtgaa 14640
gggaggacaa cctgggagag ggtgggagga gggagctggg gtctcctgcg taggacaggg 14700
ctgtgagacg gacagagggc tcctgttgga gcctgaatag gaaagagg~c atcagagagg 147 60
gac~,ggagtc acaccagaaa aatcaaattg aactggaatt ggaaaggggc aggaaaacct 14820
caacagttct attttcctag ttaattgtca ctggccacta cgtttttasa aatcataata 14880
actgcatcag atgacacttt aaataaaaac ataaccaggg catgaaacac tgtcctcatc 14940
cgcctaccgc ggacattgga aaataagccc caggctgtgg agggccctgg gaaccctcat 15000
gaactcatcc acaggaatct gcagcctgtc ccaggcactg gggtgcaacc aagatc 15056
193
CA 02404060 2002-09-23
WO 01/72341 PCT/USO1/09042
Mucin-THE SEQ ID NO:-
cgagcggccc ctcagcttcg gcgcccagcc ccgcaaggct cccggtgacc actagagggc 60
gggaggagct cctggccagt ggtggagagt ggcaaggaag gaccctaggg ttcatcggag 120
cccaggttta ctcccttaag tggaaatttc ttcccccact cctccttggc tttctccaag 180
gagggaaccc aggctgctgg aaagtccggc tggggcgggg actgtgggtt caggggagaa 240
cggggtgtgg aacgggacag ggagcggtta gaagggtggg gctattccgg gaagtggtgg 300
ggggagggag cccaaaacta gcacctagtc cactcattat ccagccctct tatttctcgg 360
ccgctctgct tcagtggacc cggggagggc ggggaagtgg agtgggagac ctaggggtgg 420
gcttcccgac cttgctgtac aggacctcga cctagctggc tttgttcccc atccccacgt 480
2~ tagttgttgc cctgaggcta aaactagagc ccaggggccc caagttccag actgcccctc 540
ccccctcccc cggagccagg gagtggttgg tgaaaggggg aggccagctg gagaacaaac 600
gggtagtcag ggggttgagc gattagagcc cttgtaccct acccaggaat ggttggggag 660
gaggaggaag aggtaggagg taggggaggg ggcggggttt tgtcacctgt cacctgctcg 720
ctgtgcctag ggcgggcggg cggggagtgg ggggaccggt ataaagcggt aggcgcctgt 780
3~ gcccgctcca cctctcaagc agccagcgcc tgcctgaatc tgttctgccc cctccccacc 840
catttcacca ccaccatg 858
aFP-THE SEQ ID NO:-
gaattcttag aaatatgggg gtaggggtgg tggtggtaat tctgttttca bcccataggt 60
4~
gagataagca ttgggttaaa tgtgctttca cacacacatc acatttcata agaattaagg 120
aacagactat gggctggagg actttgagga tgtctgtctc ataacacttg ggttgtatct 180
gttctatggg gcttgtttta agcttggcaa cttgcaacag ggttcactga ctttctcccc 240
aagcccaagg tactgtcctc ttttcatatc tgttttgggg cctctggggc ttgaatatct 300
gagaaaatat aaacatttca ataatgttct gtggtgagat gagtatgaga gatgtgtcat 360
5~
tcatttgtat caatgaatga atgaggacaa ttagtgtata aatccttagt acaacaatct 420
gagggtaggg gtggtactat tcaatttcta tttataaaga tacttatttc tatttattta 480
tgcttgtgac aaatgttttg ttcgggacca caggaatcac aaagatgagt ctttgaattt 540
aagaagttaa tggtccagga ataattacat agcttacaaa tgactatgat ataccatcaa 600
194
CA 02404060 2002-09-23
WO 01/72341 PCT/USO1/09042
acaagaggtt ccatgagaaa ataatctgaa aggtttaata agttgtcaaa ggtgagaggg 660
ctcttctcta gctagagact aatcagaaat acattcaggg ataattattt gaatagacct 720
taagggttgg gtacattttg ttcaagcatt gatggagaag gagagtgaat atttgaaaac 780
attttcaact aaccaaccac ccaatccaac aaacaaaaaa tgaaaagaat ctcagaaaca 840
1~ gtgagataag agaaggaatt ttctcacaac ccacacgtat agctcaactg ctctgaagaa 900
gtatatatct aatatttaac actaacatca tgctaataat gataataatt actgtcattt 960
tttaatgtct ataagtacca ggcatttaga agatattatt ccatttatat atcaaaataa 1020
acttgagggg atagatcatt ttcatgatat atgagaaaaa ttaaaaacag attgaattat 1080
ttgcctgtca tacagctaat aattgaccat aagacaatta gatttaaatt agttttgaat 1140
ctttctaata ccaaagttca gtttactgtt ccatgttgct tctgagtggc ttcacagact 1200
tatgaaaaag taaacggaat cagaattaca tcaatgcaaa agcattgctg tgaactctgt 1260
acttaggact aaactttgag caataacaca catagattga ggattgtttg ctgttagcat 1320
acaaactctg gttcaaagct cctctttatt gcttgtcttg gaaaatttgc tgttcttcat 1380
ggtttctctt ttcactgcta tctatttttc tcaaccactc acatggctac aataactgtc 1440
3~ tgcaagctta tgattcccaa atatctatct ctagcctcaa tcttgttcca gaagataaaa 1500
agtagtattc aaatgcacat caacgtctcc acttggaggg cttaaagacg tttcaacata 1560
caaaccgggg agttttgcct ggaatgtttc ctaaaatgtg tcctgtagca catagggtcc 1620
tcttgttcct taaaatctaa ttacttttag cccagtgctc atcccaccta tggggagatg 1680
agagtgaaaa gggagcctga ttaataatta cactaagtca ataggcatag agccaggact 1740
gtttgggtaa actggtcact ttatcttaaa ctaaatatat ccaaaactga acatgtactt 1800
agttactaag tctttgactt tatctcattc ataccactca gctttatcca ggccacttat 1860
ttgacagtat tattgcgaaa acttcctaac tggtctcctt atcatagtct tatccccttt 1920
tgaaacaaaa gagacagttt caaaatacaa atatgatttt tattagctcc cttttgttgt 1980
ctataatagt cccagaagga gttataaact ccatttaaaa agtctttgag atgtggccct 2040
tgccaacttt gccaggaatt cccaatatct agtattttct actattaaac tttgtgcctc 2100
ttcaaaactg cattttctct cattccctaa gtgtgcattg ttttccctta ccggttggtt 2160
tttccaccac cttttacatt ttcctggaac actataccct ccctcttcat ttggcccacc 2220
tctaattttc tttcagatct ccatgaagat gttacttcct ccaggaagcc ttatctgacc 2280
cctccaaaga tgtcatgagt tcctcttttc attctactaa tcacagcatc catcacacca 2340
___. 195
CA 02404060 2002-09-23
WO 01/72341 PCT/USO1/09042
tgttgtgatt actgatacta ttgtctgttt ctctgattag gcagtaagct caacaagagc 2400
tacatggtgc ctgtctcttg ttgctgatta ttcccatcca aaaacagtgc ctggaatgca 2460
gacttaacat tttattgaat gaataaataa aaccccatct atcgagtgct actttgtgca 2520
agacccggtt ctgaggcatt tatatttatt gatttattta attctcattt aaccatgaag 2580
gaggtactat cactatcctt attttatagt tgataaagat aaagcccaga gaaatgaatt 2640
aactcaccca aagtcatgta gctaagtgac agggcaaaaa ttcaaaccag ttceccaact 2700
ttacgtgatt aatactgtgc tatactgcct ctctgatcat atggcatgga atgcagacat 2760
ctgctccgta aggcagaata tggaaggaga ttggaggatg acacaaaacc agcataatat 2820
cagaggaaaa gtccaaacag gacctgaact gatagaaaag ttgttactcc tggtgtagtc 2880
gcatcgacat cttgatgaac tggtggctga cacaacatac attggcttga tgtgtacata 2940
ttatttgtag ttgtgtgtgt atttttatat atatatttgt aatattgaaa tagtcataat 3000
ttactaaagg cctaccattt gccaggcatt tttacatttg tcccctctaa tcttttgatg 3060
agatgatcag attggattac ttggccttga agatgatata tctacatcta tatctatatc 3120
tatatctata tctatatcta tatctatatc tatatctata tatgtatatc agaaaagctg 3180
aaatatgttt tgtaaagtta taaagatttc agactttata gaatctggga tttgccaaat 3240
gtaacccctt tctctacatt aaacccatgt tggaacaaat acatttatta ttcattcatc 3300
aaatgttgct gagtcctggc tatgaaccag acactgtgaa agcctttggg atattttgcc 3360
catgcttggg caagcttata tagtttgctt cataaaactc tatttcagtt cttcataact 3420
aatacttcat gactattgct tttcaggtat tccttcataa caaatacttt ggctttcata 3480
tatttgagta aagtccccct tgaggaagag tagaagaact gcactttgta aatactatcc 3540
tggaatccaa acggatagac aaggatggtg ctacctcttt ctggagagta cgtgagcaag 3600
gcctgttttg ttaacatgtt ccttaggaga caaaacttag gagagacacg catagcagaa 3660
aatggacaaa aactaacaaa tgaatgggaa ttgtacttga ttagcattga agaccttgtt 3720
tatactatga taaatgtttg tatttgctgg aagtgctact gacggtaaac cctttttgtt 3780
taaatgtgtg ccctagtagc ttgcagtatg atctattttt taagtactgt acttagctta 3840
tttaaaaatt ttatgtttaa aattgcatag tgctctttca ttgaagaagt tttgagagag 3900
agatagaatt aaattcactt atcttaccat ctagagaaac ccaatgttaa aactttgttg 3960
tccattattt ctgtctttta ttcaacattt tttttagagg gtgggaggaa tacagaggag 4020
gtacaatgat acacaaatga gagcactctc catgtattgt tttgtcctgt ttttcagtta 4080
196
CA 02404060 2002-09-23
WO 01/72341 PCT/USO1/09042
acaatatatt atgagcatat ttccatttca ttaaatattc ttccacaaag ttattttgat 4140
ggctgtatat caccctactt tatgaatgta ccatattaat ttatttcctg gtgtgggtta 4200
tttgatttta taatcttacc tttagaataa tgaaacacct gtgaagcttt agaaaatact 4260
ggtgcctggg tctcaactcc acagattctg atttaactgg tctgggttac agactaggca 4320
ttgggaattc aaaaagttcc cccagtgatt ctaatgtgta gccaagatcg ggaacccttg 4380
tagacaggga tgataggagg tgagccactc ttagcatcca tcatttagta ttaacatcat 4440
catcttgagt tgctaagtga atgatgcacc tgacccactt tataaagaca catgtgcaaa 4500
taaaattatt ataggacttg gtttattagg gcttgtgctc taagttttct atgttaagcc 4560
atacatcgca tactaaatac tttaaaatgt accttattga catacatatt aagtgaaaag 4620
tgtttctgag ctaaacaatg acagcataat tatcaagcaa tgataatttg aaatgaattt 9680
attattctgc aacttaggga caagtcatct ctctgaattt tttgtacttt gagagtattt 9740
gttatatttg caagatgaag agtctgaatt ggtcagacaa tgtcttgtgt gcctggcata 4800
tgataggcat ttaatagttt taaagaatta atgtatttag atgaattgca taccaaatct 4860
gctgtctttt ctttatggct tcattaactt aatttgagag aaattaatta ttctgcaact 4920
3Q tagggacaag tcatgtcttt gaatattctg tagtttgagg agaatatttg ttatatttgc 4980
aaaataaaat aagtttgcaa gttttttttt tctgccccaa agagctctgt gtccttgaac 5040
ataaaataca aataaccgct atgctgttaa ttattggcaa atgtcccatt ttcaacctaa 5100
ggaaatacca taaagtaaca gatataccaa caaaaggtta ctagttaaca ggcattgcct 5160
gaaaagagta taaaagaatt tcagcatgat tttccatatt gtgcttccac cactgccaat 5220
aaca 5224
197
